Application of allele-specific RNAi in hepatitis B virus lamivudine resistance.

Understanding the consequences of mutation in the tyrosine-methionine-aspartate-aspartate (YMDD) motif of hepatitis B virus (HBV) genome on replication is critical for treating chronic hepatitis B with lamivudine. Allele-specific gene silencing by RNAi (allele-specific RNAi: ASP-RNAi) is an advanced application of RNAi techniques. Use of this strategy as a means for specifically inhibiting an allele expression of interest suggested that it can specifically suppress the expression of alleles causing disease without inhibiting the expression of corresponding wild-type alleles. However, no studies have used ASP-RNAi to address the issue of HBV lamivudine resistance. In this study, we applied ASP-RNAi into two long-term eukaryotic cell lines of full-length HBV containing either lamivudine-resistant mutants (HBV-YIDD) or wild type (HBV-WT) which we generated in previously. The designed siRNAs were also used in this eukaryotic expression system together with lamivudine. ELISA and real-time PCR were performed to monitor virus-specific protein synthesis and viral DNA replication. The results showed that the base substitutions conferring marked ASP-RNAi appeared to be largely present in positions 1, 3, 6, 11, 12, 15 and 19 of the sense strand of siRNAs which were different from the most sensitive positions of this application in eukaryotes. In addition, siRNA-lamivudine combinations did not possess the prominent anti-HBV activity we expected because of some unknown mechanisms. These findings recapitulated many of the features of ASP-RNAi in hepadnaviruses which provided a new insight into the development of a potent strategy against HBV drug resistance.

Hepatitis B virus subgenotype C2 is the most prevalent subgenotype in northeast China.

The geographical distribution of hepatitis B virus (HBV) subgenotypes and their clinical implications in patients with acute and chronic hepatitis B in the Heilung-kiang province of northeast China were investigated. Nested PCR and multiplex PCR were performed with genotype-specific primers and with subgenotype-specific primers to identify genotypes and subgenotypes from serum samples of 412 HBV infections including 69 with acute self-limited hepatitis (ASH) and 343 with chronic hepatitis (CH). A total of 361 samples were genotyped and 304 were further subgenotyped. The most common HBV genotype was C (93.63%, 338/361), with subgenotype group C2 (83.73%, 283/338) predominating. Genotype B was also found and subgenotype B2 predominated within this genotype. Out of 69 infected patients with ASH, 48 were identified as genotype C and all belonged to subgenotype C2. Of 343 infected patients with CH, 313 were genotyped and 256 were subgenotyped; amongst these, C2 (91.80%, 235/256), B2 (7.42%, 19/256) and mixed subgenotypes B2 and C2 (0.78%, 2/256) were found. In HBV subgenotype C2 infections, ASH had a higher ratio of women than CH patients. These results show that HBV subgenotypes C2 and B2 were found in Heilung-kiang province of northeast China. In ASH and CH groups, the distributions of subgenotypes were coincident with C2, the predominant subgenotype. Analysis of the association between subgenotype and the outcomes of HBV infection was inconclusive in our study.

[Comparative study on antibody levels of vaccination group and control group after 4 years of immunized with type B inactivated vaccine against hemorrhagic fever with renal syndrome].

OBJECTIVE: To compare the antibody level of vaccine group and control group in a randomized, controlled trial of type B inactivated vaccine against hemorrhagic fever with renal syndrome(HFRS) in Jiande County of Zhejiang Province and to find out if there is enhancement of subclinical infection about type B inactivated vaccine against HFRS. METHODS: The number of sera collected (July 1994-July 1998) in vaccine group immunized type B inactivated vaccine against HFRS and control group were 305 and 283 persons in Jiande County respectively, and indirect immunofluorescent antibody method(IFAT) was used for testing the titer of HFRS IgG in sera. The cut-off values were determined on IFA titer of the second sera in control group with positive and negative IFA before vaccine was given respectively, and the subclinical infection of positive and negative IFA of the first sera in vaccine group was evaluated. RESULTS: When taking different cut-off values of subclinical infection, there were no statistical differences in HFRS IFA positive rates between vaccine group and control group of positive IFA in the first sera. In vaccine group and control group of negative IFA before vaccine given, also the IFA positive rates were not statistically different in these groups. There were no statistical differences of IFA GMT and IFA sero-negative rate between vaccine group and control group. CONCLUSIONS: The investigation showed there was no statistical difference in antibody level between vaccine group and control group, so there could not be enhancement of subclinical infection of vaccine group receiving type B inactivated vaccine against HFRS.

[An epidemiological study on influenza pandemic].

