To understand the current epidemiological status and biological characteristics of avian paramyxoviruses(APMV)in wild birds in China,a total of 1 384 fecal samples of wild birds were collected in eight provinces(autonomous regions),including Ningxia,in 2023,to detect avian pa-ramyxovirus infections by viral isolation and RT-PCR.Positive samples were subjected to F gene sequence amplification and genetic evolutionary analyses.The results showed that 10 strains of APMV were isolated and identified from 1 384 wild bird feces samples with a positive rate of 0.72％.Out of the 10 strains,4 strains were APMV-1,which was in the same branch to the Ameri-can goose APMV-1 strain and had the homology ranging from 93％to 97.3％.Three strains of APMV-4 were in the same branch with the Russian duck APMV-4 strain and the Russian pintail APMV-4 strain,with homology ranging from 99.1％to 99.5％.Three strains were APMV-6,they were in the same branch with the Russian ruddy bladdered duck APMV-6 strain,with homology ranging from 98.7％to 99.20％.The intracerebral inoculatable pathogenicity index(ICPI)of the four strains for 1-day-old chicks ranged from 0 to 0.48,which was low in pathogenicity for chick-ens.The above results enriches the epidemiological information and the biological characteristics of avian paramyxovirus in wild birds in China,which provides a reference for the early warning,scien-tific prevention and control of this disease.

To comprehend the genetic evolutionary characteristics and biological properties of the H4N1 subtype avian influenza virus(AIV)in China,this study conducted whole genome sequen-cing,genetic evolutionary analysis,and pathogenicity test in BALB/c mice of a duck-derived H4N1 subtype AIV strain(DK/GX/E1424/20)isolated from the live poultry market in the southern re-gion in 2020.The results indicated that the cleavage site motif of the HA protein was PEKASR/GLF,which conformed to the characteristics of low pathogenic AIV.All the eight gene fragments were situated in the Eurasian lineage,and the homology of AIV-related genes of the H1N1,H3N8,H4N6,H6N1,and H10N8 subtypes isolated from wild waterfowl was the highest,representing a recombinant virus strain.Without prior adaptation,it replicated effectively in the lungs and turbi-nates of mice,with viral titers of 3.00 and 2.08 log10EID50/mL respectively,and induced weight loss in infected mice.This study suggested that this virus had significant genetic diversity and low pathogenicity in mice,posing a potential risk for mammalian infection.

To establish an indirect enzyme linked immunosorbent assay(ELISA)method for the detection of Zaire Ebola virus(ZEBOV)specific antibodies,the full-length of ZEBOV GP1 gene was amplified by PCR and cloned into pET-30a(+)vector to generate the pET-30a(+)-GP1 plasmid.After expressed in the E.coli expression system,the purified GP1 protein was used as coating antigen to establish the indirect ELISA method for detection of ZEBOV antibody.The con-ditions including concentration of coating antigen and serum dilution were determined by chess-board titration.Specificity,sensitivity,and reproducibility of the established ELISA detection meth-od were evaluated.GP1 protein was successfully prepared by prokaryotic expression,and was used as the coatingantigen for indirect ELISA.By optimizing the reaction conditions,the optimal concen-tration of the coating antigen was determined to be 0.5 g/L;the optimal dilution of serum was cal-culated to be 1∶3 200;the optimal dilution of enzyme-labeled secondary antibody was measured to be 1∶20 000.The established method exhibited excellent specificity,sensitivity,and reproducibili-ty.In the present study,the GP1 protein was successfully expressed in the E.coli expression sys-tem and the high purity GP1 protein was used as the coating protein to establish an indirect ELISA assay for ZEBOV antibody.This method is highly specific,sensitive,and reproducible,which provides technical support for the fur-ther study of the biological function of GP1 and the detection of ZEBOV antibody in serum.

Moloney leukemia virus 10(MOV10)gene knockout(MKO)mouse neuro 2a(N2a)cell lines was constructed by CRISPR/Cas9(clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9)gene editing technology.First,a recombinant plasmid pMD18T-U6-sgRNA expressing MOV10 gene-specific guide RNA(sgRNA)was constructed,and then pMD18T-U6-sgRNA and pMJ920-Cas9-eGFP were co-transfected into N2a.The results showed that the MKO N2a cell line had normal cell activity and cell proliferation ability.The infection test of the MKO N2a cell line was carried out using the rabies virus(RABV)and vesicular stomatitis virus(VSV)of the Rhabdoviridae family.The results showed that the replication level of the Rhabdoviridae virus in the MKO N2a cell line was significantly enhanced.The results showed that a MKO N2a cell line was successfully constructed in this study,which provided a preliminary basis for the exploration of the biological function and antiviral mechanism of MOV10 and the develop-ment of a recombinant viral vector vaccine with RABV/VSV as the vector.

