Virology Journal - Most viewed articles

Replicative Homeostasis: A fundamental mechanism mediating selective viral replication and escape mutation

Richard Sallie — 2005-02-11

Hepatitis C (HCV), hepatitis B (HBV), the human immunodeficiency viruses (HIV), and other viruses that replicate via RNA intermediaries, cause an enormous burden of disease and premature death worldwide. These viruses circulate within infected hosts as vast populations of closely related, but genetically diverse, molecules known as "quasispecies". The mechanism(s) by which this extreme genetic and antigenic diversity is stably maintained are unclear, but are fundamental to understanding viral persistence and pathobiology. The persistence of HCV, an RNA virus, is especially problematic and HCV stability, maintained despite rapid genomic mutation, is highly paradoxical. This paper presents the hypothesis, and evidence, that viruses capable of persistent infection autoregulate replication and the likely mechanism mediating autoregulation – Replicative Homeostasis – is described. Replicative homeostasis causes formation of stable, but highly reactive, equilibria that drive quasispecies expansion and generates escape mutation. Replicative homeostasis explains both viral kinetics and the enigma of RNA quasispecies stability and provides a rational, mechanistic basis for all observed viral behaviours and host responses. More importantly, this paradigm has specific therapeutic implication and defines, precisely, new approaches to antiviral therapy. Replicative homeostasis may also modulate cellular gene expression.

Replicative homeostasis II: Influence of polymerase fidelity on RNA virus quasispecies biology: Implications for immune recognition, viral autoimmunity and other "virus receptor" diseases

Richard Sallie — 2005-08-22

Much of the worlds' population is in active or imminent danger from established infectious pathogens, while sporadic and pandemic infections by these and emerging agents threaten everyone. RNA polymerases (RNApol) generate enormous genetic and consequent antigenic heterogeneity permitting both viruses and cellular pathogens to evade host defences. Thus, RNApol causes more morbidity and premature mortality than any other molecule. The extraordinary genetic heterogeneity defining viral quasispecies results from RNApol infidelity causing rapid cumulative genomic RNA mutation a process that, if uncontrolled, would cause catastrophic loss of sequence integrity and inexorable quasispecies extinction. Selective replication and replicative homeostasis, an epicyclical regulatory mechanism dynamically linking RNApol fidelity and processivity with quasispecies phenotypic diversity, modulating polymerase fidelity and, hence, controlling quasispecies behaviour, prevents this happening and also mediates immune escape. Perhaps more importantly, ineluctable generation of broad phenotypic diversity after viral RNA is translated to protein quasispecies suggests a mechanism of disease that specifically targets, and functionally disrupts, the host cell surface molecules – including hormone, lipid, cell signalling or neurotransmitter receptors – that viruses co-opt for cell entry. This mechanism – "Viral Receptor Disease (VRD)" – may explain so-called "viral autoimmunity", some classical autoimmune disorders and other diseases, including type II diabetes mellitus, and some forms of obesity. Viral receptor disease is a unifying hypothesis that may also explain some diseases with well-established, but multi-factorial and apparently unrelated aetiologies – like coronary artery and other vascular diseases – in addition to diseases like schizophrenia that are poorly understood and lack plausible, coherent, pathogenic explanations.

Prevalence of Influenza A viruses in wild migratory birds in Alaska: Patterns of variation in detection at a crossroads of intercontinental flyways

2008-06-04

Background: The global spread of the highly pathogenic avian influenza H5N1 virus has stimulated interest in a better understanding of the mechanisms of H5N1 dispersal, including the potential role of migratory birds as carriers. Although wild birds have been found dead during H5N1 outbreaks, evidence suggests that others have survived natural infections, and recent studies have shown several species of ducks capable of surviving experimental inoculations of H5N1 and shedding virus. To investigate the possibility of migratory birds as a means of H5N1 dispersal into North America, we monitored for the virus in a surveillance program based on the risk that wild birds may carry the virus from Asia. Results: Of 16,797 birds sampled in Alaska between May 2006 and March 2007, low pathogenic avian influenza viruses were detected in 1.7% by rRT-PCR but no highly pathogenic viruses were found. Our data suggest that prevalence varied among sampling locations, species (highest in waterfowl, lowest in passerines), ages (juveniles higher than adults), sexes (males higher than females), date (highest in autumn), and analytical technique (rRT-PCR prevalence = 1.7%; virus isolation prevalence = 1.5%). Conclusion: The prevalence of low pathogenic avian influenza viruses isolated from wild birds depends on biological, temporal, and geographical factors, as well as testing methods. Future studies should control for, or sample across, these sources of variation to allow direct comparison of prevalence rates.

