Virulence Of Avian Influenza Viruses Research Articles

Epidemiology and molecular characterization of avian influenza virus in backyard poultry of Chattogram, Bangladesh.

Ducks, the natural reservoir of avian influenza virus (AIV), act as reassortment vessels for HPAI and low pathogenic avian influenza (LPAI) virus for domestic and wild bird species. In Bangladesh, earlier research was mainly focused on AIV in commercial poultry and live bird markets, where there is scanty literature reported on AIV in apparently healthy backyard poultry at the household level. The present cross-sectional study was carried out to reveal the genomic epidemiology of AIV of backyard poultry in coastal (Anowara) and plain land (Rangunia) areas of Bangladesh. We randomly selected a total of 292 households' poultry (having both chicken and duck) for sampling. We administered structured pre-tested questionnaires to farmers through direct interviews. We tested cloacal samples from birds for the matrix gene (M gene) followed by H5 and H9 subtypes using real-time reverse transcriptase-polymerase chain reaction (rRT-PCR). All AIV-positive samples were subjected to four-gene segment sequencing (M, PB1, HA, and NA gene). We found that the prevalence of AIV RNA at the household level was 6.2% (n=18; N=292), whereas duck and chicken prevalence was 3.6% and 3.2%, respectively. Prevalence varied with season, ranging from 3.1% in the summer to 8.2% in the winter. The prevalence of subtypes H5 and H9 in backyard poultry was 2.7% and 3.3%, respectively. The phylogenetic analysis of M, HA, NA, and PB1 genes revealed intra-genomic similarity, and they are closely related to previously reported AIV strains in Bangladesh and Southeast Asia. The findings indicate that H5 and H9 subtypes of AIV are circulating in the backyard poultry with or without clinical symptoms. Moreover, we revealed the circulation of 2.3.2.1a (new) clade among the chicken and duck population without occurring outbreak which might be due to vaccination. In addition to routine surveillance, molecular epidemiology of AIV will assist to gain a clear understanding of the genomic evolution of the AIV virus in the backyard poultry rearing system, thereby facilitating the implementation of effective preventive measures to control infection and prevent the potential spillover to humans.

Avian influenza viruses suppress innate immunity by inducing trans-transcriptional readthrough via SSU72

Innate immunity plays critical antiviral roles. The highly virulent avian influenza viruses (AIVs) H5N1, H7N9, and H5N6 can better escape host innate immune responses than the less virulent seasonal H1N1 virus. Here, we report a mechanism by which transcriptional readthrough (TRT)-mediated suppression of innate immunity occurs post AIV infection. By using cell lines, mouse lungs, and patient PBMCs, we showed that genes on the complementary strand (“trans” genes) influenced by TRT were involved in the disruption of host antiviral responses during AIV infection. The trans-TRT enhanced viral lethality, and TRT abolishment increased cell viability and STAT1/2 expression. The viral NS1 protein directly bound to SSU72, and degradation of SSU72 induced TRT. SSU72 overexpression reduced TRT and alleviated mouse lung injury. Our results suggest that AIVs infection induce TRT by reducing SSU72 expression, thereby impairing host immune responses, a molecular mechanism acting through the NS1-SSU72-trans-TRT-STAT1/2 axis. Thus, restoration of SSU72 expression might be a potential strategy for preventing AIV pandemics.

Molecular Analysis of the Avian H7 Influenza Viruses Circulating in South Korea during 2018-2019: Evolutionary Significance and Associated Zoonotic Threats.

Avian influenza virus (AIV) subtypes H5 and H7, possessing the ability to mutate spontaneously from low pathogenic (LP) to highly pathogenic (HP) variants, are major concerns for enormous socio-economic losses in the poultry industry, as well as for fatal human infections. Through antigenic drift and shift, genetic reassortments of the genotypes pose serious threats of increased virulence and pathogenicity leading to potential pandemics. In this study, we isolated the H7-subtype AIVs circulating in the Republic of Korea during 2018–2019, and perform detailed molecular analysis to study their circulation, evolution, and possible emergence as a zoonotic threat. Phylogenetic and nucleotide sequence analyses of these isolates revealed their distribution into two distinct clusters, with the HA gene sharing the highest nucleotide identity with either the A/common teal/Shanghai/CM1216/2017, isolated from wild birds in Shanghai, China, or the A/duck/Shimane/2014, isolated from Japan. Mutations were found in HA (S138A (H3 numbering)), M1 (N30D and T215A), NS1 (P42S), PB2 (L89V), and PA (H266R and F277S) proteins—the mutations had previously been reported to be related to mammalian adaptation and changes in the virulence of AIVs. Taken together, the results firmly put forth the demand for routine surveillance of AIVs in wild birds to prevent possible pandemics arising from reassortant AIVs.

