| In recent years,serious outbreaks of diarrhea in pigs have been reported in China,causing severe economic losses to China's pig-breeding industry.Diarrhea in pigs is caused by the infection of viruses,bacteria and other pathogenic microorganisms,poor environment,and improper feeding conditions.And virus infection is a major cause of diarrhea in pigs.The common viruses that cause diarrhea in pigs are porcine epidemic diarrhea virus(PEDV),porcine rotavirus(RV)and porcine transmissible gastroenteritis virus(TGEV).However,in recent years,the bigeminal vaccines and rubella vaccines which were commonly used in the swine industry have frequently experienced immune failure.Some new viruses,such as porcine deltacoronavirus(PDCoV)and porcine enteric alphacoronavirus(PEAV),were found that could also cause diarrhea in pigs.In addition,porcine kobuvirus(PKV)might be associated with diarrhea in pigs.PKV is the member of the large and growing family Picornaviridae and is a genomic positive single-stranded RNA virus without envelope.In 2016,the International Committee on Taxonomy of Viruses(ICTV)formally established it as a member of the Kobuvirus.It was first detected in Hungary in 2007,and since then was detected worldwide with high detection rate.However,the relationship between the virus and disease was still unclear.It was reported in a large number of studies that the detection rate of PKV in diarrheic pigs was higher than that of non-diarrheic pigs.Most researchers speculated that the virus might be related to diarrhea in pigs.However,some studies have pointed out that the detection rate of the virus in non-diarrheic pigs was higher than that of diarrheic pigs.In this study,a pair of primers was designed in the conserved sequence of PKV 3D regions,and a total of 634 samples including swine intestine contents and feces were detected by RTPCR,which were collected from 43 pig farms in 11 cities of Shandong Province.A statistics was performed to investigate PKV infection in Shandong Province.The results showed that in634 samples,the positive rate of PKV was 39.91%(253/634),of which the positive rate of diarrheic pigs was 28.90%(63/218),and the positive rate of non-diarrheic pigs was 45.67%(190/416).The detection rate of PKV was high in the collected samples,indicating that the infection of PKV in Shandong Province was very common,and the positive rate of nondiarrheic pigs was much higher than that of diarrheic pigs.This study showed that there wasno necessary relation between PKV infection and porcine diarrhea in Shandong province.In other words,PKV might not be the dominant virus in porcine diarrhea.The positive rate of PKV in suckling piglets,weaning piglets,fattening pigs and adulthood pigs was 48.31%(100/207),34.78%(48/138),and 27.75%(63/227)and 67.74%(42/62),which was shown in the statistics of the different growth stages of the collected pigs,indicating that PKV infected all of age groups in Shandong pigs,and the infection rate was generally high.The positive rate of PKV in pigs in Shandong Province had obvious season differences in the statistics of PKV detection in different seasons of pigs,and the positive rate of PKV in summer and autumn(53.42%,117/219)was significantly higher than that in winter and spring(32.77%,136/415),contrary to the outbreak season of porcine epidemic diarrhea,which deeply explained that PKV might not play a dominant role in porcine diarrhea.Statistical analysis of PKV results of different breeds of pigs found that the local breeds had the highest positive rate with 69.06%(96/139),followed by the outer sanyuan with 33.73%(140/415),the inner sanyuan has the lowest positive rate,which was 21.25%(17/80).PEDV,TGEV,RV,PDCoV and PEAV,which could cause porcine diarrhea,were also detected in this study,and the co-infection of PKV and these viruses was counted,showing a clear mixed infection between PKV and PEDV,TGEV.The rate of PKV single-infected in non-diarrheic pigs was significantly higher than that of diarrheic pigs,but the co-infection rate of PKV with PEDV and TGEV in nondiarrheic pigs was significantly lower than that in diarrhea pigs.Since both PEDV and TGEV were the main and common causes of diarrhea in pigs,it is not possible to determine whether PKV played a role in the process of causing diarrhea in pigs.It was speculated that PKV might act as a synergistic pathogen in the process of diarrhea in pigs,and there was no ability to cause diarrhea in pigs with PKV singleinfected in general.In this study,we designed a pair of primers containing VP1 gene by comparing the sequences of the two ends of VP1 gene of PKV,and 11 VP1 gene sequences were obtained with a length of 762-765 bp.By comparison with reference strains,there was a 3 nt insertion between 713-715 bp in the 11 VP1 sequences and a 3 nt deletion between 727-729 bp in Liaocheng/160112,resulting in different lengths.The 11 VP1 sequences obtained in this study were compared with reference strains in GenBank.The results showed that the homology between the 11 VP1 sequences was between79.3-100%,among which,the homology between Ji'nan/Jiyang/171226 and Tai'an/171222 was 100%,most likely the same strain.The homology between the 11 VP1 sequences and the reference strains was between 78.0-88.6% and the amino acid homology was between 79.9-97.6%,indicating that there was a certain variation between the isolated VP1 sequence and the reference strain.According to PKV VP1 phylogenetic tree,it was divided into 5 large groups.All of the 11 VP1 sequences were in the Group 1 and were located on three branches,indicating that the PKV strains popular in Shandong Province might evolve from one or several original strains after a long period of variation. |
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