2006 Vol.21(4)
|
The significance and preM sequence analysis of two different mutant strains of Japanese encephalitis virus during persistent infection was studied. Two wild strains of Japanese encephalitis viruses (JaGAr-01 and Nakayama) were used to infect a human hepatoma cell line. Persistent infection was established after the cells were subcultured several times. Mutant viruses in persistently infected cells were collected by freezing and thawing of the cells. Viral titers were examined by plaque assys in BHK cells. The preM coding region of four Japanese encephalitis strains, two wild-type and two mutant viruses from infected cells were amplified by RT-PCR. The PCR products were sequenced and the preM sequences of the four strains were compared. The results showed that there was a single aminot acid mutation (E9 Leu→Arg) in the JaGAr-01 persistently infecting mutant in comparison to wild-type JaGAr-01. Two amino acid replacements (E9 Leu→Arg and E13 Val→Ile) were noted in the persistently infecting Nakayama and its wild-type strains. The amino acid sequences of the mutant strains JaGAr-01 and Nakayama were completely the same. We inferred here that genotypic variation existed in preM region of all mutant viruses and that genotypic variation of protein encoded by preM region may play a role in persistent infections and may contribute to the maintenance of the characters of Japanese encephalitis virus and replication.
The epitopes of SARS-CoV were screened from a 12-mer phage display random peptide library using anti- SARS-CoV horse polyclonal antibodies of as a target. The phage clones enriched in biopannings were sequenced. Two consensus sequences were obtained, DXXDP and TXTLL, which had close identity to the 341-345 aa and 392-396 aa of SARA-CoV N protein sequences,respectively. The phage clones with consensus sequences and the N protein were both recognized and bound by the antibodies in a competitive-inhibition ELISA test. The consensus sequence peptides were cloned and displayed on the bacterial flagellin. Balb/c mice were vaccinated using the reconstruted bacteria and the collected serum was shown to speccifically combined with SARS-CoV N protein. This confirmed that DXXDP and TXTLL are two continuous epitopes of SARS-CoV N protein.
The structural protein of SARS-associated coronavirus (SARS-CoV) responsible for the activation of hfgl2 gene was investigated. Gene fragments encoding the nucleocapsid, the membrane, and spike proteins were amplified by RT-PCR, and they were cloned into the eukaryotic expression vector pcDNA3.1（+）. The plasmids were verified by restriction endonuclease analysis and sequencing. Chinese hamster ovary (CHO) cells were co-transfected with a plasmid carrying the hfgl2 promoter/luciferase and with one of the recombinant plasmids containing the genes enconding the N, M and S structural proteins. Luciferase activity was assayed as a measure of promoter function. Immunohistochemistry showed that genes encoding the structural protein of SARS-CoV were successfully expressed only in those cells transfected with the recombinated plasmids. Co-transfection of N gene expression construct with the reporter construct containing hfgl2 promoter in CHO cells showed a remarkable increase in luciferase activity compared with nontransfected cells. However, there was no significant difference in luciferase activity in cells cotransfected with pcDNA3.1-M and with pcDNA3.1-S2 along with hfgl2 promoter/ luciferase reporter gene. We conclude that the nucleocapsid protein of SARS-CoV upregulates the transcription of hfgl2 prothrombinase/fibroleukin gene.
The biological Characteristics and partial S4 gene sequence of a new Reovirus isolate was determined. Cell sensitivity testing on four cell lines was conducted by using the new isolate and the data showed that L929 cells and LLC-MK2 cells were highly permissive. The reovirus was resistant to ether, non-resistant to acid and heat in the physicochemical characteristics. It also had hemagglutination activity to human O-erythrocyte, and the titer reached up 1:32. Electron microscopic (EM) examination of LLC-MK2 cells infected with the reovirus revealed characteristic reovirus particles of about 70nm~80nm in diameter. Aggregates of reovirus particles showed in the cytoplasm of L929 cells. The putative S4 gene was amplified by PCR and the product had sequence homology to 3 prototypic strains T1L, T2J and T3D was 84%, 78%, 85%, respectively. All the results suggested that the new reovirus isolate is closely related to the known clusters of Reoviridae.
