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[Objective] To understand the molecular genetic evolution characteristics of H3N8 subtype avian influenza virus isolated from waterfowl in Guangxi. [Method] The cotton swab samples collected from live poultry market were treated and inoculated into SPF chicken embryos. The collected allantoic fluid was serologically identified by hemagglutination test and hemagglutination inhibition test, and then the whole gene sequence and genetic evolution analysis of the isolated avian influenza virus were carried out. [Result] Serological HA and NA gene sequencing results showed that the isolated strain was H3N8 subtype avian influenza virus, named A/ duck / Guangxi/ 446D28 / 2021 (H3N8). Genetic evolution analysis of the whole gene showed that there was no continuous basic amino acid insertion in HA protein, which accorded with the characteristics of typical low pathogenic avian influenza virus. NA protein appeared drug-resistant amino acid site mutation;The results of comparative analysis of eight gene fragments of this strain in GenBank showed that the strains with the highest homology mainly came from Vietnam, Ningxia and Jiangxi provinces, which are located on the migratory route of migratory birds in China. [Conclusion] The isolated strain may be a gene recombinant formed by gene exchange among different subtypes of avian influenza viruses. [ABSTRACT FROM AUTHOR]

[Objective] To study the effects of protein X (pX) on Toll-like receptors after fowl adenovirus serotype 4 (FAdV-4) infection of LMH cells, the present paper provided data reference for further explaining the pathogenic mechanism and immune response mechanism of FAdV-4 infection. [Method] FAdV-4 X gene coding sequence (CDS) was amplified by PCR, and linked to pEFl α-HA vector, and the recombinant expression plasmid pEFlα-HA-X was constructed. The recombinant expression plasmid pEFlα-HA-X and empty vector pEFlα-HA-were transfected into LMH cells respectively, after 24 hours, the transfected cells were stimulated with FAdV-4, and the treatments were marked as pEFlα-HA-X+ and pEFlα-HA+, without virus stimulation after transfection was set up as control and marked as pEFlα-HAX- and pEFlα-HA-. Western-blotting and indirect immunofluorescence were used to verified expression of recombinant protein X, real-time florescence quantitative PCR assays was used to detected the mRNA expression levels of Toll-like receptors (chTLRla, chTLRlb, chTLR2a, chTLR2b, chTLRJ, chTLR4, chTLR5, chTLR7, chTLR15, chTLR21) and effect factors (JFN-α, INF-β, IL-1β, IL-6, IL-8, IL-15). [Result] The open reading frame (ORF) of X gene was 540 hp, encoding 179 amino acids residues. Western-blotting showed that recombinant X protein reacted with HA tag mouse-derived monoclonal antibody with a single band at 27 kD and green fluorescence visualized by indirect immunofluorescence. Real-time florescence quantitative PCR showed that, compared with pEFlα-HA-group the mRNA transcription levels of chTLRla and chTLRl b in pEFl α-HA-X-group were extremely significantly up-regulated (P < 0. 01, the same as below), as 9. 76 and 12. 34 times as that of pEFl α-HA-group, mRNA transcription levels of chTLR2a, chTLR2b, chTLR5, chTLR7, chTLRl 5 and chTLR21 were significantly up-regulated (P < 0. 05, the same as below). After virus infection, the mRNA transcription levels of chTLRl a and chTLRl b in pEFlα-HA-X+ group were extremely significantly down-regulated by 60. 0% and 66. 7% compared with pEFlα-HA-X-group, and the mRNA transcription levels of chTLR2a, chTLR2b and chTLRJ were significantly increased by 2. 91, 2. 01 and 1. 47 times respectively, the transcription levels of other chTLRs were down-regulated but not significantly ( P > 0. 05). At the same time, the mRNA transcription levels of IFN-α, IFN-β, IL-1β in pEFlα-HA-X + group were significantly up-regulated compared with pEFlα-HA-X-group, mRNA transcription level of IL-8 was extremely significantly down-regulated. [Conclusion] Overexpression of pX in LMH cells and after infection with FAdV-4, pX can activate host immune response system, mRNA transcription level of Toll-like receptors (chTLR2a, chTLR2b, chTLRJ) and effector factors (JFN-α, INF-β, IL-1β) significantly up-regulate to inhibit viral replication, indicating that pX has the function of activating the natural immune system of host cells. [ABSTRACT FROM AUTHOR]

