The expression of the hemagglutinins of Avian influenza virus H5 H7and H9 subtypes was studied in this article by Pichia pastoris, one of the eukaryotis expression systems. Three reconstructed expression plasmids and engineering strains, named pPIC9K-H5HA, pPIC9K-H7HA, pPIC9K-H9HA and GS115/pPIC9K-H5HA, GS115/pPIC9K-H7HA, GS115/pPIC9K-H9HA repectively, were obtained. The reconstructed yeast engineering strains were identified by MD and MM plate selecting and PCR. The induced interests proteins were examined by SDS-PAGE and Western-bloting,the results showed that the interest genes were expressed exactly. And this will be helpful in the future study of antigen detection and antibody detection kit, as well in the subunit vaccines developing.
Animals ; Hemagglutinin Glycoproteins, Influenza Virus ; biosynthesis ; genetics ; Influenza A Virus, H5N1 Subtype ; genetics ; Influenza A Virus, H7N7 Subtype ; genetics ; Influenza A Virus, H9N2 Subtype ; genetics ; Pichia ; genetics ; metabolism

BACKGROUNDTo understand the characterization of genome of a strain of avian influenza A H9N2 virus repeatedly isolated from a child with influenza illness. Thereafter to reveal the origin of this H9N2 virus.

METHODSViruses were passed in embryonated hen eggs and virion RNA was extracted from allantoic fluid and reverse transcribed to synthesize cDNA. cDNA was amplified by PCR and the PCR product was purified with a purification kit. Afterwards RNA sequence analysis was performed by dideoxynucleotide chain termination and a cloning method. Finally, phylogenetic analysis of the sequencing data was performed with MegAlign (Version 1.03) and Editseg (Version 3.69) softwares.

RESULTSGenome of A/Guangzhou/333/99 (H9N2) virus was closely related to avian influenza A H9N2 virus, but obvious difference from that of A/Duck/Hong Kong/Y439/97(H9N2) virus, as well as its genome did not include any RNA segment derived from human influenza A virus. However, the genes encoding the HA,NA,NP and NS proteins of A/Guangzhou/333/99 virus were derived from those of G9 lineage virus, the rest genes encoding the M and three polymerase (PB2,PB1 and PA) proteins were derived from G1 lineage strain.

CONCLUSIONSA/Guangzhou/333/99 virus was a reassortant derived from reassortment betweenG9 and G1 lineages of avian influenzaA(H9N2) viruses. Therefore, the most possibility is that it is derived from avian influenza A virus directly. The results do not only demonstrate that avian influenza A (H9N2) virus could infect men, but also firstly prove that the genetic reassortment could be occurred between different genetic lineages of avian influenza A (H9N2) viruses in the nature.

Animals ; Base Sequence ; Chick Embryo ; Child ; Genome, Viral ; Humans ; Influenza A Virus, H9N2 Subtype ; Influenza A virus ; genetics ; Influenza, Human ; virology ; Phylogeny

Samples of chicken, duck, quail, and pigeon were collected from Jiangsu, Anhui, and Hebei in 2009-2011, and sixteen H9N2 subtype isolates of avian influenza virus (AIV) were identified. The eight full-length genes of 16 AIV isolates were amplified by RT-PCR and sequenced. Genome sequence analysis showed that the amino acid motif of cleavage sites in the HA gene was P-S-R/K-S-S-R, which was consistent with the characterization of the LPAIV, and the Leucine (L) at the amino acid position 226 in the HA genes of all isolates indicated the potential of binding with SAalpha, 2-6 receptor. All isolates had a S to N substitution at residue 31 in the M2 gene, which is related to the resistance phenotype of adamantanes. The key molecular features of 16 AIV isolates from different hosts were same. Genome phylogenetic analysis revealed that all 16 H9N2 subtype AIVs originated from F98-like virus as backbone and formed two new genotypes through reassortment with HA gene of Y280-like virus and PB2 and M genes of G1-like virus. Our findings suggest that more attention should be paid to the surveillance of H9N2 influenza virus and its direction of reassortment.
Genome, Viral ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; Influenza A Virus, H9N2 Subtype ; classification ; genetics ; Neuraminidase ; genetics ; Phylogeny ; Sequence Analysis, DNA

LG1 strain of avian influenza virus H9N2 was passaged continuously for 40 generations in chicken embryos with anti-LG1 maternal antibodies in 4 parallel experiments, of which 3 experiments had a stable mutation of "G" to "A" at #99 of the neuraminidase gene(NA)from the 20th passage resulting in a change of Met to Ile and 2 had a stable mutation of "A" to "G" at #473 of the NA gene from the 30th passage resulting in a change of Asn to Ser which occurred in the 50th passage of another experiment. Eighty continuous passages in chicken embryos without antibody did not have the same mutation, indicating that the mutations of the 2 positions were associated with selective pressure of antibodies. Analysis of the ratios of nonsynonium (NS) vs synonium (S) mutations of nucleic acids demonstrated that NS/S of 4 parallel experiments with antibodies was 4.6 (32/7) compared with 2.0 (16/8) of the 2 experiments without antibodies and this significant difference implied the selective pressure of antibodies.
Animals ; Antibodies, Viral ; immunology ; Chick Embryo ; Influenza A Virus, H9N2 Subtype ; genetics ; immunology ; Mutation ; Neuraminidase ; genetics

BACKGROUNDTo understand the characteristics of internal genes of two strains of influenza A(H9N2) virus isolated from men and on the basis of these to reveal the origin of these two strains of influenza A(H9N2)virus.