An eukaryotic expression vector phIE composed of pcDNA3 skeleton and the herpes simplex virus immediate early gene promoter was constructed to provide a new choice for ectopic expression. The plasmid phIE-Us1 was constructed by inserting the us1 cDNA downstream of the promoter to analyze the function of ICP22 protein very early in the infection process. Furthermore, the gene encoding the chloramphenicol acetyl-transferase was inserted into the multiple cloning site of the expression vector to yield the phIE-CAT. In functional analysis of viral protein ICP22 and Vp16, phIE-CAT was confirmed to be capable of evaluating the mechanism of the genome of herpes simplex virus 1 transcriptional regulation.
The aim of this study was to investigate the variation of the second envelope glycoprotein E2 of Hepatitis C Virus strains from the south of Jiangsu province and to learn about the relationship between HCV quasispecies and alanine aminotransferase (ALT). Identification genotypes of sera samples obtained from 166 donors who were HCV positive was carried our by the PCR with type specific primers based on the sequence of 5′non-coding region (5′NCR). Forty-three chronic 1b type hepatitis C patients who had not received any antiviral therapy were categorized into two groups according to the ALT level. HCV quasispecies diversity were determined by RT-PCR single-strand conformational polymorphism analysis of E2/NS1. Ten strains of HCV E2 were sequenced. A phylogenetic tree of HCV E2 sequences was constructed by using CLUSTAL W and PHYLIP. Out of total 166 patients, 102 were HCV RNA positive in which 86 patients had genotype 1b(86/102; 84.3%), 6 patients had genotype 2 (6/102; 5.9%), 5 patients was1b/2 mixed types (5/102; 4.9%) and a genotype could not be determined in 5 patients. Phylogeny confirmed that variation in the HCV E2 region was not random but with several conservative gene sequences and amino acid in fixed sites. The quasispecies bands numbers for 43 strains were significantly different with 2.54±1.05 and 4.48±2.14, respectively. Logistic regression analysis also found the quasispecies diversity was an influencing factor in ALT level. Type 1b was the predominant HCV genotype in this area. Phylogenetic trees of HCV E indicated that HCV strains in south of JIANGSU province was close to the ones from SHANGHAI and JAPAN. Some conservative sites were fixed in HCV E region. Quasispecies diversity was correlated with ALT level.
To examine whether the recombinant protein Hsp70-NP could induce the N-specific CTLs, we immunized C57/BL6 mice with purified protein Hsp70-NP, NP or Hsp70. The lymphoproliferative responses and CTL activities of spleen cells from vaccinated mice were determined. Accordingly, in order to get the target cells for cytotoxic assay, we tranfected pcDNA3.1/S into mice melanoma cells B16 with Lipofectamine2000, and selected stable transfectants with G418. RT-PCR, Western blots and immunofluorescence were used to identify the B16-N. The lymphoproliferative responses of spleen cells of mice vaccinated with Hsp70-NP, NP clearly showed proliferation in the presence of the N protein. But the stimulation indexes of splenocytes to NP of Hsp70-NP group were significantly higher than that of NP group. The CTL results showed that the cytotoxic effect of lymphocyte of the Hsp70-NP group was higher than that of the NP gorup. The results showed that Hsp70 could enhance the potency of NP to elicit HTNV NP specific cellular immune responses in vivo.
The aim of this study was to characterize functions of the unknown open-reading frames, Orf7, Orf8 and Orf9 within the Severe acute respiratory syndrome coronavirus (SARS-CoV) genome. We cloned the cDNAs of Orf7, Orf8, Orf9 from the SARS-CoV BJ01 strain were cloned by RT-PCR and constructed into five expression vectors designated as pEGFP-Orf7, pEGFP-Orf8, pEGFP-Orf79, pcDNA3-Orf7 and pcDNA3-Orf8. Hela cells were transfected with the constructs and analyzed for transient expressions by fluorescent analysis. We observed that the expression of pEGFP was enhanced when pcDNA3-Orf7 and pEGFP were co-transfected into Hela cells. Also the expression of pDsRed was increased with pEGFP-Orf7 and pDsRed. More cells which expressed green and red fluorescence at the same time were detected. Based on these results, we demonstrated for the first time that the 63 amino acid protein encoded by Orf7 served as a transcriptional transactivator in SARS-CoV.
