H9N2 influenza virus acquires intravenous pathogenicity on the introduction of a pair of di-basic amino acid residues at the cleavage site of the hemagglutinin and consecutive passages in chickens

Abstract

BackgroundOutbreaks of avian influenza (AI) caused by infection with low pathogenic H9N2 viruses have occurred in poultry, resulting in serious economic losses in Asia and the Middle East. It has been difficult to eradicate the H9N2 virus because of its low pathogenicity, frequently causing in apparent infection. It is important for the control of AI to assess whether the H9N2 virus acquires pathogenicity as H5 and H7 viruses. In the present study, we investigated whether a non-pathogenic H9N2 virus, A/chicken/Yokohama/aq-55/2001 (Y55) (H9N2), acquires pathogenicity in chickens when a pair of di-basic amino acid residues is introduced at the cleavage site of its HA molecule.ResultsrgY55sub (H9N2), which had four basic amino acid residues at the HA cleavage site, replicated in MDCK cells in the absence of trypsin after six consecutive passages in the air sacs of chicks, and acquired intravenous pathogenicity to chicken after four additional passages. More than 75% of chickens inoculated intravenously with the passaged virus, rgY55sub-P10 (H9N2), died, indicating that it is pathogenic comparable to that of highly pathogenic avian influenza viruses (HPAIVs) defined by World Organization for Animal Health (OIE). The chickens inoculated with the virus via the intranasal route, however, survived without showing any clinical signs. On the other hand, an avirulent H5N1 strain, A/duck/Hokkaido/Vac-1/2004 (Vac1) (H5N1), acquired intranasal pathogenicity after a pair of di-basic amino acid residues was introduced into the cleavage site of the HA, followed by two passages by air sac inoculation in chicks.ConclusionThe present results demonstrate that an H9N2 virus has the potential to acquire intravenous pathogenicity in chickens although the morbidity via the nasal route of infection is lower than that of H5N1 HPAIV.

Immunization with live nonpathogenic H5N3 duck influenza virus protects chickens against highly pathogenic H5N1 virus

“…This virus acquired partial virulence in 2-dayold chickens only after 11 passages through air sacs (Ito et al, 2001). Introduction of four basic amino acid residues at the HA cleavage site and ten subsequent consecutive passages in air sacs of chickens were needed for acquisition of intravenous pathogenicity of a non-pathogenic H9N2 virus in chickens (Soda et al, 2011). In our study we show that A/duck/Moscow/4182/2010 virus also poorly replicated in the internal organs of chickens and was not transmitted to contact birds.…”

“…Reassortment with field strains or reverse mutation could repair the virulence of a virus. Because of the presumable risks of such reversions to pathogenicity, live vaccines against H5N1 virus are not approved for use anywhere except China (Spackman & Swayne, 2013).…”

“…Nevertheless, "regulatory barriers are probably insurmountable for avian influenza viruses based live vaccine in poultry" (Spackman and Swayne, 2013).…”

“…Most commonly used are inactivated oil-in-water emulsionadjuvanted whole virus vaccines based on reverse genetically engineered (rg) reassortants with H5 hemagglutinin (HA) and N1 neuraminidase (NA) from H5N1 viruses, and the remaining genes from A/PuertoRico/8/1934 (PR8). Tens of billions of doses of such vaccines were used in affected countries, primarily in China (Chen, 2009;Spackman and Swayne, 2013).…”

“…Compared with inactivated vaccines, potential advantages of live vaccines are the possibility of mass inoculation and a potentially increased immune response to viral replication in relevant tissues involving cellular immunity to influenza (Spackman and Swayne, 2013). To date, a number of live recombinant H5N1 influenza vaccines have been developed using other virus vectors, such as fowl pox virus (FPV), Newcastle disease virus (NDV), turkey herpes virus (HVT), duck enteritis virus (DEV), and infectious laryngotracheitis virus (ILTV).…”