Avian Influenza Virus Surveillance in Wild Birds in Georgia: 2009-2011

The Caucasus, at the border of Europe and Asia, is important for migration and over-wintering of wild waterbirds. Three flyways, the Central Asian, East Africa-West Asia, and Mediterranean/Black Sea flyways, converge in the Caucasus region. Thus, the Caucasus region might act as a migratory bridge for influenza virus transmission when birds aggregate in high concentrations in the post-breeding, migrating and overwintering periods. Since August 2009, we have established a surveillance network for influenza viruses in wild birds, using five sample areas geographically spread throughout suitable habitats in both eastern and western Georgia. We took paired tracheal and cloacal swabs and fresh feces samples. We collected 8343 swabs from 76 species belonging to 17 families in 11 orders of birds, of which 84 were real-time RT-PCR positive for avian influenza virus (AIV). No highly pathogenic AIV (HPAIV) H5 or H7 viruses were detected. The overall AIV prevalence was 1.6%. We observed peak prevalence in large gulls during the autumn migration (5.3-9.8%), but peak prevalence in Black-headed Gulls in spring (4.2-13%). In ducks, we observed increased AIV prevalence during the autumn post-moult aggregations and migration stop-over period (6.3%) but at lower levels to those observed in other more northerly post-moult areas in Eurasia. We observed another prevalence peak in the overwintering period (0.14-5.9%). Serological and virological monitoring of a breeding colony of Armenian Gulls showed that adult birds were seropositive on arrival at the breeding colony, but juveniles remained serologically and virologically negative for AIV throughout their time on the breeding grounds, in contrast to gull AIV data from other geographic regions. We show that close phylogenetic relatives of viruses isolated in Georgia are sourced from a wide geographic area throughout Western and Central Eurasia, and from areas that are represented by multiple different flyways, likely linking different host sub-populations

Evolution of infectious bronchitis virus in the field after homologous vaccination introduction

International audienceAbstractDespite the fact that vaccine resistance has been typically considered a rare phenomenon, some episodes of vaccine failure have been reported with increasing frequency in intensively-raised livestock. Infectious bronchitis virus (IBV) is a widespread avian coronavirus, whose control relies mainly on extensive vaccine administration. Unfortunately, the continuous emergence of new vaccine-immunity escaping variants prompts the development of new vaccines. In the present work, a molecular epidemiology study was performed to evaluate the potential role of homologous vaccination in driving IBV evolution. This was undertaken by assessing IBV viral RNA sequences from the ORF encoding the S1 portion of viral surface glycoprotein (S) before and after the introduction of a new live vaccine on broiler farms in northern-Italy. The results of several biostatistics analyses consistently demonstrate the presence of a higher pressure in the post-vaccination period. Natural selection was detected essentially on sites located on the protein surface, within or nearby domains involved in viral attachment or related functions. This evidence strongly supports the action of vaccine-induced immunity in conditioning viral evolution, potentially leading to the emergence of new vaccine-escape variants. The great plasticity of rapidly-evolving RNA-viruses in response to human intervention, which extends beyond the poultry industry, is demonstrated, claiming further attention due to their relevance for animal and especially human health

The Replicase Gene of Avian Coronavirus Infectious Bronchitis Virus Is a Determinant of Pathogenicity

We have previously demonstrated that the replacement of the S gene from an avirulent strain (Beaudette) of infectious bronchitis virus (IBV) with an S gene from a virulent strain (M41) resulted in a recombinant virus (BeauR-M41(S)) with the in vitro cell tropism of the virulent virus but that was still avirulent. In order to investigate whether any of the other structural or accessory genes played a role in pathogenicity we have now replaced these from the Beaudette strain with those from M41. The recombinant IBV was in effect a chimaeric virus with the replicase gene derived from Beaudette and the rest of the genome from M41. This demonstrated that it is possible to exchange a large region of the IBV genome, approximately 8.4 kb, using our transient dominant selection method. Recovery of a viable recombinant IBV also demonstrated that it is possible to interchange a complete replicase gene as we had in effect replaced the M41 replicase gene with the Beaudette derived gene. Analysis of the chimaeric virus showed that it was avirulent indicating that none of the structural or accessory genes derived from a virulent isolate of IBV were able to restore virulence and that therefore, the loss of virulence associated with the Beaudette strain resides in the replicase gene

Cleavage of the SARS Coronavirus Spike Glycoprotein by Airway Proteases Enhances Virus Entry into Human Bronchial Epithelial Cells In Vitro

Background: Entry of enveloped viruses into host cells requires the activation of viral envelope glycoproteins through cleavage by either intracellular or extracellular proteases. In order to gain insight into the molecular basis of protease cleavage and its impact on the efficiency of viral entry, we investigated the susceptibility of a recombinant native full-length S-protein trimer (triSpike) of the severe acute respiratory syndrome coronavirus (SARS-CoV) to cleavage by various airway proteases. Methodology/Principal Findings: Purified triSpike proteins were readily cleaved in vitro by three different airway proteases: trypsin, plasmin and TMPRSS11a. High Performance Liquid Chromatography (HPLC) and amino acid sequencing analyses identified two arginine residues (R667 and R797) as potential protease cleavage site(s). The effect of protease-dependent enhancement of SARS-CoV infection was demonstrated with ACE2 expressing human bronchial epithelial cells 16HBE. Airway proteases regulate the infectivity of SARS-CoV in a fashion dependent on previous receptor binding. The role of arginine residues was further shown with mutant constructs (R667A, R797A or R797AR667A). Mutation of R667 or R797 did not affect the expression of S-protein but resulted in a differential efficacy of pseudotyping into SARS-CoVpp. The R667A SARS-CoVpp mutant exhibited a lack of virus entry enhancement following protease treatment. Conclusions/Significance: These results suggest that SARS S-protein is susceptible to airway protease cleavage and, furthermore, that protease mediated enhancement of virus entry depends on specific conformation of SARS S-protein upon ACE2 binding. These data have direct implications for the cell entry mechanism of SARS-CoV along the respiratory system and, furthermore expand the possibility of identifying potential therapeutic agents against SARS-CoV. © 2009 Kam et al.published_or_final_versio