In this study, psiHBs was constructed through the backbone of psiRNA-vector to transcribe a small hairpin RNA(shRNA) corresponding to the messenger RNA of the HBV small surface antigen. After transfection of HepG2 cells or Balb/c mice with psiHBs and pHBV1.3, a plasmid containing 1.3-genomic-length replicative-competent HBV DNA, the viral antigens were evaluated by ELISA, Western blotting, or immunohistochemistry and the viral replicative intermediates were detected by Southern hybridization. The data indicated that after transfection of HepG2 cells with pHBV1.3 and psiHBs, the HBsAg and HBeAg levels were reduced by over 90%, and the replicative intermediates in cells were decreased to 10% relative to control. In hydrodynamic injection mouse model, Balb/c mice were co-injected with pHBV1.3 and psiHBs. The HBsAg levels in mice sera were reduced by 80% relative to the control group, whereas the number of HBcAg positive hepatocytes in the mouse liver sections was decreased substantially by 75.1%. Our study showed that shRNA inhibits the HBV replication and gene expression substantially and effectively. The RNAi-based approach might become a pentential and novel tool for medical therapeutics in the future.
The rationale of this study was to elucidate the etiological agent characteristics and pathogenic mechanisms of the Avian H5N1 influenza virus in mammals, and to offer a platform for evaluating vaccines and drugs against AIV. Male BALB/c mice (18~20g) were anesthetized by inhalation of ether and inoculated intranasally with 0.05mL of infectious virus (A/Tiger/Harbin/01/2002) isolating from a tiger. The dose lethal to 50% of mice (MLD50) was determined by infecting groups of ten with serial 10-fold viral dilutions. Mice were observed daily for 14 days for clinical signs of infection and day of survival. The mice showed the typical clinical symptoms, pathological changes and death rates. The MLD50 was about 10-7.1/0.05mL. The data demonstrate that infecting BALB/c mice with the avian H5N1 could serve as a successful model to assess viral infectivity.
To study the specific subcellular localization of different regions of Pseudorabies virus (PrV) VP22, a series of mutants with the C-terminal truncations were amplified from a plasmid containing the full-length VP22 gene of the Ea strain. The resulting truncation constructs were fused to ORF encoding the green fluorescent protein (GFP) to generate eukaryotic expression plasmids expressing VP22 mutants and GFP fusion proteins. HeLa cells were transiently transfected with these fusion expression plasmids. By detecting the GFP fluorescence localization, several potential regions of PrV VP22 for specific subcellular localization were determined as follows: 60-90 amino acids (aa) of VP22 are required for nuclear targeting; 111-159 aa may be involved with the formation of particles in the nucleus; 187-241 aa are needed for binding to microtubules. These results lay foundation for further study on the structure and function of PrV VP22.
The GP5 gene of Porcine reproductive and respiratory syndrome virus (PRRSV) S1 strain was amplified by RT-PCR, which was digested with KpnI and XhoI and cloned into pShuttle-CMV plasmid. Sequence analysis showed that GP5 gene was cloned in correctly. Then the recombinant plasmid pShuttle-CMV-GP5 was linearized , and co-transformed into E.coli BJ5183 strain together with adenovirus backbone vector pAdEasy-1. Recombinant adenovirus plasmid pAd-GP5 was linearized with PacI, and transferred into HEK293-A cells to obtain recombinant adenovirus. The obtained recombinant adenovirus rAd-GP5 was identified with RT-PCR and IFA. The result showed that the recombinant adenovirus expressed GP5 protein of PRRSV successfully. Virus neutralization assay was carried out after callection of serum, the result demonstrated that the recombinant adenovirus can elicit effective neutralization antibody against of GP5 PRRSV, therefore, the recombinant adenovirus may be a candidate for gene engineering vaccines.
