Dianjun Cao

Molecular biology and replication of hepatitis E virus

Hepatitis E virus (HEV), a single-stranded, positive-sense RNA virus, is responsible for acute hepatitis E epidemics in many developing countries, and the virus is also endemic in some industrialized countries. Hepatitis E is a recognized zoonotic disease, and several animal species, including pigs, are potential reservoirs for HEV. The genome of HEV contains three open reading frames (ORFs). ORF1 encodes the nonstructural proteins, ORF2 encodes the capsid protein, and ORF3 encodes a small multifunctional protein. The ORF2 and ORF3 proteins are translated from a single, bicistronic mRNA. The coding sequences for these two ORFs overlap each other, but neither overlaps with ORF1. Whereas the mechanisms underlying HEV replication are poorly understood, the construction of infectious viral clones, the identification of cell lines that support HEV replication, and the development of small animal models have allowed for more detailed study of the virus. As result of these advances, recently, our understanding of viral entry, genomic replication and viral egress has improved. Furthermore, the determination of the T=1 and T=3 structure of HEV virus-like particles has furthered our understanding of the replication of HEV. This article reviews the latest developments in the molecular biology of HEV with an emphasis on the genomic organization, the expression and function of genes, and the structure and replication of HEV.

Simian Rotaviruses Possess Divergent Gene Constellations That Originated from Interspecies Transmission and Reassortment

ABSTRACT Although few simian rotaviruses (RVs) have been isolated, such strains have been important for basic research and vaccine development. To explore the origins of simian RVs, the complete genome sequences of strains PTRV (G8P[1]), RRV (G3P[3]), and TUCH (G3P[24]) were determined. These data allowed the genotype constellations of each virus to be determined and the phylogenetic relationships of the simian strains with each other and with nonsimian RVs to be elucidated. The results indicate that PTRV was likely transmitted from a bovine or other ruminant into pig-tailed macaques (its host of origin), since its genes have genotypes and encode outer-capsid proteins similar to those of bovine RVs. In contrast, most of the genes of rhesus-macaque strains, RRV and TUCH, have genotypes more typical of canine-feline RVs. However, the sequences of the canine and/or feline (canine/feline)-like genes of RRV and TUCH are only distantly related to those of modern canine/feline RVs, indicating that any potential transmission of a progenitor of these viruses from a canine/feline host to a simian host was not recent. The remaining genes of RRV and TUCH appear to have originated through reassortment with bovine, human, or other RV strains. Finally, comparison of PTRV, RRV, and TUCH genes with those of the vervet-monkey RV SA11-H96 (G3P[2]) indicates that SA11-H96 shares little genetic similarity to other simian strains and likely has evolved independently. Collectively, our data indicate that simian RVs are of diverse ancestry with genome constellations that originated largely by interspecies transmission and reassortment with nonhuman animal RVs.

Establishment of a DNA-launched infectious clone for a highly pneumovirulent strain of type 2 porcine reproductive and respiratory syndrome virus: identification and in vitro and in vivo characterization of a large spontaneous deletion in the nsp2 region.

A highly pneumovirulent strain of porcine reproductive and respiratory syndrome virus (PRRSV), ATCC VR2385, was isolated from a pig exhibiting typical PRRS in the early 90s. While passaging the virus in monkey kidney cells, we identified a large spontaneous deletion of a 435-bp in the nsp2 gene. To assess the biological significance of this spontaneous deletion, we first determined the full-length genomic sequence of this virus and established a DNA-launched infectious clone of the passage 14 virus containing the 435-bp nsp2 deletion (designated as pIR-VR2385-CA). The full-length viral genome engineered with two ribozyme elements at both ends was placed under the control of the eukaryotic CMV promoter. The infectious virus was successfully rescued from pIR-VR2385-CA DNA-transfected BHK-21 cells. To characterize the biological and pathological significance of this large nsp2 deletion, we subsequently constructed another DNA-launched infectious clone, pIR-VR2385-R, in which we restored the deleted 435-bp nsp2 sequence back to the pIR-VR2385-CA backbone. The growth characteristics of the two rescued viruses (VR2385-CA and VR2385-R) were compared, and the results showed that the VR2385-CA virus with the nsp2 deletion replicated more efficiently in vitro (1.0-1.5 log titer higher) than the VR2385-R virus with the restored nsp2 sequence but the VR2385-CA virus exhibited a significantly reduced serum viral RNA load in vivo. A comparative pathogenicity study in pigs (n=10) revealed that the nsp2 deletion had no effect on virus virulence, and the restored nsp2 sequence in the VR2385-R virus remains stable during virus replication in pigs. The results from this study indicates that the spontaneous nsp2 deletion plays a role for enhanced PRRSV replication in vitro but has no effect on the pathogenicity of the virus.

