H9N2 AIV Research Articles

Evaluation of RT-PCR for the Detection of Influenza Virus Serotype H9N2 among Broiler Chickens in Pakistan

A Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) was standardized and compared with virus isolation techniques for the detection of avian influenza virus serotype H9N2 among Broilers suffering from respiratory tract infection. In this regard 50 tissues and cloacal swabs were tested for the isolation of avian influenza viruses. Out of this, 17 samples from tissues and 3 from cloacal swabs showed the presence of AIV H9N2 through virus isolation test while six tissue samples showed the presence of NDV. Out of the negative samples another 15 samples were positive for AIV H9N2 when tested by RT-PCR while seven samples were positive for NDV by RT PCR. Detection of AIV from the cloacal swabs by RT-PCR also confirmed the shedding of AIV among the infected flocks. Apart from detecting the presence of AIV H9N2 from clinical specimens, the RT-PCR was able to detect higher number of AIV, from healthy flocks, which would otherwise have been missed by routine lab methods or wrongly diagnosed and treated adversely.

Sequence and Phylogenetic Analysis of Three Isolates of Avian Influenza H9N2 from Chickens in Southern China

Three isolates of H9N2 subtype of influenza virus have been isolated from chickens in Guangxi province. In this study, eight full-length genes of each of the H9N2 isolates, A/Chicken/Guangxi/1/00, A/Chicken/Guangxi/14/00, and A/Chicken/Guangxi/17/00, were obtained. Sequence analysis and phylogenetic studies were conducted by comparing eight genes of each isolate with those of the available H9N2 strains at GenBank. Results showed a high degree of homology between the Guangxi isolates and isolates from Guangdong and Jiangsu provinces, suggesting that Guangxi isolates originated from the same source. However, the eight genes of these three isolates from Guangxi were not in the same sublineages in the phylogenic trees, which suggest that they were products of natural reassortment between H9N2 avian influenza viruses from different sublineages. The 9 nucleotides ACAGAGATA encoding amino acids T, E, I were absent between nucleotide 205 and 214 in the open reading frame of NA genes in the Guangxi isolates. AIV strains that infect human have in their HA proteins, Leucine at position 226. The analysis of deduced amino acid sequence of HA proteins of C/GX/1/00, C/GX/14/00, and C/GX/17/00 showed that the position 226 of these isolates was glycine instead of leucine, suggesting that these three isolates differ from H9N2 AIV strains isolated from human infections.

Characterization of an H5N1 avian influenza virus from Taiwan

In 2003, an avian influenza (AI) virus of H5N1 subtype (A/Duck/China/E319-2/03; Dk/CHN/E319-2/03) was isolated from a smuggled duck in Kinmen Island of Taiwan. Phylogenetic analysis and pairwise comparison of nucleotide and amino acid sequences revealed that the virus displayed high similarity to the H5N1 viruses circulating in Asia during 2004 and 2005. The hemagglutinin (HA) protein of the virus contained multiple basic amino acid residues (-RERRRKR-) adjacent to the cleavage site between the HA1 and HA2 domains, showing the highly pathogenic (HP) characteristics. The HP phenotype was confirmed by experimental infection of chickens, which led up to 100% mortality within 24–72 h postinfection. The virus replicated equally well in the majority of organs of the infected chickens with titers ranging from 10 7.5 to 10 4.7 50% embryo lethal dose (ELD 50) per gram of tissue. In a mouse model the virus exhibits low pathogenic characteristics with a lethal infection observed only after applying high inoculating dose (≥10 7.6 ELD 50) of the virus. The infectious virus particles were recovered only from the pulmonary system including trachea and lungs. Our study suggests that ducks infected with H5N1 AIV of HPAI pathotype showing no disease signs can carry the virus silently and that bird smuggling represent a serious risk for H5N1 HPAI transmission.

Different neutralization efficiency of neutralizing monoclonal antibodies against avian influenza H5N1 virus to virus strains from different hosts

BALB/c mice were immunized with formalin-treated influenza A/CK/Hubei/327/2004 virus. Six monoclonal antibodies specific to HA were selected, designed 1H8, 1D11, 2B7, 2C9, 2H4 and 4C9, respectively. The six Mabs probed linear epitopes by western blot assays. In ELISA additivity assays, the low additivity indexes (≤28.3) of each pair Mabs indicated that the epitopes recognized by the six Mabs were located on the globular head of HA1. The neutralization activity of anti-HA1 Mabs and chicken polyclonal sera to various AIV H5N1 strains from different hosts was followed by virus neutralization with MDCK cells. All Mabs except 2C9 and chicken polyclonal serum showed highest neutralizing activity to lowly virulent A/Duck/XF/XFY/2004 from different phylogenetic lineage, and lowest neutralization efficiency to highly virulent A/CK/XF/XFJ/2004. For the other two highly virulent viruses, 1D11, 2H4, 4C9 and chicken polyclonal sera had higher neutralization to A/Goose/ZF/ZFE/2004 than A/CK/Hubei/327/2004, and 1H8 and 2B7 had considerable level of neutralizing efficiency to them. These findings suggested that the neutralizing antibodies showed lower neutralization efficiency to highly virulent virus strains than lowly virulent virus strains and strong cross-neutralizing reaction between virus strains located in different phylogenetic lineages. Moreover, the neutralizing Mabs could more efficiently neutralize AIV H5N1 strains from the natural hosts generally, such as waterfowl.

Construction and immunogenicity of recombinant fowlpox vaccines coexpressing HA of AIV H5N1 and chicken IL18

cDNAs of the HA genes of subtype H5N1 AIV were fused to form a single open reading frame, designated H5HA-H7HA. The H5HA-H7HA cDNA and chicken Interleukin-18 (IL18) were inserted into the fowlpox virus (FPV) expression vector pUTA-16-LacZ to produce pUTAL-H5-H7-IL18. cDNA of H5N1 AIV HA was inserted into the FPV expression vector pUTA2 to create the recombinant expression plasmid pUTA2-H5. Plasmids were then co-transected into CEF cells. The two recombinant fowlpox viruses (rFPV) were produced by three cycles with the BrdU and verified by RT-PCR, IFA and Western blotting. One-day-old specific pathogen free (SPF) chickens and 7-day-old commercial Leghorn egg-laying chickens were inoculated with 10 6 PFU recombinant or parental fowlpox vaccine viruses by wing-web puncture. Hemagglutination inhibition (HI) antibody titer and nonspecific cellular immunity level were assessed after 1–3 weeks post-immunization. We found that all rFPV-vaccinated groups produced HI-specific antibodies, and the level of cellular immunity induced by the rFPV-H5-H7-IL18 strain was significantly higher than that induced by rFPV-H5HA. At 3 weeks post-inoculation, immunized SPF and Leghorn chickens were challenged with H5N1 HP AIV. The rFPV-H5-H7-IL18 vaccine strains were able to induce complete (10/10) protection, while the rFPV-H5HA vaccine strain induced (9/10) protection. Cloacal swabbing samples were collected from immunized leghorn chickens during the first week post-challenge; no shedding was found in the rFPV-H5-H7-IL18 vaccinated group. The rFPV-H5-H7-IL18 vaccinated group displayed significantly increased weight gain relative to the rFPV-H5HA group. This study reports a significant step in the further development of new AIV vaccines.