Crimson Publishers High Impact Journals

Tuesday, May 31, 2022

Temperature Influence on the Process of the Gelation of the Engine Oil_Crimson Publishers

Temperature Influence on the Process of the Gelation of the Engine Oil by Liang Hong in Progress in Petrochemical Science_Journal of Petroleum Science and Engineering


Abstract

When a cool N2-CO2 co-gas stream flows over superheated activated carbon (AC) flakes, a vortex field near the interface is induced as a consequence of the confrontation of the cool co-gas stream and the hot vaporized carbon stream, where the pre-deposited Pt atom clusters on AC catalyze gasification of AC by CO2 to produce CO, and the CO undergoes immediate disproportionation to release carbon atoms in vapor form. The vortex field functions as a dynamic template for deposition of carbon vapor, leading to the proliferation of nano-sized carbon needles with characteristic spikes (ca. 1µm) and short in length through an anisotropic assembling of carbon atoms. A trace amount of Pt pre-coated on the AC flakes is sufficient to catalyze gasifying AC by CO2. This phenomenon is the first observation over the surface of amorphous carbon via catalytic pyrolysis without electric potential assistance.

Keywords: Nano carbon spines; Polypyrrole; HEC polymer; Raman spectra; Dendritic nano carbons; AC catalyze gasification; catalytic pyrolysis; Pt atom; CO gas treatment; Reverse boudouard reaction; AC flakes

Introduction

Dendritic growth is characterized by the presence of side branches that evolve under two different ways when the latent heat of fusion is removed from the interface [1]. Growth resulting from an undercooled melt (usually in alloys) results in equiaxed dendritic crystal formations when latent heat is dissipated through the cooler fluid at the interface whilst directional solidification or constrained growth results when the latent heat is dissipated swiftly.

This study unveils that the Pt atom clusters assist the generation of carbon atoms forming a vapor stream via a two-step reaction mechanism, which is responsible for the growth of dendritic dense carbon nanofibers from a porous carbon flakes. Such dendritic growth has been observed previously in cells [2-5], crystals [6,7] and metal alloys [8-13] with a characteristic tree-like structure, which is considered as the result of mass transfer under meta-stable thermodynamic state. Namely, the growth happens through a series of thermodynamic instabilities when the growth rate is limited by the rate of diffusion of solute atoms to the interface and the material is supercooled at the same time [14]. Dendrites have shapes that are most suitable for heat and mass transfers at small scales and hence, are highly attractive for applications seeking these properties. Numerous studies undertaken over the years offered the insights in dendritic growth of crystals [7], as well as mathematical models and simulations [6, 12-13,15-18] about the growth.

These growths are a result of faster material packing along energetically favorable crystallographic directions and may be due to anisotropy in the surface energy. In trying to minimize the area of these surfaces with the highest surface energy, the dendrite would exhibit a sharper and sharper tip as it grows [19]. When the crystallization front becomes morphologically unstable, small perturbations at the interface will lead to the formation of various polycrystalline structures, especially so for dendritic growth. The dendritic growth theory using Ivantsov transport theory relating to the dendrite tip radius and velocity of growth to the tip has been found to predict the growth rates and limitation of the existence of dendrites in 2D fairly accurately [1,14].

To the best of our knowledge, no reports or discussions on the dendritic carbon spinal growth in N2-CO2 co-gas atmosphere have been published nor observed before. Contrary to the growth of porous carbon fibers described explicitly in our previous work [20] caused by the random stacking of polyaromatic hydrocarbons (PAH) in the axial direction leading to the formation of fibers, this work proposes a different gowth path catalyzed by platinum atom clusters that assist with generation of CO via the reverse Boudouard reaction [21], which subsequently releases carbon atoms in vapor form that condense to form dense dendritic structures in the vortex field as illustrated in Figure 1. Although the incubation environment of this study is similar to that reported in [20], the resulting growth looks drastically different due to mediation by Pt atom clusters or colloids spread on the surface of the sample prior to the co-gas treatment. This paper aims to report and explain the growth mechanism we observed in detail.

Figure 1:


Experimental Procedure

Preparation of AC Flakes

An initial sample of 2-Hydroxyethyel cellulose (HEC) is carbonized by the method discussed elsewhere [22]. The resulting carbonaceous material from HEC was then activated at 700 oC under CO2 for 1 hour and cooled in an Ar purging stream. The carbon powder obtained was washed in water until the filtrate became colorless. This protocol resulted in an AC powder consisting of dense carbon flakes, which was used as the starting material for the preparation of the carbon needles.

Carbon Spinal Growth and Characterizations

Two separate, independent methods were employed to incorporate platinum onto the samples.

  1. Microwave approach: 0.2M Na2PtCl6.6H2O (Aldrich) was mixed with 0.2M SnCl2.2H2O (Aldrich) and 0.8M NaOH with ethylene glycol (Merck) and sonicated for 10 mins in an ultrasonic bath. The AC flakes (10g) was then added and the mixture sonicated for a further 10 minutes. Once this was complete, the mixture was placed in microwave and treated at high power for a minute and dried in a vacuum oven overnight [23]. The sample was subsequently treated at 800 °C under N2-CO2 co-gas atmosphere (50 cm3 min-1, 50 vol.% co-gas feed) for 5h to grow carbon needles.
  2. Sputtering approach: The AC flakes (10g) were coated with platinum via sputtering (JEOL JFC-1300 Auto Fine Coater, 90s, 30mA). The powder was then subjected to the N2-CO2 co-gas treatment as described above for 5h.

The final carbon samples obtained from both Pt-deposition methods were characterized by electron microscopy (JSM-6700F Field Emission Scanning Electron Microscope (FESEM), JEOL), Raman spectroscopy (Renishaw in Via Raman Microscope), and X-ray Diffraction (XRD, Bruker D8 Advance, Cu Ka radiation, l=1.54Å) using Cu target Ka-ray (40kV and 30mA) as X-ray source, respectively.

Result and Discussion

Formation of Dendritic Structures Over the Surface of AC Under Purge of CO-Gas

Under close examination using the transmission electron microscope (TEM), tree-like dendritic structures are observed to have formed from the sputtering coated AC specimen, with needle-like spinal growth sprouting forth from the “main vines” of the dendrites (Figure 2 & 3). Closer examination under a higher magnification reveals that these spines are dense and closely packed of different carbon blocks from base to tip (Figure 4). This type of growth is expected under supercooled condition, which presents itself when the elevated temperature at the Pt- deposited sites on AC (i.e. activated sites) comes into contact with the cool co-gas purge entering the reactor (Figure 1). During this process, Pt metal atom clusters deposited onto AC initiate conversion of PAH of AC to carbon atom species, which simultaneously experience a “supercooling effect” due to the contact with the entering co-gas stream. The tree-like dendritic needle structures start to grow in a specific radial direction due to the morphological instability [19] over the interface between AC and co-gas. The cool entering gas would form vortices upon reaching the region of superheated vaporized carbon species, leading to sporadic areas of cold fronts. The “supercooling effect” that takes place when these fronts meet therefore lead to the formation of the dendritic structure.

Figure 2: TEM image of dendrite produced by sputtered Pt; enlarged image of carbon spines (inset).


Figure 3: The tree-like growth from a main stem observed from the Pt sputtered AC flakes.


Figure 4: TEM image of a tip of a carbon spine.


After a time of exposure, the platinum atom clusters were gradually converted to volatile carbonyl complexes in the CO atmosphere that vaporize and are removed together with effluent gas stream, thereby abruptly terminating the growth. This was verified by the EDS elemental analysis that showed no platinum content remained within the sample after the treatment was complete. Based on the observations from the micrograph in Figure 5, the treated sample displays dense, needle-like dendritic structures growing radially from the AC flakes in massive quantities; atom clustered around the surfaces of the flakes (Figure 5 magnified view). The needles were short, dense and thin, with no apparent pores on their surfaces. They are also split driven by crystallization [24]. Since the “supercooling” was limited only to the activated sites catalyzed by the platinum atom clusters, resulting in short ranged dendritic growth because the Pt atom clusters were likely vaporized before long in the form of carbonyl complexes.

Figure 5: Dense needle-like spines growing radially from the Pt-sputtered AC flake sample.


When the activated carbon is sputtered with platinum, most of the platinum atom cluster are miniature and uniformly spread over the surface of AC. It is found that the polymer precursor contributes significantly towards the development of the cactus-like structures with further branches growing outwardly from a main stem (Figure 2). A previous study done [22] by our group on the effects of HEC side chain groups on the final structure formed after carbonization and activation has shown the structural differences in PAH flakes, which would profoundly affect the final porous structures of the AC obtained. This phenomenon of similar dendritic growth had previously been observed in CO2-cyclopentane hydrates in solution [25] but neither in gaseous nor solid environments. Furthermore, an attempt to cultivate these fibers using polypyrrole as carbon source was unsuccessful due to a different PAH flake structure.

Formation of Carbon Spinal Structures

It is noteworthy that although similar dendritic growth resulted under both Pt-deposition conditions, growth of carbon needle-like spines could only be resulted from the sputtering approach. On the contrary, the microwave approach laid a less uniform covering of Pt colloids on AC and hence resulted coarse dendritic tree structure (Figure 6) due to dilute nucleation spots. The discussion, therefore, focuses the incubation of the Pt-sputtered sample.

Figure 6: The tree-like growth on the AC flakes prepared by the microwave-aided deposition approach.


When scrutinizing the dark dots seen in the micrograph (Figure 5), it is a sodium chloride crystal resting on a small carbon center and not a platinum particle. Interestingly, it was apparent the branch-like dendritic structures exhibiting carbon needle-like outward growth originated from the activated flakes that the NaCl grains rested upon (Figure 7). The NaCl was known to leave behind from HEC polymer and, more importantly, this observation supports the view that PAH of AC were etched by CO2 mediated by the Pt atom clusters, namely, this gasification of AC concentrates NaCl distributing in AC simultaneously. Therefore, each NaCl grains represents a Pt-catalyzing gasification site. These sites occurred across the whole sample, but growth stopped a short distance radially away from the flakes (Figure 5). These NaCl grains hence label the original locations of Pt atom clusters before they were eventually vaporized as carbonyl complexes, terminating the growth of dendritic needles.

Figure 7: Carbon spines growing radially from a carbon flake center.


Besides, the surface analysis performed on the incubated sample shows a surface area of 951m2/g with pore volume of ca. 0.4cm3/g of sample. This concurs with our previous observation [20] that a larger surface area gained, compared to AC flake, can be attributed to the growth of needles whilst it is dense and hence, has a lower pore volume. Moreover, to understand whether the polymer precursor of AC affects dendric growth, glucose polymer and polypyrrole were in place of HEC, respectively, as the precursor for the AC flakes. The results were highly encouraging as thick, dense fibers were observed to grow on the surface of the AC flakes from the glucose polymer. However, no any spine was found on the polypyrrole-based AC flakes possibly due to a lack of oxygenated groups, which are present abundantly in AC prepared from glucose polymer as well as HEC.

Structural Characteristics of the Carbon Spines

We then used XRD method to examine possible crystalline structures of all the samples related to this study (Figure 8). It includes the following information:

Figure 8: XRD plot comparing the various samples related to this study. The x sign labels the crystalline NaCl phase.


  1. HEC polymer (pink) and AC flakes (red) are amorphous;
  2. Incubation without Pt-coating under different atmospheres: N2 (black) and co-gas (blue) for 5h. Both samples reveal different XRD patterns. This signifies two vaporization mechanisms, namely, sublimation of PAH in N2 to form C fibers [20] and the participation of the uncatalyzed reverse Boudouard reaction in co-gas to shape the C fibers;
  3. Extension of the un-catalyzed incubation in co-gas for 19h (green) resulted in removal of the crystalline carbon phase as reported in [20];
  4. Incubation of the AC flake with sputtered Pt for 5h (purple) presents a series of diffraction peaks due to generation of dendritic carbon spines displayed in Figures [2-5]. As stated above, this sample does not include Pt species. However, it also does not show the typical NaCl-XRD pattern, implying that the previously observed NaCl grains should have an alternative crystalline structure represented by the XRD pattern (green) owing to the entering of carbon atoms into NaCl lattice.

Raman spectroscopy characterization technique was employed to characterize the carbon skeleton structures in the two incubated samples differentiated by the different Pt-deposition methods. Both spectra obtained (Figure 9) display the D-band, a band around 1330cm-1 that is the result of a hybridized vibrational mode associated with the edges of graphene sheets, and G-band, a band around 1550-1580cm-1 arising from in-plane stretching of graphene sheets. The spectrum of the microwaved sample shows a peak ratio of the D-band to the G-band of about 1.7, while the same ratio of the sputtered sample is slightly greater (c.a. 1.9) than the above one. This implies that the sputtered sample has a higher quantity of edges of graphene than the microwaved sample [26]. This on the other hand proves the more delicate carbon spines that certainly contains a larger portion of edge of the stacked graphene sheets. In addition, the G band of the sputtered sample occurs at 1583cm-1, which is higher than the microwaved sample at 1556cm-1. This difference proposes that the graphene sheets of the later sample is relatively movable in-plane displacement, which may be interpreted as the result of containing more defects or a lower density of sheet stacking. Moreover, the presence of a broad band at 2750cm-1 is the 2nd-order “G” band, the overtone of the D-band frequency [27]. The relative peak intensity of this band relative to the respective G band is slightly stronger in the spectrum of the microwaved sample than in the sputtered sample, which coincides the above analysis about the difference in the G-band frequencies of these two samples.