The Nucleotides on the Stem-Loop RNA Structure in the Junction Region of the Hepatitis E Virus Genome Are Critical for Virus Replication

ABSTRACT The roles of conserved nucleotides on the stem-loop (SL) structure in the intergenic region of the hepatitis E virus (HEV) genome in virus replication were determined by using Huh7 cells transfected with HEV SL mutant replicons containing reporter genes. One or two nucleotide mutations of the AGA motif on the loop significantly reduced HEV replication, and three or more nucleotide mutations on the loop abolished HEV replication. Mutations on the stem and of the subgenome start sequence also significantly inhibited HEV replication. The results indicated that both the sequence and the SL structure in the junction region play important roles in HEV replication.

Computer-aided codon-pairs deoptimization of the major envelope GP5 gene attenuates porcine reproductive and respiratory syndrome virus.

Synthetic attenuated virus engineering (SAVE) is an emerging technology that enables rapid attenuation of viruses. In this study, by using SAVE we demonstrated rapid attenuation of an arterivirus, porcine reproductive and respiratory syndrome virus (PRRSV). The major envelope GP5 gene of PRRSV was codon-pair deoptimized aided by a computer algorithm. The codon-pair deoptimized virus, designated as SAVE5 with a deoptimized GP5 gene, was successfully rescued in vitro. The SAVE5 virus replicated at a lower level in vitro with a significant decrease of GP5 protein expression compared to the wild-type PRRSV VR2385 virus. Pigs experimentally infected with the SAVE5 virus had significantly lower viremia level up to 14 days post-infection as well as significantly reduced gross and histological lung lesions when compared to wild-type PRRSV VR2385 virus-infected pigs, indicating the attenuation of the SAVE5 virus. This study proved the feasibility of rapidly attenuating PRRSV by SAVE.

Complete Genome Sequence of Hepatitis E Virus from Rabbits in the United States

ABSTRACT Hepatitis E virus (HEV) is a single-strand positive-sense RNA virus in the family Hepeviridae. The disease caused by HEV, hepatitis E, is an important public health problem in developing countries of Asia and Africa and is also endemic in many industrialized countries, including the United States. HEV has been identified from several other animal species in addition to humans, including the pig, chicken, mongoose, deer, rabbit, ferret, bat, and fish. Here we report the complete genome sequence of the first strain of HEV from rabbits in the United States. Sequence and phylogenetic analyses revealed that the U.S. rabbit HEV is a distant member of the zoonotic genotype 3 HEV, thus raising a concern for potential zoonotic human infection. A unique 90-nucleotide insertion within the X domain of the ORF1 was identified in the rabbit HEV, and this insertion may play a role in the species tropism of HEV.

DNA shuffling of the GP3 genes of porcine reproductive and respiratory syndrome virus (PRRSV) produces a chimeric virus with an improved cross-neutralizing ability against a heterologous PRRSV strain.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important swine pathogen. Here we applied the DNA shuffling approaches to molecularly breed the PRRSV GP3 gene, a neutralizing antibodies inducer, in an attempt to improve its heterologous cross-neutralizing ability. The GP3 genes of six different PRRSV strains were bred by traditional DNA shuffling. Additionally, synthetic DNA shuffling of the GP3 gene was also performed using degenerate oligonucleotides. The shuffled-GP3-libraries were cloned into the backbone of a DNA-launched PRRSV infectious clone pIR-VR2385-CA. Four traditional-shuffled chimeras each representing all 6 parental strains and four other synthetic-shuffled chimeras were successfully rescued. These chimeras displayed similar levels of replication both in vitro and in vivo, compared to the backbone parental virus, indicating that the GP3 shuffling did not impair the replication capability of the chimeras. One chimera GP3TS22 induced significantly higher levels of cross-neutralizing antibodies in pigs against a heterologous PRRSV strain FL-12.