Replicative Homeostasis III: implications for antiviral therapy and mechanisms of response and non-response

Richard Sallie — 2007-03-13

While improved drug regimens have greatly enhanced outcomes for patients with chronic viral infection, antiviral therapy is still not ideal due to drug toxicities, treatment costs, primary drug failure and emergent resistance. New antiviral agents, alternative treatment strategies and a better understanding of viral pathobiology, host responses and drug action are desperately needed. Interferon (IFN) and ribavirin, are effective drugs used to treat hepatitis C (HCV), but the mechanism(s) of their action are uncertain. Error catastrophe (EC), or precipitous loss of replicative fitness caused by genomic mutation, is postulated to mediate ribavirin action, but is a deeply flawed hypothesis lacking empirical confirmation. Paradoxically ribavirin, a proven RNA mutagen, has no impact on HCV viraemia long term, suggesting real viruses, replicating in-vitro, as opposed to mathematical models, replicating in-silico, are likely to resist EC by highly selective replication of fit (~consensus sequence) genomes mediated, in part, by replicative homeostasis (RH), an epicyclic mechanism that dynamically links RNApol fidelity and processivity and other viral protein functions. Replicative homeostasis provides a rational explanation for the various responses seen during treatment of HCV, including genotype-specific and viral load-dependent differential response rates, as well as otherwise unexplained phenomena like the transient inhibition and rebound of HCV viraemia seen during ribavirin monotherapy. Replicative homeostasis also suggests a primarily non-immunological mechanism that mediates increased immune responsiveness during treatment with ribavirin (and other nucleos(t)ide analogues), explicating the enhanced second-phase clearance of HCV ribavirin promotes and, thus, the apparent immunomodulatory action of ribavirin. More importantly, RH suggests specific new antiviral therapeutic strategies.

Herpes simplex virus type-1(HSV-1) oncolytic and highly fusogenic mutants carrying the NV1020 genomic deletion effectively inhibit primary and metastatic tumors in mice

2008-06-02

Background: The NV1020 oncolytic herpes simplex virus type-1 has shown significant promise for the treatment of many different types of tumors in experimental animal models and human trials. Previously, we described the construction and use of the NV1020-like virus OncSyn to treat human breast tumors implanted in nude mice. The syncytial mutation gKsyn1 (Ala-to-Val at position 40) was introduced into the OncSyn viral genome cloned into a bacterial artificial chromosome using double-red mutagenesis in E. coli to produce the OncdSyn virus carrying syncytial mutations in both gB(syn3) and gK(syn1). Results: The OncdSyn virus caused extensive virus-induced cell fusion in cell culture. The oncolytic potential of the OncSyn and OncdSyn viruses was tested in the highly metastatic syngeneic mouse model system, which utilizes 4T1 murine mammary cancer cells implanted within the interscapular region of Balb/c mice. Mice were given three consecutive intratumor injections of OncSyn, OncdSyn, or phosphate buffered saline four days apart. Both OncSyn and OncdSyn virus injections resulted in significant reduction of tumor sizes (p < 0.05) compared to control tumors. Virus treated mice but not controls showed a marked reduction of metastatic foci in lungs and internal organs. Mouse weights were not significantly impacted by any treatment during the course of the entire study (p = 0.296). Conclusion: These results show that the attenuated, but highly fusogenic OncSyn and OncdSyn viruses can effectively reduce primary and metastatic breast tumors in immuncompetent mice. The available bac-cloned OncSyn and OncdSyn viral genomes can be rapidly modified to express a number of different anti-tumor and immunomodulatory genes that can further enhance their anti-tumor potency.

Recombination in feline immunodeficiency virus from feral and companion domestic cats

Jessica J Hayward and Allen G Rodrigo — 2008-06-17

Background: Recombination is a relatively common phenomenon in retroviruses. We investigated recombination in Feline Immunodeficiency Virus from naturally-infected New Zealand domestic cats (Felis catus) by sequencing regions of the gag, pol and env genes. Results: The occurrence of intragenic recombination was highest in env, with evidence of recombination in 6.4% (n = 156) of all cats. A further recombinant was identified in each of the gag (n = 48) and pol (n = 91) genes. Comparisons of phylogenetic trees across genes identified cases of incongruence, indicating intergenic recombination. Three (7.7%, n = 39) of these incongruencies were found to be significantly different using the Shimodaira-Hasegawa test.Surprisingly, our phylogenies from the gag and pol genes showed that no New Zealand sequences group with reference subtype C sequences within intrasubtype pairwise distances. Indeed, we find one and two distinct unknown subtype groups in gag and pol, respectively. These observations cause us to speculate that these New Zealand FIV strains have undergone several recombination events between subtype A parent strains and undefined unknown subtype strains, similar to the evolutionary history hypothesised for HIV-1 "subtype E".Endpoint dilution sequencing was used to confirm the consensus sequences of the putative recombinants and unknown subtype groups, providing evidence for the authenticity of these sequences. Endpoint dilution sequencing also resulted in the identification of a dual infection event in the env gene. In addition, an intrahost recombination event between variants of the same subtype in the pol gene was established. This is the first known example of naturally-occurring recombination in a cat with infection of the parent strains. Conclusion: Evidence of intragenic recombination in the gag, pol and env regions, and complex intergenic recombination, of FIV from naturally-infected domestic cats in New Zealand was found. Strains of unknown subtype were identified in all three gene regions. These results have implications for the use of the current FIV vaccine in New Zealand.