A cross-sectional epidemiological study on "the relationship between five variables [Fish, Pig, Duck, Chicken and Integrated Fish Farming (IFF)] and influenza pandemic" was carried out in 697 disease surveillance spots in the rural areas of Shandong & Henan provides. Results suggested that ducks might have played an important role in the ecology of influenza. Pigs did not seem to be a greater culprit in ecology of influenza but gave the inconsistent view with the hypothesis that IFF could lead to human influenza pandemic.

[The relationship between integrated fish farming and human influenza pandemic].

The study was carried out for reviewing the hypothesis on the relationship between integrated fish farming (IFF) and human influenza A pandemic using study methods of longitudinal epidemiology, ecology and serology. Four rural villages with IFF, non-IFF and non-fish farming, selected from two provinces (Henan and Shandong of PRC) were investigated for the status about human influenza A epidemic and infections with human, duck, swine influenaz A viruses in those communities. The results put forward views on that there is no relation between IFF and the increase of incidence rate, infection rate and antibodies level to human influenza A virus. Duck may play an important role in the increase of infection rate to human influenza A viruses among human communities. Pigs seem not to play obvious role in the increase of infection rate to human influenza A viruses among human communities. Human, duck, swine influenza A viruses may spread among human duck and pigs communities interchangably.

Arboviruses as aetiological agents of encephalitis in the People's Republic of China.

A serological study was undertaken to determine the role of arboviruses as etiological agents of encephalitis in the People's Republic of China (PRC). Paired sera were collected during mosquito seasons in 1988-1990 from 614 patients with possible viral encephalitis in 15 regions of PRC and tested for haemagglutination inhibiting antibodies to selected arboviruses. Seroconversions were documented to alphavirus and flavivirus antigens in 13.0 and 18.7% of patients respectively in most of the study areas. No California group seroconversion was detected. The age of alphavirus seroconvertors ranged from 2 months to 32 years and of flavivirus seroconvertors from 6 months to 50 years, with higher numbers in males. Serious central nervous system manifestations were seen more commonly in flavivirus seroconvertors. This study affirms the importance of flavivirus as causative agents of encephalitis in PRC and provides evidence that one or more alphaviruses are causing symptomatic infections with neurological involvement in PRC.

Feasibility of UV-inactivated vaccinia virus in the modification of tumor cells for augmentation of their immunogenicity.

We compared the immunity induced by tumor cells modified with UV-inactivated purified vaccinia virus (UV-VV) and with live purified vaccinia virus (L-VV). C3H/HeN mice were inoculated i.p. with UV-VV or L-VV after whole-body irradiation with 150 rads of X-rays (priming). After 3 weeks the mice were immunized i.p. 3 times at weekly intervals with syngeneic X5563 or MH134 cells that had been absorbed in vitro with UV-VV or infected with L-VV and subsequently irradiated with 10(4) rads of X-rays. Then 1 week after the last immunization, the mice were challenged s.c. with X5563 viable tumor cells or challenged i.p. with MH134 viable tumor cells. The 50% lethal dose (TLD50) of X5563 in mice primed and immunized with UV-VV (UV-VV group) on s.c. challenge (10(6.06)) was the same as for mice treated with L-VV (L-VV group), whereas the TLD50 of unprimed or nonimmunized mice (control group) was 10(2.61). The TLD50 of MH134 in the UV-VV treated group on i.p. challenge (10(6.48)) was similar to that of the L-VV treated group (10(6.54)), while the TLD50 of the control group was 10(1.00). The difference between the TLD50 values of X5563 on s.c. challenge of mice primed and immunized with UV-VV or L-VV and control mice was 10(3.4). The difference between the TLD50 values of MH134 on i.p. challenge of primed and immunized mice and control mice was 10(5.5). These results indicate that the in vivo helper function of UV-VV is similar to that of L-VV and that the augmenting effect of this protocol depends on the kind of tumor.

Serological evidence of infection with California serogroup viruses (family Bunyaviridae) in residents of Long Hua, suburb of Shanghai, People's Republic of China.

Sera from 126 residents of Long Hua, a suburb of Shanghai, in the People's Republic of China, were studied. Sera were tested for haemagglutination inhibiting antibodies to alphavirus (eastern equine encephalitis, western equine encephalitis), flavivirus (St. Louis encephalitis, Powassan, dengue) and California serogroup (snowshoe hare) antigens. Flavivirus antibodies were found in 14 (11.1%) and California serogroup antibodies in 5 (3.9%) individuals. Neutralizing antibodies with highest titres to snowshoe hare virus were demonstrated in 3 of the 5 California serogroup reactors. We believe this to be the first report of California serogroup virus antibodies in Chinese residents and the first evidence to suggest that California serogroup viruses may be circulating in the Orient.