Attenuation of Porcine Reproductive and Respiratory Syndrome Virus by Molecular Breeding of Virus Envelope Genes from Genetically Divergent Strains

ABSTRACT Molecular breeding via DNA shuffling can direct the evolution of viruses with desired traits. By using a positive-strand RNA virus, porcine reproductive and respiratory syndrome virus (PRRSV), as a model, rapid attenuation of the virus was achieved in this study by DNA shuffling of the viral envelope genes from multiple strains. The GP5 envelope genes of 7 genetically divergent PRRSV strains and the GP5-M genes of 6 different PRRSV strains were molecularly bred by DNA shuffling and iteration of the process, and the shuffled genes were cloned into the backbone of a DNA-launched PRRSV infectious clone. Two representative chimeric viruses, DS722 with shuffled GP5 genes and DS5M3 with shuffled GP5-M genes, were rescued and shown to replicate at a lower level and to form smaller plaques in vitro than their parental virus. An in vivo pathogenicity study revealed that pigs infected with the two chimeric viruses had significant reductions in viral-RNA loads in sera and lungs and in gross and microscopic lung lesions, indicating attenuation of the chimeric viruses. Furthermore, pigs vaccinated with the chimeric virus DS722, but not pigs vaccinated with DS5M3, still acquired protection against PRRSV challenge at a level similar to that of the parental virus. Therefore, this study reveals a unique approach through DNA shuffling of viral envelope genes to attenuate a positive-strand RNA virus. The results have important implications for future vaccine development and will generate broad general interest in the scientific community in rapidly attenuating other important human and veterinary viruses.

A dual vaccine candidate against norovirus and hepatitis E virus.

Norovirus (NoV) and hepatitis E virus (HEV) are both enterically-transmitted viruses causing gastroenteritis and hepatitis, respectively, in humans. While a vaccine against HEVs recently became available in China, there is no prophylactic or therapeutic approach against NoVs. Both NoV and HEV have surface protrusions formed by dimers of the protruding (P) domains of the viral capsids, which is responsible for virus-host interactions and eliciting viral neutralizing antibody. We developed in this study a bivalent vaccine against the two viruses through a recently developed polyvalent complex platform. The dimeric P domains of NoV and HEV were fused together, designated as NoV P(-)-HEV P, which was then linked with the dimeric glutathione-S-transferase (GST). After expression and purification in E. coli, the GST-NoV P(-)-HEV P fusion protein assembled into polyvalent complexes with a mean size of 1.8μm, while the NoV P(-)-HEV P formed oligomers ranging from 100 to 420kDa. Mouse immunization study demonstrated that both GST-NoV P(-)-HEV P and NoV P(-)-HEV P complexes induced significantly higher antibody titers to NoV P(-) and HEV P, respectively, than those induced by a mixture of the NoV P(-) and HEV P dimers. Furthermore, the complex-induced antisera exhibited significantly higher neutralizing activity against HEV infection in HepG2/3A cells and higher blocking activity on NoV P particles binding to HBGA receptors than those of the dimer-induced antisera. Thus, GST-NoV P(-)-HEV P and NoV P(-)-HEV P complexes are promising dual vaccine candidates against both NoV and HEV.

Porcine Rotavirus Bearing an Aberrant Gene Stemming from an Intergenic Recombination of the NSP2 and NSP5 Genes Is Defective and Interfering

ABSTRACT Serial undiluted passage of a porcine rotavirus in MA104 cells yielded three distinct virus populations, each of which bore different rearranged genes. Sequencing revealed that each of two populations bore a distinct intragenic recombinant NSP3 gene consisting of a partial duplication in a head-to-tail orientation without altering the NSP3 open reading frame and the third population carried both an intragenic recombinant NSP3 gene and an intergenic recombinant gene (1,647 nucleotides in length) which contained a truncated NSP2 gene inserted into the NSP5 gene at residue 332. The former two populations were viable, whereas the latter population was defective and interfering.