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Avian influenza (AI) virus (AIV) subtype H9N2 continues to cause significant outbreaks among commercial and backyard poultry in Pakistan. Despite this, the characterization of H9N2 viruses in avian hosts other than chickens in Pakistan has not been thoroughly investigated. In this study, 12 low pathogenicity avian influenza viruses subtype H9N2 were isolated from peacocks ( n = 4), ducks ( n = 4), pheasants ( n = 2), geese ( n = 1), and black swans ( n = 1) in Pakistan during 2016 and were characterized on the basis of the hemagglutinin (HA) and neuraminidase genes. All of the viruses possessed an amino acid substitution Q226L in the receptor-binding site of the HA protein, which is known to contribute to increased viral replication and virulence in mammals. In addition, phylogenetic studies showed that these H9N2 AIVs belonged to the Middle East B genetic group of sublineage G1 and were very similar to viruses isolated from an outbreak in chickens in Pakistan in 2017. This demonstrates an epidemiologic link between poultry and other avian species, which is a fact to consider in future H9N2 disease management programs.

This paper describes the results of the molecular and phylogenetic analysis of seven highly pathogenic avian influenza (HPAI) H5N1 strains isolated in 2006 (n = 5) and 2007 (n = 2) from wild birds and poultry in Poland. The whole genome sequence of these isolates was determined. All of the isolates possessed the hemagglutinin (HA) cleavage site sequence PQGERRRKKR*GLF typical of HPAI. Molecular markers associated with increased adaptation and virulence in mammals, as well as susceptibility to neuraminidase inhibitors, were revealed in the HA, neuraminidase (NA), and PB2 proteins. Based on the sequencing results related to the HA and NA genomic segments, H5N1 viruses circulating in Poland all belong to lineage 2.2. However, isolates isolated in 2006 were genetically distinct from those isolated in 2007 and grouped in different sublineages. H5N1 viruses isolated from wild birds in 2006 are almost identical to each other (99.9% HA; 99.6%-100% NA), and they are grouped within a cluster of viruses isolated in Germany from wild and domestic birds and mammals in 2006. Isolates from 2007 are also closely related to each other (nucleotide homologies 99.9% and 100% for HA and NA, respectively), and they are grouped together with isolates from wild and domestic birds collected in Eastern and Central Europe (Romania, Germany), and the Middle East (Kuwait, Saudi Arabia). Phylogenetic analysis of the sequences related to the internal proteins confirmed the results obtained for the HA and NA genes. Overall, the results indicate that HPAI H5N1 in Poland in 2006-07 was caused by at least two separate incursions of genetically distinct viruses.

The production and supply of reference reagents for the diagnosis of avian influenza (AI) is one of the duties of the World Organization for Animal Health reference laboratories. The lyophilized reagents are routinely shipped on dry ice to both national and international clients. In order to determine the effect of different short-term storage temperatures on the activity of AI reference reagents, vials containing lyophilized avian influenza A antigens and antisera preparations were maintained at 4 C, 22 C, and 37 C over a 21-day period. At days 0, 3, 7, 14, and 21 the reagents were titrated using the hemagglutination test, the hemagglutination inhibition test, or the agar gel immunodiffusion test (AGID). All of the AI antigens that were kept at 4 C and 22 C retained hemagglutinating activity for at least 21 days, but when they were stored at 37 C several lost their hemagglutinating activity. All of the reference antisera tested were still able to inhibit hemagglutination after 21 days, and the antigen used in AGID also gave clear results after 21 days even after incubation at 37 C. Our results therefore indicate that lyophilized avian influenza antigens maintain their hemagglutinating activity at temperatures between 4 C and 22 C for at least 21 days, and both antisera and antigens prepared for AGID remain stable for 21 days between 4 C and 37 C. This information will allow for alternative shipping temperatures than those presently recommended, in addition to the short-term storage of these reagents at nonrefrigerated temperatures.

In order to support eradication efforts of avian influenza (AI) infections in poultry, the implementation of "differentiation of infected from vaccinated animals" (DIVA) vaccination strategies has been recommended by international organizations. These systems enable the detection of field exposure in vaccinated flocks, and through this detection, infected flocks may be properly managed, thus interrupting the perpetuation of the infectious cycle. A promising system, based on the detection of antibodies to the nonstructural 1 (NS1) protein of AI, has been deemed a good candidate. However, there are presently no data available, in support of this DIVA system, with regard to the kinetics of antibody production against the NS1 proteins in poultry following infection. The present investigation was undertaken to establish the dynamics of the appearance of anti-NS1 antibodies in a naïve population. Following experimental infection of turkeys, antibodies to a peptide spanning the c-terminal of the NS1 protein were detected by enzyme-linked immunosorbent assay (ELISA) starting between day 3 and day 5 postinfection. In contrast, no antibodies to the NS1 peptide could be detected in chickens over the test period. In addition, the turkeys and chickens reacted differently at a clinical level to the infection by the H9N2 challenge virus. Taken together, these findings indicate that there is a significant difference in the viral replication in turkeys and chickens, resulting in a variation in the production of antibodies to NS1, as detected by the peptide-based ELISA used. This fact must be taken into consideration when using a DIVA system based on the identification of antibodies to the NS1 protein.