The Development And Characterization Of Chimeric Arterivirus

The Arteriviridae which was established in 1996 has four members: equine arteritis virus (EAV), porcine reproductive and respiratory syndrome virus (PRRSV), lactate dehydrogenase-elevating virus (LDV), and simian hemorrhagic fever virus (SHFV). Arterivirus can cause either acute or persistent infections in respective host animals. PRRSV infection (so called porcine reproductive and respiratory syndrome, PRRS), in particular, often leads to high-mortality disease outbreaks and mass economic loss to the swine industry worldwide. Until now, the detail molecular mechanism of how the arterivirus replicate in cell, especially the relationship of structure-function of envelope proteins, is poorly understood. In this study, based on infectious full-length cDNA clones of PRRSV and EAV, the envelope protein functions and properties of transcription regulation are dissected. This will lay foundation to better understand of arterivirus and to develop efficient ways to prevent arterivirus infection.1. The development and characterization of intra-genotypic chimeric PRRSVPRRSV is classified into two genotypes, type 1 and type 2, between which share only approximate 60% genetic identity. Bioinformatics analysis showed that the envelope proteins GP2a, E, GP3, GP4, GP5 between vSHE (type 1 strain) and vAPRRS (type 2 strain) share 53?76% identities. Taking the pAPRRS, the full-length cDNA clone of type 2 PRRSV, as backbone, an Asc I restriction enzyme recognition site was introduced immediately upstream of the ORF2 start codon to construct clone pAPRRSasc. Then based on pAPRRSasc, the chimeric full-length clone pAPRRS-SHE23, pAPRRS-SHE234, pAPRRS-SHE2345, pAPRRS-SHE5 were constructed by ORF2?3, ORF2?4, ORF2?5, ORF5 respective substitution with correspond ORFs of vSHE. The viable progeny viruses vAPRRSasc, vAPRRS-SHE234, vAPRRS-SHE2345, vAPRRS-SHE5 were rescued from MARC-145 cells which were transfected with corresponding clones. IFA and complete genome sequencing analysis confirmed the chimeric genomic structures of these rescued viruses. The sequences analysis of passage 8 viruses showed that the chimeric viruses displayed robust genetic stability in vitro. Utilizing antibodies specific for type 1 envelope proteins, further IFA results indicated that these heterologous genes were expressed in type 2 PRRSV backbone. The analysis of virological characteristics showed that all the chimeric viruses displayed similar plaque morphology, except for vAPRRS-SHE5, and similar replication ability in MARC-145 with parental viruses. These results suggested that the envelope proteins of type 1 PRRSV can play full function and work in concert in type 2 virus. Utilizing RT-PCR with specific primers, the subgenomic mRNAs (sg mRNAs) were amplified from intracellular RNA pool and subjected to sequencing. The sequences analysis showed that the TRS-Bs from type 1 can join with TRS-L of type 2 PRRSV to synthesis hybrid sg mRNAs. In addition, some small sg RNAs were generated as the imprecise base paring between the type 2 TRS-L and type 1 TRS-Bs. In this study, the viable intra-genotypic chimeric PRRSVs were fistly successfully rescued in the world, and this study found that the type 1 envelope proteins can play full function in backbone of type 2. This indicated that the recombination between the two genotypic PRRSV is possible, and more importantly, this paved the way to further elucidate the structure-function relationship of PRRSV envelope proteins, and may enable the development of novel gene marker vaccines.2. The development and characterization of intra-species chimeric arterivirusThe identities of arterivirus cellular receptor and viral attachment proteins are in poor understand. The most well accepted viral attachment protein is GP5, while it is being challenged by the minor envelope proteins. However, both of the two hypotheses are only supported by biochemical evidences, lacking of genetic and virological evidences. In vitro, PRRSV can only infect MARC-145 cell line while EAV display a more broaden cell tropism such as MARC-145, BHK-21, Vero and so on. In this study, taking the previously constructed pAPRRSasc as backbone, the chimeric full-length cDNA clones pAPRRS-EAV234 and pAPRRS-EAV5 were constructed through ORF2?4 and ORF5 substitutions respective with counterparties of EAV. The viable progeny virus vAPRRS-EAV234 was rescued from pAPRRS-EAV234 transfected MARC-145 cells. Trough IFA and complete genome sequencing analysis confirmed that the viable progeny virus has a chimeric genomic structure, and the chimeric virus is genetic stable in vitro. The results of IFA using antibody specific to EAV GP2b indicated that the genes from EAV were translated in PRRSV backbone. The more important was that the chimeric virus vAPRRS-EAV234 obtained the cellular tropism of donor parental virus EAV, it can infect MARC-145, BHK-21 and Vero cell lines, while lost the infectivity to the PRRSV target primary porcine alveolar macrophages (PAM). The vAPRRS-EAV234 can replicate well in MARC-145 and BHK-21, and its'peak titer in BHK-21 was 10 times higher than that in MARC-145. The analysis of transcriptional profiles of APRRS-EAV234 showed that the EAV TRS-Bs join with PRRSV TRS-L, although the base pairs did not match well between them and lead to the down-regulation of sg mRNA2?4. In addition, some novel sg RNAs were generated in chimeric virus which may be translated into unknown proteins. This study first rescued viable chimeric virus in which the intact minor envelope proteins ORFs were substituted by counterparties of EAV, and more importantly, this study provided first genetic and virological evidences to prove that the minor envelope proteins play major role in arterivirus entry into cells. This study also developed a new method to produce high titer PRRSV, and it will be of great significance to vaccine productivity.3. The development and characterization of chimeric PRRSV containing ORFs from EAVThe above study has shown that the ORF2?4 of EAV can stably exist in PRRSV genome and play function in virus replication. And it is known that another arterivirus member, SHFV, has two panel of minor envelope protein ORFs in genome. In this study, taking the pAPRRSasc as backbone, the ORF2?4 of EAV were inserted between ORF1b and ORF2a of PRRSV backbone to generate chimeric clone pAPRRS(EAV234). Viable progeny virus vAPRRS(EAV234) was rescued from MARC-145 cells transfected with pAPRRS(EAV234). IFA and viral complete genome sequencing analysis showed that the progeny virus shared a chimeric genomic structure and the inserted EAV ORFs were translated in chimera infected cells. The inserted heterologious ORFs stably exist in chimeic virus vAPRRS(EAV234), however, the partial sequences of ORF2?4 of PRRSV were found to be deleted. The vAPRRS(EAV234) can infect both MARC-145 and BHK-21cells. This study indicated that PRRSV, and even other arterivirus, can contain two panel of minor envelope protein ORFs in genome, although its'genetic stability was frustrated. As one potential vector, PRRSV can deliver heterologious genes which were inserted between ORF1b and ORF2a. This paved the way to further identify the arterivirus attachment proteins, dissect the protein-protein interaction and virion assembly mechanism, and develop PRRSV vector.