ObjectiveTo genotype the specimens from hand-foot-and-mouth disease (HFMD) cases in Dehong Prefecture, Yunnan Province in 2023 by gene sequencing, and to analyze the genetic characteristics of Coxsackievirus A4 (CVA4), CVA6, CVA10, CVA16 and enterovirus A71 (EV-A71), so as to clarify the serotypes of enteroviruses causing endemic HFMD and the genotype composition of CVA4, CVA6, CVA10, CVA16 and EV-A71 prevalent in HFMD, and to provide baseline genotype data for monitoring the dynamic changes of the five virus genotypes. MethodsStool specimens from HFMD cases in Dehong Prefecture in 2023 that tested positive for EV-A71, CVA16 and other enteroviruses by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) were first subjected to gene sequencing and typing of the VP4/VP2 junction region to determine the viral types. Subsequently, the complete VP1 gene of each virus was amplified in two segments using group-specific VP1 gene primers corresponding to enterovirus groups (i.e., enterovirus group A, B, and C) and sequenced (group A virus: primers 486/488 and 487/489, group B virus: primers 490/492 and 491/493, group C virus: primers 494/496 and 495/497). The representative reference sequences of VP1 genes for CVA4, CVA6, CVA10, CVA16 and EV-A71 were downloaded from the literature, and the VP1 gene phylogenetic tree was constructed using MEGA 6.0 software for genetic characterization. ResultsA total of 256 stool specimens were collected from HFMD cases in Dehong Prefecture in 2023, of which 94 tested positive for nucleic acid by RT-qPCR, yielding a positive rate of 36.72%. Of the 94 positive specimens, 83 were identified as group A viruses, and were classified into 5 serotypes (17 strains of CVA4, 3 of CVA6, 6 of CVA10, 49 of CVA16, and 8 of EV-A71). Nine strains were group B viruses, comprising 2 serotypes: 2 strains of echovirus type 3 (E3) and 7 strains of Coxsackievirus B type 5 (CVB5). The remaining 2 strains were group C viruses, both identified as poliovirus type 3 (PV3). Genetic analyses of the VP1 region showed that all 17 CVA4 strains were identified as subgenotype C2, all 3 CVA6 strains as subgenotype D3a, all 6 CVA10 strains as genotype C. Among the 49 CVA16 strains, 46 were identified as subgenotype B1a and 3 as subgenotype B1b, and all 8 EV-A71 strains were identified as subgenotype C4a. ConclusionIn 2023, group A viruses dominated the pathogenic spectrum of HFMD in Dehong Prefecture. Among them, CVA16 had the highest positivity rate. Genetic characteristic analyses showed that CVA4, CVA6, CVA10 and EV-A71 were each composed of a single genotype (subgenotype), while CVA16 was composed of two subgenotypes (B1a and B1b), with B1a being the predominant one. It is recommended that molecular epidemiological surveillance of HFMD pathogens, particularly group A viruses, be continuously strengthened in Dehong Prefecture.

To develop vaccines for the prevention and control of porcine circovirus 2d genotype(PCV2d)and pseudorabies virus(PRV),the PCV2d ORF2 gene was amplified by PCR,and cloned into the BamH Ⅰ site of PRV transfer plasmid pG vector harboring the enhanced green fluorescent protein(EGFP)gene.The resulting recombinant transfer plasmid pG-PCV2d-EGFP was transfect-ed into ST cells infected with the three gene deleted PRV variant strain gE-/g-/TK-PRV NY to generate a recombinant virus rPRV-PCV2d-EGFP+,and then the EGFP gene was knocked out to harvest the rPRV-PCV2d using gene-editing technology termed CRISPR/Cas9 system.The recom-binant virus rPRV-PCV2d had similar genetic stability to the parental PRV as indicated by PCR and one-step growth curve test,and the expression of PCV2d capsid(Cap)protein was validated by Western blot.In animal experiment,higher PCV2-specific ELISA antibodies and detectable PCV2-specific neutralizing antibodies could be elicited in mice immunized with the recombinant vi-rus rPRV-PCV2d compared to commercial PCV2 inactivated vaccine.rPRV-PCV2d significantly re-duced the PCV2d loads in tissues such as the heart,liver and spleen of mice following virulent PCV2d challenge.Moreover,rPRV-PCV2d elicits PRV-specific immune responses in mice and can prevent PRV virulent infection in mice,indicating the recombinant virus rPRV-PCV2d has strong immunogenicity.

To develop vaccines for the prevention and control of porcine circovirus 2d genotype(PCV2d)and pseudorabies virus(PRV),the PCV2d ORF2 gene was amplified by PCR,and cloned into the BamH Ⅰ site of PRV transfer plasmid pG vector harboring the enhanced green fluorescent protein(EGFP)gene.The resulting recombinant transfer plasmid pG-PCV2d-EGFP was transfect-ed into ST cells infected with the three gene deleted PRV variant strain gE-/g-/TK-PRV NY to generate a recombinant virus rPRV-PCV2d-EGFP+,and then the EGFP gene was knocked out to harvest the rPRV-PCV2d using gene-editing technology termed CRISPR/Cas9 system.The recom-binant virus rPRV-PCV2d had similar genetic stability to the parental PRV as indicated by PCR and one-step growth curve test,and the expression of PCV2d capsid(Cap)protein was validated by Western blot.In animal experiment,higher PCV2-specific ELISA antibodies and detectable PCV2-specific neutralizing antibodies could be elicited in mice immunized with the recombinant vi-rus rPRV-PCV2d compared to commercial PCV2 inactivated vaccine.rPRV-PCV2d significantly re-duced the PCV2d loads in tissues such as the heart,liver and spleen of mice following virulent PCV2d challenge.Moreover,rPRV-PCV2d elicits PRV-specific immune responses in mice and can prevent PRV virulent infection in mice,indicating the recombinant virus rPRV-PCV2d has strong immunogenicity.