Large felids, such as tigers, lions and leopards are often infected with Canine Distemper (CD). Blood samples from pre-immune Amur tiger, African lion and lynx were collected and the antibody against CDV was measured by neutralization tests. The results showed that their CDV antibody level were all below 1:5. After inoculated with attenuated CD vaccine, the young animals produced antibody in 14 days but the antibody levels of the adults increased rapidly within two months and maintained at titer of 1: 64, then gradually dropped after 9 to 11 months. After booster immunizing, antibody level went up rapidly to≥1:112. The vaccination was safe to the animals. Achroia Indian tiger, however, could not produce CDV neutralizing antibody after vaccination.
One set of primers and a TaqMan probe were designed based on the nucleotide sequence within ORF2 of Porcine circovirus type 2 (PCV2). A quantitative TaqMan real-time PCR was developed for detecting PCV2 with highly specificity, reproducibility and more sensitivity than conventional PCR. Standard dilutions allowed absolute quantification of the amount of viral DNA to 1copy/μL, which is 106 times more sensitive than conventional PCR. The result of detecting PCV2 from tissues and sera of pigs with PMWS also confirms the high sensitivity and specificity of this method. This method is probably a powerful enough tool for early diagnosis of PMWS.
he RdRp gene of Foot-and-mouth disease virus was amplified by reverse transcription polymerase chain reaction (RT-PCR) using pfuultraTM High-Fidelity DNA polymerase, and was then cloned into the prokaryotic expression vector pET-28a (+). E.coli BL-21 cells were transformed with the recombinant plasmid and the target protein was expressed in soluble form. The purified recombinant protein was confirmed to be expressed correctly by Western-blotting. The RNA in vitro replication assay was used to determine the high activity of purified RdRp. The product of replication system was quantified by strand-specific real-time RT-PCR. These results suggested that the purified RdRp could initiate the de-novo RNA synthesis but was mainly in a primer-dependent manner, which shows high activity of RNA-dependent RNA polymerase. The Vpg protein primer can dramatically improve the capacity of RNA synthesis.
The nucleocapsid protein gene (N) located 108 to 1679 situation of Canine distemper virus (CDV) genome and was immunogenicity protein with strong conservatism, so N gene was choosed as aim gene. A shuttle plasmid pVAXΔE3LPN containing the gene encoding the nucleocapsid protein of CDV was constructed by restriction endonuclease digestion and ligation. A recombinant plasmid pCAV-2-CDVLPN was constructed with pPoly2-CAV-2 vector containing CAV-2 SY genome. The plasmid pCAV-2-CDVLPN was transfected into MDCK cells by Lipofectamine and after three transfections, a typical CAV-2 cytopathic effect (CPE) was observed. We demonstrated that a recombinant canine adenovirus type-2 expressing nucleocapsid protein of canine distemper virus was successfully constructed and observed by electron microscopy, enzyme digestion, PCR and sequencing. The molecular mass of nucleocapsid protein was 58 kDa.
The VP1 gene of Swine vesicular disease virus (SVDV) was amplified by reverse transcription-polymerase chain reaction (RT-PCR) yielding a product of 849bp cDNA fragment. Using T-A cloning technique, the PCR product was cloned into pMD18-T vector. The purified VP1 gene was subcloned into the pBAD/Thio TOPO vector and the plasmid was identified by PCR. It was sequenced to confirm the authenticity of the sequence and orientations. SDS-PAGE and Western blotting revealed that the VP1 protein was expressed in Escherichia coli LGM194 at a high level and the recombinant fusion protein contained a N-terminal HP-Thioredoxin and a C-terminal polyhistidine tag. The optimal amount of the expressed fusion protein was 16% of total bacterial protein after being induced with L-arabinose at 0.002%concentration for 5 hours. It had a molecular mass of approximately 47.13 kDa and was immunologically reactive. The recombinant protein will be characterized and tested in an enzyme-linked immunosorbent assay (ELISA) format for potential application in the serodiagnosis of swine vesicular disease.