Characterization of the low-pathogenic H7N7 avian influenza virus in Shanghai, China

H7N7 avian influenza virus (AIV) can divided into low-pathogenic AIV and high-pathogenic AIV groups. It has been shown to infect humans and animals. Its prevalence state in wild birds in China remains largely unclear. In this study, a new strain of H7N7 AIV, designated CM1216, isolated from wild birds in Shanghai, China, was characterized. Phylogenetic and nucleotide sequence analyses of CM1216 revealed that HA, NA, PB1, NP, and M genes shared the highest nucleotide identity with the Japan H7 subtype AIV circulated in 2019; the PB2 and PA genes shared the highest nucleotide identity with the Korea H7 subtype AIV circulated in wild birds in 2018, while NS gene of CM1216 was 98.93% identical to that of the duck AIV circulating in Bangladesh, and they all belong to the Eurasian lineage. A Bayesian phylogenetic reconstruction of the 2 surface genes of CM1216 showed that multiple reassortments might have occurred in 2015. Mutations were found in HA (A135 T, T136S, and T160 A [H3 numbering]), M1 (N30D and T215 A), NS1 (P42S and D97 E), PB2 (R389 K), and PA (N383D) proteins; these mutations have been shown to be related to mammalian adaptation and changes in virulence of AIVs. Infection studies demonstrated that CM1216 could infect mice and cause symptoms characteristic of influenza virus infection and proliferate in the lungs without prior adaption. This study demonstrates the need for routine surveillance of AIVs in wild birds and detection of their evolution to become a virus with high pathogenicity and ability to infect humans.

A panel of anti-influenza virus nucleoprotein antibodies selected from phage-displayed synthetic antibody libraries with rapid diagnostic capability to distinguish diverse influenza virus subtypes

Immunoassays based on sandwich immuno-complexes of capture and detection antibodies simultaneously binding to the target analytes have been powerful technologies in molecular analyses. Recent developments in single molecule detection technologies enable the detection limit of the sandwich immunoassays approaching femtomolar (10–15 M), driving the needs of developing sensitive and specific antibodies for ever-increasingly broad applications in detecting and quantifying biomarkers. The key components underlying the sandwich immunoassays are antibody-based affinity reagents, for which the conventional sources are mono- or poly-clonal antibodies from immunized animals. The downsides of the animal-based antibodies as affinity reagents arise from the requirement of months of development timespan and limited choices of antibody candidates due to immunodominance of humoral immune responses in animals. Hence, developing animal antibodies capable of distinguishing highly related antigens could be challenging. To overcome the limitation imposed by the animal immune systems, we developed an in vitro methodology based on phage-displayed synthetic antibody libraries for diverse antibodies as affinity reagents against closely related influenza virus nucleoprotein (NP) subtypes, aiming to differentiating avian influenza virus (H5N1) from seasonal influenza viruses (H1N1 and H3N2), for which the NPs are closely related by 90–94% in terms of pairwise amino acid sequence identity. We applied the methodology to attain, within four weeks, a panel of IgGs with distinguishable specificities against a group of representative NPs with pairwise amino acid sequence identities up to more than 90%, and the antibodies derived from the antibody libraries without further affinity refinement had comparable affinity of mouse antibodies to the NPs with the detection limit less than 1 nM of viral NP from lysed virus with sandwich ELISA. The panel of IgGs were capable of rapidly distinguishing infections due to virulent avian influenza virus from infections of seasonal flu, in responding to a probable emergency scenario where avian influenza virus would be transmissible among humans overlapping with the seasonal influenza infections. The results indicate that the in vitro antibody development methodology enables developing diagnostic antibodies that would not otherwise be available from animal-based antibody technologies.

