The Field Investigation Of Main Subtypes Of Avain Influenza In A Northern Province And Research On RT-LAMP Detection Method

Avian influenza virus(AIV), especially the highly pathogenic avian influenza virus, is a causative agent of a zoonotic disease. The outbreaks of AIV have devastated the poultry industry worldwide and also brought a serious threat to public health. The high-pathogenicity H5N1 and a subtype lately emerged, H7N9, have overcome the species barrier to infect humans and led to the death of infected people.H9N2,a subtype avian influenza virus with low pathogenicity,is widespread exist in China. Since it provides relatively lower hazards to the poultry industry and public health compared with the highly pathogenic avian influenza virus, H9N2 is not given enough attention, as a subtype of highly pathogenic avian influenza,H5N1 is During the widespread and continuous evolution of H9N2, the subtype of AIV has not only been isolated from poultry and wild birds, but also has been able to infect mammal and even human beings. In 2013, the epidemic of H7N9 in humans and the event of H10N8 infecting humans in China have gained wide attention from numerous researchers as well as the government. Recent study shows that the internal genes of H7N9 and H10N8 influenza virus is originally from H9N2 subtype AIV, which indicates that H9N2 virus has become a provider of new types of influenza virus and plays an important role in the production of new viruses, so H9N2 AIV is a potential and a great threat to public health. Until now, all the researches on avian influenza and the virus have been focused on epidemiological investigation, molecular biology, pathogenesis, pathogenic diagnosis as well as vaccine development. Although previous studies have made a lot larger number of achievements, there are still many unresolved problems on avian influenza, especially very less reports of systematic research on a specific region of avian influenza.Therefore, this study utilized molecular biological technology, immunologic methods and serological methods to investigate the epidemic situation of chicken avian influenza in poultry farms and markets from 14 cities in one province localized in North China between 2012 and 2014. This study also carried out researches and comprehensive system analysis on the related gene sequences of AIV and the pathogenicity on mice caused by the isolated strains. In addition, we established a fast, accurate and in-field detection method--avian influenza RT-LAMP--to provide technical support and scientific basis for exploring the epidemic situation of AIV in the province as well as for the prevention and control of avian influenza virus(AIV). The main research contents and results are listed as follows:(1) The epidemic investigation of avian influenza on chickens in poultry farms in the northern provinceReal-time fluorescent PCR was used to examine 17,940 clinical swab samples and 315 clinical tissue and organ samples from the farms mentioned before. Among the 315 clinical tissue and organ samples, two samples were identified to be positive for H5N1 avian influenza, and all 2,110 swab samples were confirmed to be negative for H7 avian influenza. Among the 1,560 chicken swab samples, five swab samples from the same farm were identified to be positive for H9N2 avian influenza, which made the positive rate at 1.92% for the farms and 0.32% positive rate for all the samples. In contrast to the swab samples, among the 83 examined avian tissue and organs samples, 35 samples from 10 farms were detected to be positive for H9N2 avian influenza, which made the positive rate at 12.05% for the detected farms, and the positive rate was 11.11% for all the detected samples. Although no swab samples were detected to be positive for H5 avian influenza virus, these results did suggest that there existed certain infections of H5 and H9 avian influenzal virus in the province.The Hemagglutinin inhibition(HI) assay results showed that the immunization coverage rate of the immune antibody against H5 avian influenza virus was over than 80% in the farms, suggesting that the H5 vaccine can provide proper protection against H5 avian influenza virus in the province. The immunization coverage rate of H9 subtype avian influenza vaccine was only 30.78% in 52 farms, while the positive rate of H9 subtype avian influenza virus was up to 75% in the chickens without immunized H9 vaccine, which further confirmed that the potential risk of outbreaking H9 avian influenza exists.(2) The establishment of evaluation indicators for chicken avian influenza immunization and its significanceIn order to scientifically evaluate the performance of chicken immune antibody, studies were conducted to establish slip circle immunity(Q) based on the monitoring data of immune antibody to avian influenza on feeding chicken in the province in the past three years. When the Q value is larger or equal to 50% in chicks, the chicks were in relatively stable healthy status. For the laying hens, when Q is ≥100%, the laying hens were in stable healthy status and the immunization had little influence on laying eggs. Although there showed an egg-laying ability decrease because of some factors, the egg-laying ability recovered soon. When Q is <50%, the health status of chicken was very unstable, which indicated that the vaccination should be conducted soon. The evaluation indicators can reflect the performance of chicken immunization, the general health status and possible disease risks in a more comprehensive way compared to the current indicators. This evaluation system is of great significance in understanding the chicken’s avian influenza immunization status and appropriate vaccination time.