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Characterization and evolution of NS5A quasispecies of hepatitis C virus genotype 1b in patients with different stages of liver disease.

The quasispecies nature of hepatitis C virus (HCV) is thought to play a central role in modulating viral functions. Recent work has linked NS5A protein with viral replication, resistance to interferon (IFN), and control of cellular growth, probably through the interaction of its protein kinase R (double stranded RNA-activated protein kinase, PKR) binding domain (PKR-bd) with cellular PKR, but knowledge of how PKR-bd viral population evolves during disease progression is limited. Since we have previously described an association between amino acid composition of the PKR-bd and the presence of HCC, in this report we further investigated the dynamic behavior of viral population parameters by sequencing an average of 20 clones per sample in 27 samples from 19 untreated patients with different degrees of liver disease, 8 of whom were followed over time. Viral population parameters varied widely from patient to patient, but no differences were observed in the complexity, diversity, types of nucleotide changes, or evolutionary pattern of the quasispecies according to the stage of liver disease. In five samples, we detected "quasispecies-tails"; that is, clones whose minimum genetic distance to the remaining clones of their own quasispecies were higher than the maximum genetic distance found between any other two clones of the same sample. In summary, independent of the degree of liver disease, or the mutations detected within the consensus sequence of the PKR-bd, the NS5A of HCV presents a flexible and variable quasispecies structure that remains largely stable during the natural course of an HCV infection, highlighting the central role of NS5A protein in viral life cycle.

Amino Acid Sequence↗

Sequence evolution of the hypervariable region in the putative envelope region E2/NS1 of hepatitis C virus is correlated with specific humoral immune responses.

Sequence evolution of the hypervariable region 1 (HVR1) in the N terminus of E2/NS1 of hepatitis C virus (HCV) was studied retrospectively in six chimpanzees inoculated with the same genotype 1b strain, containing a unique predominant HVR1 sequence. Immediately after inoculation, all animals contained the same HVR predominant sequence. Two animals developed an acute self-limiting infection. Anti-HVR1 immunoglobulin G (IgG) was produced 40 to 60 days after inoculation and rapidly disappeared after normalization of transaminases. Another chimpanzee, previously infected with human immunodeficiency virus type 1, showed a delayed response to HVR1 epitopes after superinfection with HCV. No sequence variation of HVR1 was observed in these two animals during the transient viremia in the acute phase. Three other chimpanzees developed a chronic HCV infection. During follow up, sequence evolution occurred in two animals and their anti-HVR1 response remained at varying but detectable levels. The first mutations occurred immediately after the production of anti-HVR1 during the acute phase. However, IgM anti-HVR1 was not detectable. Remarkably, HVR1 sequences remained conserved for more than 6 years in another chronically infected animal. This correlated with the complete absence of detectable anti-HVR1 during this period. Seven years after inoculation, anti-HVR1 IgG was produced and coincided with an HVR1 alteration. These results strongly suggest the involvement of neutralizing anti-HVR antibodies in sequence evolution of HVR1 through immune selection.

Amino Acid Sequence↗

Evolution of surface and nonstructural-1 genes of influenza B viruses isolated in the Province of Québec, Canada, during the 1998-2001 period.

After 2 minor winter seasons, influenza B viruses were predominantly isolated in the Province of Quebec, Canada, during the 2000-2001 season representing 74% of laboratory-confirmed influenza viruses. We performed an antigenic study of the hemagglutinin (HA) protein and a molecular characterization of the HA1 region, nonstructural-1 (NS1) and neuraminidase (NA)/NB genes of 20 influenza B strains isolated in the Province of Quebec during the 1998-2001 period. Our isolates were compared to recent vaccine strains (B/Harbin/7/94 in 1998-1999, B/Yamanashi/166/98 in 1999-2000 and 2000-2001, and B/Sichuan/379/99 in 2001-2002). The hemagglutination inhibition (HI) test revealed that all isolates were different from B/Harbin/7/94 and were more related to the 2 other vaccine strains although precise identification was often impossible. Molecular analysis of the HA1 gene revealed that both B/Yamanashi/166/98-like and B/Sichuan/379/99-like isolates co-circulated during the 1998-1999 season whereas isolates from the 2 subsequent years were more related to B/Sichuan/379/99. Most isolates (8/9) of the 2000-2001 season contained a N126D substitution recently associated with altered antigenicity in recent influenza B/Yamagata/16/88-related viruses. Although the HA1 and NS1 protein sequences of viruses isolated during the 1998-1999 season were clearly different from those of the respective vaccine strain (B/Harbin/7/94), the NA protein sequence of those isolates was slightly more related to B/Harbin/7/94 than B/Yamanashi/166/98 suggesting distinct patterns of evolution for these genes. This study confirms the importance of a detailed molecular analysis for understanding the evolution of influenza B viruses.