ANALYSIS OF GENETIC CHARACTERISTICS OF INFLUENZA VIRUS A/CHICKEN/CHELYABINSK/30/2019 H9N2 ISOLATED IN CHELYABINSK OBLAST

The paper presents data on the study of genetic characteristics of the infl uenza virus A/chicken/ Chelyabinsk/30/2019 H9N2 isolated from pathological material (chicken internal organs) in February 2019 and received from the poultry farm in the Chelyabinsk Oblast. The H9N2 subtype of the isolated virus was identifi ed based on virological analysis. Sequencing of the hemagglutinin gene segment revealed that the amino acid sequence at the cleavage site was RSSR/GLF, which is characteristic of a low virulent avian infl uenza virus. Phylogenetic analysis of the obtained nucleotide sequences of the hemagglutinin gene fragment (1–1539 bp open reading frame) showed that the A/chicken/Chelyabinsk/30/2019 H9N2 isolate belongs to the G1 genetic group of the low virulent infl uenza virus A/H9, the representatives of which are widely spread in the Middle Eastern and Central Asian countries. The complete nucleotide genome sequence of the studied pathogen was determined. The comparative analysis of all genomic segments using the GenBank database revealed a close relationship (over 99%) between the A/chicken/Chelyabinsk/30/2019 H9N2 virus and the A/H9 infl uenza virus isolates circulating in Israel in 2006–2012. According to the analysis of the predicted amino acid sequence of the studied isolate, the positions of some molecular markers that determine the biological properties of the virus have been identifi ed. Most amino acid positions of hemagglutinin (according to H3 subtype sequence numbering) suggest affi nity for the ACA2-3Gal-receptors of avian epithelial cells. Amino acid substitutions were detected at the site within the receptor-binding domain as compared to the A/H9N2 infl uenza virus isolates obtained in Russia in 2018. The primary structure of the A/chicken/Chelyabinsk/30/2019 H9N2 isolate demonstrates a very high level of genetic similarity to the infl uenza virus isolate A/chicken/ Israel/215/2007 H9N2 used as a vaccine strain.

Insights into the Acquisition of Virulence of Avian Influenza Viruses during a Single Passage in Ferrets

Circulating avian influenza viruses pose a significant threat, with human infections occurring infrequently but with potentially severe consequences. To examine the dynamics and locale of the adaptation process of avian influenza viruses when introduced to a mammalian host, we infected ferrets with H5N1 viruses. As expected, all ferrets infected with the human H5N1 isolate A/Vietnam/1203/2004 showed severe disease and virus replication outside the respiratory tract in multiple organs including the brain. In contrast infection of ferrets with the avian H5N1 virus A/Chicken/Laos/Xaythiani-26/2006 showed a different collective pattern of infection; many ferrets developed and cleared a mild respiratory infection but a subset (25–50%), showed extended replication in the upper respiratory tract and developed infection in distal sites. Virus from these severely infected ferrets was commonly found in tissues that included liver and small intestine. In most instances the virus had acquired the common virulence substitution PB2 E627K but, in one case, a previously unidentified combination of two amino acid substitutions at PB2 S489P and NP V408I, which enhanced polymerase activity, was found. We noted that virus with high pathogenicity adaptations could be dominant in an extra-respiratory site without being equally represented in the nasal wash. Further ferret passage of these mutated viruses resulted in high pathogenicity in all ferrets. These findings illustrate the remarkable ability of avian influenza viruses that avoid clearance in the respiratory tract, to mutate towards a high pathogenicity phenotype during just a single passage in ferrets and also indicate a window of less than 5 days in which treatment may curtail systemic infection.

Identification the Presence of Clade 2.3.2.1c-Avian Influenza H5N1, a Highly Pathogenic Virus in Iraq, 2018

Abstract For the first time in Iraq, we identified in March, 2018 the presence of a highly virulent avian influenza virus (AIV), H5N1 (Clade 2.3.2.1c), causing highly pathogenic avian influenza (HPAI) in poultry farms, Iraq,. The identification of the virus was done using a rapid serological test, a real time-qPCR, and glycoprotein gene sequencing. Using sequencing and phylogenetic analyses, the clade 2.3.2.1c virus was recorded to be clustered, with high similarity to Asian and West African AIV, HPAI H5N1 from Ivory Coast identified in 2015. According to our knowledge, there was no previous detection of the clade 2.3.2.1c made in Iraq. Our results provide evidence that high risk of HPAI H5 outbreaks might be present in Iraq, and this needs to lead to high quality surveillance targeting of wild and domestic birds for early diagnosis of HPAI. The current work provides feasible and accurate approaches for understanding the evolution of HPAI H5 virus in different countries around the world.

