Chuyu Zhang

Attenuated lapinized chinese strain of classical swine fever virus: complete nucleotide sequence and character of 3'-noncoding region.

The complete nucleotide sequence including precise 5′- and 3′-terminal non-coding regions (NCRs) of the attenuated lapinized Chinese strain (HCLV) of Classical Swine Fever Virus (CSFV) was determined from overlapping cDNA clones constructed by separated RT-PCR and rapid amplification of cDNA ends (RACE) methods. The genomic RNA of the HCLV strain consists of 12,310 nucleotides (nts) including 374 nts and 242 nts in the 5′- and 3′-NCRs, respectively. It contains one large open reading frame (ORF) encoding a polyprotein of 3,898 amino acids with a calculated molecular weight of 437.6 kDa. There is one notable insertion of 12 continuous nts, CTTTTTTCTTTT in the 3′-NCR of HCLV genomic cDNA when compared with its parental virulent Shimen strain. Sequence alignment of partial 3′-NCR reveals two groups of CSFV vaccine strains carrying similar T-rich insertions at different positions in this region. Computer-predicted secondary structures suggest that T-rich insertion greatly change the structures and thus decrease the promoter functions of 3′-NCRs during the replications of these two groups of CSFV vaccine strains.

De novo RNA synthesis and homology modeling of the classical swine fever virus RNA polymerase

Abstract Classical swine fever virus (CSFV) non-structural protein 5B (NS5B) encodes an RNA-dependent RNA polymerase (RdRp), a key enzyme which initiates RNA replication by a de novo mechanism without a primer and is a potential target for anti-virus therapy. We expressed the NS5B protein in Escherichia coli. The rGTP can stimulate de novo initiation of RNA synthesis and mutation of the GDD motif to Gly–Asp–Asp (GAA) abolishes the RNA synthesis. To better understand the mechanism of viral RNA synthesis in CSFV, a three-dimensional model was built by homology modeling based on the alignment with several virus RdRps. The model contains 605 residues folded in the characteristic fingers, palm and thumb domains. The fingers domain contains an N-terminal region that plays an important role in conformational change. We propose that the experimentally observed promotion of polymerase efficiency by rGTP is probably due to the conformational changes of the polymerase caused by binding the rGTP. Mutation of the GDD to GAA interferes with the interaction between the residues at the polymerase active site and metal ions, and thus renders the polymerase inactive.

Construction of cytopathic PK-15 cell model of classical swine fever virus

No cytopathic effect (CPE) can be observed on classical swine fever virus (CSFV) infected cell culture in vitro. This brings an obstacle to the researches on reciprocity between CSFV and host cells. Based on the construction of full-length genomic infectious cDNA clone of Chinese CSFV standard virulent Shimen strain, partial deletion is introduced into genomic cDNA to obtain a 7.5 kb subgenomic cDNA. A new subgenomic CSFV is derived from transfection with the subgenomic cDNA on PK-15 cells pre-infected by CSFV Shimen virus. Typical CPE induced by this subgenomic virus is observed on PK-15 cells. Coexistence of wildtype and subgenomic virus in cytopathic cell culture is demonstrated by RT-PCR detection in cytopathic cells. For conclusion, the construction of cytopathic cell model exploited a new way for researches on the molecular mechanism of CSFV pathogenesis.

Qualitative, quantitative and structural analysis of non- coding regions of classical swine fever virus genome

Classical swine fever virus (CSFV) is the pathogen of the swine fever. Understanding of the replication and expression of its genome is the basis for research of the pathogenicity for CSFV and development of antiviral drug. The noncoding regions (NCRs) of CSFV are the main regulatory regions for replication and expression. Qualitative, quantitative and structural analysis of 3′ NCRs and 5′ NCRs was done in order to locate the regulatory region in the NCRs and to character the NCRs. The sites, conserved sequences and structural elements related to the initiation of replication and expression were extracted from 17 3′ NCRs and 56 5′ NCRs. Those ciselements may be initial recognition sites for replication, binding sites for transcription factors of host cell and interacting sites for initiation of protein synthesis, based on which a mechanism for the replication and expression of CSFV was brought forth. This research offers the direction for further experiment and lays down a basis for the research on hepatitis C virus (HCV), other pestiviruses and plus-strand RNA viruses.

Thymidine kinase gene mutation leads to reduced virulence of pseudorabies virus

To explore correlation between the tk gene structure of pseudorabies virus (PRV) and its virulence, to study the effect of the gene mutation on PRV biological properties, and to investigate mechinism of reduced virulence, thymidine kinase (TK)-deficient mutant of pseudorabies virus strain Hubei (PRV HB) was isolated by selection for resistance to 5-bromodeoxyuridine. The tk genes of PRV HB and its TK− mutant were cloned and sequenced. 1587 base pairs of the tk gene and flanking regions of wild-type (wt) virus were sequenced, which included an open reading frame (ORF) of 1098 bp encoding a protein of 366 amino acids. The ORF contained two 137-bp repeated sequences, which were connected by an adenosine. 1458 bp of the tk and flanking regions of TK− mutant were sequenced. Analysis of the tk gene sequence of TK− mutant indicated that one of 137 bp repeated sequence and the connecting adenosine in the tk gene of the wt virus was deleted and a repeated sequence of 8 nucleotides (GCGCGCC) was inserted. All other nucleotides of TK− mutant were identical to that of wt virus. Deletion and insertion of the nucleotide sequence resulted in a frameshift and a premature chain termination, and the resultant TK protein was not active. Analysis of the amino acid sequence revealed that TK protein of PRV HB contained the conserved consensus sequence of herpesviral TKs and an additional conserved-DHR-motif. The results of this work also indicated that TK− mutant was genetically stable. Compared to PRV HB, virulence of TK− mutant was greatly decreased. Mice vaccinated with TK− mutant were completely protected against a lethal challenge with virulent PRV (HB).

Construction of cDNA library, nucleotide sequence and analysis of entire genome of classical swine fever virus strain Shimen

According to the previously published CSFV sequences, 18 pairs of primers have been designed and synthesized, which cover the entire genome of CSFV strain Shimen. Each cDNA fragment has been amplified by RT-PCR from the anticoagulant blood of strain Shimen infected pig. The PCR products have been cloned respectively and sequenced. Results show that the cDNA library of strain Shimen and its nucleotide sequence have been obtained. The genomic RNA of strain Shimen is 12 298 nucleotides in length, containing a 5′ and a 3′ noncoding region 373 and 231 nt long respectively. The center of genome is a single large open reading frame of 11 697 nt which encodes a polyprotein of 3 898 amino acids. The entire sequence of strain Shimen has also been compared with that of other CSFV strains.