Figure 9: Raman Spectra of the incubated samples initially prepared by the Pt-sputtering and (Sputtered Pt) and microwave depositing methods (Microwaved Pt).


Conclusion

This study demonstrates a finding of the growth of dendritic nanocarbon needles over the surface of an activated carbon flakes, which is pre-coated with a layer of platinum atom clusters, under a flow of N2-CO2 co-gas. This phenomenon has never been observed anywhere else before for a solid-gaseous interface nor ever for a solid carbon substance. The gas purge plays a vital role in this g-s reaction as it promps vortex field when meeting the hot carbon stream rising from carbon surface, but more importantly, the Pt atom clusters catalyze gasification of carbon (the reverse Boudouard reaction) by CO2 to produce CO. The instant disproportionation of CO provides carton atom stream for the in-situ growth of carbon dendrites. There are three conditions affecting this growth, firstly, the co-gas; secondly, the method to lay the Pt coating on carbon flakes; and thirdly, the polymer precursor to make the activated carbon flakes. The amount of platinum coating is also pivotal in the final structure of dendritic growth. Whilst treatment with lower concentrations of platinum colloids resulted in bushy tree-like structures, increasing the platinum atom cluster loading results in spikey cactus-like growth of carbon needle-like spines. As for the last factor, it requires the carbon flakes to be produced from a high oxygen-content polymer, e.g. hydroxyethyl cellulose or glucose polymer, but rather polypyrrole since the carbon flakes must be able to be gasified by CO2 under the catalysis of Pt atom clusters.

References

  1. Trivedi R, Kurz W (1994) Dendritic growth. International Materials Reviews 39(2): 49-74.
  2. Jan YN, Jan LY (2010) Branching out: Mechanisms of dendritic arborization. Nat Rev Neurosci 11(5): 316-328.
  3. Wong ROL, Ghosh A (2002) Activity-dependent regulation of dendritic growth and Nat Rev Neurosci 3(10): 803-812.
  4. Nacher J, Guirado R, Castillo GE (2013) Structural plasticity of interneurons in the adult brain: Role of PSA-NCAM and implications for psychiatric disorders. Neurochemical Research 38(6): 1122-1133.
  5. Shirao T, González-Billault C (2013) Actin filaments and microtubules in dendritic spines. Journal of Neurochemistry 126(2): 155-164.
  6. Miyata Y, Shima Y (1994) Self-consistent analytical theory of dendritic growth with Advanced Materials 93, Elsevier pp. 621-624.
  7. Haxhimali T, Karma A, Gonzales F, Rappaz M (2006) Orientation selection in dendritic evolution. Nat Mater 5(8): 660-664.
  8. Acer E, Erol H, Gündüz M (2013) Relationship between growth rates and dendritic microstructure parameters in Al-5wt. Zn binary alloy. Materials Science Forum 765: 215-219.
  9. Chen M, Hu XD, Ju DY, Zhao HY (2013) The microstructure prediction of magnesium alloy crystal growth in directional solidification. Computational Materials Science 79: 684- 690.
  10. Kaldre I, Fautrelle Y, Etay J, Bojarevics A, Buligins L (2013) Thermoelectric current and magnetic field interaction influence on the structure of directionally solidified Sn-10 wt.%Pb alloy. Journal of Alloys and Compounds 571: 50-55.
  11. Patakham U, Kajornchaiyakul J, Limmaneevichitr C (2013) Modification mechanism of eutectic silicon in Al-6Si-0.3Mg alloy with scandium. Journal of Alloys and Compounds 575: 273-284.
  12. Wu MW, Xiong SM (2012) Modeling of equiaxed and columnar dendritic growth of magnesium alloy. Transactions of Nonferrous Metals Society of China 22(9): 2212-2219.
  13. Zhao P, Vénere M, Heinrich JC, Poirier DR (2003) Modeling dendritic growth of a binary alloy. Journal of Computational Physics 188(2): 434-461.
  14. Criscione A, Kintea D, Tuković Z, Jakirlić S, Roisman IV, et al. (2013) Crystallization of supercooled water: A level-set-based modeling of the dendrite tip velocity. International Journal of Heat and Mass Transfer 66: 830-837.
  15. Akolkar R (2013) Mathematical model of the dendritic growth during lithium Journal of Power Sources 232: 23-28.
  16. Sun DK, Zhu MF, Pan SY, Yang CR, Raabe D (2011) Lattice Boltzmann modeling of dendritic growth in forced and natural convection. Computers & Mathematics with Applications 61(12): 3585-3592.
  17. Hunt JD, Lu SZ (1993) Numerical modelling of cellular and dendritic array growth: Spacing and structure predictions. Materials Science and Engineering: A 173(1-2): 79-83.
  18. Miyata Y, Shima Y (1994) Self-consistent analytical theory of dendritic growth with Advanced Materials 93, Elsevier pp. 621-624.
  19. Haxhimali T, Karma A, Gonzales F, Rappaz M (2006) Orientation selection in dendritic evolution. Nat Mater 5: 660-664.
  20. Zhou YE, Hong L (2013) The growth of porous carbon fibres through in-situ vapour RSC Advances 3: 19769-19773.
  21. Liu YY, Liu L, Hong L (2017) Gasification of char with CO2 to produce CO impact ofcatalyst carbon interface. Catalysis Today 281: 352-359.
  22. Sun M, Hong L (2011) Impacts of the pendant functional groups of cellulose precursor on the generation of pore structures of activated carbons. Carbon 49(7): 2173-2180.
  23. Liu Z, Lee JY, Chen W, Han M, Gan LM (2004) Physical and electrochemical characterizations of microwave-assisted polyol preparation of carbon-supported PtRu nanoparticles. Langmuir 20(1): 181-187.
  24. Kharissova OV, Kharisov BI (2010) Less-common nanostructures in the forms of Ind Eng Chem Res 49(22): 11142-11169.
  25. Lim YA, Babu P, Kumar R, Linga P (2013) Morphology of carbon dioxide-hydrogen-cyclopentane hydrates with or without sodium dodecyl sulfate. Cryst Growth Des 13(5): 2047-2059.
  26. Costa S, Borowiak PE, Kruszyñska M, Bachmatiuk A, Kaleńczuk RJ (2008) Characterization of carbon nanotubes by raman spectroscopy. Materials Science Poland 26(2): 433-441.
  27. Dresselhaus MS, Pimenta MA, Eklund PC, Dresselhaus G (2000) Raman scattering in fullerenes and related carbon-based materials, in: W Weber, R Merlin (Eds.) Raman Scattering in Materials Science, Springer Berlin Heidelberg pp. 314-364.

Publishers: https://crimsonpublishers.com/

For more articles in Journal of Petroleum Science and Engineering
Please click on below link: https://crimsonpublishers.com/pps/

Monday, May 30, 2022

Candida Tropicalis Infection of the Knee Joint in an Immunocompromised Pediatric Patient: A Case Report_Crimson Publishers

Candida Tropicalis Infection of the Knee Joint in an Immunocompromised Pediatric Patient: A Case Report by Benjamin Sookhoo in Surgical Medicine Open Access Journal_journal of Surgical Medicine


Abstract

Background: Candida septic arthritis is a debilitating condition affecting joint function. Candida tropicalis, an organism found in normal human flora is noted to be the third most common pathogenic yeast in the elderly and immunosuppressed population. Infections are rare in the US, typically limited to the neonate and elderly populations. Most infections occur in the south America and southeast Asian regions. In the last 2 decades, Tropialis infection rates have risen impart due to antifungal resistance. We present a case of Candida tropicalis infection in the knee of a 13-year-old female with a past history of relapsed Acute Myeloid Leukemia following bone marrow transplant, pancytopenia and graft vs. host disease.

Methods: Patient case is thoroughly discussed, and a review of the current literature performed.

Result: Patient underwent multiple open irrigation and sharp debridement as well as arthroscopic complete synovectomy in addition to medical management. Patient remained pancytopenic throughout her course and eventually underwent granulocyte transfusion. During her course, she developed a relapse of her AML and subsequently expired. Limited case reports exist in the literature, with no recent reports of Candida arthritis in an adolescent in the United States.

Discussion: Candida septic arthritis remains a rare but potentially devastating condition, particularly in the immunocompromised patient population. Candida arthritis should be high on the treating physician’s suspicion in immunocompromised patients presenting with new onset joint pain. Aggressive medical and surgical management is recommended, and current literature has shown a high cure rate with early treatment.

Introduction

Septic arthritis due to Candida species is a relatively rare infection typically seen in patients with immunocompromising conditions. While C. albicans is the most commonly encountered fungal species affecting humans, C. tropicalis has demonstrated increasing resistance to anti-fungal medications leading to persistent and difficult to control infections [1-15]. C. tropicalis is part of the normal human microbiota and is typically found on the skin and mucosal surfaces of the body. It is the third most common Non-Candida albicans Candida species (NCAC) isolated in clinical practice and primarily found in tropical regions, particularly Asia and South America [1,8]. Few reports of Tropicalis septic arthritis in North America have been published. We present a case report of a confirmed monoarticular C. tropicalis septic arthritis in a pediatric patient with chemotherapy induced granulocytopenia the setting of relapsed acute myeloid leukemia that lead to systemic candidiasis and patient mortality.

Case Report

Our patient is a 13-year-old Caucasian female who presented to our institution with relapsed AML following bone marrow transplant with clinical signs and symptoms concerning for graft vs. host disease. She had undergone previous chemotherapy treatment a little over a month prior to her admission and had an indwelling catheter. The patient was scheduled for intrathecal chemotherapy administration and was undergoing preoperative evaluation when it was noticed she had a diffuse rash and endorsing febrile episodes as well as diffuse joint pain. Pre-operative labs showed of white count of 0.1 cells/mm3 as well as elevated inflammatory markers. She was admitted to our Children’s hospital for further work up and evaluation. 2 days into her hospital stay, patient awoke in the middle of the night new onset severe left knee pain and swelling with inability to bear weight to the extremity. An MRI of the knee (Figure 1), showed a moderate effusion to the joint. An arthrocentesis was performed and synovial fluid culture demonstrated fungal growth that was identified as C. tropicalis.

Figure 1: MRI of the knee showing moderate effusion.


Figure 2: TArthroscopic views of the knee showing plaque formation on the femoral condyles.


Patient was started on IV Micafungin and was taken to the operating room where a formal irrigation and debridement was performed. A large amount of dark, viscous and serosanguinous fluid was encountered and evacuated from the joint along with a large of necrotic looking synovial tissue in the lateral gutter of the knee joint. Following initial I&D, patient continued to have febrile episodes and rapidly progressive skin rash. Initial medical work up including echocardiogram and CT scans of the abdomen and pelvis showed no evidence of vegetations or hepato-splenic candidiasis. Over the next 2 weeks, our patient subsequently underwent 3 additional open knee irrigations with sharp debridement of the synovium due to failure of response, despite adjustments in anti-fungal medications, including addition of Amphotericin B. Patient subsequently underwent a complete an arthroscopic synovectomy to better reduce her fungal load. An abundance of white plaques was noted on her femoral condyles that were determined to be yeast colonies on culture, (Figure 2).

In the two weeks following her final surgery, patient continue to have significant pain and swelling to the left knee, however, she remained afebrile. Her overall clinical picture soon began to decline, and repeat echocardiogram demonstrated a pericardial effusion with loculations that was not present on initial examination. Chest radiographs taken 2 weeks apart, (Figure 3), show enlarging of the cardiac silhouette. Her renal function subsequently began to decline as well, secondary to the Amphotericin B. Amphotericin B was discontinued and she was started on Voriconazole. Her oncology and pediatric medical teams decided to initiate a granulocyte transfusion to due to her continued pancytopenia and poor response to medical and surgical treatment of her infection. Her granulocyte transfusion was not well tolerated, and the patient developed respiratory distress in addition to worsening kidney function. CBC following her granulocyte transfusion showed peripheral blasts cells that were confirmed by flow cytometry to be relapsed AML. After a family meeting with the medical teams, a decision was made to place the patient on hospice and comfort measures. She was discharged from the hospital and expired at home the following day.

Figure 3: Chest radiographs obtained 2 weeks apart demonstrating enlarged cardiac silhouette.


