The Journal of Degenerative Diseases Vol 1. May 1999
Viteria: Bacterial Sequences in Animal and Human Viruses
Viteria: Bacterial Sequences in Animal and Human Viruses
W. John Martin, M.D., Ph.D.
Address for Correspondence:
DNA sequencing studies have identified bacterial sequences within portions of an atypically structured cytopathic virus isolated from a patient with chronic fatigue syndrome. The virus has retained several features indicating an original derivation from an African green monkey simian cytomegalovirus (SCMV). While it lacks critical antigens required to evoke an effective anti-viral cellular immune defense, it has gained additional genes through genetic recombination with normal cellular genes, and as reviewed in this article, with numerous genes of bacterial origins. The term viteria has been coined to describe a virus infectious for eukaryotic cells that has acquired bacterial genes. The diagnostic, therapeutic and epidemiological implications of viteria as pathogenic agents are discussed. The need for urgent studies on these agents is underscored by their capacity to acquire cellular genes with potential oncogenic activity.
A Brief Overview of Biology
Life forms can be classified as viral, bacterial, fungal, plant and animal. While the origins of viruses is unsettled, evolutionary data indicate that bacteria predate the other life forms. Bacterial cells are termed prokaryotic, in contrast to cells of fungi, plants and animals, which are termed eukaryotic. Multiple characteristics distinguish prokaryotic from eukaryotic cells. These include chromosomal structure, the absence of a defined nucleus, the presence of an outer cell wall, and overall organization of both ribosomal RNA and transfer RNA (tRNA) sequences.
Viruses differ from both prokaryotic and eukaryotic cells in having a much smaller genome comprising, at least at one stage of their life cycle, DNA or RNA, rather than both DNA and RNA. Viruses are capable of only limited intrinsic enzymatic activity and require the complex metabolic machinery of a living cell for complete replication. Bacterial viruses utilize prokaryotic cells as their hosts, while fungal, plant and animal viruses, replicate in the corresponding eukaryotic cell type.
As with most classification schemes, not all replicating life forms fit neatly into the above groups. For example, obligate intracellular bacteria depend on a host eukaryotic cell for replication. Many bacteria can function normally only with the concomitant metabolic activity of resident viruses (commonly referred to as plasmids). Mitochondria in eukaryotic cells and chloroplasts in plant cells, have an ancestral origin as intracellular bacteria that have establish symbiotic and dependent relationships with eukaryotic cellular metabolism.
Reliance on "foreign" microorganisms for specific metabolic needs is reflected in the genetic streamlining that has allowed different life forms to dispense with genes coding for a variety of products that are readily available from alternative symbiotic sources. The process has been referred to as "reductive evolution" and is seen, for example, in the requirement of humans to ingest so called "essential metabolites" that can no longer be synthesized by our own cells.
Life itself can be viewed as the initial transformation of various inorganic molecules into organic substrates for use as building blocks and as energy sources; to be followed ultimately by the breakdown of organic compounds to replace the consumed inorganic molecules. The wide diversity of life forms helps to segregate various aspects of what amounts to interlacing chains of cooperative, progressive, metabolic inter-conversions. The multiplicity of life's players allows for fine tuning the multiple complex metabolic pathways, in response to either the over or under abundance of specific products. It also avoids the necessity of evolving life forms to carry all of the genetic baggage of their ancestors.
Bacterial Viruses as Vectors for the Transfer of Genetic Information
The role of viruses in genetic evolution is less appreciated than the genetic changes resulting from stochastic errors in the fidelity of DNA replication. Potentially, viruses have the capacity to bypass the slow selective process of Darwinian evolution, by "en bloc" transfer of ready made genes between different organisms. The genetic benefits of viral infection are established for bacteria, although this process can also occur in animals and plants.
Bacterial viruses include plasmids that pass directly between coupled bacteria, and bacteriophages that can persist in the external environment before reentering a susceptible bacterium. As is the situation with animals and plants, not all viral infections of bacteria are beneficial. For example, bacteriophage viruses are named for their ability to destroy bacteria. Furthermore, bacteria have protective mechanisms that will prevent certain infections from occurring. For example, restriction enzymes can recognize sequence differences and altered methylation patterns that may exist between bacterial DNA and that of an invading plasmid or bacteriophage. Such protective mechanisms do not typically operate against plasmids from closely related bacteria. Indeed many bacteria are particularly receptive to becoming infected with plasmids that will enhance their metabolic competitiveness in a changing environment. The widespread use of antibiotics for example has been matched by the dissemination among various bacterial species of plasmids coding for genes enabling antibiotic resistance.