Antigenic Variation↗

The evolution of porcine reproductive and respiratory syndrome virus: quasispecies and emergence of a virus subpopulation during infection of pigs with VR-2332.

GP5, the principal envelope glycoprotein of porcine reproductive and respiratory syndrome virus (PRRSV), contains a hypervariable region within the ectodomain which is responsible for generating diversity in field isolates. The purpose of this study was to gain insight into the possible origin of this diversity by following GP5 sequence changes in pigs exposed to PRRSV strain VR-2332 in utero. A region of the PRRS virus genome containing portions of ORF4 and ORF5 was amplified directly from tissues of infected pigs from birth to 132 days of age. We observed the emergence of a new PRRSV population, identified by a single nucleotide change in the ectodomain. The Asp to Asn change at amino acid 34 was also found as a minor component in pigs that expressed the wild-type sequence. The results from this study suggest that the variability in the ectodomain of ORF5 is the result of positive or negative selection, of which the mechanism remains to be determined.

Amino Acid Sequence↗

The sequential introduction of HIV-1 subtype B and CRF01AE in Singapore by sexual transmission: accelerated V3 region evolution in a subpopulation of Asian CRF01 viruses.

The rapid spread of the human immunodeficiency virus type 1 (HIV-1) circulating recombinant form (CRF) 01AE throughout Asia demonstrates the dynamic nature of emerging epidemics. To further characterize the dissemination of these strains regionally, we sequenced 58 strains from Singapore and found that subtype B and CRF01 were introduced separately, by homosexual and heterosexual transmission, respectively. Protein similarity scores of the Singapore CRF01, as well as all Asian strains, demonstrated a complex distribution of scores in the V3 loop--some strains had very similar V3 loop sequences, while others were highly divergent. Furthermore, we found a strong correlation between the loss of a V3 glycosylation site and the divergent strains. This suggests that loss of this glycosylation site may make the V3 loop more susceptible to immune surveillance. The identification of a rapidly evolving population of CRF01AE variants should be considered when designing new candidate vaccines and when evaluating breakthrough strains from current vaccine trials.

Acquired Immunodeficiency Syndrome↗

Evolution of post-natal herpes simplex virus encephalitis to multicystic encephalopathy.

A 3-week-old, previously healthy infant developed biopsy-proven Herpes virus type 2 (HSV-2) encephalitis. The encephalitis was characterized by cells having intranuclear inclusions and was without evidence of inflammation or hemorrhage. Neuroimaging studies did not show any destructive lesions in the brain. In spite of antiviral therapy, the infant's neurological conditions deteriorated, and the patient died at the age of 18 weeks. Post-mortem examination showed that most of the cerebral hemispheres were replaced by multiloculated cystic cavities of various sizes, typical of multicystic encephalopathy (MCE). The cystic lesions were randomly distributed and were not confined to any vascular territory. By light microscopy, there were no features of viral infection in the brain. Although in situ hybridization of the biopsy specimen taken during the acute phase of the disease demonstrated abundant HSV genome, this same method failed to detect HSV on the post-mortem specimen. These findings suggest that HSV-2 can induce MCE. Furthermore, the absence of histological features of viral encephalitis and the failure to demonstrate viral genome in the brain at autopsy does not exclude an infectious etiology in certain cases of MCE.

Autopsy↗

Infections associated with medical intervention: hepatitis viruses and HGV.

Transfusion and other medical interventions, as well as their non-medical derivatives, have a lot to answer for. Much of HCV infection can be traced back to this route, HGV is still being transmitted in this way and a substantial proportion of HBV in Africa has been spread around by use of contaminated skin-piercing manoeuvres, medical or otherwise. Many practitioners can still remember the days when blood-borne viruses were not a major feature of medical curricula. Unfortunately, patients currently subjected to cost-cutting medical practices, for example use of unscreened blood and re-use of needles, will have to pay the price in years to come. The evolution of these viruses is linked to their route of introduction, but the relationship between variability and clinical outcome is less certain. Nevertheless, variability is important for clinical practice, especially for the design of sensitive diagnostics and highly effective vaccines.