METHODSThe target gene was amplified by RT-PCR,the PCR product was ligated with P GEM-T Vector (Promega Company, USA) at 4 degrees, the recombined plasmid was transferred into dH5a bacteria, and the positive colonies were selected and identified with restriction enzyme. Afterwards, they were sent to Liu He Tong Company in Beijing for nucleotide sequencing. Finally, phylogenetic analysis of the sequencing data was performed with MegAlign (version 1.03)and Editseq (Version 3.69) softwares.

RESULTSInternal genes of the two strains of H9N2 virus were G9 lineage. There was a slight difference in nucleotide sequence in PA gene between the two strains, whereas another five gene segments were identical to each other.

CONCLUSIONThe genomes of the two strains of influenza A(H9N2)virus were G9 lineage.They were transmitted to men separately from different avian sources with different characteristics of gene of influenza A(H9N2) virus, respectively.

Animals ; Chick Embryo ; China ; Humans ; Influenza A Virus, H9N2 Subtype ; classification ; genetics ; isolation & purification ; Influenza, Human ; virology ; Phylogeny ; Viral Proteins ; genetics

A quail-origin subtype of the influenza virus was isolated from a human-infecting H7N9 subtype of the avian influenza virus found in a live poultry market and was given the name A/Quail/Hangzhou/1/ 2013 (H9N2). We analyzed the whole genome of this virus and its biologic characteristics. Sequence analyses suggested that the: HA and NS genes belonged to a CK/BJ/1/94-like lineage; NA, NP, PA and PB1 genes belonged to a SH/F/98-like lineage; M and PB2 genes belonged to a G1-like lineage. Analyses of key amino acids showed that the cleavage site in HA protein was PSRSSR ↓ GL, and that the HA protein had a human receptor-binding site with Leu226. Deletion of amino acids 69 - 73 was detected in the stalk of NA protein, the M2 protein had an Asn31 mutation, and the NS1 protein had two mutations at Ser42, Ala149. The intravenous pathogenicity of this virus was 0.36. A study in chickens suggested that all inoculated birds shed the virus from the trachea and cloaca on the third day post-infection (p. i. ) until 11 days. All chickens that had direct contact shed the virus on the second day p. i. until 8 days. Results of virus reisolation suggested that lung and tracheal tissues could shed the virus in 5 days, whereas the other organs could shed the virus in 3 days. These results suggest that this virus strain is H9N2 subtype LPAIV, whose lineage is prevalent in mainland China. This research provides evidence on how to monitor and prevent the H9N2 subtype of the avian influenza virus.
Animals ; Chick Embryo ; Chickens ; China ; Genotype ; Influenza A Virus, H9N2 Subtype ; classification ; genetics ; isolation & purification ; Influenza in Birds ; virology ; Phylogeny ; Quail ; virology

This paper aims to explore the effects of chicken interferon-γ (ChIFN-γ) and interleukin-2 (ChIL-2) on type 1 helper (Th1) T lymphocyte differentiation. To be specific, ChIFN-γ and ChIL-2 were first expressed in Escherichia coli competent cells and then purified by Ni-NTA affinity chromatography. Different concentration of ChIFN-γ and ChIL-2 were employed to stimulate the lymphocytes in chicken peripheral blood which had been activated by concanavalin A (Con A), and the mRNA levels of cytokines related to Th1 cell differentiation were detected by real-time quantitative PCR (RT-qPCR). The results showed that both ChIFN-γ and ChIL-2 can significantly up-regulate mRNA levels of cytokines related to Th1 cell differentiation and the optimal concentration was 12.5 μg/mL and 25.0 μg/mL, respectively. In addition, specific-pathogen-free (SPF) chickens were immunized with ChIL-2 or ChIFN-γ together with H9N2 vaccine, or H9N2 vaccine alone by oral administration or intramuscular injection, respectively. The mRNA levels of cytokines related to Th1 cell differentiation were detected after immunization. The results showed that ChIFN-γ and ChIL-2 significantly up-regulated the mRNA levels of cytokines related to Th1 cell differentiation induced by H9N2 vaccine compared with H9N2 vaccine alone, and that the intramuscular injection was better than oral administration. In this study, we verified that ChIFN-γ and ChIL-2 can significantly enhance mRNA levels of cytokines related to Th1 cell differentiation induced by ConA or H9N2 vaccine in vitro and in vivo. The results of this study can lay a theoretical basis for using ChIFN-γ and ChIL-2 as vaccine adjuvants.
Animals ; Cell Differentiation ; Chickens ; Concanavalin A ; Cytokines/genetics* ; Influenza A Virus, H9N2 Subtype/genetics* ; Interferon-gamma/metabolism* ; Interleukin-2/genetics* ; RNA, Messenger