Seen differently, the antibiotic-challenged bacteria have provided a mechanism for the expansion of certain plasmids. Moreover, through genetic recombinations, composite plasmids can provide their bacterial hosts not only wide ranging antibiotic resistance, but also disease associated functions such as tissue invasiveness and toxin production. Some of the genes that these complex plasmids shuttle between bacteria are of bacterial origins. In this context, plasmids are acting as natural vector for the transfer of bacterial genes, sometimes even between evolutionary distant bacterial species.
Animal Viruses as Vectors for the Transfer of Cellular Genetic Information
Evidence that animal viruses have participated in the horizontal transfer of genes has been obtained with retroviruses. Genetic sequences of retroviral origins comprise a significant proportion (at least 0.1%) of the entire genomes of many animals, especially mammals. Whole copies, as well as derivative mutated forms, of the genes coding the nucleocapsid (gag) protein, envelope glycoprotein and reverse transcriptase enzyme, are commonly transcribed as part of normal embryonic development, and also in response to exogenous viral infections and various cellular cytokines. Both cellular and exogenous RNA molecules can potentially be converted by reverse transcriptase to DNA capable of being inserted into the cellular genome. The insertion process itself, and the genetic information coded by the inserted sequences, can conceivably lead to profound cellular modifications, including cancer development.
While much of the inheritance of retroviral related sequences has followed the pattern of long term evolutionary relationships between species, several examples point to a more recent cross species transfers, presumably a result of the horizontal spread of a retroviral infection. For a retroviral infection to become embedded in a species, it needs to infect either the male or female germ cell, or, if immunogenic, to be passed during the embryonic and/or perinatal period when the immune system is less vigilant in responding to foreign invaders.
Much of the early discoveries on retroviruses stemmed from their capacity to assimilate, replicate and horizontally transfer cellular genes that can induce the recipient cell to become malignant. The cell growth regulatory genes and their viral counterparts became known as oncogenes. Retroviral transmitted cancers are not uncommon in animals and include leukemias in chicken, mice, cats and cattle, breast cancers in mice and lung cancers in sheep. A human T lymphotropic virus (HTLV-I) infrequently causes leukemia in those infected. Other viruses associated with human and animal tumors include the papovaviruses (polyoma, SV-40 and papillomaviruses); hepatitis B and C viruses; and various herpesviruses. An oncogenic role for herpesviruses has been accepted in the causation of human Burkitt's lymphoma and nasopharyngeal carcinoma (Epstein-Barr virus); and of lymphoma in Old World monkeys inoculated with Herpesvirus saimiri, a virus derived from New World monkey). Human herpesvirus-8 has been associated with Kaposi's sarcoma and body cavity lymphomas in humans, while renal cell cancer in frogs can be caused by Leuke herpesvirus.
Sequencing studies on traditional herpesviruses suggest that, like retroviruses, they have managed to incorporate certain genes that were originally of cellular origin. The viruses probably use some of these genes to promote cell growth and thereby induce a more permissive environment for virus replication. Providing the infected cells also expressed antigens recognized by the cellular immune system, the actual outgrowth of proliferating virus infected cells in the form of a cancer is unlikely to occur. This formulation is consistent with immune suppression being a major co-factor in the development of human lymphomas with Epstein-Barr virus. Similarly, the unique susceptibility of Old World monkeys to leukemia induced by herpesvirus saimiri has been traced to their sluggish immune response to viral antigens, when compared to that of New World monkeys, which are the natural hosts for this infection.
In addition to oncogenes, animal viruses can presumably convey other genes that can control cellular functions. A sine qua nom of viral pathogenicity is the capacity of at least some of the viral genes to influence cell function by masquerading as cellular genes. The implicit homologies between various viral and cellular genes, are readily identified for those viruses for which sequence data are available. The issue of whether a particular virus originally contributed some of its genes to an infected cell, rather than incorporating a cellular gene into its own genome, can be partially addressed by tracing the evolutionary origins of the gene. Viral related genes that suddenly appear as cellular sequences restricted to a limited set of species are presumably of viral origin. Conversely, genes restricted to a narrow subset of viruses, yet widely represented throughout the animal Kingdom, are likely to be intrinsically cellular in origin. This paradigm will find increasing use as progress continues with sequencing entire genomes of many microorganisms as well as that of humans and other animal species.
Stealth Virusees of Eukaryotic Cells as Possible Vectors for Genes of Bacterial Origin
What had previously not been considered is the potential for recombination between the genetic sequences of a virus that replicates in eukaryotic cells and sequences of bacterial and/or bacterial plasmid origin. Genetic evidence for such recombinants has been obtained from sequencing studies on atypically structured cytopathic viruses that are termed stealth because they bypass the cellular immune system. The lack of effective immune recognition of these viruses is attributed to the loss of genes coding for the major antigens required for cellular immunity. This hypothesis is still viewed skeptically by many virologists who intuitively believe that all viruses possess ample targets for immune recognition and/or that viruses without critical elements would be unable to replicate, cause cell damage or pass between individuals. Sequence studies on a prototype stealth-adapted virus has nonetheless continued to provide data consistent with the stealth adaptation hypothesis.
