Kaposi’s sarcoma-associated herpes-virus (KSHV), also known as Human herpesvirus 8 (HHV-8) is etiologically linked to the development of Kaposi’s sarcoma(KS), the most commonly found cancers in patients with AIDS. Extensive epidemiology studies have shown that the epidemiology of KSHV mimics that of KS displaying distinct geographic distribution patterns with low prevalence in North America and Europe, medium prevalence in Eastern European and Mediterranean regions, and high prevalence in some regions in Africa. However, the prevalence of KSHV infection in Asia, especially in China remains undefined. To investigate the prevalence of KSHV infection from the general population in the Han nationality from Hubei Province, we examined 560 sera specimens using an ELISA-based assay that measured antibodies to KSHV lytic antigen small capsid protein ORF65. The overall KSHV seroprevalence in this population was 5.2%. There was no difference between male and female (5.7% vs 4.5%, P = 0.542). On the other hand, the age distribution pattern was peculiar. KSHV-seropositive rate was significantly higher in subjects younger than 10 years old (P = 0.006, OR = 6.692, 95% CI: 1.710-26.198). Excluding this group, KSHV-seropositive rate showed a trend of increase with age but it was not statistically significant. Our results indicate that the prevalence of KSHV infection is similar to those reported in Western countries in the adult population. However, the relative high prevalence of KSHV infection among the young children is reminiscent of some African regions, and might implicate specific mode of transmission in this population.
To develop national reference of HIV-1 P24 antigen and establish the corresponding standard, specimens collected from normal population, HIV cultivated cell lysates and patients infected with HIV or suspected infection, were detected for HIV antibody, HIV P24 antigen, HCV antibody and HBsAg. 21 specimens positive for HIV P24 antigen, from patients infected with HIV or suspected infection, were detected for HIV RNA and, at the same time, HIV genotypes of these samples positive for HIV RNA were analyzed. The national references of HIV P24 consisted of 20 negative samples, 10 positive samples, 10 sensitivity samples and 2 precision samples, were developed. After analysed the p24 references with the P24-detecting reagents from different manufactures, the corresponding standards were established. It also showed that 3 times freeze-thawing did not affect the stability of the references. The national references of HIV-1 P24 antigen would provide important guides on quality control of HIV P24-detecting reagents.
The adenovirus subgenus C serotypes 5 (Ad5) vectors which have been used to transduce epithelial cells have very limited ability to infect hematopoietic cells. For feasible generation of fiber-retargeted adenoviral vectors, we have modified the versatile AdEasy system with a chimeric fiber gene encoding the Ad5 fiber tail domain and Ad11p fiber shaft and knob domains. An Ad5-based vector encoding the green fluorescent protein (GFP) gene under the control of the CMV promoter with Ad11p fiber receptor specificity was generated (Ad5F11p-GFP). The Ad5F11p-GFP vector-mediated gene transfer efficiency for some committed hematopoietic cell lines such as myeloblasts (K562) and monocytes (U937) was evaluated using flow cytometry and compared to that of Ad5-GFP, which also encodes the GFP gene under the control of the CMV promoter. The results showed that these cell lines were superiorly transduced by the Ad5F11p-GFP vector at the same MOI (multiplicity of infection, MOI) compared with the Ad5-GFP vector，more than 90% tested cells were infected by Ad5F11p-GFP adenovirus while less than 30% cells were infected by Ad5-GFP at 10 MOI.
Respiratory syncytial virus(RSV) was isolated from nasopharyngeal aspirates(NPA) collected of children suffering acute lower respiratory tract infection in the Children’s hospital of Zhejiang province in the winter of 2004. The isolated strains were identified by RT-PCR. The whole region of the G gene of the isolates was sequenced and phylogenetic trees were constructed by comparison with RSV standard strains and reference strains. Results showed that four RSV strains were isolated. The identities of the G gene and the G protein among the four isolates were 98.05% and 97.06%, respectively. The identity of the G gene of the four isolated strains with the standard A type of RSV strain was 95.69% and the identity was 78.7% with the standard B type of RSV strain; the sequence of the 3’hypervariable region of the G gene was identified with the GA2 subtype of RSV. It was suggested that the GA2 subtype of RSV was predominant in Zhejiang province during the winter of 2004.