Cross Infection↗

Cocirculation and evolution of two lineages of influenza B viruses in europe and Israel in the 2001-2002 season.

Forty-nine influenza B virus isolates collected in Belgium, Finland, Spain, and Israel during the 2001-2002 winter season were categorized into either of two lineages, B/Yamagata/16/88 or B/Victoria/2/87, based on the phylogenetic studies of HA1 sequences. The data trace the geographic spread of B/Victoria/2/87-like viruses and support the emergence of B/Hong Kong/1351/02-like viruses, possibly due to selective advantages of reassortment.

Amino Acid Sequence↗

[Retrospective study of 149 cases of hepatitis B virus infections. Study of markers and of evolution].

A retrospective study has been performed in 149 subjects with present or past HBs antigenemia. The group consisted of 8 asymptomatic carriers, 90 with acute hepatitis, 7 with fulminating hepatitis, 27 with chronic hepatitis, 16 with cirrhosis and 1 with hepatoma. The changes from one clinical condition to another, the sources of infection, the percentage of acute hepatitis in the history of chronic hepatitis cases and the working capacity an average of two years after the infection were studied. HBe antigen and the corresponding antibody were detected by immunodiffusion and the results compared with the clinical course.

Acute Disease↗

Review: molecular evolution and the feasibility of an avian influenza virus becoming a pandemic strain--a conceptual shift.

During recent years, a conceptual shift took place with respect to the genetic dynamics of influenza A viruses. In difference of the widely accepted approach that avian viral strains have the capacity to infect man only after undergoing genetic reassortment within pigs, it is now contended that direct transfection of man by intact avian-harbored viral genotypes is an actual, recurrent move, which may bring bout the generation of a new pandemic strain. This cardinal conceptual shift has been propelled by the appearance in 1997 of the zoonotic avian influenza H5N1 virus--a virulent, not yet contagious strain for humans--and ostensibly followed a genuine, unprecedented path within the evolutionary paradigm of Influenza A virus. This paper suggests that direct avian-human genetic interface is a pristine fundamental within the natural history of this protean pathogen, points at earlier as well as corroborative findings leading to such postulation, and regards the course of the H5N1 virus (and alike), as a readily detectable and traceable one, presently, rather then a novel development It further examines the general feasibility of various components of that interface at large, such that give rise--whether gradually or abruptly--to pandemic genotypes, in terms of infectivity, pathogenicity and contagiousness. Within that context, the anticipated involvement of certain human-adapted antigenic subtypes is referred to, extrapolatively. Connectedly, the significance of natural ice as plausible regenerator of influenza A viruses, and its possible contribution to the emergence and reemergence of pandemic strains are accentuated.

Animals↗

Morbillivirus infections, with special emphasis on morbilliviruses of carnivores.

Morbilliviruses infections cause significant mortality in human beings and animals. Measles virus is responsible for up to two million childhood deaths annually in the developing world, while rinderpest and peste des petits ruminants cause severe epizootics in domestic and wild ruminants in areas of the world where they remain endemic. Canine distemper virus (CDV) is a cause of fatal disease in many species of carnivores. Distemper is controlled by vaccination in domestic dogs and farmed mink, but it may be impossible to eradicate the virus because of its global distribution and wide variety of susceptible host species, which includes both freshwater and marine seals. Research is currently under way to develop new recombinant vaccines, since the currently available live attenuated vaccines for CDV are not safe for use for all species and many valuable zoo animals need to be protected from CDV. New morbilliviruses with potentially disastrous ecological consequences for marine mammals have been discovered in the past decade; phocid distemper virus (PDV) in seals and the cetacean morbillivirus (CMV) has been found in dolphins, whales and porpoises. Reverse transcription, coupled with the polymerase chain reaction (RT/PCR) and nucleic acid sequencing, has been used to characterise the morbilliviruses and has given insights into the evolution of this virus genus.

Animals↗

Efficient infection mediated by viral receptors incorporated into retroviral particles.

