The Sequence Analysis Of A Swine H5 Subtype Strains Of Avian Influenza Viruses And Pathogenicty Test

In this study, 500 parts of nasal swabs and 210 parts of lung were collected from some fattening pig in pig farms and slaughtered pig in slaughterhouses in southern China from June 2014 to October 2015. After the diseased inoculated in aseptic processing the allantoic cavity of the 9 to 11 days age chick embryos, and then the inoculated chick embryo harvested under sterile conditions solution, 1% chicken erythrocytes determine if there hemagglutination activity, urine hemagglutination activity cyst fluid with H1, H3, H5, H6, H7 and H9 subtypes of influenza positive serum, NDV positive serum hemagglutination inhibition. Then sequenced and was isolated as a H5N6 subtype Avian influenza virus, named A/Avian/Huanan/C135/2015?H5N6? referred C135.Whole genome sequencing C135 strains, phylogenetic analysis showed that NA gene ORF frame of 1380 nucleotides in length, neck deletion of 33 nucleotides on the gene does not exist E119 V, R152 K, H275 Y, R293 K, N295 S and other resistance locus mutation. HA gene total length of 1776 nucleotides containing a complete ORF box contains 1704 nucleotides encoding 567 amino acids can be deduced, the 5' untranslated region and 3' untranslated regions, respectively containing 28 and 44 nucleotides not found insertions and deletions of nucleotides; cleavage site RRRKR?G, in line with the characteristics of highly pathogenic virus gene. N-glycosylation sites, 26, 27, 39, 181, 302, 499 and 558 the presence of glycosylation sites. After the M gene analysis found, H5N6 subtype SIV full-length 1027 nucleotides, the complete ORF frame consists of two parts, M1 length of 759 nucleotides, M2 length of 294 nucleotides, encoding a deduced 97 amino acids. PB1 gene series analysis: total length of 2341 bp nucleotides contain a complete ORF frame length of 2274 nucleotides encoding 757 amino acids deduced, including the 5' untranslated region and 3' untranslated regions each containing 24 and 43 nucleotides, no insertions and deletions of nucleotides; the virus mutated to all 13 P, 678 bit is not mutated were S. PB2 gene from the 2341 bp nucleotides and complete ORF frame length 2280 bp nucleotides encoding 759 amino acids can be deduced, the 5' untranslated region and 3' untranslated regions, respectively containing 27 and 34 nucleotides. PB2 gene analysis found that 627 of E, there is no mutation; virulence is still closely related to the 701 is D, these genes are not mutated. PA gene fragment 2233 nucleotides in length, comprising a length of 2151 nucleotides complete ORF frame encoding 716 amino acids can be deduced, the 5 'untranslated region and 3' untranslated regions, respectively containing 24 and 58 nucleotides, insertion or deletion of amino acids not found, but it happened N615 K mutations mutated to K; with the virus in the cell growth-related 638 is R, and the replication of 539 to K, associated with the polymerase activity the 510 is H, and associated with the pathogenicity 224 and 383 respectively, S and D, there is no mutation. NP gene is 1565 nucleotides in length, containing a full length ORF of 1497 nucleotides frame encoding 498 amino acids can be deduced not found insertions and deletions of nucleotides. 5' untranslated region and 3' untranslated regions, respectively containing 45 and 23 nucleotides. Strains site analysis, the impact of the test animals lung virus titers 479 related to the presence L479 F mutation; 319 no mutation, still N. NS gene ORF box divided into two parts, ORF1 from 27 to 704 nucleotides, is a continuous fragment containing 678 nucleotides, NS1 encoding 225 amino acids can be deduced, ORF2 and contains two non-contiguous portions, part from 27 to 56 nucleotides, in part, from 514 to 849 nucleotides, 366 nucleotides, located at the 5' end of ORF1 and ORF2 30 nucleotides overlap portions spaced 457 336 nucleotide after the second part of the OFR is NS2 box, NS2 is derived may be encoded 121 amino acids. Site analysis found that C135 of 42 mutations S, 92 bits mutation E, 103 bits non-pathogenic mutation K, 106 bits mutation V, 149 bits are G; at the end of NS1 is ESEV residues.To further understand the swine avian influenza virus pathogenicity tests on animals, swine with avian influenza virus C135 to 106EID50 ice dose of anesthesia, infection intranasal way 12 to 14 grams of female BALB/c mice; to 105EID50 dose by intranasal eye of way infection in 4 to 6 weeks old SPF chickens. The results showed that mice poison attack mortality rate of 75%. Mouse Brain virus was not detected, the higher the lung virus titers is 4.75 to 6.5log₁₀EID₅₀/m L, indicating that the virus may be in the proliferation of mouse lungs, but do not have the ability to enter the brain; the mice was observed during physical weight loss, messy hair, and other neurological symptoms, the autopsy found internal organs appeared pathological changes, tests showed that the strain of mouse has a strong pathogenicity. Virus enables all SPF chickens died within 2 days after infection, detected high titers of virus in the brain is 4.91 to 7.08log₁₀EID₅₀/m L, heart, liver, spleen, lung and kidney these six various tissues; cohabitation group brain, heart, liver, spleen, lung and kidney these tissue organs virus was not detected, indicating that the virus in SPF chickens with a high mortality rate.