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Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) have recently been identified as cytoplasmic sensors for RNA viruses. The RLR signaling cascades induce the production of type I interferons and pro-inflammatory cytokines, which are rigorously regulated by the host. On other side, RNA viruses have evolved strategies to evade RLR signaling. In this review, we present our current understanding of RLR signaling in RNA virus recognition and antiviral innate immunity.

Objective: To investigate the effect of the well-defined RNA elements (VR, RCS2, CS2, CS1 and SL) within the 3'untranslated region (UTR) on dengue virus (DEN) translation., Methods: We constructed a virus induced reporter gene (VIRG) by inserting the firefly luciferase (LUC) gene between 5'- and 3'-UTRs of DEN2-43 genome. Subsequently, a series of modified VIRGs consisting of different RNA elements in the 3'UTR were constructed. A 3'UTR-deficient VIRG was also constructed. The translational efficiency of all VIRGs was then analyzed by LUC detection, real time RT-PCR and Western blot assays., Results: The translation of 3'UTR-deficient VIRG was abolished. The translational efficiency of VIRG with RNA element VR was comparable with that of VIRG with unmodified 3'UTR. The translational efficiency of VIRG with RNA elements RCS2 or CS2 was significantly higher while the translational efficiency of VIRG with RNA elements CS1 or SL was substantially lower than that of VIRG with RNA element VR., Conclusion: These results suggested that 3'UTR was indispensable for DEN translation, and some RNA elements within 3'UTR might either up-regulate or down- regulate translation.

Objective: To study pharmacokinetics of honokiol in Cortex of Magnolia officinalis and its compound prescription Houpu Sanwu Decoction (HPSWD) in rat, and discuss the change on pharmacokinetic process affected by other components., Methods: The rats were divided into two groups, one was supplied with HPSWD and the other was administrated with Cortex of Magnolia officinalis. Concentrations of honokiol in rat plasma were then determined using HPLC method and main pharmacokinetic parameters were estimated., Results: The plasma concentrations of honokiol of both groups were conformed to the two-compartment model with first order absorption. There existed significant differences in AUC, C(max), T(max), CL/F among Cortex of Magnolia officinalis and HPSWD., Conclusion: The results indicate that Rhubarb and Immature Orange Fruit in HPSWD can influence the asorption, distribution and elimination of honokiol.

Using recombination analysis, we identified a recombinant dengue virus type 1 strain, namely, GD23/95, with three recombination regions, located within the sequences of the prM/E junction, NS1, and NS3, respectively. The recombinant dengue virus was further confirmed by phylogenetic analysis based on its recombination and non-recombination regions. This appears to be the first study to confirm the existence of three recombination regions in a single dengue virus isolate and to report recombination between parent virus strains isolated from the same geographic area (Guangdong province, China). It is also the first to report breakpoints within the NS3 gene of dengue viruses.

In the present study, the domain IIIs of all four dengue virus (DENV) serotypes were connected sequentially to construct the tandem domain III. The resulting DNA fragment was then cloned into pBAD/Topo ThioFusion plasmid. After induction, the Escherichia coli expression protein was purified and used to immunize BALB/c mice by subcutaneous route. The sera from mice immunized with the purified protein were confirmed to contain specific high antibody titers against DEN1, DEN2, and DEN4, and moderate antibody titer against DEN3. In suckling mouse model, 70% of the mice challenged with DEN1, DEN2, and DEN4 in combination with sera from mice immunized with the purified protein were protected, and 18% of the mice challenged with DEN3 in combination with the same sera were protected. Our data suggest that the tandem domain III of the envelope protein can be used as a potential tetravalent dengue vaccine based on a single antigen.

A full-length cDNA clone (pD212) of dengue virus type 2 isolated in China (DEN2-43) was constructed. Based on this, we constructed several mutants with deletions in capsid protein C using fusion PCR. These deletions removed part or almost all of the internal stretch of hydrophobic amino acid residues that is probably involved in virion assembly. We thus obtained viable mutant viruses. The propagation capacity of the mutant viruses in cell culture was impaired in parallel with the increasing size of the deletion, and the infectivity of mutant C(Delta42-59), from which all of helix III of capsid protein C was removed, was completely abolished. More importantly, the mutant viruses were highly attenuated in suckling mice but induced high levels of antibodies in adult mice. This study indicates that the structural and functional flexibility of capsid protein C make it a candidate target for the attenuation of dengue virus, which could open a promising new avenue for the development of live attenuated dengue vaccines.