As part of our ongoing influenza surveillance program in South China, 19 field strains of H9N2 subtype avian influenza viruses (AIVs) were isolated from dead or diseased chicken flocks in Guangdong province, South China, between 2012 and 2013. Hemagglutinin (HA) genes of these strains were sequenced and analyzed and phylogenic analysis showed that 12 of the 19 isolates belonged to the lineage h9.4.2.5, while the other seven belonged to h9.4.2.6. Specifically, we found that all of the viruses isolated in 2013 belonged to lineage h9.4.2.5. The lineage h9.4.2.5 viruses contained a PSRSSRdownward arrowGLF motif at HA cleavage site, while the lineage h9.4.2.6 viruses contained a PARSSRdownward arrowGLF at the same position. Most of the isolates in lineage h9.4.2.5 lost one potential glycosylation site at residues 200-202, and had an additional one at residues 295-297 in HA1. Notably, 19 isolates had an amino acid exchange (Q226L) in the receptor binding site, which indicated that the viruses had potential affinity of binding to human like receptor. The present study shows the importance of continuing surveillance of new H9N2 strains to better prepare for the next epidemic or pandemic outbreak of H9N2 AIV infections in chicken flocks.
Animals ; *Chickens ; China ; Hemagglutinin Glycoproteins, Influenza Virus/chemistry/*genetics/metabolism ; Influenza A Virus, H9N2 Subtype/*genetics/metabolism ; Influenza in Birds/virology ; Phylogeny ; Poultry Diseases/*virology ; Sequence Analysis, RNA/veterinary

Avian influenza virus subtype H9N2 has been circulating in multiple terrestrial birds and repeatedly infecting mammals, including swines and humans to pose a significant threat to public health. The cross-species infection of human, replication activity and tissue tropism of avian influenza virus H9N2 was evaluated in this study. The results showed that surgically removed human lung tissue samples were infected ex vivo by avian influenza virus subtype H9N2 (Ck/GX/1875/04, Ck/GX/187/05) and seasonal human influenza virus H3N2 (A/ST/602/05). Examination of nucleoprotein expression replication in the infected human lung tissue samples showed that the replication of avian influenza virus H9N2 and seasonal human influenza virus H3N2 were mainly prevalent in alveolar epithelial cells, respiratory bronchiole epithelial cells and bronchial epithelial cells. Double-immunostaining for viral antigens and cellular markers indicated that avian influenza virus subtype H9N2 replicated in type 2 alveolar epithelial cells. These findings suggest that the H9N2 virus may be better adapted to the human host and replicates efficiently in human lung epithelial cells. Moreover, H9N2 avian influenza virus repeatedly infecting human, may favor gene evolution and the potential emergence of pandemic influenza virus.
Animals ; Epithelial Cells ; virology ; Humans ; Influenza A Virus, H3N2 Subtype ; genetics ; physiology ; Influenza A Virus, H9N2 Subtype ; genetics ; isolation & purification ; physiology ; Influenza, Human ; virology ; Lung ; cytology ; virology ; RNA-Binding Proteins ; genetics ; metabolism ; Viral Core Proteins ; genetics ; metabolism ; Virus Replication

The recent human infection with avian influenza virus revealed that H9N2 influenza virus is the gene donor for H7N9 and H10N8 viruses infecting humans. The crucial role of H9N2 viruses at the animal-human interface might be due to the wide host range, adaptation in both poultry and mammalian, and extensive gene reassortment. As the most prevalent subtype of influenza viruses in chickens in China, H9N2 also causes a great economic loss for the poultry industry, even under the long-term vaccination programs. The history, epidemiology, biological characteristics, and molecular determinants of H9N2 influenza virus are reviewed in this paper. The contribution of H9N2 genes, especially RNP genes, to the infection of humans needs to be investigated in the future.
Animals ; Chickens ; virology ; China ; epidemiology ; Humans ; Influenza A Virus, H7N9 Subtype ; genetics ; Influenza A Virus, H9N2 Subtype ; genetics ; immunology ; physiology ; Influenza in Birds ; epidemiology ; transmission ; virology ; Influenza, Human ; epidemiology ; transmission ; virology ; Vaccination ; Viral Proteins ; classification ; metabolism