Composition of the Hemagglutinin Polybasic Proteolytic Cleavage Motif Mediates Variable Virulence of H7N7 Avian Influenza Viruses.

Acquisition of a polybasic cleavage site (pCS) in the hemagglutinin (HA) is a prerequisite for the shift of low pathogenic (LP) avian influenza virus (AIV) to the highly pathogenic (HP) form in chickens. Whereas presence of a pCS is required for high pathogenicity, less is known about the effect of composition of pCS on virulence of AIV particularly H7N7. Here, we investigated the virulence of four avian H7N7 viruses after insertion of different naturally occurring pCS from two HPAIV H7N7 (designated pCSGE and pCSUK) or from H7N1 (pCSIT). In vitro, the different pCS motifs modulated viral replication and the HA cleavability independent on the HA background. However, in vivo, the level of virulence conferred by the different pCS varied significantly. Within the respective viral backgrounds viruses with pCSIT and pCSGE were more virulent than those coding for pCSUK. The latter showed also the most restricted spread in inoculated birds. Besides the pCS, other gene segments modulated virulence of these H7N7 viruses. Together, the specific composition of the pCS significantly influences virulence of H7N7 viruses. Eurasian LPAIV H7N7 may shift to high pathogenicity after acquisition of “specific” pCS motifs and/or other gene segments from HPAIV.

Residues in the PB2 and PA genes contribute to the pathogenicity of avian H7N3 influenza A virus in DBA/2 mice

Replication and transmission of avian influenza virus in humans poses a pandemic threat. The molecular determinants that facilitate this process are not well understood. We used DBA/2 mice to identify viral factors that mediate the difference in pathogenesis between a virulent (H7N3) and a non-virulent (H7N9) avian influenza virus from North America. In vitro and in vivo characterization of reassortant viruses identified the PB2 and PA polymerase genes as major determinants of H7N3 pathogenesis. Analysis of individual residues in the PB2 and PA genes identified position 358 (E358V) in PB2 and positions 190 (P190S) and 400 (Q400P) in PA that reduced the virulence of H7N3 virus. The E358V and P190S substitutions also caused reduced inflammation after infection. Our results suggest that specific residues in the polymerase proteins PB2 and PA are important for replication and virulence of avian influenza viruses in a mammalian host.

Prevalence of the C-terminal truncations of NS1 in avian influenza A viruses and effect on virulence and replication of a highly pathogenic H7N1 virus in chickens

abstractHighly pathogenic (HP) avian influenza viruses (AIV) evolve from low pathogenic (LP) precursors after circulation in poultry by reassortment and/or single mutations in different gene segments including that encoding NS1. The carboxyl terminal end (CTE) of NS1 exhibits deletions between amino acid 202 and 230 with still unknown impact on virulence of AIV in chickens. In this study, NS1 protein sequences of all AIV subtypes in birds from 1902 to 2015 were analyzed to study the prevalence and distribution of CTE truncation (ΔCTE). Thirteen different ΔCTE forms were observed in NS1 proteins from 11 HA and 8 NA subtypes with high prevalences in H9, H7, H6 and H10 and N9, N2, N6 and N1 subtypes particularly in chickens and minor poultry species. With 88% NS217 lacking amino acids 218–230 was the most common ΔCTE form followed by NS224 (3.6%). NS217 was found in 10 and 8 different HA and NA subtypes, respectively, whereas NS224 was detected exclusively in the Italian HPAIV H7N1 suggesting relevance for virulence. To test this assumption, 3 recombinant HPAIV H7N1 were constructed carrying wild-type HP NS1 (Hp-NS224), NS1 with extended CTE (Hp-NS230) or NS1 from LPAIV H7N1 (Hp-NSLp), and tested in-vitro and in-vivo. Extension of CTE in Hp NS1 significantly decreased virus replication in chicken embryo kidney cells. Truncation in the NS1 decreased the tropism of Hp-NS224 to the endothelium, central nervous system and respiratory tract epithelium without significant difference in virulence in chickens. This study described the variable forms of ΔCTE in NS1 and indicated that CTE is not an essential virulence determinant particularly for the Italian HPAIV H7N1 but may be a host-adaptation marker required for efficient virus replication.