Many host cell surface proteins, including viral receptors, are incorporated into enveloped viruses. To address the functional significance of these host proteins, murine leukemia viruses containing the cellular receptors for Rous sarcoma virus (Tva) or ecotropic murine leukemia virus (MCAT-1) were produced. These receptor-pseudotyped viruses efficiently infect cells expressing the cognate viral envelope glycoproteins, with titers of up to 10(5) infectious units per milliliter for the Tva pseudotypes. Receptor and viral glycoprotein specificity and functional requirements are maintained, suggesting that receptor pseudotype infection recapitulates events of normal viral entry. The ability of the Tva and MCAT-1 pseudotypes to infect cells efficiently suggests that, in contrast to human immunodeficiency virus type 1 entry, neither of these retroviral receptors requires a coreceptor for membrane fusion. In addition, the ability of receptor pseudotypes to target infected cells suggests that they may be useful therapeutic reagents for directing infection of viral vectors. Receptor-pseudotyped viruses may be useful for identifying new viral receptors or for defining functional requirements of known receptors. Moreover, this work suggests that the production of receptor pseudotypes in vivo could provide a mechanism for expanded viral tropism with potential effects on the pathogenesis and evolution of the virus.

3T3 Cells↗

Sequence analysis of the medium RNA segment of three Simbu serogroup viruses, Akabane, Aino, and Peaton viruses.

The sequence analysis was carried out for the medium (M) RNA segment of the Akabane virus (AKAV), Aino virus (AINV), and Peaton virus (PEAV) of the Simbu serogroup of the genus Orthobunyavirus of the family Bunyaviridae. The complementary sequences of the M RNA segments of AKAV, AINV, and PEAV contain a single large open reading frame (ORF), like other orthobunyaviruses. The ORFs potentially encode 1401 amino acids (aa), 1404 aa, and 1400 aa polypeptides, respectively. The identity of the M segment among these viruses is remarkably low, although previous researchers reported that the small RNA segments are highly conserved. Because the M segment codes for the viral surface glycoproteins G1 and G2, the variability of the M segment may affect the antigenicity of these viruses. Phylogenetic studies based on the M and S segment sequences suggested that genetic reassortment has been occurring among ancestral viruses of the three Simbu serogroup viruses throughout their evolution.

Animals↗

[Variability and development of viral populations: assessment and implications].

RNA virus populations consist of complex distributions of closely related but not identical genomes known as viral quasi-species. The quasi-species concept describes the dynamics of these genomes subjected to a continuous process of variation, competition, and selection. Quasi-species dynamics has broad implications not only in the understanding of the molecular mechanisms underlying adaptation of RNA viruses but also in the design of strategies for control and prevention of viral disease. Viral load and genetic heterogeneity have a determinant influence on the adaptation of RNA virus to their environment. Vaccines designed to control diseases caused by highly variable viruses must contain several B and T epitopes to provide an ample and diversified immune response. Similarly, antiviral drugs should be used in combination therapy to minimize selection of resistant viruses. The theoretical model of quasi-species has opened the way for new antiviral therapies based on augmentation of the mutation rate during replication of viral RNA. Finally the quasi-species concept provides the basis for defining the selective factors that could influence the evolution of RNA virus and promote the emergence or reemergence of viral diseases.

Antibiosis↗

Nucleotide sequences of Australian isolates of the feline immunodeficiency virus: comparison with other feline lentiviruses.

Proviral DNA from four Australian isolates of feline immunodeficiency virus (FIV) was amplified by PCR and the nucleotide sequence determined for two conserved regions within gag (p15/p24) and pol (RT) genes. Comparison with the nucleotide and deduced amino acid sequence of two previously described U.S. isolates from California (Petaluma and PPR), and a third from Maryland (MD) as well as the Japanese isolate TM2, revealed a close similarity between the Australian and Californian isolates with 95-97% nucleotide and 96-99% amino acid homologies. By contrast, the Maryland and Japanese isolates were more distantly related with only 84-87% nucleotide and 90-94% amino acid homology with either the Australian or Californian isolates. The relationship of the Australian FIV isolates to other domestic isolates as well as eight lentiviral isolates from wild felidae (panthers) published previously, was investigated further by constructing a phylogenetic tree based on the pol sequence. This revealed two subgroups of FIV, an Australian/Californian group and a less tightly clustered Maryland/Japanese group. These results suggest that the genomic variability of FIV is reflected by more than simply geographic distance. Furthermore, the relative genetic homogeneity found between Australian isolates suggest a shorter period of evolution of the virus in Australia than in North America.

Amino Acid Sequence↗