Discussion

Septic arthrosis with Candida tropicalis infection is a rare infection in the United States. Patients at risk for developing this infection include those with immunocompromising conditions including malignancy, HIV/AIDS, neonates, patients with prolonged ICU stays with or without mechanical ventilation, those with indwelling central venous catheters, prolonged broad-spectrum antibiotic use, direct inoculation or previous surgery [1-2]. Systemic infections with tropicalis are common in Asian and South American countries, where various studies have shown to be the third most commonly isolated species in patients with systemic candidemia. C. tropicalis produces more persistent infections than C. albicans leading to longer hospital stays [3-5,9]. Recent studies have also shown that C. tropicalis is becoming increasingly resistant to antifungals as a result of C. tropicalis’ various virulent factors, including biofilm formation, adhesion molecules, cell wall hydrophobicity as well as extracellular proteases and phospholipases [6]. It has been shown to have a higher mortality rate when compared to all other non-Candida Albicans candidemia (NCAC) species with mortality rates surpassing C. albicans [1,8].

Septic joint arthrosis and osteomyelitis caused by Candida species are typically seeded by hematogenous dissemination, direct inoculation or direct extension from nearby focus of infection. Knee joints are the most common site of infection, owning to the highly vascular synovium, which lacks a limiting basement membrane allowing for easy passage for joint inoculation [10]. In pediatric patients, open epiphyseal plates allow for hematogenously disseminated organisms to extend into the joint as well as the metaphysis, setting the stage for osteomyelitis [1,7]. Candida arthritis presents with a clinical picture similar to bacterial septic arthritis, however, fever may not be a presenting symptom and inflammatory markers may only be moderately elevated, particularly in immunocompromised hosts. The most common presenting symptoms include joint pain and effusion with limitations to range of motion and weightbearing secondary to pain. Surgical findings include thickened fibrotic synovium, cartilage damage and purulence [9,10].

The current literature regarding septic arthritis with C. tropicalis in pediatric populations is sparse with the vast majority of case reports emerge from outside of the United States [11-14]. Gamaletsou et al. [1] performed a systematic review of the literature of 112 cases of adult and pediatric with confirmed diagnosis of Candida arthritis. They found that C. tropicalis infection occurred in 14% of the cases identified and was the second most common recovered species behind C. albicans. 36% of the patients identified in their study pediatric, with the vast majority being neonates. 78% of the patients were cleared of the infection with either medical therapy alone or in conjunction with surgical intervention. However, while there was no significant difference between patients treated with medical therapy alone or combine with surgery, there were far fewer deaths with the latter. McCullers and Flynn reported on a case of tropicalis arthritis and osteomyelitis in a 5-year-old male following chemotherapy treatment for acute lymphocytic leukemia. This infection was successfully treated with a prolonged course of amphotericin B and rifampin. Their review of the literature found 11 additional cases involving adult and pediatric patients, with 3 originating from their institution. They reported a 11.2% risk of invasive disease among immunocompromised patients with culture positive C. tropicalis compared to 2% risk in those colonized with C. albicans.

The Infectious Disease Society of America set forth clinical practice guidelines for the treatment of Candida septic arthritis. Management of native joint septic candidiasis that received strong recommendation included fluconazole for a minimum of 6 weeks or an echinocandin such as caspofungin, micafungin or anidulafungin for 2 weeks followed by a minimum of 4 weeks of fluconazole. Surgical drainage was indicated for all cases of septic arthritis. The use of intra-articular amphotericin B has been reported, however, this modality remains controversial.

Conclusion

We present a case of monoarticular C. tropicalis arthritis in an adolescent female with relapsed acute myeloid leukemia that was unsuccessfully treated despite aggressive medical and surgical intervention. There are few reports in the literature of septic joint candidiasis caused by C. tropicalis in the pediatric population, and to our knowledge, none have been reported in North America involving the adolescent age group that resulted in patient death. Most cases reported in the literature have shown C. tropicalis septic arthritis and osteomyelitis to be successfully treated with prolonged administration of anti-fungal agents and surgical management. While this condition presents with a clinical picture similar to bacterial arthritis, the clinician should have a high suspicion for Candida arthritis in patients with immunocompromising conditions and with indwelling catheters with new onset joint pain and effusion to reduce patient morbidity and mortality.

References

  1. Gamaletsou MN, Rammaert B, Bueno MA, Sipsas NV, Moriyama B (2015) Candida arthritis: Analysis of 112 pediatric and adult cases. Open Forum of Infectious Disease 3(1): ofv207.
  2. Zuza Alves DL, Silva Rocha WP, Chaves GM (2017) An update on Candida tropicalis based on basic and clinical approaches. Front Microbiol 13(8): 1927.
  3. Kontoyiannis DP, Vaziri I, Hanna HA, Boktour M, Thornby J (2001) Risk factors for Candida tropicalis fungemia in patients with cancer. Clin Infect Dis 33(10): 1676-1681.
  4. Pappas PG, Kauffman CA, David RA, Cornelius JC, Kieren AM (2016) Clinical practice guideline for the management of candidiasis: 2016 update by the infectious disease’s society of America. Clinical Infectious Diseases (62)4: e1-e50.
  5. Sónia S, Negri M, Henriques M, Oliveira R, Williams DW (2012) Candida glabrata, Candida parapsilosis and Candida tropicalis: Biology, epidemiology, pathogenicity and antifungal resistance. FEMS Microbiology Reviews 36(2): 288-305.
  6. JA McCullers, Flynn PM (1998) Candida tropicalis osteomyelitis: Case report and review. Clinical Infectious Diseases 26(4): 1000-1001.
  7. Wang HP, Yen YF, Chen WS, Chou YL, Tsai CY (2007) An unusual case of Candida tropicalis and Candida krusei arthritis in a patient with acute myelogenous leukemia before chemotherapy. Clin Rheumatol 26(7): 1195-1197.
  8. Fanning S, Mitchell AP (2012) Fungal biofilms. PLoS Pathog 8(4): e1002585.
  9. McCarty TP, Pappas PG (2016) Invasive candidiasis. Infect Dis Clin North Am 30(1): 103-124.
  10. Krcmery V Jr, Mrazova M, Kunova A, Grey E, Mardiak J (1999) Nosocomial candidaemias due to species other than Candida albicans in cancer patients. Aetiology, risk factors, and outcome of 45 episodes within 10 years in a single cancer institution. Support Care Cancer 7(6): 428-431.
  11. Bariteau JT, Warvasz GR, McDonnell M, Fischer SA, Hayda RA (2014) Fungal osteomylietis and septic arthritis. J Am Acad Ortho Surg 22(6): 390-401.
  12. Hu XR, He JS, Ye XJ, Zheng WY, Wu WJ (2008) Candida tropicalis arthritis in a patient with acute leukemia. Zhongguo Shi Yan Xue Ye Xue Za Zhi 16(5): 1215-1218.
  13. Sim JP, Kho BC, Liu HS, Yung R, Chan JC (2005) Candida tropicalis arthritis of the knee in a patient with acute lymphoblastic leukemia: Successfully treatment with caspofungin. Hong Kong Med J 11(2): 120-123.
  14. Vicari P, Feitosa PR, Chauffaille ML, Yamamoto M, Figueiredo MS (2003) Septic arthritis as the first sign of candida tropicalis fungaemia in an acute lymphoid leukemia patient. Braz J Infect Dis 7(6): 426-428.
  15. Weisse ME, Person DA, Berkenbaugh JT (1993) Treatment of candida arthritis with flucytosine and amphotericin B. J Perinatol 13(5): 402-404.

Publishers: https://crimsonpublishers.com/

For more articles in journal of Surgical Medicine
Please click on below link: https://crimsonpublishers.com/smoaj/

Friday, May 27, 2022

Genetic Characteristics of Oocytes and Somatic Cells of Ovarian Follicles in Humans_Crimson Publishers

Genetic Characteristics of Oocytes and Somatic Cells of Ovarian Follicles in Humans by Bezerra FTG in Investigations in Gynecology Research & Womens Health_Scholarly articles for women's health journal


Mini Review

Since most of the RNAs/proteins that support the initial phases of embryogenesis are accumulated during oocyte growth [1], knowledge about the effects of oocyte quality on embryo development plays a crucial role in the development of adequate assisted reproduction techniques. In this sense, understanding the requirements of oocytes recovered from different sized follicles is very important to optimize IVM and IVF of oocytes, which can have an excellent impact on the number of embryos produced in vitro. It will also allow analysis of the developmental potential of oocytes at different stages of development, the pattern of gene expression, the epigenetic modifications and the cytogenetic disorders in various domestic species, including humans [2].

Influence of oocyte and follicle sizes on oocyte maturation

In human oocytes, gain of meiosis ability starts at the antral follicle stage, while its size reaches up to 100-120μm. Theoretically, antral follicles with diameter of 2.0-5.0mm contain oocytes with nuclear and cytoplasmic competence [3]. However, the minimum size of follicles required for developmental competence in humans is estimated to be 5.0-7.0mm in diameter [4]. It is possible that the selection of the dominant follicle induces changes in the remaining follicles that are detrimental for subsequent oocyte fertilization and embryonic development. Fadini et al. [5] reported that follicles of up to 12mm in size at the time of oocyte retrieval have not compromised their outcome. Taken together, the above information proposes that the molecular events that are triggered according to follicular and oocyte development influence oocyte maturation.

Gene expression in oocytes from follicles of different sizes

It is well known that while the oocyte progresses in growth and development, it acquires maternal stores (mRNAs and proteins) which are essential to support the development of the embryo during the early cleavage stages [6]. Under in-vitro conditions, the dynamic micro RNA (miRNA) profile changes are partly attributed to the in-vitro maturation environment or ingredients used, while under in-vivo conditions, the miRNA profile is affected by physiological conditions, like the age. In humans, treatment of metaphase I (MI) human oocytes with insulin-like growth factor 1 activates the expression of miR-133a, miR-205-5p and 145 miRNAs and suppresses 200 others, including miR-152 and miR-142-5p [7]. In the same context, [8] demonstrated that YTHDF2 post-transcriptionally regulates transcript dosage during oocyte maturation and act as determinant of mammalian egg quality. Several studies have correlated the oocyte ATP content and its developmental competence, both in humans [9].

Characteristics of somatic cells of follicles at different sizes

Within an antral follicle, the oocyte is surrounded by several layers of cumulus and mural granulosa cells. It is known that the characteristics of these cells vary according to the size of the follicle and this can directly affect the ability of the enclosed oocyte to undergo maturation. A study with humans [10] showed that the morphology of CCs collected from follicles smaller than 12.0mm is different from those retrieved from mature follicles of standard IVF. Immature oocytes rescued from smaller antral follicles are usually embedded in more compact cumulus cells [10]. The number of dispersed CCs increases with duration of human chorionic gonadotrophin (HCG) priming and growing of follicular size in IVM program [11]. The patterns of CCs are divided into three groups: dispersed, compacted and sparse. Dispersed CCs have an expanded CC and multiple layers of corona cells. A GV oocyte that is completely masked with many layers of corona cells is considered as compacted CCs, while very few coronal cells exist in sparse CCs [3]. Comparison of different patterns of COCs reveals a significant increase in COCs with dispersed cumulus cells during oocyte maturation and blastocyst formation [3].

References

  1. Biase FH, Cao X, Zhong S (2014) Cell fate inclination within 2-cell and 4-cell mouse embryos revealed by single-cell RNA sequencing. Genome Res 24(11): 1787-1796.
  2. Sirard MA, Richard F, Blondin P, Robert C (2006) Contribution of the oocyte to embryo quality. Theriogenology 65(1): 126-136.
  3. Son WY, Chung JT, Dahan M (2011) Comparison of fertilization and embryonic development in sibling in vivo matured oocytes retrieved from different sizes follicles from in vitro maturation cycles. J Assist Reprod Genet 28(6): 539-544.
  4. Trounson A, Anderiesz C, Jones G (2001) Maturation of human oocytes in vitro and their developmental competence. Reproduction 121(1): 51-75.
  5. Fadini R, Canto MB, Mignini RM, Brambillasca F, Comi R, et al. (2009) Effect of different gonadotrophin priming on IVM of oocytes from women with normal ovaries: a prospective randomized study. Reprod Biomed Online 19(3): 343-351.
  6. Li L, Zheng P, Dean J (2010) Maternal control of early mouse development. Development 137(6): 859-870.
  7. Xiao G, Xia C, Yang J, Liu J, Du H, et al. (2014) MiR-133b regulates the expression of the Actin protein TAGLN2 during oocyte growth and maturation: a potential target for infertility therapy. PLoS ONE 9(6): e100751.
  8. Ivanova I, Much C, Giacomo M, Azzi C, Morgan M, et al. (2017) The RNA m6A reader YTHDF2 Is essential for the post-transcriptional regulation of the maternal transcriptome and oocyte competence. Mol Cell 67(6): 1059-1067.
  9. Blerkom J, Davis P, Lee J (1995) ATP content of human oocytes and developmental potential and outcome after in vitro fertilization and embryo transfer. Hum Reprod 10(2): 415-424.
  10. Yang Y, Kanno C, Sakaguchi K, Yanagawa Y, Katagiri S, et al. (2017) Extension of the culture period for the in vitro growth of bovine oocytes in the presence of bone morphogenetic protein-4 increases oocyte diameter but impairs subsequent developmental competence. Anim Sci J 88(11): 1686-1691.
  11. Pincus G, Enzmann EV (1935) The comparative behavior of mammalian eggs in vivo and in vitro: I. the activation of ovarian eggs. J Exp Med 62(5): 665-675.