Prediction and Identification of T Cell Epitopes in the H5N1 Influenza Virus Nucleoprotein in Chicken

T cell epitopes can be used for the accurate monitoring of avian influenza virus (AIV) immune responses and the rational design of vaccines. No T cell epitopes have been previously identified in the H5N1 AIV virus nucleoprotein (NP) in chickens. For the first time, this study used homology modelling techniques to construct three-dimensional structures of the peptide-binding domains of chicken MHC class Ι molecules for four commonly encountered unique haplotypes, i.e., B4, B12, B15, and B19. H5N1 AIV NP was computationally parsed into octapeptides or nonapeptides according to the peptide-binding motifs of MHC class I molecules of the B4, B12, B15 and B19 haplotypes. Seventy-five peptide sequences were modelled and their MHC class I molecule-binding abilities were analysed by molecular docking. Twenty-five peptides (Ten for B4, six for B12, two for B15, and seven for B19) were predicted to be potential T cell epitopes in chicken. Nine of these peptides and one unrelated peptide were manually synthesized and their T cell responses were tested in vitro. Spleen lymphocytes were collected from SPF chickens that had been immunised with a NP-expression plasmid, pCAGGS-NP, and they were stimulated using the synthesized peptides. The secretion of chicken IFN-γ and the proliferation of CD8+ T cells were tested using an ELISA kit and flow cytometry, respectively. The significant secretion of chicken IFN-γ and proliferation of CD8+ T lymphocytes increased by 13.7% and 11.9% were monitored in cells stimulated with peptides NP89–97 and NP198–206, respectively. The results indicate that peptides NP89–97 (PKKTGGPIY) and NP198–206 (KRGINDRNF) are NP T cell epitopes in chicken of certain haplotypes. The method used in this investigation is applicable to predicting T cell epitopes for other antigens in chicken, while this study also extends our understanding of the mechanisms of the immune response to AIV in chicken.

Two Highly Pathogenic Avian Influenza H5N1 Viruses of Clade 2.3.2.1 with Similar Genetic Background but with Different Pathogenicity in Mice and Ducks

A number of genetic markers for virulence of avian influenza viruses (AIVs) in different hosts have been identified. However, we isolated two H5N1 AIVs, A/Chicken/Jiangsu/k0402/2010(CK/10) and A/Goose/Jiangsu/k0403/2010(GS/10) with similar genetic background, but most well-defined molecular markers for virulence in mammals and avian species were not found in both viral genomes. In addition, pathogenicity of this pair of viruses in different hosts remains unclear. Therefore, we evaluated their pathogenicity in chickens, mice, ducks and guinea pigs. Infection of CK/10 and GS/10 in chickens caused 100% mortality within 24 h. Mouse experiment showed that CK/10 was highly pathogenic (MLD50 = 0.33 log10 EID50), whereas GS/10 was avirulent (MLD50 > 6.32 log10 EID50). Interestingly, the virulence of CK/10 in ducks (DLD50 = 3.83 log10 EID50) was higher than that of GS/10 (DLD50 = 7.7 log10 EID50), which correlated with viral pathogenicity in mice. Although CK/10 and GS/10 showed distinct pathogenicity in mice, they both were lethal to guinea pigs, with CK/10 replicating to higher titres in airways than GS/10. Collectively, these findings suggest that AIVs with similar genetic backgrounds may exhibit distinct pathogenicity in specific hosts and that some unknown molecular markers for virulence may exist and need to be identified.

Distribution of sialic acid receptors and influenza A virus of avian and swine origin in experimentally infected pigs