Objective: To generate rescued viruses with deletion mutation of capsid protein from dengue virus type 2 isolated in China (DEN2-43)., Methods: On the basis of infectious full-length cDNA clone pD212 of DEN2-43 strain virus, the deletion mutants were constructed by fusion PCR, from which the rescued viruses with deletion mutation of capsid protein were generated by transcription in vitro and electroporation., Result and Conclusion: Sequence analysis demonstrated that the deletion mutations had been successfully inserted into the rescued viruses obtained. These mutant viruses may hold the key for elucidating the effects of deletion mutation of capsid protein on the biological characteristics of dengue virus.

Severe acute respiratory syndrome (SARS) is an important emerging infectious disease which caused by SARS coronavirus (SARS-CoV), and the study of its pathogenesis is needed for the treatment and prevention of this disease. To study the pathogenesis of SARS-CoV using reverse genetics technology, by in vitro ligation using 7 contiguous cDNAs that span the entire genome of the SARS-CoV BJ01 strain, a genomic full-length cDNA was assembled, then using T7 RiboMAX Large Scale RNA Production Systems with the genomic full-length cDNA as template, the RNA transcript was attained. The typical SARS-CoV-resulted cell pathogenic effects were observed when RNA transcript was electroporated into Vero E6 cells. The results of RT-PCR and sequencing of the rescued virus showed that it originated from transcript which derived from the full-length cDNA construct. Rescued virus-infected cells were detected by indirect fluorescent antibody staining demonstrated that it can specifically reaction with SARS-CoV. By CPE method and plaque assay, the titers of the rescued virus and wild-type virus were assessed, which demonstrated there are no significant difference between the viruses and they have similar biological characteristics. Construction of the genomic full-length cDNA of SARS-CoV BJ01 stain successfully and study of the biological characteristics of the rescued virus will provide a useful tool serving for the discovery of molecular pathogenesis and development of candidate vaccines against SARS-CoV.

Background: In 2003, severe acute respiratory syndrome (SARS) resulted in hundreds of infections and deaths globally. We aim to assess immunogenicity and protective efficacy of purified inactivated Vero-cell SARS vaccine in monkeys., Methods: The cultures of SARS coronavirus (SARS-CoV) BJ-01 strain infected Vero cells were inactivated with beta-propiolactone. Sequential procedures, including ultrafiltration, gel filtration and ion exchange chromatography, were performed to obtain purified inactivated SARS vaccine. The purified SARS vaccine was analyzed with electron microscope, HPLC and Western blotting. We immunized three groups of cynomolgus macaques fascicularis with adjuvant-containing purified vaccine, purified vaccine and unpurified vaccine, respectively, and a fourth group served as a control. Antibody titers were measured by plaque reduction neutralization test. The vaccinated monkeys were challenged with SARS-CoV BJ-01 strain to observe protective efficacy. Additionally, three groups of rhesus monkeys were immunized with different doses of the purified inactivated SARS vaccine (0.5, 1 and 2mug/time/monkey) on days 0 and 7, and the monkeys were challenged with SARS-CoV GZ-01 strain. We assessed the safety of the SARS vaccine and observed whether the antibody dependent enhancement (ADE) occurred under low levels of neutralizing antibody in rhesus., Findings: The purity of SARS vaccine was 97.6% by HPLC identification and reacted with convalescent sera of SARS patients. The purified SARS vaccine induced high levels of neutralizing antibodies and prevented the replication of SARS-CoV in monkeys. Under low levels of neutralizing antibody, no exacerbation of clinical symptoms was observed when the immunized monkeys were challenged with SARS-CoV. In this preliminary animal trial, no side effects were detected when monkeys were immunized with purified SARS vaccine either at normal or large doses., Interpretation: The purified inactivated SARS vaccine could induce high levels of neutralizing antibody, and protect the monkeys from the challenge of SARS-CoV. The SARS vaccine prepared in the study appeared to be safe in monkeys.

Capsid-targeted viral inactivation (CTVI) has emerged as a conceptually powerful antiviral strategy that exploits viral structural proteins to target a destructive enzyme specifically into progeny virions. We have recently demonstrated the principle of CTVI against dengue virus infection and observed a modest therapeutic effect in vitro (Arch Virol 2005, 150: 659-669). Here we tested a prophylactic model of CTVI, in which mammalian cells stably expressing the dengue 2 virus capsid protein fused to a nuclease were infected with dengue virus and determined the effects on progeny virion infectivity. CTVI efficiently destroyed dengue 2 virus from within and decreased the infectious titers by 10(3)- to 10(4)-fold, suggesting that CTVI has potential in the prophylactic application for dengue virus infection.

