Isolation and phylogenetic analysis of hemagglutinin gene of H9N2 influenza viruses from chickens in South China from 2012 to 2013

As part of our ongoing influenza surveillance program in South China, 19 field strains of H9N2 subtype avian influenza viruses (AIVs) were isolated from dead or diseased chicken flocks in Guangdong province, South China, between 2012 and 2013. Hemagglutinin (HA) genes of these strains were sequenced and analyzed and phylogenic analysis showed that 12 of the 19 isolates belonged to the lineage h9.4.2.5, while the other seven belonged to h9.4.2.6. Specifically, we found that all of the viruses isolated in 2013 belonged to lineage h9.4.2.5. The lineage h9.4.2.5 viruses contained a PSRSSRdownward arrowGLF motif at HA cleavage site, while the lineage h9.4.2.6 viruses contained a PARSSRdownward arrowGLF at the same position. Most of the isolates in lineage h9.4.2.5 lost one potential glycosylation site at residues 200-202, and had an additional one at residues 295-297 in HA1. Notably, 19 isolates had an amino acid exchange (Q226L) in the receptor binding site, which indicated that the viruses had potential affinity of binding to human like receptor. The present study shows the importance of continuing surveillance of new H9N2 strains to better prepare for the next epidemic or pandemic outbreak of H9N2 AIV infections in chicken flocks.

Reverse genetic platform for inactivated and live-attenuated influenza vaccine

Influenza vaccine strains have been traditionally developed by annual reassortment between vaccine donor strain and the epidemic virulent strains. The classical method requires screening and genotyping of the vaccine strain among various reassortant viruses, which are usually laborious and time-consuming. Here we developed an efficient reverse genetic system to generate the 6:2 reassortant vaccine virus from cDNAs derived from the influenza RNAs. Thus, cDNAs of the two RNAs coding for surface antigens, haemagglutinin and neuraminidase from the epidemic virus and the 6 internal genes from the donor strain were transfected into cells and the infectious viruses of 6:2 defined RNA ratio were rescued. X-31 virus (a high-growth virus in embryonated eggs) and its cold-adapted strain X-31 ca were judiciously chosen as donor strains for the generation of inactivated vaccine and live-attenuated vaccine, respectively. The growth properties of these recombinant viruses in embryonated chicken eggs and MDCK cell were indistinguishable as compared to those generated by classical reassortment process. Based on the reverse genetic system, we generated 6 + 2 reassortant avian influenza vaccine strains corresponding to the A/Chicken/Korea/MS96 (H9N2) and A/Indonesia/5/2005 (H5N1). The results would serve as technical platform for the generation of both injectable inactivated vaccine and the nasal spray live attenuated vaccine for the prevention of influenza epidemics and pandemics.

Molecular identification of the vaccine strain from the inactivated oil emulsion H9N2 low pathogenic avian influenza vaccine

In order to control the H9N2 subtype low pathogenic avian influenza (LPAI), an inactivated vaccine has been used in Korea since 2007. The Korean veterinary authority permitted the use of a single H9N2 LPAI vaccine strain to simplify the evolution of the circulating virus due to the immune pressure caused by the vaccine use. It is therefore important to determine the suitability of the vaccine strain in the final inactivated oil emulsion LPAI vaccine. In this study, we applied molecular rather than biological methods to verify the suitability of the vaccine strain used in commercial vaccines and successfully identified the strain by comparing the nucleotide sequences of the hemagglutinin and neuraminidase genes with that of the permitted Korean LPAI vaccine strain. It is thought that the method used in this study might be successfully applied to other viral genes of the LPAI vaccine strain and perhaps to other veterinary oil emulsion vaccines.

Seroprevalence of low pathogenic avian influenza (H9N2) and associated risk factors in the Gyeonggi-do of Korea during 2005-2006

Between November 2005 and March 2006, a total of 253 poultry flocks in the Gyeonggi-do of Korea were examined for seroprevalence against avian influenza (AI) using a hemagglutination inhibition (HI) test and an agar gel precipitation test. No low pathogenic avian influenza (LPAI) virus was isolated from 47 seropositive flocks that lacked clinical signs during sampling. The unadjusted percentage of seroprevalence rates of layer and broiler flocks were not significantly different, i.e., 26% (25/96) and 23% (22/97), respectively. The HI titer of the layers (mean = 89) was higher than the broilers (mean = 36; p < 0.001). A cross-sectional study was conducted for the seroprevalence of LPAI in the layers. Of 7 risk factors, farms employing one or more workers had a higher seropositive prevalence as compared to farms without hired employees (adjusted prevalence OR = 11.5, p = 0.031). Layer flocks older than 400 d had higher seropositivity than flocks younger than 300 d (OR = 4.9, p = 0.017). The farmers recognized at least one of the clinical signs in seropositive flocks, such as decreased egg production, respiratory syndromes, and increased mortality (OR = 2.3, p = 0.082). In a matched case-control study, 20 pairs of case and control flocks matched for type of flock, hired employees, age, and flock size were compared. Frequent cleansing with disinfectants was associated with a decreased risk of seropositivity (OR = 0.2, p = 0.022). Although there was a low statistical association, using a foot disinfectant when entering the building led to a decreased rate of seropositivity (OR = 0.3, p = 0.105).