To establish a dual nano-DPCR method for simultaneous detection of H7 subtype and N2 subtype avian influenza virus(AIV), in this study, the HA gene of H7 subtype AIV and the NA gene of N2 subtype AIV were obtained from GenBank database, and two pairs of specific primers were designed by Primer 7.0 and Oligo 6.0 software. The optimal reaction system was determined by optimizing the reaction conditions of nano-dPCR The optimized reaction system was used to test the specificity and sensitivity of the nano-dPCR method. Throat and cloacal swab samples were collected from poultry in live poultry markets for clinical sample testing and verification. The results showed that a nano-dPCR method was successfully established for the simultaneous detection of H7 subtype(723 bp) and N2 subtype(314 bp) AIV. This method had good specificity, no specific bands were found in other subtypes of avian influenza viruses and common avian respiratory viruses. Minimum amount of nucleic acid detection of H7 subtypes of AIV and N2 subtypes of AIV respectively reached 5×10³ and 5×10³ copies/μL, its sensitivity was 10 times of the ordinary dual PCR detection method, the coincidence rate of its detecting clinical samples was 100％, it could be used for differential diagnosis of clinical samples and daily monitoring of prevention and control． [ABSTRACT FROM AUTHOR]

【Objective】The paper aimed to express the NS1 and VP2 proteins of chicken parvovirus（ChPV） in prokaryotes and prepare polyclonal antibodies against them, which would provide material basis for further study of the protein structure and function of chicken parvovirus（ChPV） and provide materials for the establishment of serological diagnostic methods. 【Method】In the study, the sequences of NS1 and VP2 gene of chicken parvovirus GX-Ch-PV-21 strain were synthesized. The prokaryotic recombinant expression vectors pET-32a-NS1 and pET-32a-VP2 were constructed, and the correct recombinant plasmids were transformed into transsetta competent cells for prokaryotic expression. The expression conditions were optimized, and the expressions of NS1 and VP2 proteins were identified by SDS-PAGE and Western-blotting. The expression products were immunized to SPF chickens to obtain polyclonal antibodies. The titer of chicken polyclonal antibodies was determined by enzyme linked immunosorbent assay（ELISA）,and the polyclonal antibodies were identified by Western-blotting and indirect immunofluorescence assay（IFA）. 【Result】SDS-PAGE analysis showed that the size of NS1 and VP2 protein bands was about 104 and 87 kD,respectively, which was consistent with the expected size. The optimal induction conditions were screened for the recombinant plasmids pET-32a-VP2 and pET-32a-NS1. The results showed that the optimal induction conditions for the expression of NS1 protein were as follows: The final concentration of isopropyl 1-thio-β-D-galactopyranoside（IPTG） was 1. 0 mmol/mL and the expression was induced overnight at 16 ℃. The optimal conditions for the induction of VP2 protein expression were as follows: The final IPTG concentration was 0. 8 mmol/mL and the expression was induced overnight at 20 ℃. The soluble analysis showed that both NS1 and VP2 proteins mainly existed in the form of inclusion bodies. The titers of polyclonal antibodies against NS1 and VP2 by indirect ELISA were 1∶10 240 000. Western-blotting analysis showed that His tag protein and its corresponding chicken polyclonal antibody could specifically bind to NS1 and VP2 proteins. The IFA analysis showed that both VP2 and NS1 polyclonal antibodies could specifically bind to ChPV. 【Conclusion】In the study, the NS1 and VP2 proteins of chicken parvovirus were successfully expressed, and the chicken anti-polyclonal antibody was successfully prepared, which provided material basis for further study of the structure and function of chicken parvovirus protein and materials for the establishment of serological diagnostic methods. [ABSTRACT FROM AUTHOR]