Some H5 viruses isolated in poultry or wild birds between 2020 and 2021 were found to be antigenically different from the vaccine strains (H5-Re11 and H5-Re12) used in China.  In this study, we generated three new recombinant vaccine seed viruses by using reverse genetics and used them for vaccine production.  The vaccine strain H5-Re13 contains the hemagglutinin (HA) and neuraminidase (NA) genes of an H5N6 virus that bears the clade 2.3.4.4h HA gene, H5-Re14 contains the HA and NA genes of an H5N8 virus that bears the clade 2.3.4.4b HA gene, and H7-Re4 contains the HA and NA genes of H7N9 virus detected in 2021.  We evaluated the protective efficacy of the novel H5/H7 trivalent inactivated vaccine in chickens, ducks, and geese.  The inactivated vaccine was immunogenic and induced substantial antibody responses in the birds tested.  Three weeks after vaccination, chickens were challenged with five different viruses detected in 2020 and 2021: three viruses (an H5N1 virus, an H5N6 virus, and an H5N8 virus) bearing the clade 2.3.4.4b HA gene, an H5N6 virus bearing the clade 2.3.4.4h HA gene, and an H7N9 virus.  All of the control birds shed high titers of virus and died within 4 days post-challenge, whereas the vaccinated chickens were completely protected from these viruses.  Similar protective efficacy against H5 viruses bearing the clade 2.3.4.4h or 2.3.4.4b HA gene was observed in ducks and geese.  Our study indicates that the newly updated H5/H7 vaccine can provide solid protection against the H5 and H7N9 viruses that are currently circulating in nature.  

In the past decade, there has been extensive global surveillance for highly pathogenic avian influenza (HPAI) infection in both animals and humans, however, few studies on epidemiology of avian influenza in Democratic People’s Republic of Korea (DPRK) were published.  During the period 2013–2014, HPAI H5N1 viruses were detected with outbreaks in domestic poultry in DPRK.  Phylogenetic analysis revealed that the hemagglutinin gene of all samples belonged to clade 2.3.2.1c with high homology.  The HPAI H5N1 virus found in ducks at the Tudan Duck Farm in 2013 was might introduced by migratory birds and then led to the outbreaks on neighboring chicken farms in 2014.  These data provide direct evidence for the transmission of avian influenza viruses from wild birds to waterfowl to terrestrial birds.  Therefore, the monitoring and control of influenza virus in ducks must be given top priority, which are essential components to prevent and control HPAI.

H7 avian influenza viruses (AIVs) normally circulated among birds before.  From 1996 to 2012, human infections with H7 AIVs (H7N2, H7N3, and H7N7) were reported in Canada, Italy, Mexico, the Netherlands, the United Kingdom and the USA.  Until March 2013, human infections with H7N9 AIVs were reported in China.  Since then, H7N9 AIVs have continued to circulate in both humans and birds.  Therefore, the detection of antibodies against the H7 subtype of AIVs has become an important topic.  In this study, a competitive enzyme-linked immunosorbent assay (cELISA) method for the detection of antibody against H7 AIVs was established.  The optimal concentration of antigen coating was 5 μg mL^–1, serum dilution was 1/10, and enzyme-labeled antibody was 1/3 000.  To determine the cut-off value of cELISA, percent inhibition (PI) was determined by using receiver operating characteristic (ROC) curve analysis in 178 AIVs negative samples and 368 AIVs positive serum samples (n=546).  When PI was set at 40%, the specificity and sensitivity of cELISA were 99.4 and 98.9%, respectively.  This method could detect the antibodies against H7Nx (N1–N4, N7–N9) AIVs, and showed no reaction with AIVs of H1–H6 and H8–H15 subtypes or common avian viruses such as Newcastle disease virus (NDV), Infectious bronchitis virus (IBV) and Infectious bursal disease virus (IBDV), exhibiting good specificity.  This method showed a coincidence rate of 98.56% with hemagglutinin inhibition (HI) test.  And the repeatability experiment revealed that the coefficients of variation (CV) of intra- and inter-batch repetition were all less than 12%.  The data indicated that the cELISA antibody-detection method established in this study provided a simple and accurate technical support for the detection of a large number of antibody samples of H7-AIV.

   In 2013, a human influenza outbreak caused by a novel H7N9 virus occurred in China.  Recently, the H7N9 virus acquired multiple basic amino acids at its hemagglutinin (HA) cleavage site, leading to the emergence of a highly pathogenic virus.  The development of an effective diagnostic method is imperative for the prevention and control of highly pathogenic H7N9 influenza.  Here, we designed and synthesized three pairs of primers based on the nucleotide sequence at the HA cleavage site of the newly emerged highly pathogenic H7N9 influenza virus.  One of the primer pairs and the corresponding probe displayed a high level of amplification efficiency on which a real-time RT-PCR method was established.  Amplification using this method resulted in a fluorescent signal for only the highly pathogenic H7N9 virus, and not for any of the H1–H15 subtype reference strains, thus demonstrating high specificity.  The method detected as low as 39.1 copies of HA-positive plasmid and exhibited similar sensitivity to the virus isolation method using embryonated chicken eggs.  Importantly, the real-time RT-PCR method exhibited 100% consistency with the virus isolation method in the diagnosis of field samples.  Collectively, our data demonstrate that this real-time RT-PCR assay is a rapid, sensitive and specific method, and the application will greatly aid the surveillance, prevention, and control of highly pathogenic H7N9 influenza viruses.