Pocine interferon α mature protein genes were amplified by reverse transcription polymerase chain reaction (RT-PCR), and the recombinant replication-defective human adenovius serotype 5 plasmid pAd-poIFN-α was constructed. When the recombinant plasmid pAd-poIFN-α was linearized with PacI, and then transferred into HEK-293A cells, the virus plaque was isolated and purified in HEK-293A cells by three of plaque purification passages. This recombinant adenovirus rAd-poIFN-α could be stably passaged in HEK-293A cells and generated titres in the order of 107 TCID50/mL. The mRNA of the transgene in this recombinant adenovirus was detected by RT-PCR. As a result, the adenovirus exhibited high anti-swine FMDV activity in PK-15 cells and could be further studied in the control strategy of swine FMDV.
This study was aimed to analyze human rabies cases that occurred from 2004 to 2005 in Anlong County in Guizhou province, and to explore the possible factors causing the epidemics. Each case of rabies was investigated. Canine brains were collected, and an immunofluorescence assay was used to detect rabies virus antigen. The G gene was amplified by RT-PCR, and then sequenced. Phyologenetic trees were constructed to analyze the genetic relatedness of rabies viruses. From 1994 to 2003, no human rabies case occurred in Anlong. But 44 and 27 cases of human rabies were reported in 2004 and 2005, respectively. In 71 cases, 68 were attributed to dog bites, and 3 patients were reported to have been bitten by cats. The median incubation period was 47.5 days (10～282) for the 69 cases with clear history records. However, it was noticed that 13 (18.84%) and 32 (46.38%) of 69 cases developed the disease within 15 days and 30 days of exposure, respectively, and particularly, 13 of 44 cases developed the disease within 20 days in 2004. In the spring of 2005, a total of 85 dog brains were examined for rabies viral antigen by DFA and confirmed by RT-PCR, 5 (5.88%) were positive. Analysis of the sequences of G gene of the two isolates showed the identity of the nucleotides sequences was 86.5% between the two isolates. Furthermore, A13 strain has high identity with the known isolates from Guangxi, while A48 strain shares high identity with the strain SN01-23 from Indonesia. Both strains are diverse from vaccine strains, aG, PV and ERA. The phylogenetic tree constructed with the entire G gene sequence showed the two isolates were rabies virus. Comparison of the deduced amino acid sequences of the two isolates, 19 and 28 of amino acid residues including the residues in the antigenic site Ⅰ, Ⅱ and Ⅲ were substituted for A13 and A48, respectively. The results suggested that the virus caused the outbreak in Anlong is not an emerging rabies virus, and the increasing number of dogs and the high rate of virus carriers in dogs might be responsible for the outbreak.
The nucleotide sequence of segment 9 of Dendrolimus punctatus cypovirus (DpCPV) strain Hunan, which encodes the viral non-structural protein (NS5), was tagged with his-tag and expressed by pET32a (+). The recombinant protein was subsequently purified to homogeneity. By using the EMSA, which allows the detection of protein-RNA interactions in the test tube, the possible interactions were tested by the recombinant protein and the double-strand RNA (the genome of DpCPV).
By Using the modified method to extract Rice dwarf virus virions from infected rice leaves, a concentration 3.854mg/mL of purified virus virions reached, and the Rabbits was immunized with purified virions, yielding high titer antiserum; 1:20480 by the indirect enzyme-linked immunosorbent assay (I-ELISA) test. The antiserum successfully detected three rice samples from Kunming, Yuxi and Luliang by I-ELISA.Immunocapture polymerase chain reaction (IC-PCR), respectively was the utilized to show that serologic and molecular methods were successfully used for detection of Rice dwarf virus.
Three pairs of primers P1/P2, P3/P4 and P5/P6 of porcine circovirus 2 were designed and synthesized to compare the sensitivity and specialization to amplify products of 404bp, 647bp and 1773 bp. Optimum dilution of PK15 cell genome infected with PCV 2 was achieved with 10-6ng, 10-5ng to yield from the three pairs of primers. It was also confirmed that P1/P2 were 68.4% successful in detecting PCV2 in 57 swine samples from the province of Fujian, The other primers, P3/P4, P5/P6, were only 57.9% and 8.7% successful, respectively. We conclude that p1/p2 is the optimum primer pair for surveying incidents of PCV2 in pigs.
