Molecular Epidemiological Investigation On Classical Swine Fever In Parts Of Guangdong Province

Classical swine fever is a highly contagious disease of pigs caused by classicalswine fever virus (CSFV) and causes serious economic losses to pig industry annually.In1833, classical swine fever was first recognized in Ohio state of the United States.After that, there are many news about the outbreaks and prevalence of classical swinefever in succession in the major pig-raising countries in Asia, Africa, Latin Americaand Europe. The prevalence of the classical swine fever involving almost of thecountries and regions of the world except the Antarctica. It formed a great threat to thepig industry of those countries. The Office International des Epizooties (OIE) hasdesignated CSF as the class A infectious diseases and listed it into the OIE list withsome other diseases that must be notified. In China, CSF also has been classified asthe classⅠinfectious diseases.In the late1990s, Changchun Tu and his colleagues to carry out the study ofmolecular epidemiology of CSF in China. Their studies show that there are foursubsubtypes exist in China, including1.1,2.1,2.2and2.3, among which2.1subsubtype is dominant strains. Part of strains in his study which were prevalence inGuangdong province included4subsubtypes(2.1b,1.1,2.2,2.3). In recent years,Chen Ning and other academic’s studies revealed that sub-subsubtype2.1b strainsbecame predominant in southeastern China. In recent decades, A large number ofstudies confirmed that CSFV strains are developing to the group II. CSFV strains insome Asian countries or regions including China, Japan, South Korea and Taiwan, etal, there has been a switch in virus populations from subsubtype3.4to2.1as a resultof the selection forces of the vaccine or other factors. This shift may related to theadaptive abilities of CSFV trains to the selection forces within the host. The past tenyears, the prevalence trend of CSFV in Guangdong province is unclear.To fully understand the prevalence trend of CSFV and the genetic diversity ofCSFV field isolates in Guangdong province,415CSFV suspected tissue samples were collected from different farms or backyard farmers of11cities of Guangdongprovince. CSFV nucleic acid was detected by the nested RT-PCR method on the EUclassical swine fever diagnostic manual.57samples were CSFV-positive, the rates ofCSFV-positive were13.7%. In addition, another three common virus in pigs includingPRRSV, PCV-2, PRV were detected from the CSFV-positive tissue samples by PCRmethod.56out of57CSFV-positive samples were PCV-2positive,34samples werePRRSV positive,1sample was PRV positive. The results showed that the mixedinfection of CSF and PRRSV, PCV-2is very serious, the mixed infection of CSF andPRV is less serious. The multiple infection of CSF and other diseases is one of theimportant problems of the prevention and control of CSF in Guangdong province.In order to keep these precious biological materials for a longer time andaccumulate viral resources for the prevention and control of CSF of Guangdongprovince and also provide virus for the later we study the adaptive of CSFV in thePK-15cells lines and the EC cells lines.24epidemic CSFV strains were isolated from57CSF positive tissue samples. We also found that the new cell line(EC) have goodsusceptibility to the CSFV. Except16strains belong to2.1b which was the historicalCSFV strains, there are two novel sub-subgenotype s were isolated successfully,31strains belong to2.1c and4strains belong to2.1d. We provides the most basicmaterial for the study CSFV of this area in the future.A region encoding the major neutralizing epitopes at the N terminus of the E2gene was amplified by RT-PCR and then sequenced, we obtained57sequences.52reference strain sequences were got from the GenBank database and the other17reference strain sequences was previously sequenced by Changchun Tu. Phylogenetictrees were conducted by the software MEGA5.05based on the region encoding themajor neutralizing epitopes of E2gene. Phylogenetic analysis showed that4out of57isolates belonged to subgenotype1.1, and53isolate belongs to subgenotype2.1. Thesubgenotype of2.2,2.3previously prevalence in this area were not found in our study.Notably that at the CSFV monitoring regions there were two novel2.1sub-subgenotypes,2.1c and2.1d. The prevalence of these strains revealed that thegenetic diversity have changed in Guangdong province. The envelope glycoprotein E2which is one of the structural proteins of CSFV isessential for virus attachment and recognized the target cells. It also plays hinge rolesin life cycle of CSFV. E2can induce neutralizing antibodies. The variation of E2isrelated to immune escape of the virus. In order to clarify the characteristics of thevariation of E2gene of2.1c and2.1d. We selected16tissue samples representativefrom the57CSF positive tissue samples, the full-length E2gene of16CSFV isolateswas amplified by RT-PCR and then sequenced. Phylogenetic analysis based on thenucleotide sequence of E2gene showed that15out of16isolates belong tosubgenotype2.1, and one isolate belongs to subgenotype1.1. The results are the sameof that based on the region encoding the major neutralizing epitopes of E2gene.These2.1isolates can be further divided into3sub-subgenotype2.1b,2.1c and2.1daccording to the nucleotide sequence similarity, the last two sub-subgenotypes werefirst reported in this province. and there is still no evidence for2.1a in Guangdong.Multiple sequence alignment revealed that all sub-subgenotype2.1b,2.1c and2.1dcontain group specific amino acid substitutions, which further confirm the genotypingof subgenotype2.1.Overall, we carry out the molecular epidemiological investigation of CSFV ofGuangdong province, by analysising415suspected tissue samples from differentareas cover most of Guangdong province. This study provides an evidence for thepresence of different subgenotypes of CSFV field isolates in Guangdong province,especially the epidemics of2.1c and2.1d isolates, this result may be helpful for thedevelopment of prevention and control strategy of CSF in Guangdong province.