Aim: To express staphylococcus nuclease (SN) in E.coli and prepare rabbit antisera against SN., Methods: The SN gene was amplified by high-fidelity PCR from plasmid pPLC-SN and then subcloned into expression vector pLEX to obtain the recombinant plasmid pLEX-SN. The expression of recombinant protein was induced by tryptophan. The expressed SN was used to immunize a rabbit to prepare specific antibody., Results: SDS-PAGE analysis showed that the relative molecular mass (M(r)) of the expressed SN was about 17,000 and the expressed SN accounted for about 37% of total bacterial proteins. The prepared antisera were specific to react with recombinant SN., Conclusion: Expression vector of SN has been successfully constructed and rabbit antibody against SN was prepared. These results lay the foundation for developing SN as antiviral protein.

Dengue infection poses a serious public health problem in most tropical and subtropical areas. No effective antiviral drugs or vaccines are currently available against dengue infection. To explore the feasibility of using capsid-targeted viral inactivation (CTVI) as an antiviral strategy against dengue infection, we constructed a plasmid expressing a fusion protein consisting of staphylococcal nuclease (SN) fused to dengue 2 virus capsid protein (D2C), and investigated its effects on the production of infectious virions when introduced into BHK cells infected with dengue virus. The results indicated that D2C-SN can be expressed and tolerated in this mammalian cell culture. The enzymatically active SN moiety was incorporated into nascent virions during the process of viral assembly. By comparing the effects of incorporated SN and SN*, an enzymatically inactive missense mutant form of wild-type SN, on the infectivity of progeny virions, we clearly demonstrated that nucleolytic activity was the major antiviral mechanism. Expression of D2C-SN fusion protein as a therapeutic agent resulted in a reduction in infectious titers of 12- to 60-fold. Therefore, dengue virus may be particularly vulnerable to a CTVI therapeutic approach.

To explore the feasibility of capsid-targeted viral inactivation for dengue virus infection, a newly-discovered antiviral strategy, a mammalian cell line stably expressing staphylococcal nuclease fused to the capsid protein of dengue 2 virus was established and the effects on the production of infectious virus particles were examined. The results presented evidence that the enzymatically active staphylococcal nuclease fused to capsid protein could be incorporated into the nascent virions during wild virus assembly, resulting in degradation of viral genomic RNA and decrease in infectivity. Comparing the effects of incorporated SN and SN*, an enzymatically inactive missense mutant form of SN, on the infectivity of progeny virions, nucleolytic activity of incorporated SN was responsible for the major antiviral effects. These results paved the road of developing capsid-targeted viral inactivation as a new antiviral strategy against dengue.

IgG and IgM antibodies to dengue virus(DEN) in patient serum sample were detected with an indirect immunofluorescence assay. The patient's serum was inoculated into the culture of C6/36 cells to isolate dengue virus. Viral RNA was extracted from cultured supernatant and RT-PCR amplification and sequencing were carried out. The result showed that there were anti-dengue virus IgG and IgM antibodies in the serum. Both dengue type 2 and 3 virus specific sequences were confirmed by RT-PCR and sequence analysis. The results presented here demonstrated the simultaneous infection with dengue type 2 and 3 viruses in the Chinese patient.

To screen small animals susceptible to SARS-CoV, five species of animals, including guinea pig, hamster, albino hamster, chicken and rat, were experimentally infected with SARS-CoV strain BJ-01 by different routes. On the basis of this, further cynomolgus and rhesus macaques were selected and experimentally inoculated SARS-CoV, the quality they serve as animal model for SARS was evaluated. The results showed that, all five species of small animals chosed were not susceptible to SARS-CoV, no characterized changes in clinical sign and histopathology were observed after infection, but from the lung samples of large rat and pig guinea, the genomic RNA of SARS-CoV could be detected by RT-PCR at day 14 post infection, this suggested that SARS-CoV could replicate in these animals. After inoculated with SARS-CoV, all inoculated cynomolgus and rhesus macaques had developed interstitial pneumonia of differing severity. These changes on histopathology were similar to that seen in SARS patients, but the pathological lesions were less severe than that of human. Except interstitial pneumonia, no other characterized pathological changes were observed. This suggested cynomolgus and rhesus macaques were not the ideal animal model for SARS in fact, but they could serve as animal model for SARS when a more ideal animal model is absent.