Publishers: https://crimsonpublishers.com/

For more articles in Scholarly articles for women's health journal
Please click on below link: https://crimsonpublishers.com/igrwh/

Thursday, May 26, 2022

Importance of Environmental Management Systems in Ensuring Continual Environmental Protection in Offshore Upstream Oil and Gas Industry_Crimson Publishers

Importance of Environmental Management Systems in Ensuring Continual Environmental Protection in Offshore Upstream Oil and Gas Industry by Ana Rita Onodera Palmeira Nunes in Examines in Marine Biology & Oceanography_oceanography scientific articles


Abstract

The present work highlights the importance and benefits of establishing and implementing Environmental Management Systems (EMS) in offshore upstream oil and gas industry. It focuses on the priorities in pollution prevention and summarizes the pollution prevention model together with its associated selection process. It also delineates the environmental management system main elements and steps in view of ISO 14001 and how they can be used to achieve continual environmental protection in offshore upstream oil and gas industry.

Keywords: Environmental management system; Offshore oil and gas industry; ISO 14001; Upstream oil and gas industry

Introduction

The future health and well-being of the environment depends on what people do today. Unless people make drastic changes to the way they live, environment on which they depend will continue to deteriorate. Industry has long been considered the primary target of efforts to slow environmental degradation. For this reason, companies are voluntarily encouraged to have ISO 14001 to establish and maintain Environmental Management Systems (EMS). EMS standards, guidelines and operating procedures have been developed by organizations such as ISO, European Union and petroleum industry associations. The ISO, being an amalgamation of "standard bodies" from about 140 countries, has wider acceptability and it is well respected. For example, the international association of Oil and Gas Procedures (OGP), firmly supports the internationalization of standards, promotes the publication, development and use of ISO standards without modification [1]. Hence, ISO 14001 EMS is a widely recognized standard for environmental management in the petroleum and gas industry. The best practices that may ensure environmental protection in terms of environmental management procedures and practices are:

  1. Environmental Impact Assessment (EIA)/Socioeconomic Impact Assessment (SIA)/ Health, Safety, and Environmental Impact Assessment (HSEIA).
  2. Environmental Management Systems (EMS).
  3. Environmental Performance Evaluation (EPE).
  4. Environmental Monitoring and Auditing, and
  5. Environmental reporting [2]. EMS aims at achieving sustainable development which meets the needs of present without compromising ability of future generations to meet their own needs. Companies focuses on environmental management to:
  6. Comply with the applicable legislation.
  7. Avoid stakeholder pressure.
  8. Keep on image and reputation.
  9. Raise competitiveness.
  10. Achieve financial benefits.

ISO 14001 is the part of the overall company management system which includes organizational structure, planning activities, responsibilities, practices, procedures, processes, and resources for developing, implementing, achieving and maintaining the environmental policy, the main aims of a company EMS can be summarized as:

  1. Identification and control of aspects, impacts, and risks.
  2. Establishing and achieving an environmental policy, objectives, and targets including compliance with legislations.
  3. Identifying environmental opportunities.
  4. Monitoring and continual improvement of environmental performance [3]. Company environmental performance can be defined as the measurable results of the EMS, related to its control of its environmental aspects, based on its environmental policy, objectives and targets. The company environmental performance continual improvement is the process of enhancing the EMS to achieve improvements in overall environmental performance in line with the company environmental policy [3].

Objectives

The present work main objective is to evaluate EMS as a potential means of ensuring continual environmental protection from offshore upstream oil and gas industry. It also demonstrates the EMS main elements and steps in view of ISO 14001 and how EMS when adequately implemented, it should help reduce negative environmental impacts and help in achieving sustainable development in the offshore oil and gas highly important economic sector.

Methodology

This work is prepared using descriptive approach in addition to the authors knowledge and experience in the area of marine pollution and its control.

Results and Discussion

Elements of ISO 14001 according to Deming are:

Plan

Environmental policy, environmental aspects, legal requirements, objectives and targets, and environmental management program.

Do

Structure and responsibilities, training, communication, environmental management documentation, document control, operational control, and emergency preparedness.

Check/correct

Monitoring measurement, non-conformance/corrective/preventive actions.

Records and EMS audits, management review

The main components of the ISO 14001 Environmental management system are [3]:

  1. Policy.
  2. Planning.
  3. Implementation and operation.
  4. Checking and corrective actions, and
  5. Management review.

Environmental policy has to be defined by top management: Appropriate for the nature, scale, impacts of the activities, products, and services. It also has to reflect commitment for continual improvement and prevention of pollution, express commitment to comply with environmental legislation, regulations, and other requirements, highlight a framework for objectives and targets. It must be documented, implemented, maintained, communicated to all employees, suppliers and contractors in addition to its availability to the public. Planning includes preparation of:

  1. Initial review reflecting environmental aspects and impacts together with legal and other associated issues.
  2. Environmental policy.
  3. Objectives and targets, and
  4. Environmental management.

Implementation and operation implies on structure and responsibility, training, awareness and competence, communication, internal and external, document control, operational control for procedures, criteria, suppliers and contractors, and-emergency preparedness and response. Checking and corrective actions includes:

  1. Monitoring and measurement.
  2. Non-conformance and corrective actions, records, and EMS audit. Management review aims at checking suitability, adequacy and effectiveness of the EMS in view of the documentation findings and considering changes in environmental policy, objectives and targets in the light of changing circumstances and in view of performance continual improvement.

The company functions which may be affected by the development and implementation of the EMS are:

  1. Research and development.
  2. Manufacturing.
  3. Finance.
  4. Planning and development.
  5. Marketing, and management and distribution (retail and wholesale). Continual improvement in the company environmental performance can be ensured through the adequate implementation of the Deming model (Plan-Do-Check-Improve).

EMS preparation steps in view of ISO 14001 are: initial environmental review, environmental policy, environmental action plan, environmental responsibilities, environmental procedures, training, environmental auditing and management, and internal and external communication. The initial environmental review should cover laws, standards, and regulations, potential environmental issues and concerns, facility and operations description, management and operational practices, and previous environmental accidents, incidents and penalties.

The initial environmental review gives baseline for environmental management, current and future regulatory requirements, prioritization of areas of significant risk, advance identification of potential problems, and base for effective on-going appraisal of environmental performance. Environmental policy is a statement by the company of its intentions and principles in relation to its overall environmental performance. Environmental action plan is a process of narrowing down from broad, long-term goals through objectives and targets to a plan of action [3]. The plan of action meets environmental performance goals containing clear, measurable objectives and targets based on the environmental policy, identified priorities, and environmental aspects of operations.

The company can use the EMS as a dynamic tool to achieve continual environmental performance improvement through: providing resources needed, carrying out procedures and work tasks, initiating actions to prevent non-compliance with legal or policy requirements, recommending solutions to problems and verify implementation, control activities until required changes are carried out, and act in emergencies. An effective EMS must clearly define who does what in terms or structure, responsibilities, role and position of the environment, and management function.

Environmental training is needed to:

  1. Get the environmental message across the company, and
  2. Reinforce documentary and other communication initiatives of the environmental program. Types of environmental training include awareness of environmental issues, company environmental policy and program, environmental skills enhancement, environmental compliance and environmental management.

EMS environmental audit definition is a systematic documented verification process of objectively obtaining and evaluating evidence to determine whether an organization EMS, conforms with EMS audit criteria, and communicating the results of this process to the client [4]. The audit report usually includes executive summary, general information, audit findings, audit conclusion, and recommendations [4]. Problems that may hamper the implementation of an effective EMS can be summarized as follows:

  1. Incomplete identification of areas of potential environmental impact.
  2. Poor or ineffective consultation on draft procedures.
  3. Insufficient consideration of human factor.
  4. Instructions are poorly written or implemented.
  5. Insufficient or ineffective training.
  6. Out of date with organizational and operational changes.
  7. No monitoring or review.
  8. Ineffective incorporation of legal requirements, and
  9. Continuous improvement not included. Effective procedures are conversions of policy into a series of Coordinated activities and tasks which set out what people need to do, assist in identifying competencies which individuals require, and form the basis for measuring performance of the individual, group, and the company. The steps of setting and maintaining procedures are hazard identification, risk assessment, identifying risk control, preparing and implementing control and ongoing audits.

Coastal and marine environment protection aims at reducing harm to sea water, air, sediments and living organisms. The priorities and hierarchy to achieve pollution prevention in offshore areas start with:

  1. Prevent pollutants and wastes discharge followed by
  2. Eliminate and reduce waste generation.

Recycle, reuse and recover and finally treatment and waste disposal [5,6]. The conceptual design of the pollution prevention model starts with the materials procurement followed by materials utilization associated with the production and reuse and recycle practices followed by waste accumulation and on-site and off-site waste management and disposal [7].

References

  1. Alexandra SW (2002) Adoption of international standard by transnational oil companies: Reducing the impact of oil operations in emerging economies. Journal of Energy and Natural Resources 20(4): 402-434.
  2. Aboul Dahab O, Shaaban N (2017) Environmental protection and management in coastal and maritime industries. Oceanography and Fisheries Open Access Journal 5.
  3. (2015) ISO 14001. Environmental management systems-Requirements with guidance for use.
  4. (2018) ISO 19011. Guidelines for auditing management systems.
  5. Aboul Dahab O (2019 Sustainable development and waste management in offshore oil and gas drilling and production industry. Examines in Marine Biology and Oceanography. 3(2).
  6. Onwukwe SI, Nwakaudu MS (2012) Drilling wastes generation and management approach. International Journal of Environmental Science and Development 3(3).
  7. Sharif AS, Nagalak SHN, Stigowri RS, Vanth GU (2017) Drilling waste management and control the effects. Journal of Advanced Chemical Engineering 7(1).

https://crimsonpublishers.com/eimbo/fulltext/EIMBO.000565.php

Publishers: https://crimsonpublishers.com/

For more articles in oceanography scientific articles
Please click on below link: https://crimsonpublishers.com/eimbo/

Wednesday, May 25, 2022

Cellular and Bacterial Genetic Sequences in Monkey-Derived Stealth Adapted Viruses_Crimson Publishers

Cellular and Bacterial Genetic Sequences in Monkey-Derived Stealth Adapted Viruses by W John Martin in Cohesive Journal of Microbiology & Infectious Disease_open infectious diseases journal impact factor


Abstract

Stealth adapted viruses differ from the viruses from which they are derived in not being effectively recognized by the cellular immune system. This is because of the deletion or mutation of the genes coding for the relatively few virus components, which are generally targeted by cytotoxic T lymphocytes. Stealth adapted viruses do not, therefore, normally evoke inflammation, the hallmark of most infectious illnesses. A stealth adapted virus was repeatedly cultured from the blood of a patient with the chronic fatigue syndrome (CFS). Polymerase chain reaction (PCR) performed on the culture identified the virus as being derived from an African green monkey simian cytomegalovirus (SCMV). The PCR also amplified a genetic sequence closely related to a normal cellular gene. Further analysis of the viral DNA indicated that it was fragmented and genetically unstable. Moreover, additional genetic sequences have been incorporated into the replicating virus genome. Several of the additional sequences are originally of cellular origin with subsequent genetic modifications. Other incorporated sequences are of bacteria origin. PCR performed on cultures from some other CFS patients, led only to the amplification of modified cellular sequences, including sequences clearly derived from the rhesus monkey genome. It is proposed that as part of the stealth adaptation process, sequences of the original infecting virus can be largely displaced by cellular and/or bacteria sequences, which have essentially switched their affiliation to that of the stealth adapted virus. For this reason, they are referred to as renegade sequences. The term “renegade viruses.” is also proposed to describe those viruses in which the originating conventional virus sequences have yet to be detected. The findings are relevant to efforts to seek a virus cause of many common illnesses, including CFS, and to the possible misattribution of certain illnesses to bacterial infections.

