Monitoring Airborne Transmission Of H9N2Avian Influenza Virus And Detecting Its Determinants

Avian Influenza Virus (AIV) has caused serious epidemics all over the world. Notably,the low pathogenic AIV H9N2has been spreading widely, leading to enormous economiclosses to the poultry industry. More importantly, it infected human repeatly and causedinfluenza symptom. It is an animal-derived pathogen of zoonosis. So the transmission andinfection of this disease should be paid much attention. It is always agreed that avianinfluenza could transmit by three ways: direct contact, indirect contact virus contaminants andaerosols. But, the transmission state of AIV in culture environment and mechanism of itsairborne transmission in poultry are not clearly. This study established a real-time RT-PCRmethod to detect airborne H9AIV in poultry environment. The gene sequences of surfaceprotein of Shandong isolates were analyzed, and their pathogenicity to poultry and mammalsand the transmission way among mammals were also studied. The amino acids of H9N2AIVNA protein were mutated by reverse genetic technology to study their influence to aerosoltransmission and the mechanism of this transmission.There are four parts of this study.1Development of real-time RT-PCR method for rapid detection of H9avianinfluenza virus in the airTo rapidly monitor airborne H9AIVs in chicken houses and quantify its concentration,the specific primers and probe were designed according H9conserved regions, then a real-time RT-PCR method was established and used to detect air samples in some chicken housesof Shandong province, and it was also compared with the traditional RT-PCR. The resultsshowed that the real-time RT-PCR possessed high specificity and sensitivity for H9AIVs,and the sensitivity reached100copies/reaction, much higher than the traditional RT-PCR;airborne H9AIVs were found in the six chicken houses by real-time RT-PCR, and their meanconcentrations ranged from1.25×104to6.92×104copies/m3air. Overall, the real-time PCR isa valuable tool for detecting airborne H9AIVs and is important to evaluate the virustransmission.2Gene analysis of H9N2AIV isolates of Shandong province and the study of theirpathogenicitySpecificity primers were designed according the gene sequences of H9N2AIV in theGenBank for completely sequencing HA and NA gene of8H9strains isolated from chickenhouse in Shandon province. Phylogenetic tree of the isolates were constructed and their aminoacids were analyzed. The viruses were inoculated SPF chickens and guinea pigs for investigating their pathogenicity and ability to infect animals with species barrier. Thetransmission ability among mammals were accessed using guinea pigs divided into inoculategroup, direct contact group and aerosol contact group. Nasal washes were collected at twodays intervals to detect the virus shedding. Sera of animals were used to detect the titers of H9antibody. Air samples were collected by AGI-30sampler and AIV aerosol concentration weredetected by real-time RT-PCR. Results showed that8isolates were all low pathogenic avianinfluenza virus, and they had the Leu-234(226in H3number) which could bind human likereceptor in the receptor bind section. Eight isolates were belonged to HKY28097-like lineagethrough HA phylogenetic analysis and belonged to BJ194-like lineage except ACSDM isolatewhich was belonged to HKG997-like lineage through NA analysis. The isolates were notlethal to chickens while replicated efficiently to high titers in the lungs of guinea pigs withoutprior adaption. H9N2AIV could transmit among guinea pigs by direct contact not by viralaerosol, but the viruses were detected in the air of the isolate on6~10d. Experiment showedthat aerosol ID50of H9N2AIV to guinea pigs was3.58×106copies, demonstrating that theaerosols could infect guinea pigs in experiment condition which was a great thread to humans.3The sequencing analysis of SD01(H9N2AIV) complete genesSpecificity primers were designed according the gene sequences of H9N2AIV in theGenBank and the primers designed by Hoffmann et al (2001). The eight genes of Shandongisolate H9N2AIV (SD01) which could transmit among poultry by aerosol were sequencedand phylogeniticly analyzed. The amino acids of this virus were compared with tworeferences (F98and SS94). The results showed that HA gene had the highest homology withHKG997, NA gene had highest homology with HKY28097and SHF98, and PB2, PB1, NP,M and NS genes with BJ194and PA gene with SHF98. Amino acid analysis showed that thesequence at the cleavage site of HA was RSSR↓GLF and the234receptor bind site (226inH3number) was Q which could bind avian like receptor to infect birds. Three amino acids atthe sites61-63of NA stalk were deleted. The amino acids at368,402,403and432sites inHB region easily mutated and the366-373HB region was different with F98and SS94. The313,331and381amino acids in NA head activity region of SD01, F98and SS94were alsodifferent.4The influence of NA amino acids mutation on H9N2AIV transmission ability.The PolⅠ-PolⅡsystem transcription and expression plasmids of SD01eight genes andSS94NA gene were established by gene clone method using reverse genetic both-way vectorpHW2000and primers with BsmBI/AarI/BsaI restriction enzyme site. The plasmids of SD01seven genes and SS94NA gene were recombined and transfected mixed MDCK and293T cells to rescue the virus R01/NASS. The366-373,313,381,331amino acids of SD01NAgene were mutated by site-directed mutagenesis and the eight plasmids tranfected cells torescue the different mutant strains. The biological characteristics such as pathogenicity andenzyme activity of rescued strains were investigated. The transmission way of every virus inchicken groups were studied in SPF isolators by divided the chickens into three groups:inoculate group, direct contact group and aerosol contact group. Oral and cloaca swabs werecollected days interval to detect the virus shedding. Sera of animals were used to detect thetiters of H9antibody. Air samples were collected by AGI-30sampler and AIV aerosolconcentration were detected by real-time RT-PCR. The results indicated that the reassortantsR01/NASS which recombined the genes of SD01and SS94and R01/NA381which with the368E,370L,313K and381D mutations replicated more weakly than SD01in SPF chickeneggs. After inoculated chickens with R01/NASS and R01/NA381respectively, virus sheddingwas not found and seroconversion was not positive in aerosol contact chickens. Thisdemonstrated that these two viruses lost the aerosol transmission ability which was possessedby SD01. The reassortant R01/NAHB with two sites (368and370) mutations decreased theactivity of neuraminidase, but still could transmit among chickens by aerosols like SD01. It isconcluded that the amino acids at368,370,313and381sites played important role inaffecting H9N2AIV aerosol transmission among poultry.