A simple rapid detection of HIV-1 and HIV-2 antibodies was developed by using dot immunogold filtration assay. Five recombinant proteins derived from HIV env and gag regions of HIV-1 and HIV-2 (P24, GP41, GP120C, GP120V3 and GP36) were expressed in E. coli. The proteins were purified, immobilized onto the nitrocellulose membrane and used for detecting anti-HIV IgG antibodies Human IgG was used as internal control. Anti-HIV antibodies detected in sera by these antigens were observed with the naked eye with the aid of colloidal gold labeled SPA. A total of 51 sera were tested by ELISA and by our rapid HIV assay. A total of 21 HIV positive samples (containing one HIV-1 positive control serum and one HIV-2 positive control serum) confirmed by Western blots exhibited a positive reaction when tested by our assay. HIV negative sear (30 samples) were also negative by our test. These results suggested that our method can be used for detecting anti-HIV antibodies and has the advanteage of quickness, simplicity and cost effectiveness. In addition, simultaneous detection and discrimination among four HIV markers (anti-P24, GP41, GP120 and GP36 antibodies) by the rapid HIV test would be useful for increasing the sensitivity and accuracy of this rapid test and for evaluating and monitoring infected individuals and identifying the risk of imminent progression to AIDS.
Norovirus is currently the predominant human calicivirus that causes diarrhea in China. Among the documented noroviruses, types 4, 1 and 3 are the leading genetics groups. In order to improve the expression of the norovirus capsid, we re-designed and artificially synthesized the full-length norovirus capsid genes by adopting the codons preferentially used in insect cells without any changing of the amino acid sequences. The codon optimized capsid genes of Norovirus GGII1, GGII3, GGII4 and GGII7 were respectively expressed in insect cell Sf9 using baculovirus vector. The results showed that, compared to the wild type, the expression levels of the optimized genes were remarkably increased. The assembly of norovirus-like particles was observed in the Sf9 cells infected with the recombinant baculovirus expressing the capsid protein. The recombinant capsid expression peaked at 72h post infection of the recombinant baculoviruses. The results obtained in the present work will lay a foundation for the development of the immunological diagnostic reagents as well as vaccines for human caliciviruses.
HIV-protease coding sequence was amplified from HIV-1IIIB RNA by one- step RT-PCR. The PCR product was inserted into the pet28a expression vector with an additional ATG start codon before and TAA stop codon after the coding sequence. Positive clone was selected and transformed into E. coli BL21 DE3. Cells were cultured in LB medium and induced by IPTG. The recombinant protease was expressed in the form of inclusion body and was up to 40% of total bacterial protein. The inclusion body was washed by Triton X-100 before dissolved in 8M urea and then applied on sephacyl s-200 H.R column. The purity of the protease reached 90% after purification. Protease fraction was collected and diluted in refolding buffer and left at 4℃ for 24 h. The diluent was concentrated by ultrafiltration. The activity of recombinant protease and the inhibiting effect of indinavir were analyzed by FRET with synthesized fluorescence-labeled peptide. The results above indicates that the method can be used to screen inhibitor of HIV-1 protease.
The glycoprotein gene（G） of 12 Rabies virus isolates from different areas in Guangxi were analyzed and sequenced. The results indicated that all rabies virus isolates from Guangxi belong to genotype I and could be divided into 3 groups. GX01, GX08, GX09, GX014, GX091, GX195, GX260 and GXLA strains belong to group Ⅰ. GX219, GX074 and GXBM strains belong to group Ⅱ. GXN119 strain belongs to group Ⅲ. The nucleotide homologies of rabies virus isolates were 97.6% to 99.9% in group Ⅰ and were 98.2% to 99.0% in groupⅡ. The homologies of amino acid sequence were 97.7% to 100% in that the mutations of nucleotide mostly were synonymous mutations, either the main antigenic regions of GI and GⅡ or the main antigenic sites of 36, 263 and 367 amino acid were not any mutative, only the 332 site of valine mutated to isoleucine. The mutations of the deduced amino acid sequence focus on -16, -15, -14, -13, -5, -2, 90, 96, 132, 140, 156, 168, 170, 204, 241, 249, 253, 264, 289, 332, 382, 427, 436, 445, 463 and 474 amino acid on the glycoprotein, all mutations of amino acid has group specificity.