Keywords: Renegade viruses; Stealth adapted viruses; Chronic fatigue syndrome, Polio vaccine; noncoding RNA; Ochrobactrum; Mycoplasma; Cytomegalovirus; Viteria

Abbreviations: ACE: Alternative Cellular Energy; CFS: Chronic Fatigue Syndrome; CPE: Cytopathic Effect; CFS: Cerebrospinal Fluid; NCBI: National Center for Biotechnology Information; NT: Nucleotide; PCR: Polymerase Chain Reaction; RhCM: Rhesus Monkey Cytomegalovirus; SCMV:  African Green Monkey Simian Cytomegalovirus

Introduction

A stealth adapted virus was repeatedly cultured from a CFS patient [1]. It induces a foamy vacuolated cytopathic effect (CPE) when cultured on human and animal cells, including human foreskin fibroblast cells (MHRF). Electron microscopy of the infected cells indicate the presence of both herpesvirus-like particles and intracellular inclusions. The polymerase chain reaction (PCR) was employed to identify the type of virus infecting the cells. Among the sets of primers used in the PCR were a forward primer “SK43” (5’-cggatacccagtctacgtgt-3’), which matches to sequence within the tax gene of human T lymphotropic virus-1 (HTLV-I) and a backward primer “SK44” (5’-gagctgacaacgcgtccatcg-3’), which matches to a sequence within the tax gene of HTLV-II [2]. Using relatively low stringency PCR, this set of primers yielded several PCR products when performed on the infected cultures, with no identifiable products being formed when using uninfected cultures [1]. The PCR products were cloned and sequenced. A radiolabeled PCR product was also used to identify virus DNA in extracts of infected cells and in the pelleted material obtained by ultracentrifugation of filtered culture supernatant. In agarose gel electrophoresis, the virus DNA in the pelleted material migrated with an estimated size of approximately 20 kilobases (kb) [1].

Cytomegalovirus-related DNA sequences

The PCR assay yielded prominent bands in agarose gel electrophoresis of approximately 0.5 and 1.5kb [1]. The 1.5kb band was subsequently shown to contain two PCR products, measuring 1,487 and 1,510 nucleotides, respectively. The PCR amplified sequences were flanked at both ends by the SK44 primer. Using the BLASTN and BLASTX programs [3], the amplified sequences were compared with the then available genetic sequences on GenBank; a repository of nucleic acid and protein sequences maintained by the National Center of Biotechnology Information (NCBI). The analysis showed that the longest sequence (included in NCBI Accession number U09212) was related to but still different from a sequence within human cytomegalovirus. As more GenBank sequences became available for comparison, the sequence of this PCR product was shown to have far greater sequence homology with African green monkey simian cytomegalovirus (SCMV) [4]. Similarly, the other large PCR product (NCBI Accession number U09213) was subsequently shown to match closely to a sequence within SCMV with 1348/1454 (93%) identical nucleotides, with 19 gaps [5]. The next closest match of the sequence of the PCR product is to the cytomegalovirus of Mandrillus (Dril) monkey with 938/1389 (68%) identical nucleotides and 124 gaps. There was even less overlap with the best matching rhesus monkey cytomegalovirus (RhCMV,) which had 364/515 identical nucleotides. Using the standard BLASTN program, there was no significant matching of the sequence in U09213 with human cytomegalovirus.

Detection of cellular-derived DNA sequence

The smaller product that was amplified in the PCR on infected but not on uninfected cultures was sequenced (NCBI accession number AF107851.1). This product was flanked at both ends with primers in which there was a single nucleotide difference (cytosine rather than adenosine at the 10th position) than in the originally intended SK43 primer. The DNA sequence of the PCR amplified product, excluding the primers, was analyzed against the most recent “assembled” complete human genome, This showed a near identical homology with an intergenic region of the human X chromosome (NCBI accession number NC_000023.11), extending from nucleotide 30,177,214 to nucleotide 30,177,822. Within this region there were 606/609 identical nucleotides (99.51 percent identity) with 2 single nucleotide deletions and one nucleotide substitution (Figure 1). When using the nr/nt (protein and nucleotide) database in the BLASTN program, there were only two matches to the human genome (NCBI Accession numbers AC117518.3 and AC005145.1). The next 3 matches, all with 97.5% identity, were to a sequence in the chimpanzee genome. It was considered possible that the X-chromosome-related sequence may have originated in an African green monkey. On further analysis using the primates’ “assembled genomes,” there was decreasing overall relatedness of the PCR product with the human genome to the genomes of chimpanzee, rhesus, and Cercopithecus (African green) monkeys. The data are included in Table 1 and establishes the X-chromosome related genetic sequence as being human in origin.

Figure 1: Matching of the sequence of the PCR product with that of a region of the human X chromosome. What is shown is the BLASTN readout obtained by submitting to GenBank, the nucleotide sequence of a cloned PCR product generated using the SK44/SK43 primers on DNA from the stealth adapted virus infected cultures. The submitted sequence is labeled as the “Query.” The “Sbjct” is the nucleotide sequence of the subject item, which is selected by the BLASTN program as having a matching nucleotide sequence. The vertical lines between the Query and Sbjct indicate nucleotide identity. Gaps occur in regions where one or more nucleotides are in only one of the pair of sequences. The numbers refer to numbered nucleotides in the Query and Sbjct. The Sbjct in this Figure is the human X chromosome (NCBI accession number NC_000023.11).


Table 1: The relative numbers and percentages of identical nucleotides of the cloned PCR product shown in Figure 1 with matching sequences of humans and several primates.


Cloning of the virus genome

Filtered supernatant of infected cultures was ultracentrifuged as a means of pelleting extracellular virus particles. DNA was extracted from the pelleted material. In a second study, the extracted DNA was further purified by electrophoresis into agarose gel, with excision of the narrowly banded DNA. The DNA from the first study was cut using the EcoR1 restriction enzyme and cloned into pBluescript plasmids (3B series). The DNA in the second study was cut using the Sac1 restriction enzyme and was also cloned into pBluescript plasmids (C16 series). At least partial DNA sequence data were obtained from over 450 clones using the T3 and T7 promoter sites on the pBluescript plasmid [5-7].  Several of the more interesting clones were completely sequenced. The sequence data on the individual clones were provided to GenBank under the listing of stealth virus 1. Using the BLASTN and BLASTX programs of the NCBI, the sequences fell into three categories. The majority of the clones contained sequences, which matched reasonably closely to regions of the SCMV genome. Several of the clones with sequences that match to the SCMV genome have been assembled into longer stretches of non-overlapping sequences. The NCBI accession numbers of these longer stretches are listed in Table 2 and collectively they comprise 97,780 nucleotides. The overall regions of the SCMV genome (length 226,205 nucleotides) which the BLASTN program matches to the stretches of the stealth adapted virus sequences are shown in Table 2, along with the number of identical nucleotides. With sequences, U27627.2 and U27770.2, there was an intervening region in which the stealth virus and SCMV sequences did not adequately match. There are disproportionate numbers of clones, which match to certain regions of the SCMV while there is no matching to other regions. There is also a diversity in the sequences in some of the clones, which match to the same region of the SCMV genome. These differences include nucleotide substitutions, deletions, repetitions, and recombination. Nevertheless, the results are unequivocal in indicating that SCMV was the originating virus and that it has undergone substantial genetic changes.

Table 2: BLASTN matching of the cloned sequences of stealth virus-1 with the sequence of Cercopithecine herpesvirus 5 strain 2715, NCBI FJ483968.2.


Detection of additional cellular sequences related to the human genome

A second category of cloned sequences were those that could be matched to various specific regions of the human genome [8,9]. The matching cellular sequences are in different human chromosomes. While the sequences of some of the clones are identical to the matching region of the human genome, many of the clones show minor differences. The most common nucleotide changes are multiple inserts, deletions and substitutions of single nucleotides. Occasionally longer inserts and larger deletions are present. The frequency of inserted, deleted, or substituted nucleotides in the clones, compared with the best matching human sequence is quite variable, but commonly in the range of 2-4%. Figure 2 shows portions of the BLASTN readouts obtained by submitting to GenBank, the DNA sequences of three clones derived from the virus culture. With each clone, the best matching subject sequences in GenBank’s nr/nt database are different human cellular genes. It was also thought possible that some of the clones might possibly match better to the genome of African green monkeys. The sequence analysis was, therefore, occasionally repeated using the assembled genome of African green monkey. In no case so far examined, however, is the matching to the monkey genome been closer than that to the human genome. Another observation is that the regions of partial homology between the cloned sequence and that of a human cellular gene is invariably occurring within the non-coding region of the human genes. A more comprehensive analysis of the relatedness of the cloned sequences to the human and primate genomes is ongoing and will be provided in a subsequent publication

Figure 2: Matching of cloned sequences with cellular sequences. What is shown are three examples in which a region from both the T3 and the T7 partial readouts of the sequences of three clones obtained from the purified DNA from the culture supernatant is directly compared with a matching Sbjct region of a cellular sequence. The six selected cloned sequences are labeled as Query, whereas the Sbjct is best matching cellular sequence. The numbers refer to the numbered nucleotides in the clones and in the Sbjct. The Sbjct for clones C16119 , C16128, and C16261 are Homo sapiens chromosome 8 clone ABC9_45365100_H16, complete sequence AC275379.1; Homo sapiens kazrin, periplakin interacting protein (KAZN), RefSeqGene on chromosome 1 Sequence ID: NG_029844.2; and Homo sapiens isolate fa0190 immunoglobulin superfamily member 4B (IGSF4B) gene AY663433.1, respectively.


Detection of bacteria-derived sequences

The third category of sequences identified in the cloned DNA are clearly bacterial in origin [10]. Even at the initial time of submission of the sequence data to GenBank, it was apparent that several of the clones were very closely related to mycoplasma bacteria [11]. Examples of clones with near-identical mycoplasma-related sequences are 3B35, 3B512, 3B528, and 3B632. The sequences these clones closely matched to different sequences, which are essentially identical in plasmid 7 of Mycoplasma conjunctivae strain NCTC10147 and in plasmid 9 of mycoplasma fermentans strain M64 [12,13]. The numbers of identical nucleotides in these four clones with the corresponding sequences in the mycoplasma plasmids 2136/2142, 2342/2345, 2020/2044, and 1380/1384 respectively. The sequences of several additional clones still preferentially match to mycoplasma but with lower levels of nucleotide identity. An even larger number of the clones have sequences, which at the time of submission to GenBank, matched most closely to Brucella, an alphaproteobacterial [14]. These sequences have subsequently been shown to have greater homology to one or other species of Ochrobactrum [15]. Examples of these clones are NCBI accession numbers 3B23, 3B41, 3B43, 3B47, 3B534, 3B614, C1616, and C16134.The different cloned sequences tend to preferentially match to O. quorumnocens, with somewhat less matching to Ochrobactrum pituitosum and Ochrobactrum pseudogrignonense [16-18].  Yet for some of the clones, there was only minimal or no matching to O. quorumnocens. Some of these data are summarized in Table 3.  For many of the cloned sequences, the changes from the best matching Ochrobactrum species are rather minor with less than 1% nucleotide substitution and only occasional gaps. Still other clones have sequences, which, although seemingly of bacterial origin, cannot be currently assigned to a species of bacteria. Some of these clones appear to be broadly related to certain alphaproteobacteria, but only when using the BLASTX program of the NCBI. An example is clone 3B513 (NCBI accession number U27894.2), which comprises 8,106 nucleotides.  Other cloned sequences of apparent bacteria origin show a weak association with Actinobacteria.

Table 3: The relative matching of the cloned sequences from the DNA of the stealth adapted virus cultures with three species of Ochrobactrum bacteria.


Genetically unstable and fragmented virus genome

As noted above, the sequences in many of the clones correspond to diverse regions of the SCMV genome. There were many examples of significant differences between clones even when they matched to the same region of the SCMV genome. These differences were attributed to genetic instability of the virus genome, including mutations, deletions, insertions, and recombination. Furthermore, the aggregate length of non-overlapping virus sequences far exceeded the estimated 20 kb genome size based on the hybridization pattern seen in agarose gel electrophoresis. This result is consistent with a fragmented genome [19]. It is also possible that some of the DNA fragments are held together via RNA, which can be subjected to breakage from environmental sources of RNases.

PCR assays on other stealth adapted virus cultures

Figure 3: Matching of cloned sequences from culture A with a cellular sequence. What is shown is the BLASTN readout of the nucleotide matching of the “Query” sequence of a cloned PCR product from culture A with a sequence within Homo sapiens Rho guanine nucleotide exchange factor 10 (ARHGEF10) gene on chromosome 8 (NCBI accession number NG_008480.1). Note the 13-nucleotide insert into the cloned DNA sequence from nucleotide 276 to 288.


The SK43/SK44 set of PCR primers was used in PCR assays on positive stealth virus cultures obtained from several other patients. A strongly positive culture was obtained from the cerebrospinal fluid (CSF) of a comatose patient with a 4-year history of a bipolar psychosis [20]. The culture gave similarly sized PCR products as did the previous culture. Partial sequencing on one of the products from this culture confirmed a close homology to that of the earlier sequenced PCR product from the prototype SCMV-derived stealth adapted virus ((NCBI Accession number U09213).  There were still significant minor differences between the sequences of the two viruses and with the corresponding region of SCMV. Several sets of PCR primers were also developed based on sequences in the cloned region of the prototype SCMV-derived stealth adapted virus. These primers gave positive results when tested directly on the CSF obtained from the comatose patient. A positive culture from the CSF of another CFS patient, gave a negative PCR in direct testing using these primers sets. Yet the culture did show a positive PCR for SCMV-related sequences when the RNA from the culture was first transcribed into DNA using reverse transcriptase PCR.

