Complicated HCV subtype expansion among drug users in Guangdong province, China.

Guangdong Province is one of the most developed and populous provinces in southern China. The subtype situation of hepatitis C virus (HCV) in Guangdong remains unknown. The aim of this study was to investigate and estimate the HCV subtypes in drug users (DU) using a city-based sampling strategy to better understand the characteristics of HCV transmission in Guangdong. Archived plasma samples (n = 1074) from DU who were anti-HCV positive in 2014 were selected randomly from 20 cities in Guangdong Province. Subtypes were determined based on core and/or E1 sequences using phylogenetic analysis. The distributions of HCV subtypes in DU and different regions were analyzed. A total of 8 genotypes were identified. The three main HCV subtypes in DU in Guangdong were 6a (63.0%), 3a (15.2%), and 3b (11.8%). Significant differences were discovered among different registered residency and regions but not among genders, marital status, education level, or drug use patterns. HCV subtype 3b was significantly higher in Guangdong residents than in non-Guangdong residents. In contrast, HCV subtype 6a was significantly lower in Guangdong residents than in non-Guangdong residents. Subtype 1b in eastern Guangdong (eastern) was significantly lower, while 6a was significantly higher when compared with other regions. Subtype 3a in the Pearl River Delta (PRD) region was significantly higher, while 3b was significantly lower when compared with other regions. In western Guangdong, HCV subtype 3a was significantly lower when compared with other regions. Additionally, in northern Guangdong subtypes 1b and 3b were significantly higher, while 6a was significantly lower when compared with other regions. Our study revealed the diversity and distribution of HCV subtypes in DU in nearly all the cities in Guangdong. The results provide essential information that will allow the establishment of specific intervention strategies that may help prevent HCV transmission.

Establishment of HIV-1 model cell line GHOST(3) with stable DRiP78 and NHERF1 knockdown.

Chemokine receptors CXCR4 and CCR5 are indispensable co-receptors for HIV-1 entry into host cells. In our previous study, we identified that dopamine receptor-interacting protein 78 (DRiP78) and Na(+)-H(+) exchanger regulatory factor 1 (NHERF1) are the CXCR4 and CCR5 homo- or hetero-dimer-interacting proteins. DRiP78 and NHERF1 are able to influence the co-receptor internalization and intracellular trafficking. Over-expression of NHERF1 affects the ligands or HIV-1 gp120-induced CCR5 internalization and HIV-1 production. It is reasonable to speculate that DRiP78 and NHERF1, as well as the signaling pathways involved in viral replication, would probably affect HIV-1 replication through regulating the co-receptors. In this present study, we designed two short hairpin RNAs (shRNAs) targeting the DRiP78 and NHERF1, respectively, and constructed the pLenti6/BLOCK-iT-DEST lentiviral plasmids expressing DRiP78 or NHERF1 shRNA. The packaged lentiviruses were used to transduce the widely-applied HIV-1 model cell line GHOST(3). Then, cells with stable knockdown were established through selecting transduced cells with Blasticidin. This study, for the first time, reported the establishment of the GHOST(3) with DRiP78 and NHERF1 knockdown, which is the first stable cell line with HIV-1 co-receptor-interacting molecular defects.

Immune responses in Balb/c mice induced by a candidate SARS-CoV inactivated vaccine prepared from F69 strain.

The immunogenicity of a candidate-inactivated vaccine prepared from SARS-CoV F69 strain was evaluated in Balb/c mice. Potent humoral immune responses were induced under the elicitation of three times of immunizations at 2-week intervals with this vaccine, combined with three types of adjuvants (Freund's adjuvant, Al(OH)(3) adjuvant and CpG adjuvant). Titers of specific IgG antibodies in three test groups all peaked in the sixth week after first vaccination, but significant differences existed in the kinetics of specific IgG antibody levels. The strong neutralizing capacity exhibited in micro-cytopathic effect neutralization tests indicated the specific antibodies are protective. Western blot assay further demonstrated the specificity of the induced serum antibodies.

Humoral immune responses in rabbits induced by an experimental inactivated severe acute respiratory syndrome coronavirus vaccine prepared from F69 strain.