Maoecrystal V (1), a novel C(19) diterpenoid possessing a unique 6,7-seco-6-nor-15(8-->9)-abeo-5,8-epoxy-ent-kaurane skeleton, was isolated from the leaves of a Chinese medicinal herb, Isodon eriocalyx. Its structure was determined by comprehensive NMR and MS spectroscopic analysis and confirmed by single-crystal X-ray diffraction study. Compound 1 showed remarkable inhibitory activity toward HeLa cells with IC(50) = 0.02 microg/mL (cis-platin: IC(50) = 0.99 microg/mL).

To explore the potential application of capsid-targeted viral inactivation (CTVI) strategy in prophylactic model against dengue virus (DV) infection, here we fused a Ca(2+)-dependent nuclease, staphylococcal nuclease (SN), to the capsid protein of dengue 2 virus (D2C) at the carboxyl terminal, and constructed the desired expression plasmid pc/D2C-SN and control plasmids pc/D2C-SN* and pc/D2C. A mammalian cell line BHK-21 was transfected by electroporation with those plasmids and thereafter selected by 5mgr;/ml blasticidin. The resistant cell clones were then expanding cultured and screened by RT-PCR and Western Blot assays. The nuclease activity of the expressed fusion protein D2C-SN was analyzed by in vitro DNA digestion assay. It was confirmed cell lines stably expressing D2C-SN and control constructs were obtained. The intracellular expressed fusion protein D2C-SN had ideal nuclease activity and no cytotoxicity on mammalian cells. Those engineered cell lines provided the experimental system for CTVI application in prophylactic model and paved the new road for combating DV infection with CTVI.

We tested the ability of inactivated SARS-CoV vaccine to induce neutralizing antibodies in BALB/c mice. The inactivated vaccine was prepared by SARS-CoV virus propagation in Vero cells, with subsequent beta-propiolactone inactivation and Sepharose 4FF column chromatography purification. One hundred forty BALB/c female mice were divided into seven groups of 20 mice each. Of the seven groups, three groups were inoculated with 0.1, 1, and 3 microg of the vaccine without adjuvant while three other groups were inoculated at the same three dosages of vaccine with aluminum hydroxide as adjuvant, respectively. The remaining group was set up as a blank control. Each mouse was inoculated twice at an interval of 3 weeks. One week after the second immunization, mice sera were collected to detect serum neutralizing antibodies. An assay for determining neutralizing antibody titers was developed. The results can be summarized as follows: (1) higher dosages of vaccine induced higher levels of neutralizing antibody titer; (2) the level of neutralizing antibodies induced by the inoculation with aluminum hydroxide adjuvant was slightly higher than that without adjuvant, but the difference was not statistically significant.

Recently, a new coronavirus was isolated from the lung tissue of autopsy sample and nasal/throat swabs of the patients with Severe Acute Respiratory Syndrome (SARS) and the causative association with SARS was determined. To reveal further the characteristics of the virus and to provide insight about the molecular mechanism of SARS etiology, a proteomic strategy was utilized to identify the structural proteins of SARS coronavirus (SARS-CoV) isolated from Vero E6 cells infected with the BJ-01 strain of the virus. At first, Western blotting with the convalescent sera from SARS patients demonstrated that there were various structural proteins of SARS-CoV in the cultured supernatant of virus infected-Vero E6 cells and that nucleocaspid (N) protein had a prominent immunogenicity to the convalescent sera from the patients with SARS, while the immune response of spike (S) protein probably binding with membrane (M) glycoprotein was much weaker. Then, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the complex protein constituents, and the strategy of continuous slicing from loading well to the bottom of the gels was utilized to search thoroughly the structural proteins of the virus. The proteins in sliced slots were trypsinized in-gel and identified by mass spectrometry. Three structural proteins named S, N and M proteins of SARS-CoV were uncovered with the sequence coverage of 38.9, 93.1 and 28.1% respectively. Glycosylation modification in S protein was also analyzed and four glycosylation sites were discovered by comparing the mass spectra before and after deglycosylation of the peptides with PNGase F digestion. Matrix-assisted laser desorption/ionization-mass spectrometry determination showed that relative molecular weight of intact N protein is 45 929 Da, which is very close to its theoretically calculated molecular weight 45 935 Da based on the amino acid sequence deduced from the genome with the first amino acid methionine at the N-terminus depleted and second, serine, acetylated, indicating that phosphorylation does not happen at all in the predicted phosphorylation sites within infected cells nor in virus particles. Intriguingly, a series of shorter isoforms of N protein was observed by SDS-PAGE and identified by mass spectrometry characterization. For further confirmation of this phenomenon and its related mechanism, recombinant N protein of SARS-CoV was cleaved in vitro by caspase-3 and -6 respectively. The results demonstrated that these shorter isoforms could be the products from cleavage of caspase-3 rather than that of caspase-6. Further, the relationship between the caspase cleavage and the viral infection to the host cell is discussed.