The prototype stealth virus is a derivative of an African green monkey simian cytomegalovirus (SCMV). It was cultured from a patient several months after the onset of an acute illness, characterized by severe headaches, generalized muscle pain and diarrhea. Although the acute symptoms subsided during a week of hospitalization, the patient never regained normal health. Instead, the patient has been affected for over 7 years with a debilitating chronic fatigue syndrome-like illness. Clinical manifestations have included episodes of marked emotional lability and depression, unexplained erosion of nasal cartilage, cervical spondylosis which required surgical stabilization, and various additional signs and symptoms of a multi-system illness. The virus isolated from the patient was infectious for cats, causing widespread histopathological signs of cellular damage without accompanying inflammation. Clinically, the cats showed marked behavioral changes and other evidence of an encephalopathy.
DNA was extracted from the stealth virus infected cultures and cloned. Over 300 clones containing portions of viral DNA have been at least partially sequenced. The sequences were analyzed for homology with known genes in GenBank. This large database of sequences is maintained and updated daily by the National Center for Biotechnology Information (NCBI) at the National Library of Medicine. The most informative matchings were generally achieved using BlastX analysis.. This program reads the potential translation products of an unknown sequence and determines amino acid homologies with all known protein sequences. For non-coding cellular and bacterial genes, direct comparisons of nucleotide sequences had to be made using BlastN analysis.
Of the over 300 clones analyzed, 26 clones contained stretches of nucleotide sequences and/or deduced amino acid sequences that matched with a high degree of statistical probability to bacterial genes, including genes present in certain plasmids. Many of the matches were incomplete suggesting that the bacterial sequences had undergone mutational changes since being incorporated into the stealth viral genome. Alternately, some of the sequences may have arisen from related bacteria for which exact sequence data are not yet available. A partial listing of the bacterial species and the specific proteins from these bacteria, that best matched to non-overlapping sequences identified in stealth virus clones, is presented in the article entitled Bacterial-Related Sequences in a Simian Cytomegalovirus-Derived Stealth Virus Culture. Exp Mol Path 6614-18,1999.
As described in the article, multiple distinct bacterial species were identified as the potential sources of the bacterial sequences identifiable within portions of the stealth virus genomes. The various proteins potentially coded by the assimilated bacterial and bacterial plasmid sequences were also remarkable in terms of their presumed metabolic functions. Several of the genes belong to primitive metabolic pathways for energy generation while others utilize and/or synthesize uncommon metabolites.
For those species with completely sequenced genomes, the locations of the matching sequences within the genomes, were determined. The linear arrangements of various bacterial sequences within individual stealth virus clones could, therefore, be directly compared with the order of genes in the bacterial species from which the stealth virus sequences were most closely related. Even for some of the bacteria with incompletely sequenced genomes, the relative positioning certain groups of genes is known. As shown in the Table, the linear arrangement of genes in some of the clones corresponded to that in the bacteria, for example the Brucella abortus matching sequences. For many of the clones, however, the bacteria related genes matched to widely separated regions of the bacterial genome.
The term viteria has been introduced to reflect the dual contribution of genes from viruses infectious for eukaryotic cells and genes from bacteria and/or bacterial plasmids. Viteria are presumably infectious for both prokaryotic and eukaryotic cells. The diverse range of metabolic processes presumably could provide a selective growth advantages to an infected bacteria. In a preliminary study, cytopathic transmissible agents were recoverable from an extract of fecal bacteria obtained from the patient from whom the prototype stealth virus was cultured. The ability of this agent to alter the metabolic profile of normal bacteria is being assessed. Similarly, human cells infected with the agent will be tested for the expression of bacterial related sequences.
Positive preliminary findings for such agents have also been obtained in fecal extracts from several stealth virus infected patients and also in the feces of symptomatic animals. As noted above, the assimilated bacterial genes may provide selective growth advantages to an infected bacteria. Efforts are, therefore, underway to selectively propagate viteria infected bacteria using nutritionally deprived media. In related studies, the potential of fungal organisms as an additional permissive host for viteria is being explored. One of the genes identified in the prototype stealth virus does, in fact, show good homology to a gene from the fungal organism, Arabidopsis thaliana. If fungal derived genes were to be predominant and/or if the viruses were to show a propensity to replicate in fungi, the term vifungus might be preferable to viteria.