PCR assays on other stealth adapted virus cultures

The SK43/SK44 set of PCR primers was used in PCR assays on positive stealth virus cultures obtained from several other patients. A strongly positive culture was obtained from the cerebrospinal fluid (CSF) of a comatose patient with a 4-year history of a bipolar psychosis [20]. The culture gave similarly sized PCR products as did the previous culture. Partial sequencing on one of the products from this culture confirmed a close homology to that of the earlier sequenced PCR product from the prototype SCMV-derived stealth adapted virus ((NCBI Accession number U09213).  There were still significant minor differences between the sequences of the two viruses and with the corresponding region of SCMV. Several sets of PCR primers were also developed based on sequences in the cloned region of the prototype SCMV-derived stealth adapted virus. These primers gave positive results when tested directly on the CSF obtained from the comatose patient. A positive culture from the CSF of another CFS patient, gave a negative PCR in direct testing using these primers sets. Yet the culture did show a positive PCR for SCMV-related sequences when the RNA from the culture was first transcribed into DNA using reverse transcriptase PCR. SK43/SK44 primers are sometimes able to generate similarly sized, discrete PCR products when tested on positive cultures from CFS patients. Three cultures yielded a product of approximately 0.5kb, together with some smaller products. The larger product from one of the cultures was cloned into pBluescript and fully sequenced. Several PCR products from the other two cultures were similarly cloned and partially sequenced from the T3 and T7 promotor sites. The cultures were designated A, B, and C. The PCR product from culture A (NCBI AF107850.1) was flanked on one end by the SK43 primer and on the other end by the SK44 primer. The 507-nucleotide sequence between the primers was compared with the nr/nt database on GenBank using the BLASTN program. The top listed matching result was to Homo sapiens Rho guanine nucleotide exchange factor 10 (ARHGEF10), RefSeqGene (LRG_234) on chromosome 8 (NCBI accession number NG_008480.1). The matching region extended from nucleotide 88302 to nucleotide 88794 of the cellular gene. This is a non-coding region of the gene located between the 17th and 18th exons. There are 447/505 identical nucleotides (88.5%) with 16/505 gaps. A major gap occurs because of a nucleotide stretch reading “acccccccccact” in the PCR product between nucleotides 276 to 288. The matching is shown in Figure 3.

Rhesus monkey-related cellular sequences

The BLASTN program was used to further compare the sequence of the PCR product with the sequences of the assembled human genome and that of various primates. The results are presented in Table 4. The sequence of the PCR product is clearly closer to the genome of the rhesus monkey than to the human genome. There are 504/510 identical nucleotides (98.8%) with 3/505 gaps. Moreover, the stretch of nucleotides in the PCR product, which is absent in the human gene, is present along with two additional cytosines in the rhesus genome.  The matching of the PCR product with the rhesus monkey genome is shown in Figure 4. Cloning of the PCR products from the other two cultures, B and C, identified two clones from culture B (C1113 and C1132). and one clone from culture C (C1332), which all have sequences closely related to the rhesus monkey-related sequence of the PCR product from culture A. These comparisons are shown in Figure 5. Even when comparing the limited sequences, the clones are slightly different from each other (Figure 5). This even applies to the comparison of the sequences of the two related clones obtained from culture B. Four additional clones obtained from culture B and six additional clones obtained from culture C were also partially sequenced using the T3 and T7 promotors on the pBluescript plasmid. Even though the cultures were obtained from different patients, there are two sets of matching clones. The T3 and the T7 sequences beyond the primer sites of clone C1123 from culture B are like the sequences read from the T7 and the T3 promotors in clone C1313 from culture C (Figure 6). Similarly, the T3 and T7 readouts from clone C1142 from culture B are similar to the T3 and T7 readouts from clone C1335 from culture C (Figure 6). The remaining two clones from culture B and four clones from culture C are different from one another. The two clones (C1151 and C1163) from culture B matched better to the rhesus monkey genome than to either the human or African green monkey genome. The results with clone C1163 are shown in Figure 7. Yet three of the remaining four clones from culture C show closer homology to the human genome. An example with the T3 readout of clone C1311 is shown in Figure 8. The two other clones from culture C, which match more closely to the human genome are clones C1333 and C1334.

Table 4: The relative number and percentages of identical nucleotides of the cloned PCR product from culture A with matching sequences of humans and several primates.


Figure 4: Matching of cloned sequences from culture A with a rhesus monkey cellular sequence. What is shown is the BLASTN readout of the nucleotide matching of the “Query” sequence of a cloned PCR product from culture A with the Assembled genome of Macaca mulatta breed Indian rhesus macaque isolate RUp15 000818F, whole genome shotgun sequence, SBKD01000790.1. There are 504 of 510 identical nucleotides with 3 gaps. This matching is closer than that to the human cellular sequence as shown in Figure 3.


Figure 5: Alignments of the sequence of the cloned PCR product culture A with the sequences in cloned PCR products from cultures B and C. What is shown are Query sequences from the T3 and T7 sequence readouts from two clones from culture B (C1113 and C1132) and one clone from culture C (C1322) aligned with the sequence of the PCR product cloned from culture A (Sbjct). The alignments did not include the SK43 and SK44 primers.


Figure 6: Alignment of matching clones from culture B and C. What is shown are the alignments between the available T3 and T7 sequences of two sets of matching clones from culture B and culture C. These matching clones were in addition to the clones which matched to the cloned PCR product from culture A. The alignments show close similarity but not identity


Figure 7: Matching of the sequence of the cloned PCR product with primate and human genomes. What is shown is the sequence of clone C1162 from culture B as the Query, with the best matching cellular sequences (Sbjct) in the rhesus monkey, African green monkey and human genomes. The chimpanzee genome gave the same matching result as did the human genome.


Figure 8: Matching of the sequence of the cloned PCR product with rhesus monkey and human genomes What is shown is the T3 sequence of clone C1311 from culture C as the Query, with the best matching cellular sequences (Sbjct) in the assembled rhesus monkey, and human genomes. There were 235/275 (85%) identical nucleotides with the rhesus monkey genome and 266/278 (96%) identical nucleotides with the human genome.


Discussion

These results provide additional information regarding stealth adapted viruses. In addition to a mechanism for evading cellular immunity, the process of stealth adaptation can apparently lead to the substitution of some and potentially all of the originating virus genome with other genetic sequences. The substituted sequences can be cellular and bacterial in origin. These substituted sequences, as well as any remaining initial virus sequences, can undergo further changes due to the fragmentation and genetic instability of the reformed viruses. The incorporation of both cellular and bacterial sequences has occurred with an SCMV-derived stealth adapted virus [6-10]. This virus is formally referred to as stealth virus-1. It was repeatedly cultured from a CFS patient. Low stringency PCR performed on virus infected cells using a fortuitous set of SK43/ SK44 PCR primers leads to the amplification of two long SCMVrelated nucleotide sequences and a shorter sequence with near identity to an intergenic region of the human X chromosome. No discernable PCR products are generated when the PCR assay is performed using the same set of primers on uninfected cells. The X chromosome related sequence was amplified through loose crossreactions with the SK43 primer, which inadvertently had a cytosine rather than an adenosine as the tenth nucleotide. As was shown in Figure 1, the amplified sequence differs from the matching region in the human X chromosome in having two deleted nucleotides and one nucleotide substitution. The effective amplification of the sequence in the infected cells could possibly also be due to some minor nucleotide changes which would allow for more effective binding of the SK43 primer. This possibility can be addressed by further sequencing of the primer binding sites of the virus associated DNA. It is considered much more likely, however, that the amplification seen with the virus infected cells reflects a greatly increased copy number of the X chromosome-related sequence. This is consistent with the X-chromosome-related sequence having been incorporated into the virus replication process. Several additional cellular derived sequences were identified upon cloning of the DNA isolated from the supernatant of the stealth adapted virus infected culture [6]. In one of the two cloning studies, the DNA had been further purified by agarose gel electrophoresis prior to its restriction enzyme cutting and cloning into pBluescript plasmids. This was intended to reduce the possible inadvertent cloning of normal cellular DNA. Virus incorporated cellular DNA can also be distinguished from normal cellular DNA if it has undergone any mutations. Indeed, minor nucleotide differences from normal cellular DNA were seen in many of the sequenced clones with cellular-related DNA. As will be detailed in future publications, the DNA in these and in other cellular sequencerelated clones will typically match to intragenic, and occasionally to intergenic, non-coding regions within a cellular genome. The more common minor differences between the amplified sequences and their matching cellular sequence are i) added or deleted single nucleotides and ii) single nucleotide substitutions, each occurring with an overall frequency of approximately 1%. The sequences in several additional clones show far greater deviation from known cellular or viral sequences, sometimes with no convincing matching being obtainable to the human genome using the standard BLASTN program. This lack of precise matching is in spite of the human and many primate genomes having been fully sequenced. It is probably a reflection of the genetic instability of the sequences, including major insertions, deletions, substitutions, and recombination. To date, there has been no decisive example of contiguous SCMV and cellular sequences within the same clone. This type of recombination is, however, fully anticipated to be seen in further sequencing studies.

The prior incorporation of rhesus monkey cellular sequences into stealth adapted viruses has occurred in the stealth adapted viruses cultured in human cells from three CFS patients. These cultures are designated A, B, and C. They were selected for further study because in low stringency PCR assays using the SK43/SK44 primers, they yielded comparable patterns of PCR generated products using. The cloned PCR product from culture A, two of the cloned PCR products from culture B and one of the cloned PCR products from culture C all match to the same region of the rhesus monkey genome. As expected, the four PCR clones match to one another, yet show individual differences. It could either be that the three individuals were infected with the same variant strain of a stealth adapted virus or that certain viruses have a propensity to incorporate this particular rhesus cellular sequence. The finding that cultures B and C have two sets of additional shared sequences would tend to favor the first possibility. The sequence differences between the matching clones from the different cultures, and between the two matching clones from culture B, are consistent with the genetic instability of stealth adapted viruses. The fact that three of the seven cloned sequences from culture C match better to a human sequence is consistent with homologous genetic recombination. This could explain why the X-chromosome related sequence in stealth virus-1 matches better to the human genome, than to the genome of the African green monkey. The possibility exists, therefore, of the integration of monkey-derived genetic sequences into the human genome. This intriguing topic will be discussed in a subsequent article.

SCMV-derived stealth adapted viruses have presumably entered into the human population as a consequence of using SCMV infected African green monkeys to produce live polio virus vaccines [21,22]. Earlier polio virus vaccines were produced in cytomegalovirus infected rhesus monkeys. DNA from Rhesus monkey cytomegalovirus (RhCMV) is present in some earlier polio virus vaccines, including the CHAT vaccine, which was experimentally tested in African chimpanzees [23]. This is consistent with the report by Dr. Albert Sabin that the CHAT vaccine had a contaminating cytopathic virus [24]. Indeed, it is likely that stealth adapted RhCMV was involved in the transformation of simian immunodeficiency virus (SIV) to human immunodeficiency virus (HIV) [25].

A reasonable model for the incorporation of cellular sequences into the virus genome is the hybridization of a fragment from the virus genome with a molecule of single stranded cellular RNA. This could lead to RNA or DNA synthesis of the complimentary sequence. If a different virus fragment then bound to the newly synthesized nucleic acid, it could essentially mimic the PCR within the cell. What is required, therefore, is a degree of genetic complementary between a fragment of the virus genome with a sequence in cellular RNA, together with an effective polymerase enzyme. Various cellular polymerases, including endogenous reverse transcriptase enzymes [26], can potentially be involved in the replication process. The polymerases of other viruses could similarly be involved. This is relevant to the production of polio vaccine virus in SCMV infected African green monkey kidney cells since the polio virus would be a source of RNA polymerase [27]. Although, the cellular gene incorporation process for stealth virus-1 may have begun in African green monkeys, there may have been subsequent switching or exchanging of the incorporated primate cellular sequences with the related human cellular sequences. This type of exchange would evolve over time once humans became infected. The is consistent with the relatively close relationship of the PCR amplified sequence from the human X chromosome with the counterpart sequences in the genomes of primates. Stealth virus-1 induces a severe acute illness in cats [28]. To test the possibility of genetic exchange, virus passaged in cats could be periodically examined for an exchange of the human sequences with feline sequences. Similar studies can be conducted in the many species of animal cells in which the stealth adapted virus can be cultured [1].