BACKGROUND: The etiologic agent of severe acute respiratory syndrome (SARS) has been confirmed to be a novel coronavirus (CoV), namely SARS-CoV. Developing safe and effective SARS-CoV vaccines is essential for us to prevent the possible reemergence of its epidemic. Previous experiences indicate that inactivated vaccine is conventional and more hopeful to be successfully developed. Immunogenicity evaluation of an experimental inactivated SARS-CoV vaccine in rabbits was conducted and reported in this paper. METHODS: The large-scale cultured SARS-CoV F69 strain was inactivated with 0.4% formaldehyde and purified, then used as the immunogen combined with Freund's adjuvant. Eight adult New Zealand rabbits were immunized four times with this experimental inactivated vaccine. Twelve sets of rabbit serum were sampled from the third day to the seventy-fourth day after the first vaccination. The titers of specific anti-SARS-CoV IgG antibody were determined by indirect enzyme-linked immunosorbent assay, and the neutralizing antibody titers were detected with micro-cytopathic effect neutralization test. RESULTS: Rapid and potent humoral immune responses were induced by the inactivated SARS-CoV vaccine in all the eight test rabbits. Titers of both specific IgG antibody and neutralizing antibody peaked at about six weeks after first vaccination, with the maximum value of 1:81 920 and 1:20 480, respectively. After that, serum antibody levels remained at a plateau or had a slight decrease, though two boosters were given in the succedent 4 to 5 weeks. Cross neutralization response existed between SARS-CoV F69 strain and Z2-Y3 strain. CONCLUSIONS: The inactivated SARS-CoV vaccine made from F69 strain owns strong immunogenicity, and the cross neutralization response between the two different SARS-CoV strains gives a hint of the similar neutralizing epitopes, which provide stable bases for the development of inactivated SARS-CoV vaccines.

Immunogenicity of SARS inactivated vaccine in BALB/c mice.

Severe acute respiratory syndrome (SARS) is a serious infectious threat to public health. To create a novel trial vaccine and evaluate its potency, we attempted to generate a SARS inactivated vaccine using SARS coronavirus (SARS-CoV) strain F69 treated with formaldehyde and mixed with Al(OH)3. Three doses of the vaccine were used to challenge three groups of BALB/c mice. We found that the mice exhibited specific IgM on day 4 and IgG on day 8. The peak titers of IgG were at day 47 in low-dose group (1:19,200) and high-dose group (1:38,400) whereas in middle-dose group (1:19,200), the peak was at day 40. On day 63, the IgG levels reached a plateau. Neutralization assay demonstrated that the antisera could protect Vero-E6 cells from SARS-CoV's infection. Analysis of the antibody specificity revealed that the mouse antisera contained a mixture of antibodies specifically against the structure proteins of SARS-CoV. Furthermore, the mouse antisera conferred higher amount of antibodies against protein N, polypeptide S4 and S2 than those of proteins M and 3CL. These findings suggest that the inactivated SARS-CoV could preserve its antigenicity and the inactivated vaccine can stimulate mice to produce high levels of antibodies with neutralization activity. Results also suggest that polypeptides originating from protein N or S might be a potential target for the generation of a recombinant SARS vaccine.

Morphology and morphogenesis of severe acute respiratory syndrome (SARS)-associated virus.

After infecting the Vero E6 cells by nasal/throat swabs collected from SARS patients, we studied the SARS-associated virus by electron microscopy and molecular biological technique. The results show that the diameter of newly isolated virus is about 50 nm without envelope or 100 nm with envelope. The virus was proved to be a new coronavirus by RT-PCR and it responded positively to convalescent-phase serum specimen from SARS patients, which is the evidence that this new virus is etiologically linked to the outbreak of SARS. The morphogenesis and distribution of the virus are also discussed in this article.

[Isolation and identification of SARS virus in Guangdong province].

BACKGROUND: To isolate and identify pathogen of atypical pneumonia in Guangdong. METHODS: Pathogens were isolated from variety of samples collected from atypical pneumonia patient by using MDCK cells, and identified with serological and molecular methods. RESULTS: A novel coronavirus was isolated from patients with atypical pneumonia, from which an RNA fragment of 279 nt was amplified by nested RT-PCR. And sequence assay showed that only 39-65 percent of sequence of the virus was homogenous to known coronavirus, but almost 100% homogenous (with one base exception, 12a to t) to SARS-associated coronavirus isolated from patients outside Guangdong, such as in Beijing, Hong Kong, Taiwan, Germany, Italy and so on. Indirect immunofluorescence test showed a specific antigen-antibody reactivity between the coronavirus and convalescent-phase sera of SARS patients. CONCLUSION: The pathogen of the atypical pneumonia in Guangdong province was a novel type of coronavirus, which could be isolated by using MDCK cells.