A broad spectrum, one-step reverse-transcription PCR amplification of the neuraminidase gene from multiple subtypes of influenza A virus

2008-07-09

Background: The emergence of high pathogenicity strains of Influenza A virus in a variety of human and animal hosts, with wide geographic distribution, has highlighted the importance of rapid identification and subtyping of the virus for outbreak management and treatment. Type A virus can be classified into subtypes according to the viral envelope glycoproteins, haemagglutinin and neuraminidase. Here we review the existing specificity and amplification of published primers to subtype neuraminidase gene; and describe a new broad spectrum primer pair that can detect all 9 neuraminidase subtypes. Results: Bioinformatic analysis of 3,337 full-length influenza A neuraminidase segments in the NCBI database revealed semi-conserved regions not previously targeted by primers. Two degenerate primers with M13 tags, NA8F-M13 and NA10R-M13 were designed from these regions and used to generate a 253 bp cDNA product. One-step RT-PCR testing was successful in 31/32 (97%) cases using a touchdown protocol with RNA from over 32 different cultured influenza A virus strains representing the 9 neuraminidase subtypes. Frozen blinded clinical nasopharyngeal aspirates were also assayed and were mostly of subtype N2. The region amplified was direct sequenced and then used in database searches to confirm the identity of the template RNA. The RT-PCR fragment generated includes one of the mutation sites related to oseltamivir resistance, H274Y. Conclusions: Our one-step RT-PCR assay followed by sequencing is a rapid, accurate, and specific method for detection and subtyping of different neuraminidase subtypes from a range of host species and from different geographical locations.

Epitope characterization of the protective monoclonal antibody VN04-2 shows broadly neutralizing activity against highly pathogenic H5N1

2008-07-11

The monoclonal antibody VN04-2 was previously shown to protect mice against lethal A/Vietnam/1203/04 H5N1 virus challenge when administered pre- and post-infection. In this study, we characterized the binding requirements of this antibody using direct binding to hemagglutinin and neutralization assays with H5N1 virus-like particles (H5N1-VLP) of eight recent H5N1 strains representing the major mutations within the 140s antigenic loop. Binding was clade independent and 3 mutations within this antigenic region are required before escape is possible, suggesting that apart from the H5N1 viruses circulating in Indonesia, VN04-2 may provide protection against H5N1 viruses from all other regions.

Severe cytomegalovirus infection in apparently immunocompetent patients: a systematic review

2008-03-27

Background: The morbidity and mortality associated with cytomegalovirus (CMV) infection in immunocompromised patients (especially in HIV-infected patients and transplant recipients), as well as with congenital CMV infection are well known. In contrast, relatively little attention has been paid to the morbidity and mortality that CMV infection may cause in immunocompetent patients. Methods: We reviewed the evidence associated with severe manifestations of CMV infection in apparently immunocompetent patients and the potential role of antiviral treatment for these infections. We searched in PubMed, Scopus, and the Cochrane Library for the period of 1950–2007 to identify relevant articles. Results: We retrieved 89 articles reporting on severe CMV infection in 290 immunocompetent adults. Among these reports, the gastrointestinal tract (colitis) and the central nervous system (meningitis, encephalitis, transverse myelitis) were the most frequent sites of severe CMV infection. Manifestations from other organ-systems included haematological disorders (haemolytic anaemia, thrombocytopenia), thrombosis of the venous or arterial vascular system, ocular involvement (uveitis), and lung disease (pneumonitis). The clinical practice reported in the literature has been to prescribe antiviral treatment for the most severe manifestations of monophasic meningoencephalitis (seizures and coma), ocular involvement, and lung involvement due to CMV. Conclusion: Severe life-threatening complications of CMV infection in immunocompetent patients may not be as rare as previously thought.

The involvement of survival signaling pathways in rubella-virus induced apoptosis

Samantha Cooray, Li Jin and Jennifer M Best — 2005-01-04

Rubella virus (RV) causes severe congenital defects when acquired during the first trimester of pregnancy. RV cytopathic effect has been shown to be due to caspase-dependent apoptosis in a number of susceptible cell lines, and it has been suggested that this apoptotic induction could be a causal factor in the development of such defects. Often the outcome of apoptotic stimuli is dependent on apoptotic, proliferative and survival signaling mechanisms in the cell. Therefore we investigated the role of phosphoinositide 3-kinase (PI3K)-Akt survival signaling and Ras-Raf-MEK-ERK proliferative signaling during RV-induced apoptosis in RK13 cells. Increasing levels of phosphorylated ERK, Akt and GSK3β were detected from 24–96 hours post-infection, concomitant with RV-induced apoptotic signals. Inhibition of PI3K-Akt signaling reduced cell viability, and increased the speed and magnitude of RV-induced apoptosis, suggesting that this pathway contributes to cell survival during RV infection. In contrast, inhibition of the Ras-Raf-MEK-ERK pathway impaired RV replication and growth and reduced RV-induced apoptosis, suggesting that the normal cellular growth is required for efficient virus production.