[Subtype and sequence analysis of human immunodeficiency virus type-1 strains in Guangdong province].

OBJECTIVE: To investigate subtype and genetic analysis of human immunodeficiency virus type-1 (HIV-1). METHODS: DNA sequences were amplified by nested-PCR from uncultured peripheral blood mononuclear cells (PBMC) obtained from 100 HIV-1 patients from Guangdong Province. The C2 to V3 region of the envelope glycoprotein gp120 of HIV-1 was sequenced directly. The analysis of the gene-based phylogenetic tree and variation of amino acid were carried out by using Wisconsin software package or genetics computer group (GCG). RESULTS: DNA fragments were amplified from 75 PBMC samples by using nested polymerase chain reaction (PCR). Sequence analysis showed that there were 3 HIV-1 subtypes or circulating recombinant forms (CRF): CRF01-AE (n = 44), CRF-BC (n = 27) and B' (n = 4). CONCLUSIONS: Three HIV-1 subtypes or circulating recombinant forms: CRF01-AE, CRF-BC and B' might be circulating in Guangdong Province. Findings from this study suggested that several subtypes might exist in Guangdong Province and the epidemic situation of AIDS be serious. It should be a challenge for Guangdong Province in treating patients, preventing and controlling AIDS in the future.

Treatment of severe acute respiratory syndrome with glucosteroids: the Guangzhou experience.

STUDY OBJECTIVE: To investigate the efficacy and safety profiles of corticosteroid therapy in severe acute respiratory syndrome (SARS) patients. DESIGN: Four hundred one of 1,278 SARS cases treated in Guangzhou China between December 2002 and June 2003 fulfilled the diagnostic criteria issued by the World Health Organization for confirmed identification of SARS. Among them, the diagnosis of critical SARS was defined by criteria of SARS guidelines incorporated with a low oxygenation index (OI) [< 300 mm Hg]. Data of these patients retrieved from a database were retrospectively analyzed by logistic regression and Cox regression for the effect of corticosteroid therapy on death, hospitalization days, and complication presentation. RESULTS: Among the 401 SARS patients studied, 147 of 249 noncritical patients (59.0%) received corticosteroids (mean daily dose, 105.3 +/- 86.1 mg) [+/- SD], and all survived the disease; 121 of 152 critical patients (79.6%) received corticosteroids at a mean daily dose of 133.5 +/- 102.3 mg, and 25 died. Analysis of these 401 confirmed cases did not show any benefits of corticosteroid on the death rate and hospitalization days. However, when focused on 152 critical SARS cases, factors correlated with these end points indicated by univariate analysis included use of corticosteroid, age, rigor at onset, secondary respiratory infections, pulmonary rales, grading of OI, and use of invasive ventilation. After adjustment for possible confounders, treatment with corticosteroid was shown contributing to lower overall mortality, instant mortality, and shorter hospitalization stay (p < 0.05). Incidence of complications was significantly associated with the need for invasive ventilation but not with use of corticosteroids. CONCLUSION: This Guangzhou retrospective study revealed that proper use of corticosteroid in confirmed critical SARS resulted in lowered mortality and shorter hospitalization stay, and was not associated with significant secondary lower respiratory infection and other complications.

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.

Nucleocapsid protein as early diagnostic marker for SARS.

Serum samples from 317 patients with patients with severe acute respiratory syndrome (SARS) were tested for the nucleocapsid (N) protein of SARS-associated coronavirus, with sensitivities of 94% and 78% for the first 5 days and 6-10 days after onset, respectively. The specificity was 99.9%. N protein can be used as an early diagnostic maker for SARS.

[Distribution of recombinant human immunodeficiency virus type-1 CRF01_AE strains in China and its sequence variations in the env V3-C3 region].

OBJECTIVE: To characterize CRF01_AE strains of recombinant human immunodeficiency virus type-1 (HIV-1) found in the Second National Molecular Epidemiology Study on HIV in China and to analyze its sequence variation in the env V3-C3 region during the First National Molecular Epidemiology Study (NMES1, 1996 - 1998) to the Second National Molecular Epidemiology Study (NMES2, 2001 - 2002). METHODS: DNA was extracted from peripheal blood mononuclear cells of the subjects with HIV infection. The env C2-V4 region of HIV-1 was amplified with nested polymerase chain reaction (n-PCR). PCR products were directly sequenced using ABI 377 DNA sequencer, then the gene-based phylogenetic tree was constructed and its variation of amino acids was analyzed with GCG software. RESULTS: Totally, 169 strains of recombinant HIV-1 CRF01_AE were identified from blood samples collected from different high risk groups in 17 of 31 provinces, municipalities and autonomous regions all over China by the end of 2002. Although sexual transmission still dominated during NMES1 (62.2%, 23/37) and NMES2 (55.3%, 73/132), prevalence of HIV-1 CRF01_AE in intravenous drug users (IDUs) increased to 41.6% (57/137) during NMES2 from 27% (10/37) during NMES1. Phylogenetic tree analysis showed that HIV-1 CRF01_AE strains prevalent in IDUs during NMES2 did not cluster with those prevalent in the subjects infected by sexual transmission during NMES2 and those in IDUs during NMES1. The amino acid residues of V3 region of HIV-1 CRF01_AE in IDUs were relatively conservative, but the sixth, eighth, ninth, tenth, twelfth, fifteenth, sixteenth amino acid residues of C3 region displayed regular changes. CONCLUSIONS: HIV-1 CRF01_AE strain has been introduced into inland provinces from southeastern coast areas and southwestern border areas, with an increasing prevalence in IDUs. The sequence of env V3-C3 region of recombinant HIV-1 CRF01_AE strains prevalent in IDUs during NMES2 was obviously different from that during NMES1, suggesting that HIV-1 CRF01_AE strains prevalent in IDUs during NMES2 might come from a new source and have a potential to spread.

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.

Rapid and efficient preparation of monoclonal antibodies against SARS-associated coronavirus nucleocapsid protein by immunizing mice.

OBJECTIVE: To develop a rapid and efficient method for preparing monoclonal antibodies (mAb) against SARS-associated coronavirus (SARS-Cov) nucleocapsid (N) protein. METHODS: BALB/c mice were injected with the recombinant N protein of SARS-Cov into the foot-pads for the immunization, and the popliteal lymph nodes were isolated 15 d later for mAb-producing hybridomas, from which the mAbs against the N protein of SARS-Cov were screened. The identification of the mAb against the N protein of SARS-Cov was performed using indirect enzyme-linked immunosorbent assay (ELISA), indirect fluorescent-antibody assay (IFA), and Western immunoblotting. RESULTS: Four strains of hybridomas were obtained that produced the mAb specific to the N protein without detectable cross-reactivity with other pathogens. Of the 4 strains, 2 were identified as the immunoglobulin G1 (IgG1) isotype, 1 IgG2a, and the other IgG2b, with affinity constants (Ka) of 2 of the strains being 4.14 x 10(-9)M and 3.19 x 10(-9)M respectively. CONCLUSION: This is the first report on the preparation of mAb that is specific to the SARS-Cov, and the high-specificity and high-affinity mAb produced by the 4 strains of hybridomas provide a basis for further researches on the pathogenesis and early diagnosis of SARS.

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.