To understand the current epidemiological status and biological characteristics of avian paramyxoviruses(APMV)in wild birds in China,a total of 1 384 fecal samples of wild birds were collected in eight provinces(autonomous regions),including Ningxia,in 2023,to detect avian pa-ramyxovirus infections by viral isolation and RT-PCR.Positive samples were subjected to F gene sequence amplification and genetic evolutionary analyses.The results showed that 10 strains of APMV were isolated and identified from 1 384 wild bird feces samples with a positive rate of 0.72％.Out of the 10 strains,4 strains were APMV-1,which was in the same branch to the Ameri-can goose APMV-1 strain and had the homology ranging from 93％to 97.3％.Three strains of APMV-4 were in the same branch with the Russian duck APMV-4 strain and the Russian pintail APMV-4 strain,with homology ranging from 99.1％to 99.5％.Three strains were APMV-6,they were in the same branch with the Russian ruddy bladdered duck APMV-6 strain,with homology ranging from 98.7％to 99.20％.The intracerebral inoculatable pathogenicity index(ICPI)of the four strains for 1-day-old chicks ranged from 0 to 0.48,which was low in pathogenicity for chick-ens.The above results enriches the epidemiological information and the biological characteristics of avian paramyxovirus in wild birds in China,which provides a reference for the early warning,scien-tific prevention and control of this disease.

To comprehend the genetic evolutionary characteristics and biological properties of the H4N1 subtype avian influenza virus(AIV)in China,this study conducted whole genome sequen-cing,genetic evolutionary analysis,and pathogenicity test in BALB/c mice of a duck-derived H4N1 subtype AIV strain(DK/GX/E1424/20)isolated from the live poultry market in the southern re-gion in 2020.The results indicated that the cleavage site motif of the HA protein was PEKASR/GLF,which conformed to the characteristics of low pathogenic AIV.All the eight gene fragments were situated in the Eurasian lineage,and the homology of AIV-related genes of the H1N1,H3N8,H4N6,H6N1,and H10N8 subtypes isolated from wild waterfowl was the highest,representing a recombinant virus strain.Without prior adaptation,it replicated effectively in the lungs and turbi-nates of mice,with viral titers of 3.00 and 2.08 log10EID50/mL respectively,and induced weight loss in infected mice.This study suggested that this virus had significant genetic diversity and low pathogenicity in mice,posing a potential risk for mammalian infection.

To establish an indirect enzyme linked immunosorbent assay(ELISA)method for the detection of Zaire Ebola virus(ZEBOV)specific antibodies,the full-length of ZEBOV GP1 gene was amplified by PCR and cloned into pET-30a(+)vector to generate the pET-30a(+)-GP1 plasmid.After expressed in the E.coli expression system,the purified GP1 protein was used as coating antigen to establish the indirect ELISA method for detection of ZEBOV antibody.The con-ditions including concentration of coating antigen and serum dilution were determined by chess-board titration.Specificity,sensitivity,and reproducibility of the established ELISA detection meth-od were evaluated.GP1 protein was successfully prepared by prokaryotic expression,and was used as the coatingantigen for indirect ELISA.By optimizing the reaction conditions,the optimal concen-tration of the coating antigen was determined to be 0.5 g/L;the optimal dilution of serum was cal-culated to be 1∶3 200;the optimal dilution of enzyme-labeled secondary antibody was measured to be 1∶20 000.The established method exhibited excellent specificity,sensitivity,and reproducibili-ty.In the present study,the GP1 protein was successfully expressed in the E.coli expression sys-tem and the high purity GP1 protein was used as the coating protein to establish an indirect ELISA assay for ZEBOV antibody.This method is highly specific,sensitive,and reproducible,which provides technical support for the fur-ther study of the biological function of GP1 and the detection of ZEBOV antibody in serum.