The process by which cellular sequences have become part of the virus replication process can seemingly also apply to the incorporation of bacterial sequences. There is no evidence that the stealth adapted cultures were infected with any bacteria. Specifically, on several occasions, subcultures were maintained for extended periods in antibiotic free media. Regular microscopy, including the high-power examination of hematoxylin and eosin (H&E) stained cells, showed no bacteria, nor did electron microscopic examination of the infected cells. Rather it appears that the incorporation of bacteria-derived sequences was a prior event occurring during or shortly after the stealth adaptation process. The terms “viteria” and “vifungus” were suggested to describe eukaryotic viruses with incorporated bacterial or fungal sequences, respectively [10,29]. Of interest are the potential locations at which virus and bacterial sequences would have the opportunity to interact. For intracellular bacteria, such as mycoplasma, borrelia, and brucella, it could be within a dually infected eukaryotic cell. The incorporation of ochrobactrum sequences into a stealth adapted virus probably requires a different explanation. Conventional eukaryotic viruses do not ordinarily grow in bacteria. The genes coding for the growthrestrictive elements may, however, be deleted during the stealth adaptation process. Stealth virus-1 is cytopathic for cells of multiple species, including insects. There is also evidence of stealth adapted viruses affecting bacteria. For example, atypical bacteria were cultured from the feces of the stealth virus-1 infected CFS patient. Moreover, transmissible cytopathic activity was subsequently retrieved from the atypical bacterial colonies (unpublished). Ochrobactrum bacteria are generally regarded as being soil-based, although Ochrobactrum anthropi can occasionally infect humans [30]. Humans can, however, be exposed to these bacteria in consumed food [31]. These bacteria may also potentially reside in the gut microbiota. Unlike the non-coding cellular sequences, many of the identified bacteria sequences have open reading frames and would presumably lead to the formation of proteins. The bacterial proteins, including enzymes, may potentially contribute to the virus replicative process and to the induced cytopathology. The proteins could also potentially function as a makeshift capsid for the intercellular transfer of replicated nucleic acids. To do so, the bacterial proteins would presumably need to self-assemble into cage-like structures, which are able to enclose strands of nucleic acids. Although speculative as to its relevance, it is interesting to relate the incorporation of bacterial sequences to a self-healing process, which occurs during the culturing of stealth adapted viruses. The healing process is associated with the production of self-assembling aliphatic and aromatic chemical compounds. The assembled materials are referred to as alternative cellular energy (ACE) pigments [32]. These materials can attract an external force called KELEA (Kinetic Energy Limiting Electrostatic Attraction), which drives the ACE pathway [33-37]. Enhancing the ACE pathway can lead to the suppression of the virus induced CPE [32]. It would not be surprising, therefore, if some of the bacterial gene-coded proteins were to be subsequently identified as contributing to the formation of ACE pigments. These pigments could potentially prolong the infection process. Bacterial proteins might also be expected to lead to intracellular protein aggregates and to trigger the unfolded protein response [38]. This response can also potentially lead to a delay in virus-induced cell death, thereby, allowing for a more prolonged symbiotic relationship. A diverse array of intracellular and extracellular aggregates can be seen by electron microscopy in stealth adapted virus-infected cultured cells and in the biopsied tissues from infected individuals.

The relocation of genetic sequences from their normal cellular or bacteria origin to a virus can be viewed as a passive hijacking of the sequences by the virus or as a loss of the cell’s or the bacteria’s capacity to restrain some of their own genetic sequences from deserting and moving elsewhere. In this context and to help in conveying an understanding of the process, the relocating sequences are being referred to as renegade sequences. The capturing of cellular sequences by viruses is not a new phenomenon [39]. Indeed, even regular SCMV encodes five copies of a G-protein coding gene, which was presumably originally cellular in origin [40]. What is different with regards to the present findings is the concept that the incorporated genes could potentially come to comprise an essentially autonomous, replicating, cytopathic, renegade virus. In other words, once the replicative process involving cellular sequences is underway, there may be no longer a need for remaining sequences of the initiating stealth adapted virus. In this regard, stealth virus-1 may be relatively early in its evolution with its retention of extensive SCMV-related sequences.

Viruses are known to engage with noncoding RNA elements within the cell. The interactions can either be advantageous or inhibitory to the growth of the virus, depending on what particular RNA element is being affected [41-46]. These previous reports describe the cell and the viruses as separate and discrete, although interactive, entities. A fundamentally different concept is the switching of certain noncoding DNA or RNA elements from primarily belonging to the cell to becoming part of the replicating and transmissible virus genome. Since noncoding sequences have specificity in their activities [47,48], the type or types of incorporated sequences could have a bearing on the cytotoxic or tumorigenic effects of the modified (renegade) virus.

Major substitutions of renegade sequences for the original virus sequences can have important diagnostic implications. On the one hand, the presence of bacteria-derived sequences in stealth adapted viruses can potentially lead to mistaking a virus infection for a bacterial infection. Examples where this error is probably occurring include i) Attributing so called chronic Lyme disease to an active infection by Borrelia burgdorferi [49]. Indeed, there is good unpublished evidence from performing stealth virus cultures in many patients diagnosed with chronic Lyme disease, that the individuals are virally infected. It remains to be shown that the viruses contain borrelia-related genetic sequences. ii) Also mistaking Morgellons skin disease for an infection with Borrelia, Agrobacteria, or other parasites [49-51]. There have been no examples of actually culturing bacteria from these patients. Many of those tested, however, have had positive stealth virus cultures. iii) Linking CFS with mycoplasma [52,53] or with brucella infection. An interesting personal communication from a mycoplasma researcher was that the mycoplasma sequences he was identifying were actually slightly different from those of the known mycoplasma species. Some CFS patients have been tested for brucella antibodies and found to be positive. iv) Defining PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections) as a bacteria-induced disease [54,55]. Again, on close analysis there is often a discrepancy between the anti-streptolysin O and anti-DNase B antibody testing in patients diagnosed with PANDAS (personal observation). v) Associating bacteria, such as Porphyromonas gingivalis [56,57] or chlamydia [58-60] with Alzheimer disease and with rheumatoid arthritis [61,62]. The prospect of viteria infections may help in reorienting the thinking about these associations. An even bigger issue is the many illnesses in which a virus infection is somewhat suspected, yet the regular molecular and serological screenings for known viruses yield negative results. There is typically no accompanying inflammation associated with these illnesses. Prominent examples of these illnesses include CFS, fibromyalgia, autism, multiple sclerosis, amyotrophic lateral sclerosis, bipolar depression, schizophrenia, and Alzheimer disease. Some of the controversies related to viruses causing such illnesses are covered in the references [63-78]. Glioblastoma is a further example of a disease with suggestive evidence of an underlying virus infection [79-81]. A few cases tested by stealth virus culture did give positive results. Until disproven, many persisting illnesses, including cancers, should be studied for evidence of a possible contribution by an ongoing infection with stealth adapted/renegade viruses.

The brain is particularly susceptible to symptomatic illness due to stealth adapted viruses [76-78]. This is because of the spatial distribution of the various aspects of brain functioning. It is unlike other organs in which limited localized cell damage can be easily compensated by overactivity elsewhere in the same organ. There has been an overall increase in mental illness, and this may well be due to an epidemic of stealth adapted virus infections.

Stealth adapted/renegade viruses can best be detected using virus cultures that consider the production of ACE pigments. The presence of disease-causing viruses can be further confirmed by inoculation of the cultured material into animals [28]. The detection of stealth adapted induced illnesses is important in disease prevention and is relevant to the safety of the nation’s blood supply. It is also relevant to the risk of vaccine triggering of a tissue damaging immune response against some of the normally non-immunogenic components in these viruses. Fortunately, the body’s ACE pathway has the capacity to suppress stealth adapted viruses. Clinical studies are needed to evaluate the effectiveness of the various means of enhancing the ACE pathway in the therapy of stealth adapted/renegade virus infections [82-90].

Summary

The process of stealth adaptation of viruses has been extended beyond the deletion or mutation of the genes coding for the relatively few components normally targeted by the cellular immune system. Stealth adaptation can also involve the incorporation of added sequences, which can be cellular and microbial in origin. The incorporated sequences can lead to the further elimination of the originating virus sequences, with the reformed virus retaining the capacity for replication and transmission and, thereby, the ability to cause diseases. The switching of cellular and bacterial sequences into the virus world is likened to the actions of a renegade. The term renegade is, therefore, being introduced as an added description of stealth adapted viruses. Conventional serological and molecular assays for viruses are inadequate as a screening method for renegade viruses. Many chronic illnesses, including some cancers, are likely caused by renegade viruses. They are best treated by enhancing the alternative cellular energy (ACE) pathway.

Acknowledgement

I am grateful to the individuals who were unswayed when learning that senior Public Health officials had declared that stealth adapted viruses do not exist and that the clinical testing for the viruses had put the Nation’s health in Immediate Jeopardy. This article with a slight change in the Title has also been published in the Journal of Human Virology and Retrovirology. Duplication is intended and agreed upon to help reach as wide a readership as possible given the significance of infectious monkey-derived cellular sequences being transmitted to humans.

