Influenza A viruses occur worldwide in wild birds and are occasionally associated with outbreaks in commercial chickens and turkeys. However, avian influenza viruses have not been isolated from wild birds or poultry in South America. A recent outbreak in chickens of H7N3 low pathogenic avian influenza (LPAI) occurred in Chile. One month later, after a sudden increase in deaths, H7N3 highly pathogenic avian influenza (HPAI) virus was isolated. Sequence analysis of all eight genes of the LPAI virus and the HPAI viruses showed minor differences between the viruses except at the hemagglutinin (HA) cleavage site. The LPAI virus had a cleavage site similar to other low pathogenic H7 viruses, but the HPAI isolates had a 30 nucleotide insert. The insertion likely occurred by recombination between the HA and nucleoprotein genes of the LPAI virus, resulting in a virulence shift. Sequence comparison of all eight gene segments showed the Chilean viruses were also distinct from all other avian influenza viruses and represent a distinct South American clade.

Outbreaks of avian influenza due to an H7N1 virus of low pathogenicity occurred in domestic poultry in northern Italy from March 1999 until December 1999 when a highly pathogenic avian influenza (HPAI) virus emerged. Nucleotide sequences were determined for the HA1 and the stalk region of the neuraminidase (NA) for viruses from the outbreaks. The HPAI viruses have an unusual multibasic haemagglutinin (HA) cleavage site motif, PEIPKGSRVRRGLF. Phylogenetic analysis showed that the HPAI viruses arose from low pathogenicity viruses and that they are most closely related to a wild bird isolate, A/teal/Taiwan/98. Additional glycosylation sites were present at amino acid position 149 of the HA for two separate lineages, and at position 123 for all HPAI and some low pathogenicity viruses. Other viruses had no additional glycosylation sites. All viruses examined from the Italian outbreaks had a 22 amino acid deletion in the NA stalk that is not present in the N1 genes of the wild bird viruses examined. We conclude that the Italian HPAI viruses arose from low pathogenicity strains, and that a deletion in the NA stalk followed by the acquisition of additional glycosylation near the receptor binding site of HA1 may be an adaptation of H7 viruses to a new host species i.e. domestic poultry.

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The declaration of the human influenza A pandemic (H1N1) 2009 (H1N1/09) raised important questions, including origin and host range [1], [2]. Two of the three pandemics in the last century resulted in the spread of virus to pigs (H1N1, 1918; H3N2, 1968) with subsequent independent establishment and evolution within swine worldwide [3]. A key public and veterinary health consideration in the context of the evolving pandemic is whether the H1N1/09 virus could become established in pig populations [4]. We performed an infection and transmission study in pigs with A/California/07/09. In combination, clinical, pathological, modified influenza A matrix gene real time RT-PCR and viral genomic analyses have shown that infection results in the induction of clinical signs, viral pathogenesis restricted to the respiratory tract, infection dynamics consistent with endemic strains of influenza A in pigs, virus transmissibility between pigs and virus-host adaptation events. Our results demonstrate that extant H1N1/09 is fully capable of becoming established in global pig populations. We also show the roles of viral receptor specificity in both transmission and tissue tropism. Remarkably, following direct inoculation of pigs with virus quasispecies differing by amino acid substitutions in the haemagglutinin receptor-binding site, only virus with aspartic acid at position 225 (225D) was detected in nasal secretions of contact infected pigs. In contrast, in lower respiratory tract samples from directly inoculated pigs, with clearly demonstrable pulmonary pathology, there was apparent selection of a virus variant with glycine (225G). These findings provide potential clues to the existence and biological significance of viral receptor-binding variants with 225D and 225G during the 1918 pandemic [5].

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Real time reverse transcriptase (RRT)-polymerase chain reaction (PCR) for the detection of Eurasian H5 avian influenza virus (AIV) isolates was adapted from an existing protocol, optimized, and validated using a number of genetically diverse H5 isolates (n = 51). These included 34 "Asian lineage" H5N1 highly pathogenic avian influenza (HPAI) viruses (2004-2006), plus 12 other H5 isolates from poultry outbreaks and wild birds in the Eastern Hemisphere (1996-2005). All 51 were positive by H5 Eurasian RRT-PCR. Specificity was assessed by testing representative isolates from all other AL virus subtypes (n = 52), non-AI avian pathogens (n = 8), plus a negative population of clinical specimens derived from AI-uninfected wild birds and poultry (n = 604); all were negative by H5 Eurasian RRT-PCR. RNA was directly extracted from suspect HPAI H5N1 clinical specimens (Africa, Asia, and Europe; 2005-2006; n = 58) from dead poultry and wild birds, and 55 recorded as positive by H5 Eurasian RRT-PCR: Fifty-one of these 55 were in agreement with positive AIV isolation in embryonated chickens' eggs. H5 Eurasian RRT-PCR was invaluable in H5 outbreak diagnosis and management by virtue of its rapidity and high degree of sensitivity and specificity. This method provides a platform for automation that can be applied for large-scale intensive investigations, including surveillance.

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Background  Avian influenza (AI) caused by H7 AI viruses (AIVs) of both low pathogenicity (LP) and high pathogenicity (HP) are notifiable poultry diseases. Objectives  Design and validate two RealTime reverse transcriptase polymerase chain reactions (RRT PCRs) for Eurasian H7 AIV detection and pathotyping. Methods  The H7 RRT PCRs amplified within the (i) HA2 and (ii) cleavage site CS regions of the haemagglutinin gene. Both were validated against 65 H7 AIVs, 57 non‐H7 AIVs and 259 poultry swabs in comparison to M gene (AI generic) RRT PCR and virus isolation (VI). An additional 38 swabs and 20 tissue specimens extended validation against M gene RRT PCR. Results  Both H7 RRT PCRs amplified all 61 Eurasian lineage H7 AIVs and none of 57 non‐H7 AIVs. A total of 297 poultry swabs were used to determine diagnostic sensitivity and specificity relative to M gene RRT PCR, sensitivity was 95·4% and 64·6% for the HA2 and CS RRT PCRs respectively, and specificity 97·9% and 99·6% respectively. The H7 HA2 RRT PCR was more sensitive than VI. This was emphasized by analysis of 37 swabs from turkeys infected experimentally with HPAI H7N1 virus sampled at 24 hours post‐inoculation and LPAI H7N1 chicken infections sampled at 40–64 hours. Although less sensitive, usefulness of the H7 CS RRT PCR was confirmed by the correct molecular pathotyping for all 61 Eurasian lineage H7 AIVs tested. Conclusions  The high sensitivity of H7 HA2 RRT PCR confirms its suitability for use in poultry surveillance and disease diagnosis. H7 CS RRT PCR provides an opportunity for rapid pathotyping of H7 AIVs.