In order to understand the genetic diversity and distribution of Hantavirus (HV) in different tissues of wild brown rats, we amplified the partial M segment of HV from various organs of 20 hantavirus positive Rattus norvegicus individuals which were collected from Beijing. Then the amplicons were sequenced directly and the genetic variation analysis was made with DNASTAR software. The “A→G” transitional mutations were detected only in the infected lung tissue. We also evaluated the HV quantities in these organs with real time PCR. Significant difference of HV prevalence rates was found among different organs of R. norvegicus （χ2=16.10，P=0.003）. The virus-loads in lung tissues of natural infected rats were significantly higher than those in other studied tissues. These results suggested that lung tissue of natural infected rat serves as the main target site for HV maintenance and replication; and HV mutation was more prone to occur in lungs. The findings are useful for further research in HV epidemiology and reservoir–HV relationship.
In order to find the cause of different transactivate abilities among JDV, BIV and HIV-1 Tat, four chimeric Tat cDNA expression constructs, JH, HJ, JB and BJ, were generated with the crossover points at the boundary of activation and binding domains based on functional domain division of HIV-1 Tat and amino acid sequence comparision among HIV-1, JDV and BIV Tat. These chimeric Tat proteins were co-transfected with JDV, BIV and HIV-1 LTR report plasmids in Hela. Transient assay showed that different transactivate abilities among JDV, BIV and HIV-1 Tat were mainly caused by different binding abilities of their binding domains. We further excluded some possibilities that may cause the poor transactivate ability of JH on HIV-1 LTR, such as incomplete functional domains and the lack of related cytokines.
In this study, the open reading frame of PPV vp2 was amplified by PCR, sequenced, cloned and expressed both in prokaryotic and insect cells. In E. coli, by using pET-32(a) expression vector, VP2 was expressed as a Trx-His-S Tag fusion protein with the size of 84 kDa and the protein was used to immunize rabbits for generating specific antibody. Moreover, vp2 gene was cloned into pFastBacDUAL and expressed in baculovirus/insect cell expression system: AcMNPV Bac-to-Bac system. SDS-PAGE and Western-blot analysis showed a single band with the size of 64 kDa. In Sf-21 cells, virus like particles could be observed under electron microscope.
To investigate the characteritics and the mechanism of bluetongue virus strain HbC3 (BTV-HbC3) infecting MA782 malignancy in vitro，BTV-HbC3 was used to infect MA782 cell, and the cytopathic effects（CPE）was observed.Transmission electron microscope（TEM）was introduced to study changes of ultrastructure. Immunohistochemical staining was used to observe the expressed viral protein in tumors and organs. TUNEL staining was taken to detect the apoptosis of MA782 cells induced by BTV-HbC3. The results indicated that MA782 cell was sensitive; Lots of virtu particles were found in cytoplasm by TEM ;Viral protein expressed in cell membrane and cytoplasm; and early apoptopsis cells were found by TUNEL staining. All these resucss showed that BTV-HbC3 could infect and replicate in MA782 cells efficiently in vitro.
To understand the interaction between Marek’s disease viruses (MDV) and Reticuloendothe -liosis viruses (REV) in co-infected chickens, their mutual influences on vriremia levels and specific antibody titiers were studied in REV maternal antibody positive (REV-ab+) or negative (REV-ab-) commercial broilers vaccinated with or without MDV vaccine CVI988. The results indicated that REV viremia did not influence the MDV viremia level and antibody titers after inoculation with virulent MDV in unvaccinated chickens. In chickens vaccinated with CVI988, however, REV viremia could significantly inhibit antibody titers (p0.01) and resistance to MDV, and compromised protective effect of CVI988 vaccine from MDV viremia in vaccinated birds. In another hand, MDV infection could decrease the resistance to REV infection in REV-ab+ chickens. REV inoculation caused no REV viremia in REV-ab+ chickens without MDV viremia but caused high titers of REV viremia in REV-ab+ chickens with MDV viremia. MDV viremia also decreased antibody responses to REV inoculation in REV-ab+ chickens. Analysis of the data suggested that co-infection of MDV and REV could influence each other’s pathogenicity mainly through their inbibitory effects on chicken’s immune functions.
Seventy serum samples were collected from adult brown ducks in Hubei were collected in order to clarify the natural Duck hepatitis B virus (DHBV) infection in and to characterize the genome structure of DHBV. The complete genome of the DHBV strain was amplified by polymerase chain reaction (PCR) and cloned into T vector and sequenced. The results showed that the natural infection rate of DHBV in Hubei was 10%. The DHBV genome isolated by PCR (GenBank accession number DQ276978 ) had 3 024 nucleotides with three overlapping reading frames encoding the surface, core and the polymerase proteins, respectively. Comparison of genome sequences of this strain with those of 17 DHBV strains from the GenBank revealed an identity from 89.3% to 93.5% at the nucleotide level. The amino acid sequences of the S protein，core protein，and functional domain of the Pol protein were highly reserved among all of these DHBV strains. This Hubei strain was found to share more signature amino acids in the polymerase genes with the “Chinese” DHBV strains than those of the “Western” country strains. This finding was also corroborated by a phylogenetic tree analysis. Therefore, this Hubei DHBV strain should be classified to a subtype of the Chinese strains.