The potential of viteria being harbored within the bacteria and/or fungal flora of the body has obvious diagnostic, therapeutic and epidemiological implications. Diagnostically, the presence of bacterial sequences in viteria can help reconcile the inconsistent reports of positive mycoplasma and chlamydia PCR assays in patients with CFS and, with the related Gulf war syndrome. Similarly, the laboratory findings suggesting that patients labeled as having chronic Lyme disease are infected with Borrelia, could be explained by viteria expressing amino acids and/or nucleotide sequences cross reactive with those of Borrelia. Indeed, the majority of chronic Lyme disease patients so far tested have yielded a positive result in assays for stealth viruses.
Therapeutically, it is useful to determine the antibiotic susceptibility of any bacteria cultured from the bowel, mouth or other locations that can be shown to be viteria infected. This should help control possible relapses in a patient treated with anti-viral agents, and more importantly, help control the spread of infection within households and within larger community groups. Antibiotic suppression of metabolically aberrant, viteria-infected bacteria, may also reconcile reports of clinical improvements occasionally seen in patients diagnosed as having CFS, Gulf war syndrome, Lyme disease, etc. To rationalize such therapies, it will be useful, however, to determine the specific antibiotic susceptibilities of viteria infected bacteria and/or fungi isolated from a patient. Analysis of viteria load can also help direct various dietary and other interventions that can help influence the bacterial composition of the bowel and other body sites.
Potentially Oncogenic Viteria
Stealth viruses have the capacity to assimilate various cellular genes, including genes with potential oncogenic activities. For example, multiple copies of a melanoma associated oncogene were detectable in the prototype SCMV-derived viteria. Positive stealth virus cultures have now been obtained in patients with various malignancies, including multiple myeloma, lymphomas, gliomas, breast cancers, lung cancers, melanomas and salivary gland tumors. Many of the patients describe disabling neurocognitive and mood disorders in themselves prior to the diagnosis of malignancy, and importantly, in some of their family members. As an example, a mother who struggles with a chronic fatigue syndrome, and whose son developed an acute and persisting learning disorder as a Junior in high school, reported that after years of being depressed, her father had been diagnosed with multiple myeloma. The mother subsequently developed a uterine malignancy and was incidentally found to give a false positive antibody test for HIV. All of family members are positive by stealth virus culture. Sequencing studies on the virus isolates from these patients may help establish a common source with subsequent differences in assimilated additional genes. Sequencing studies also need to be performed on markedly cytopathic agents cultured from malignant breast tissue, bone marrow, lymph nodes, brain biopsies, etc., of various cancer patients tested over the last several years. It is predicted that such studies will reveal additional examples of stealth adapted viruses containing cellular genes with oncogenic activity. The possibility that such viruses might also be carried by bacteria has ominous public health implications. It may, however, hasten the testing of various therapeutic strategies with a dual focus on their effects on the cancer and on any underlying neurological/neuropsychiatric disorders. An awareness of potentially oncogenic viteria may also help curtail the spread and possible further introduction of viteria into humans and animals.
The Origins of Viteria
A critical question is whether viteria are products of the late 20th century, or have been in existence for eons and have simply previously gone unrecognized. As documented above, the alignments of various bacterial genes within several of the clones of the prototype stealth virus is quite different than that so far encountered in known bacteria. This points to a more recent origin of the stealth virus. Overall, the available sequencing data are indicative of a complex progressive process of genomic recombinations between portions of the original SCMV genome and genes of diverse bacterial species. If this conclusion is substantiated with additional isolate, extremely urgent consideration must be given to the potential capacity of viteria to drastically reformat the genomic structures of both prokaryotic and eukaryotic organisms.
As discussed elsewhere, the SCMV sequences in the prototype stealth virus point to an African green monkey as the primary source of this particular isolate. These monkeys were widely used, and indeed are still used to produce an attenuated live polio virus vaccine. Detailed information on the process of vaccine production and on the results of Government safety testing of vaccines is shielded from public inquiry as a protection of proprietary interests. For inexplicable reasons, molecular assays for SCMV contamination of production lots of polio vaccines have not been employed. Unquestionably, an open discussion of the present and prior risks inherent in the production and use of polio vaccines, and of other live vaccines administered to human and animal, could shed useful insights into a possible calamity of modern vaccine technology.
Animals were presumably also used in the production and testing of biological agents for military purposes. As detailed in a recently published book, Brucella bacteria have been considered as potential pathogens for germ warfare. This observation is intriguing since nucleotide and amino acid sequences from this bacteria are present within the cloned genes derived from the viteria infected patient. Records for biological studies performed with these and other bacteria and viruses should also be made available for scientific review.
The existence of viteria extends the unsettling notion that stealth viruses represent "Nature's Biological Weapons Program." Continued, unchecked replication of genetically diversified, potentially oncogenic, pathogens, that can pass between species, yet bypass cellular immune defenses, could have devastating consequences. A program to combat viteria through a better understanding of their origins and biological activities is urgently required. Further information on stealth-adapted viruses, and on the viteria sequencing program, is available at www.ccid.org