Isolation And Molecular Epidemiological Investigation Of Porcine Parvovirus 7

Seven species of Porcine parvovirus PPV have been identified,namely PPV1 to PPV7.PPV1 is one of the important pathogens causing reproductive disorders in sows,with the main clinical symptoms leading to infertility,abortion,mummified fetus,stillbirth and neonatal fetal death in primiparous sows,posing a great threat to the health of pigs.In recent years,PPV has been reported to increase year by year,with PPV7 being the most prominent and has become widely prevalent in swine herds.Current research on PPV7 has focused on pathogen detection and epidemiological investigation,and little is known about the pathogenicity,genetic evolution,transmission routes,and pathogenesis of PPV7.Therefore,in this study,we conducted isolation and identification,genome sequencing,and molecμLar characterization of the prevalent strains of PPV7 in order to lay the foundation for subsequent studies on pathogenesis,genetic evolution,and transmission pathways.Clinical PPV7-positive lung samples were inocμLated on primary porcine alveolar macrophage(PAM)and swine testicle cells ST and passaged.The longest sequence of PPV7 in the NCBI is 4331 bp,without stem-loop structure at both ends,which is not a complete sequence.The four PPV7 strains and eight clinical positive samples were sequenced to obtain the near-fμLl-length sequences(4579bp),and the sequences were compared with the NCBI sequences from PPV1 to PPV7 for homology analysis.12 near-fμLl-length sequences were all on the same branch as the PPV7 sequences,and the stem-loop structure existed at both ends of the gene,and the stem-loop structure at the 5’ end was 204 bp long,of which the stem was 27 bp long and the loop was 150 bp long.27 bp and 150 bp ring length;the stem-loop structure at the 3’ end is 410 or 454 bp long,with 45 bp stem length and 320 or 364 bp ring length;the NS1 gene is 2019 bp long(692-2710);the Cap gene is 1425 bp long(2728-4152),which is the first time in the world to obtain the stem-loop structure sequence and near-fμLl-length sequence of both ends of PPV7.The structure of its viral particles was hexagonal or circμLar under electron microscope,and the size was between 20 and 25 nm.Western-blot verification revealed that there was and only one band with a size of about 55 k Da matching the size of the Cap protein,which clearly established that the Cap protein is a structural protein of PPV7.In addition,the resμLts of PPV7 testing of 550 samples(including 90 sera,335 lungs and 125 anal swabs)from 14 pig farms in Hunan Province from 2019 to 2021 showed a positive rate of 41.64%(229/550),including 67.14%(141/210)for lung samples from slaμghterhouses and 25.88%(88/340)for clinical samples sent for testing.25.88%(88/340).Sixty-five positive samples were taken from which the whole genome of Cap was sequenced,and the gene sequences were compared with those of other reference strains of Cap.The resμLts showed that the Cap protein of PPV7 encodes 466-474 amino acids;its antigenic epitope prediction showed the presence of 11-24 antigenic determinants clusters;recombination analysis showed that65 Cap of PPV7 gene had 15 recombination events(including 21 recombinant sequences);the resμLts of stress selection analysis showed that purifying selection(negative selection)played a dominant role in nucleotide mutation selection of PPV7,and only a small number of positive selection sites were found.This study successfully isolated PPV7 for the first time,laying a solid foundation for the study of the pathogenicity and other biological characteristics of PPV7;and successfully obtained the full-length sequence including the stem-loop structure at both ends of its genome,whereas the previous sequences did not include the stem-loop structure at both ends;through investigating the infection of PPV7 in some pig herds in Hunan,and conducting antigenic epitope prediction,gene recombination analysis and selective pressure analysis on the Cap gene of PPV7,we provided a new perspective for the epidemiological pattern and characteristics of PPV7 in pig herds.By investigating the infection of PPV7 in some pig populations in Hunan,and by performing antigenic epitope prediction,recombination analysis and selective pressure analysis on the Cap gene of PPV7,we provided new insights into the epidemiological pattern and characteristics of PPV7 in pig populations,and laid a solid foundation for the molecular epidemiological study and molecular genetic characterization of PPV7.