BackgroundPigs are considered susceptible to influenza A virus infections from different host origins because earlier studies have shown that they have receptors for both avian (sialic acid-alpha-2,3-terminal saccharides (SA-alpha-2,3)) and swine/human (SA-alpha-2,6) influenza viruses in the upper respiratory tract. Furthermore, experimental and natural infections in pigs have been reported with influenza A virus from avian and human sources.MethodsThis study investigated the receptor distribution in the entire respiratory tract of pigs using specific lectins Maackia Amurensis (MAA) I, and II, and Sambucus Nigra (SNA). Furthermore, the predilection sites of swine influenza virus (SIV) subtypes H1N1 and H1N2 as well as avian influenza virus (AIV) subtype H4N6 were investigated in the respiratory tract of experimentally infected pigs using immunohistochemical methods.ResultsSIV antigen was widely distributed in bronchi, but was also present in epithelial cells of the nose, trachea, bronchioles, and alveolar type I and II epithelial cells in severely affected animals. AIV was found in the lower respiratory tract, especially in alveolar type II epithelial cells and occasionally in bronchiolar epithelial cells. SA-alpha-2,6 was the predominant receptor in all areas of the respiratory tract with an average of 80-100% lining at the epithelial cells. On the contrary, the SA-alpha-2,3 was not present (0%) at epithelial cells of nose, trachea, and most bronchi, but was found in small amounts in bronchioles, and in alveoli reaching an average of 20-40% at the epithelial cells. Interestingly, the receptor expression of both SA-alpha-2,3 and 2,6 was markedly diminished in influenza infected areas compared to non-infected areas.ConclusionsA difference in predilection sites between SIV and AIV virus was found, and this difference was in accordance with the distribution of the SA-alpha-2,6 and SA-alpha-2,3 receptor, respectively. The results indicated that the distribution of influenza A virus receptors in pigs are similar to that of humans and therefore challenge the theory that the pig acts as a mixing vessel between human and avian influenza viruses. Furthermore, it was shown that AIV prefers to infect alveolar type II epithelial cells in pigs. This corresponds with findings in humans emphasising the resemblance between the two species.

Amino acids adjacent to the haemagglutinin cleavage site are relevant for virulence of avian influenza viruses of subtype H5

The prime virulence determinant of highly pathogenic avian influenza viruses (HPAIVs) is the polybasic haemagglutinin (HA) cleavage site. However, engineering of a polybasic cleavage site into an avian influenza virus of low pathogenicity does not result in transformation into an HPAIV, indicating the importance of other adaptations. Here, the influence of amino acids adjacent to the HA cleavage site on virulence was studied. Most HPAIVs of subtype H5 carry serine or threonine at position 346 (corresponding to position 323 according to H3 numbering), whereas almost all low-pathogenic H5 viruses have valine. Moreover, all H5 low-pathogenic strains carry threonine at position 351 (corresponding to position 328 according to H3 numbering), suggesting that acquisition of a polybasic cleavage site involves several steps. This study generated a virus mutant derived from HPAIV A/Swan/Germany/R65/06 H5N1 (R65) with a monobasic cleavage site, R65(mono)-S-ER, and the following additional mutants: R65(mono)-V-ER with serine changed to valine at position 346, and R65(mono)-S-ETR and R65(mono)-V-ETR with threonine inserted at position 351. Moreover, in the R65 HA, serine was replaced with valine at position 346 (R65-V). Infection of chickens with R65(mono)-S-ETR or R65(mono)-S-ER led to slight transient respiratory symptoms, whereas R65-infected animals died within 2 days. However, chickens infected with R65-V survived longer than R65-infected animals, indicating that serine 346 in R65 HA contributes to virulence. These data suggest that evolution of H5 HPAIVs from low-pathogenic precursors, besides acquisition of a polybasic cleavage site, involves adaptation of neighbouring regions.

Novel Genotypes of H9N2 Influenza A Viruses Isolated from Poultry in Pakistan Containing NS Genes Similar to Highly Pathogenic H7N3 and H5N1 Viruses

The impact of avian influenza caused by H9N2 viruses in Pakistan is now significantly more severe than in previous years. Since all gene segments contribute towards the virulence of avian influenza virus, it was imperative to investigate the molecular features and genetic relationships of H9N2 viruses prevalent in this region. Analysis of the gene sequences of all eight RNA segments from 12 viruses isolated between 2005 and 2008 was undertaken. The hemagglutinin (HA) sequences of all isolates were closely related to H9N2 viruses isolated from Iran between 2004 and 2007 and contained leucine instead of glutamine at position 226 in the receptor binding pocket, a recognised marker for the recognition of sialic acids linked α2–6 to galactose. The neuraminidase (NA) of two isolates contained a unique five residue deletion in the stalk (from residues 80 to 84), a possible indication of greater adaptation of these viruses to the chicken host. The HA, NA, nucleoprotein (NP), and matrix (M) genes showed close identity with H9N2 viruses isolated during 1999 in Pakistan and clustered in the A/Quail/Hong Kong/G1/97 virus lineage. In contrast, the polymerase genes clustered with H9N2 viruses from India, Iran and Dubai. The NS gene segment showed greater genetic diversity and shared a high level of similarity with NS genes from either H5 or H7 subtypes rather than with established H9N2 Eurasian lineages. These results indicate that during recent years the H9N2 viruses have undergone extensive genetic reassortment which has led to the generation of H9N2 viruses of novel genotypes in the Indian sub-continent. The novel genotypes of H9N2 viruses may play a role in the increased problems observed by H9N2 to poultry and reinforce the continued need to monitor H9N2 infections for their zoonotic potential.