(3) Isolation, identification and gene sequence analysis of H5 and H9 subtype avian influenza virus1) Isolation, identification and gene sequence analysis of H5 subtype avian influenza virusTwo strains of H5N1 subtype avian influenza virus named CK/LN/DL02/12 and CK/LN/HS 03/05H5N1 were obtained with virus isolation and identification, and also the whole genome sequences of these two strains were determined and analyzed separately. The results showed that both cleavage sites on HA proteins of the two strains were 4-6 continuous basic amino acids, such as RRRKKR*G and RRRKR*G, which were characterized as H5 subtype highly pathogenic avian influenza virus. Phylogenetic tree analysis showed that CK/LN/DL02/12 belongs to clade 2.3.2.1c, while CK/LN/HS03/05 belongs to Calde2.2. For the reason that HA and NA genes of these two strains were highly homologous with HA and NA genes of the virus from wild birds, we can infer that these two virus strains may be derived from wild birds. In addition, there was a significant difference at 627 amino acid of PB2 protein between two strains, in which 627 amino acid of PB2 in CK/LN/DL02/12 was E and it was K in CK/LN/HS03/05.2) Isolation, identification and gene sequence analysis of H9 subtype avian influenza virusTen strains of H9N2 subtype avian influenza virus were obtained through virus isolation and identification in the H9 positive samples. The whole genome sequence analysis showed that the cleavage sites of HA protein were all characterized as typical cleavage site of the low pathogenic avian influenza virus, but the 226 a.a of HA proteins in these ten strains of H9N2 subtype avian influenza virus were all found to be mutated to leucine(L) instead of Glutamine(Q). And there was no mutation found at 627 a.a and 701 a.a, of PB2 and these sites are considered to play an important role in the pathogenicity and transmission ability of the mammalian proteins. The 292 a. a of NA proteins were all identified as arginine(R), but there were mutations found at 31 a.a of M2 protein in several strains. The internal gene of the virus was highly homologous to the H7N9 virus in 2013. According to the genetic evolutionary relationship, these 10 strains of H9N2 virus could be divided into 5 genotypes including 4 strains in genotype I, one strain in genotype II, two strains in genotype III, two strains in genotype IV and one strain in genotype V.(4) The pathogenicity in mice of H5 and H9 subtype avian influenza virus1) The pathogenicity in mice of H5 subtype avian influenza virusIn order to study the pathogenicity of Clade 2.2 and 2.3.2.1c Clade of H5N1 in mammals, the BALB/c mice were infected with 106EID50/50 u L. The results showed that these two strains were able to replicate in the lungs of infected mice. The weight of infected mice was greatly decreased, which also led to death. According to viral replication in the brains, spleens and kidneys, the CK/LN/HS03/05 strain presented higher pathogenicity to mice than CK/LN/DL02/12 strain. After 3 days infection, the CK/LN/HS03/05 strain was not only effectively replicated in the lungs, but also in the brains, kidneys and spleens, while The CK/LN/DL02/12 strain was only found to replicate in the lungs after 3 days. These results suggested that both two strains of H5N1 subtype avian influenza virus were able to infect mice, but there were some differences in their pathogenicity.2) The pathogenicity in mice of H9 subtype avian influenza virusIn order to study the potential infecting ability of chicken H9N2 subtype avian influenza virus from this province to mammals and humans, the BALB/c mice were infected with 106EID50/50 u L through nasal cavity. The results showed that the CK/LN/SY03/12, CK/LN/FX04/12, CK/LN/ PJ03/12 and CK/LN/LY04/12 strains were able to be stably replicated in the lungs of mice, while the CK/LN/SN02/12 and A/CK/SZ02/13 strains were found weakly replicated in the lungs after 3 day infection and the replicated products were cleaned in day 6 after infection. The CK/LN/A10/14 and CK/LN/TL01/13 strains were found to be unable to infect mice. These results indicated that the pathogenicity of these 10 isolated strains of H9N2 subtype avian influenza viruses to mice were not identical. These strains presented the biological characteristics of low pathogenic avian influenza virus, which includes weak infection in lung only or no infection to mice at all.(5) Establishment and application of general loop mediated isothermal amplification(RT-LAMP) detection for avian influenza virusAccording to the principle of loop-mediated isothermal amplification method, 4 specific primers were designed to amply the conserved region of AIV M gene and the optimized condition of the reaction was identified: the molar ratio of inner and outer primer is 8:1; the optimal Mg2+ concentration is 8 mmol/L; the suitable reaction time is 30 min. The above reaction was utilized to amply the M gene of H1~H16 in H9N2 subtype avian influenza virus and the results of all the reactions were positive. In addition, there was no cross reaction with other susceptible viruses of avian, such as NDV、IBV、IBDV、ILTV and ALV, which confirmed that this method with the optimized conditions was specific to AIV. Real-time turbidity detection system was used to detect common H1、H5、H7、H9 subtype avian influenza virus and the sensitivity of the method was 10-6. A comparative test was carried out with routine RT-PCR and Real-time RT-PCR and the results indicated that the detection limit of the loop-mediated isothermal amplification method was 13.43EID50/mL, which was 10 times sensitive than the routine RT-PCR method. For the AIV suspected samples, the total coincidence rate between loop-mediated isothermal amplification method and routine RT-PCR or Real-time RT-PCR was 81.8%. All these results suggested that RT-LAMP provides a rapid, convenient and practical method for the clinic test and epidemiological investigation of AIV in chicken flocks.