Viral protein VP22 is indispensable for the growth and replication of Marek’s disease virus serotype 1(MDV-1). In this study, vp22 gene was amplified from and avirluent CVI988/Rispens strain. Antibodies specific to the carboxyl terminus of VP22 expressed in E. coli were generated by immunizing Balb/C mice 3 times. The antibodies reacted with the complete VP22 expressed in Chick Embryo Fibroblast (CEF) cells infected with CVI988 virus. It is interesting that the VP22 protein could be found in normal CEF cells around MDV plagues. The results suggested that VP22 may be transported among cwlls in MDV infection, which is vital to the studies on functions of VP22.
A severe contagious disease broke out in several duckeries in Ningbo, Zhejiang province with the symptoms of diarrhea and grasping. The liver tissues from the dead ducks were collected and inoculated into the allantoic cavity of the 11-day-old embryos. Through purification by sucrose density gradient centrifugation, a diameter of 20nm virions with typical duck parvovirus (DPV) characters was observed by electron microscopy. In the test of agar-gel immunodiffusion, the reaction between the stuff from dead embryos and positive serum was obvious. Through SDS-PAGE, three main segments were visible which coincided with DPV. In animal test, similar symptoms were also observed. Using the primers designed according to DPV genome, we acquired an anticipative 600bp DNA fragment, which was cloned into E.coli and sequenced. The sequence shared 98% identity with the representative DPV strain. From all above results, the pathologic agent was diagnosed as Duck Parvovirus. To acquire more information of the rep gene, we sequenced and aligned it with 4 other sequences of DPV or goose parvovirus (GPV) from GenBank. The result indicated the homology of rep gene was about 98% with DPV and 81% with GPV.
A pair of primers was designed according to the sequence of vp28 gene of White spot syndrome virus (WSSV) in the GenBank. The vp28 DNA fragment was amplified by PCR and cloned into E. coli expression vector pET- 22b(+) successfully. Then pET22b-vp28 was transformed into E. coli. After IPTG induction at 37℃, the fusion protein with 32kDa was expressed,which was conformed by Western-blot and SDS-PAGE analysis. The envelope protein VP28 purified by Ni2+-column chromatography was injected into crayfish or used as a supplement to feed crayfish. The result showed that the engineered protein VP28 expressed in E.coli could improve the immunity ability of crayfish to resist WSSV infection.
The phospholipase A2 functional domain of PfDNV capsid gene VP1 was obtained by RT-PCR amplification. The amplified fragment was ligated into a pMD18-T vector and sub-cloned into prokaryotic expression vector pET28a and pET26b. The recombinant plasmid pET28a-PLA and pET26b-PLA were used to transform E. coli BLl21-codonplus（DE3）-RIL competent cells. After induction by IPTG, SDS-PAGE indicated that the highly expressed fusion protein was produced. The fusion protein was purified with Ni-NTA affinity columns and analyzed by Western blot using mouse anti-His monoclonal antibodies. The results demonstrated that the recombinant PLA2 protein of PfDNV capsid gene was successfully expressed, which should facilitate further studies on the biological properties of the enzyme and its function in virus infection.
To understand the dual infection by Hepatitis B virus (HBV) and Hepatitis C virus (HCV), and the viral interference between HBV and HCV infections, biochemical and virological markers of 196 patients were detected. Among 196 patients tested, 76 patients were only infected by HBV, 72 patients were only infected by HCV, and 48 patients were co-infected by HBV and HCV. There were no striking association of ALT in patients with HBV, HCV or co-infection. The positive rate of HBsAg and HBV DNA in patients with co-infection was obviously lower than that of HBV infection only. These results suggested that disease was more severe in patients with co-infection, prevalence of LC and HCC in patients with co-infection was higher than either infection alone. HBV and HCV inhibit the replication with each other, but HCV seems to suppress HBV replication more evidently. Key words：Hepatitis B virus(HBV); Hepatitis C virus(HCV); Co-infection