References

  1. Martin WJ, Zeng LC, Ahmed K, Roy M (1994) Cytomegalovirus-related sequence in an atypical cytopathic virus repeatedly isolated from a patient with chronic fatigue syndrome.The American Journal of Pathology 145(2): 440-451.
  2. Ehrlich GD, Glaser JB, LaVigne K, Quan D, Mildvan D, et al. (1989) Prevalence of human T-cell leukemia lymphoma virus (HTLV) type II infection among high-risk individuals: Type-specific identification of HTLVs by polymerase chain reaction. Blood 74: 1658.
  3. Johnson M, Zaretskaya I, Raytselis Y,Merezhuk Y, McGinnis S, et al. (2008) NCBI BLAST: a better web interface. Nucleic Acids Research 36: 5-9.
  4. Martin WJ, Ahmed KN, Zeng LC, Olsen JC, Seward JG, et al. (1995) African green monkey origin of the atypical cytopathic “stealth virus” isolated from a patient with chronic fatigue syndrome.Clinical and Diagnostic Virology 4(1): 93-103.
  5. Martin WJ (1999) Stealth adaptation of an African green monkey simian cytomegalovirus.Experimental and Molecular Pathology 66(1): 3-7.
  6. Martin WJ (2014) Stealth adaptation of viruses: Review of earlier studies and updated molecular analysis of a stealth adapted African green monkey simian cytomegalovirus. In Stealth Adapted Viruses; Alternative Cellular energy pp. 3-30.
  7. Martin WJ (2014) Stealth adaptation of viruses: Review and updated molecular analysis on a stealth adapted African green monkey simian cytomegalovirus (SCMV). Journal of Human Virology & Retrovirology 1(4): 00020.
  8. Martin WJ (1998) Cellular sequences in stealth viruses.Pathobiology 66: 53-58.
  9. Martin WJ (1999) Melanoma growth stimulatory activity (MGSA/GRO-α) chemokine genes incorporated into an African green monkey simian cytomegalovirus-derived stealth virus.Experimental and Molecular Pathology. 66(1): 15-18.
  10. Martin WJ (1999) Bacteria-related sequences in a simian cytomegalovirus-derived stealth virus culture. Experimental and Molecular Pathology. 66(1): 8-14.
  11. JohannsonKE, PetterssonB (2002)Taxonomy of mollicutes. Molecular Biology and Pathogenicity of Mycoplasmas,
  12. Calderon CS, Wigger G, Wunderlin C, Schmidheini T, Frey J, et al. (2009) The Mycoplasma conjunctivae genome sequencing, annotation and analysis. BMC Bioinformatics. 10(6): S7.
  13. Lo SC, Hayes MM, Tully JG, Wang RY, Kotani H, et al. (1992)Mycoplasma penetrans sp. nov., from the urogenital tract of patients with AIDS. Int J Syst Bacteriol 42(3): 357-364.
  14. Paulsen IT, Seshadri R, Nelson KE, Eisen JA, Heidelberg JF, et al. (2002) The Brucella suis genome reveals fundamental similarities between animal and plant pathogens and symbionts. Proceedings of the National Academy of Sciences 99(20): 13148-13153.
  15. Scholz HC, Dahouk S, Tomaso H, Neubauer H, Witte A, et al. (2008) Genetic diversity and phylogenetic relationships of bacteria belonging to the Ochrobactrum Brucella group by recA and 16S rRNA gene-based comparative sequence analysis. Syst Appl Microbiol 31(1): 1-16.
  16. Krzyżanowska DM, Maciąg T, Ossowicki A, Rajewska M, Kaczyński Z, et al. (2019) Ochrobactrum quorumnocens sp nov a quorum quenching bacterium from the potato rhizosphere, and comparative genome analysis with related type strains. PLoS ONE 14(1): e0210874.
  17. Huber B, Scholz HC, Kämpfer P, Falsen E, Langer S, et al. (2016) Ochrobactrum pituitosum sp nov isolated from an industrial environment. Int J Syst Evol Microbiol 60(2): 321-326.
  18. Kampfer P (2007) Ochrobactrum haematophilum sp nov and Ochrobactrum pseudogrignonense sp nov isolated from human clinical specimens. International Journal of Systematic and Evolutionary Microbiology 57(11): 2513-2518.
  19. Martin WJ (1996) Genetic instability and fragmentation of a stealth viral genome.Pathobiology 64(1): 9-17.
  20. Martin WJ (1996) Simian cytomegalovirus-related stealth virus isolated from the cerebrospinal fluid of a patient with bipolar psychosis and acute encephalopathy. Pathobiology 64(2): 64-66.
  21. Sierra HA, Krause M, Philip R (2002) Live oral poliovirus vaccines and simian cytomegalovirus. Journal of the International Association of Biological Standardization 30(3): 167-174.
  22. Baylis SA, Shah N, Jenkins A, Berry NJ, Minor PD (2003) Simian cytomegalovirus and contamination of oral poliovirus vaccines. Biologicals 31(1): 63-73.
  23. Berry N, Davis C, Jenkins, A, Wood D, Minor P, et al. (2001) Vaccine safety: Analysis of oral polio vaccine CHAT stocks. Nature 410(6832): 1046-1047.
  24. Sabin AB (1959) Present position on immunizing against poliomyelitis with live virus vaccines. Brit Med J 1(5123): 663-80.
  25. Martin WJ (2015) Chimpanzees inoculated with cytomegalovirus contaminated polio vaccines may explain origin of HIV-1. Journal of Human Virology & Retrovirology 2(2): 00035.
  26. Abraham GN, Khan AS (1990) Human endogenous retroviruses and immune disease. Clin Immunol Immunopathol 56(1): 1-8.
  27. Barton DJ, Flanegan JB (1993) Coupled translation and replication of poliovirus RNA in vitro: synthesis of functional 3D polymerase and infectious virus. The Journal of Virology 67(2): 822-82231.
  28. Martin WJ, Glass RT (1995) Acute encephalopathy induced in cats with a stealth virus isolated from a patient with chronic fatigue syndrome. Pathobiology 63: 115‑118.
  29. Martin WJ (2005) Alternative cellular energy pigments mistaken for parasitic skin infestations. Exp Mol Path 78(3): 212-214.
  30. Holmes B, Popoff M, Kiredjian M, Kersters K (1988) Ochrobactrum anthropigen nov sp nov from human clinical specimens and previously known as group Vd. Int J Syst Bacteriol 38: 406-416.
  31. Jaykus LA, Wang HH, Schlesinger LS (2009) Food borne microbes: shaping the host ecosystem. ASM Press, USA.
  32. Martin WJ (2003) Stealth virus culture pigments: a potential source of cellular energy. Exp Mol Path 74: 210-223.
  33. Martin WJ (2015) KELEA: A natural energy that seemingly reduces intermolecular hydrogen bonding in water and other liquids. Open Journal of Biophysics 5(3): 69-79.
  34. Martin WJ (2015) Interacting light paths attract KELEA (kinetic energy limiting electrostatic attraction) and can lead to the activation of water. Open J Biophysics 5(4): 115-121.
  35. Martin WJ (2015) Interacting electric fields attract KELEA (kinetic energy limiting electrostatic attraction) and can lead to the activation of water. International J Complementary & Alternative Medicine 1(6): 00034.
  36. Martin WJ (2016) KELEA (kinetic energy limiting electrostatic attraction) may add to the measured weight of an object. J Modern Physics 7(6): 461-472.
  37. Martin WJ (2016) KELEA cosmic rays cloud formation and electromagnetic radiation: Electropollution as a possible explanation for climate change. Atmospheric and Climate Sciences. 6(2): 174-179.
  38. Cao SS, Kaufman RJ (2012) Unfolded protein response. Current Biology 22(16): R622-R626.
  39. Dagna L (2007) Virus-encoded chemokines, chemokine receptors and chemokine-binding proteins: new paradigms for future therapy. Future Virol 2(4): 353-368.
  40. Sahagun RA, Sierra HA, Krause P, Murphy PM (2004) Simian cytomegalovirus encodes five rapidly evolving chemokine receptor homologues. Virus Genes 28(1): 71-83.
  41. Wang P, Xu J, Wang Y, Cao X (2017) An interferon-independent lncRNA promotes viral replication by modulating cellular metabolism. Science 358(6366): 1051-1055.
  42. Goodwin CM, Xu S, Munger J (2015) Stealing the keys to the kitchen: viral manipulation of the host cell metabolic network. Trends Microbiol 23(12): 789-798.
  43. Mesquita I, Estaquier J (2018) Viral manipulation of the host metabolic network. Metabolic Interaction in Infection Experientia Supplementum 109: 899-989.
  44. Powdrill MH, Desrochers GF, Singaravelu R, Pezacki JP (2016) The role of microRNAs in metabolic interactions between viruses and their hosts. Curr Opin Virol 19: 71-76.
  45. Ghosh Z, Mallick B, Chakrabarti J (2009) Cellular versus viral microRNAs in host-virus interaction. Nucleic Acids Res 37(4): 1035-1048.
  46. Fiorucci G, Chiantore MV, Mangino G, Romeo G (2015) MicroRNAs in virus-induced tumorigenesis and IFN system. Cytokine Cell Growth Rev 26(2): 183-194.
  47. Mohebbi A, Tahamtan A, Eskandarian S, Askari FS, Shafaei M, et al. (2018) Viruses and long non-coding RNAs: implicating an evolutionary conserved region. Virusdisease 29(4): 478-485.
  48. Slack FJ, Chinnaiyan (2019) The role of noncoding RNA in oncology. Cell 177(5): 1033-1055.
  49. Middelveen MJ, Bandoski C, Burke J (2015) Exploring the association between Morgellons disease and Lyme disease: identification of Borrelia burgdorferi in Morgellons disease patients. BMC dermatology 15(1): 1-1.
  50. Middelveen MJ Stricker RB (2016) Morgellons disease: a filamentous borrelial dermatitis. International Journal of General Medicine 9: 349-354.
  51. Middelveen MJ, Filush KR, Bandoski C, Kasliwala RS, Melillo A, et al. (2019) Mixedborrelia burgdorferi and helicobacter pylori biofilms in morgellons disease dermatological specimens. Healthcare (Basel) 7(2): 70.
  52. Nijs Jo, Nicolson GL, De Becker P, Coomans D, De Meirleir K (2002) High prevalence of Mycoplasma infections among European chronic fatigue syndrome patients. Examination of four Mycoplasma species in blood of chronic fatigue syndrome patients. FEMS Immunology and Medical Microbiology 34(3): 209-214.
  53. Endresen G (2003) Mycoplasma blood infection in chronic fatigue and fibromyalgia syndromes. Rheumatology International 23(5): 211-215.
  54. Swedo SE, Leonard HL, Garvey M, Mittleman B, Allen AJ, et al. (1998) Pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections: clinical description of the first 50 cases. Am J Psychiatry 155(2): 264-271.
  55. March JS (2004) Pediatric autoimmune neuropsychiatric disorders associated with streptococcal infection (PANDAS): implications for clinical practice. Arch Pediatr Adolesc Med 158(9): 927-929.
  56. Dominy SS, Lynch C, Ermini F (2019) Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors. Science advances. 5(1): 3333.
  57. Singhrao SK, Harding A, Poole S, Kesavalu, L, Stjohn C (2015) Porphyromonas gingivalis periodontal infection and its putative links with Alzheimer’s disease. Mediators of Inflammation
  58. Shima K, Kuhlenbäumer G, Rupp J (2010) Chlamydia pneumoniae infection and Alzheimer’s disease: a connection to remember? Medical Microbiology and Immunology 199(4): 283-289.
  59. Appelt DM, Howanski RJ, Hallock LR, Hammond CJ, Scott LC et al. (2010) Immunohistological detection of Chlamydia pneumoniae in the Alzheimer’s disease brain. BMC Neuroscience 11(1): 121.
  60. Gérard HC. Dreses WU, Wildt KS (2006) Chlamydophila (Chlamydia) pneumoniae in the Alzheimer’s brain. FEMS Immunology & Medical Microbiology 48(3): 355-366.
  61. Loyola RJP, Martinez MRE, Abud MC, Patiño MN, Seymour GJ (2010) Rheumatoid arthritis and the role of oral bacteria.Journal of Oral Microbiology 2(1): 1-8.
  62. Andersen I, Olesen HP (1990) Anaerobic bacteria in rheumatoid arthritis.Annals of the Rheumatic Diseases 49(7): 568.
  63. Kendell R (1991) Chronic fatigue, viruses, and depression.Lancet 337: 160-162.
  64. Branco JC, Tavares V, Abreu I, Humbel RL (1994) Viral infection in fibromyalgia. Acta Med Port 7(6): 337-341.
  65. Sprott H (2011) Is fibromyalgia a viral disease? Z Rheumatol 70(8): 637-638.
  66. Virtanen JO, Jacobson S (2016) Viruses and Multiple Sclerosis. CNS Neurol Disord Drug Targets 11(5): 528-544.
  67. Kakalacheva K, Comabella M (2010) Epstein barr virus and multiple sclerosis: causation or association? Future Microbiology 5(11): 1617-1619.
  68. Yolken RH, Torrey EF (1995) Viruses, schizophrenia, and bipolar disorder. Clin Microbiol Rev 8(1): 131-145.
  69. Libbey JE, Sweeten TL, McMahon WM, Fujinami RS (2005) Autistic disorder and viral infections. J Neurovirol 11(1): 1-10.
  70. Martin WJ (1995) Stealth virus isolated from an autistic child. J Autism Dev Disord 25(2): 223-224.
  71. Weiner PL, Stohlman,AS, Davis, LR (1980) Attempts to demonstrate virus in amyotrophic lateral sclerosis. Neurology 30(12): 1319-1322.
  72. Corcia P, Giraud P, Guennoc AM, Toffol B, Autret A (2003) Acute motor axonal neuropathy, enterovirus and amyotrophic lateral sclerosis: can there be a link? Revue Neurologique 159(1): 80-82.
  73. Xue Y, Chao FR, Cashman N, Luo H (2018) Enteroviral infection: The forgotten aink to amyotrophic lateral sclerosis? Frontiers in Molecular Neuroscience 11: 63.
  74. Torrey EF (1988) Stalking the schizovirus. Schizophrenia bulletin 14(2): 223-229.
  75. Libikova H (1983) Schizophrenia and viruses: principles of etiologic studies. Advances in Biological Psychiatry 12: 20-51. 
  76. Peacrce DB (2003) Can a virus cause schizophrenia? KUWER Academic Publishers, London, UK.
  77. Crow TJ (1978) Viral causes of psychiatric disease. Postgrad Med J 54: 763-767.
  78. Hare EH (1979) Schizophrenia as an infectious disease. British Journal of Psychiatry 135: 468-470.
  79. Cosset E, Petty TJ, Dutoit V, Cordey S, Padioleau I, et al. (2014) Comprehensive metagenomic analysis of glioblastoma reveals absence of known virus despite antiviral‐like type I interferon gene response. International Journal of Cancer 135(6): 1381-1389.
  80. Cobbs CS, Harkins L, Samanta M (2002) Human cytomegalovirus infection and expression in human malignant glioma. Cancer Res 62(12): 3347-3350.
  81. Scheurer ME, Bondy ML, Aldape K (2008) Detection of human cytomegalovirus in different histological types of gliomas. Acta Neuropathol 116(1): 79-86.
  82. Martin WJ (1996) Severe stealth virus encephalopathy following chronic fatigue syndrome‑like illness: Clinical and histopathological features. Pathobiology 64: 1‑8.
  83. Martin WJ (2015) Stealth adapted viruses -possible drivers of major neuropsychiatric illnesses including Alzheimer’s disease. J Neurol Stroke 2(3): 00057.
  84. Martin WJ (2015) Stealth adapted viruses: a bridge between molecular virology and clinical psychiatry. Open Journal of Psychiatry5(4): 311-319.
  85. Martin WJ (2015) Therapeutic potential of KELEA activated water. International Journal of Complementary & Alternative Medicine 1(1): 00001.
  86. Martin WJ (2017) Cancer is treatable via the alternative cellular energy (ACE) pathway. J Cancer Therapy 8(13): 1279-1290.
  87. Martin WJ (2016) The ACE pathway in comparison to the immune system in the defense against infectious diseases. J Human Virology & Retrovirology 3(5): 00124.
  88. Martin WJ (2016) Insufficiency of cellular energy (ICE) the basis for many illnesses potentially correctable using KELEA activated water. International J Complementary & Alternative Medicine 4(1): 00106.
  89. Martin WJ (2015) Stealth adaptation of viruses: implications for therapy and for potential toxicity of vaccines. JOJ Immuno Virol 1: 555551.
  90. Martin WJ (2020) Enhancing the alternative cellular energy (ACE) pathway with KELEA activated water as therapy for infectious diseases. Infectious Disorders Drug Targets.

Publishers: https://crimsonpublishers.com/

For more articles in open infectious diseases journal impact factor
Please click on below link: https://crimsonpublishers.com/cjmi/

A Close Look at the Application of the Yin-Yang- Based Acupoint Pairs_Crimson Publishers

A Close Look at the Application of the Yin-Yang- Based Acupoint Pairs by Tong Zheng Hong in Advancements in Bioequivalence & Bioavailabi...