Influence of avian influenza virus NS1 protein on the expression of IP-10 in BEAS-2B cells

To investigate the influence of avian influenza virus (AIV) NS1 protein on the expression of interferon-inducible protein 10 (IP-10). NSI gene from virus A/Anhui/1/2005 (H5N1), NS1 gene inserted with 80-84 amino acids from virus A/Anhui/1/2005 (H5N1) and NS1 gene from virus A/Puerto Rico/8/1934 (H1N1) were cloned into the eukaryotic expression vector pEGFP-N1, and transfected into BEAS-2B cells, IP-10 expression level in transfected cells was detected by flow cytometry. Compared with the control group pEGFP-N1, expression of these three different NS1 genes can down-regulate the expression of IP-10 in BEAS-2B cells, but there is no significant difference as to the lower level among them. NS1 protein of A/Anhui/1/2005 (H5N1) can down-regulate the expression level of IP-10, but this may not clarify its relationship with the virulence of AIV.

IS THE OCCURRENCE OF AVIAN INFLUENZA VIRUS IN CHARADRIIFORMES SPECIES AND LOCATION DEPENDENT?

Birds in the order Charadriiformes were sampled at multiple sites in the eastern half of the continental USA, as well as at Argentina, Chile, and Bermuda, during 1999-2005, and tested for avian influenza virus (AIV). Of more than 9,400 birds sampled, AIV virus was isolated from 290 birds. Although Ruddy Turnstones (Arenaria interpres) comprised just 25% of birds sampled, they accounted for 87% of isolates. Only eight AIV isolations were made from birds at four locations outside of the Delaware Bay, USA, region; six of these were from gulls (Laridae). At Delaware Bay, AIV isolations were predominated by hemagglutinin (HA) subtype H10, but subtype diversity varied each year. These results suggest that AIV infection among shorebirds (Scolopacidae) may be localized, species specific, and highly variable in relation to AIV subtype diversity.

Genetic insight of the H5N1 hemagglutinin cleavage site.

The cleavability of the hemagglutinin (HA) plays a major role in virulence of avian influenza viruses. Detailed analyses of the cleavage sequences and their evolution would give insights into the high pathogenicity of the H5N1 virus. HA segments were visually identifiable in the cellular automata (CA) image, and a feature gene segment (FGS) was only found in H5N1 rather than any other subtype. This FGS is a 30-bp gene segment mainly consisting of ‘A’ and ‘G’. When translated into amino acids the FGS converted into a sequence of mainly basic amino acids with positive charges. This feature amino acid segment (FAAS) was located in the cleavage site loop of HA which was potentially cleavable by various proteases. The 3D structure of H5N1 HA was reconstructed using homology modelling. It was found that the cleavage site loop was well exposed to potential proteases. The molecular surfaces were reconstructed to study how mutation and deletion of some amino acids in the FAAS affected the charge distribution. It was found that some mutations had severely changed the landscape of the charge distribution. Statistical analyses of FAAS were made with respect to when and where the H5N1 viruses were found. In 2005, there were less un-mutated FAAS than the other years according to temporal evolution, and more mutated FAAS appeared in China than other regions according to geographic distribution. These results are helpful for exploring the evolution of virus high pathogenicity.

Differentiation of highly pathogenic strain H5N1 of AIV by Real Time PCR in the first outbreak in Romania

Fast diagnosis of Avian Influenza is a prerequisite for confining outbreaks. Diagnosis implies the differentiation of virulent and non-virulent Avian Influenza virus. After starting with PCR screening for matrix protein followed by identifying the presence of H5 and N1 genes, the diagnosis methodology within Romanian Institute for Diagnosis and Animal Health has moved to rapid molecular tests for detecting the virulent and nonvirulent strains. During October-December 2005 over 3400 biological specimens from 16 affected poultry backyards from Eastern Romania were tested by Real- Time PCR in the first Romanian outbreak. Over 3000 specimens have been tested in 2006, in the second Romanian outbreak when commercial poultry farms from central and southern Romania were affected.