Moloney leukemia virus 10(MOV10)gene knockout(MKO)mouse neuro 2a(N2a)cell lines was constructed by CRISPR/Cas9(clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9)gene editing technology.First,a recombinant plasmid pMD18T-U6-sgRNA expressing MOV10 gene-specific guide RNA(sgRNA)was constructed,and then pMD18T-U6-sgRNA and pMJ920-Cas9-eGFP were co-transfected into N2a.The results showed that the MKO N2a cell line had normal cell activity and cell proliferation ability.The infection test of the MKO N2a cell line was carried out using the rabies virus(RABV)and vesicular stomatitis virus(VSV)of the Rhabdoviridae family.The results showed that the replication level of the Rhabdoviridae virus in the MKO N2a cell line was significantly enhanced.The results showed that a MKO N2a cell line was successfully constructed in this study,which provided a preliminary basis for the exploration of the biological function and antiviral mechanism of MOV10 and the develop-ment of a recombinant viral vector vaccine with RABV/VSV as the vector.

African swine fever(ASF),caused by the African swine fever virus(ASFV),is a highly contagious infectious disease of pigs.This disease has been spread rapidly in China since 2018,po-sing a huge threat to China's pig farming industry.To rapid detect the ASFV,a loop-mediated iso-thermal amplification(LAMP)combined with the disposable nucleic acid visualization test strip was established for visual detection of the nucleic acid of ASFV B646L gene.The method was easy to operate without special instruments and equipment,while it effectively avoided the disadvantage of false positives caused by aerosol contamination.The method was able to detect 1.16 copies/μL of the recombinant plasmid in 50 min at 65 ℃.In addition,the method was specific with no cross-re-action with classical swine fever virus,porcine reproductive and respiratory syndrome virus,por-cine parvovirus,transmissible gastroenteritis virus.The results in this study provides a rapid,con-venient,sensitive and reliable method for early diagnosis and screening for ASFV suspected infec-tion cases.

In order to explore the effects of Coptis detoxification powder on immune suppression and inflammatory response caused by heat stress in animals,Bama pigs were used as experimental animals to establish an animal model of heat stress,and the effects of Coptis detoxification powder on transcriptional activation of TLRs mRNA in PBMC induced by heat stress and its mediated ex-pression of inflammatory factors were studied in vivo.The animal model of heat stress was estab-lished by artificial climate greenhouse,Chinese medicine"Coptis detoxification powder"was pre-pared,and its inhibitory effect on heat stress and inflammation was studied by preventive and therapeutic administration.Real-time fluorescence quantitative PCR was used to determine the transcriptional changes of HSP70,TLRs and IL-17 mRNA,and ELISA was used to determine the dynamic changes of serum IL-2,IL-6 and TNF-α.Western blot and indirect immunofluorescence(IFA)were used to determine the nuclear translocation and protein expression of p65 phosphoryl-ated protein in NF-κβ inflammatory factor pathway.The transcription of PBMC TLRs mRNA and its mediated inflammatory factor expression and function in Bama pigs under heat stress were sys-tematically analyzed.The results showed that the experimental animal model of Bama pigs under heat stress was successfully established,and heat stress significantly up-regulated the mRNA tran-scription levels of HSP70,TLR2,TLR4 and inflammatory factor IL-17,resulting in significantly enhanced expressions of serum inflammatory factors IL-2,IL-6 and TNF-α.The NF-κβ pathway significantly promoted the level of p65 nuclear phosphorylation and the expression of cytoplasmic protein.The Chinese medicine"Coptis detoxification powder"could significantly reduce the upreg-ulation effect of heat stress conditions on the transcription levels of HSP70 and TLRs mRNA,thus inhibiting the mRNA transcription of inflammatory factor IL-17 and the expression of IL-2,IL-6 and TNF-α in serum,and significantly antagonizing the transcriptional activity of NF-κβ caused by heat stress.It showed good anti-heat stress effect.The anti-inflammatory effects of Chinese veteri-nary medicine prescription"Coptis detoxification powder",its pathway of action and its effectors were investigated,the expression and secretion levels of the regulatory factors related to the path-way associated with TLRs mRNA inhibition were analyzed,and the functional effects and mecha-nism of its clinical anti-heat stress and anti-inflammatory effects were clarified,which laid a data foundation for the promotion and application of this prescription and related mechanism research.