Background: The widespread threat of severe acute respiratory syndrome (SARS) to human life has spawned challenges to develop fast and accurate analytical methods for its early diagnosis and to create a safe antiviral vaccine for preventive use. Consequently, we thoroughly investigated the immunoreactivities with patient sera of a series of synthesized peptides from SARS-coronavirus structural proteins., Methods: We synthesized 41 peptides ranging in size from 16 to 25 amino acid residues of relatively high hydrophilicity. The immunoreactivities of the peptides with SARS patient sera were determined by ELISA., Results: Four epitopic sites, S599, M137, N66, and N371-404, located in the SARS-coronavirus S, M, and N proteins, respectively, were detected by screening synthesized peptides. Notably, N371 and N385, located at the COOH terminus of the N protein, inhibited binding of antibodies to SARS-coronavirus lysate and bound to antibodies in >94% of samples from SARS study patients. N385 had the highest affinity for forming peptide-antibody complexes with SARS serum., Conclusions: Five peptides from SARS structural proteins, especially two from the COOH terminus of the N protein, appear to be highly immunogenic and may be useful for serologic assays. The identification of these antigenic peptides contributes to the understanding of the immunogenicity and persistence of SARS coronavirus.

Beijing has been one of the epicenters attacked most severely by the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) since the first patient was diagnosed in one of the city's hospitals. We now report complete genome sequences of the BJ Group, including four isolates (Isolates BJ01, BJ02, BJ03, and BJ04) of the SARS-CoV. It is remarkable that all members of the BJ Group share a common haplotype, consisting of seven loci that differentiate the group from other isolates published to date. Among 42 substitutions uniquely identified from the BJ group, 32 are non-synonymous changes at the amino acid level. Rooted phylogenetic trees, proposed on the basis of haplotypes and other sequence variations of SARS-CoV isolates from Canada, USA, Singapore, and China, gave rise to different paradigms but positioned the BJ Group, together with the newly discovered GD01 (GD-Ins29) in the same clade, followed by the H-U Group (from Hong Kong to USA) and the H-T Group (from Hong Kong to Toronto), leaving the SP Group (Singapore) more distant. This result appears to suggest a possible transmission path from Guangdong to Beijing/Hong Kong, then to other countries and regions.

Objective: Electron microscopical study of infected cells to identify the pathogenic agent of SARS., Methods: Vero E6 cells infected with lung autopsy samples or nasopharyngeal swabs from SARS patients of Beijing and Guangzhou were inoculated. The supernatant and cultured cells exhibiting identifiable cytopathic effect (CPE) were prepared for electron microscopic study., Results: Examination of CPE cells on thin-section revealed characteristic coronavirus particles within the cisternae of endoplasmic reticulum, Golgi apparatus, vesicles and extracellular space. They were mainly spherical or oval in shape, annular or dense, about 80 nm in diameter. Negative-stain electron microscopy identified coronavirus particles in culture supernatant, 80 - 120 nm in diameter, with club-shaped surface projections. Elongated, rod-, kidney- or other irregular shaped virons with the size of 100 - 200 nm by 60 - 90 nm were also found in the cultured cells infected with the lung samples from the Guangdong patients. Infectious virons entered cells by endocytosis or membrane fusion and released through a budding process., Conclusion: These data indicate a novel coronavirus as the causative agent of SARS. Most viral particles showed typical characteristics of coronavirus. The potential role of special shape viruses is expected to be further investigated.

We report a complete genomic sequence of rare isolates (minor genotype) of the SARS-CoV from SARS patients in Guangdong, China, where the first few cases emerged. The most striking discovery from the isolate is an extra 29-nucleotide sequence located at the nucleotide positions between 27,863 and 27,864 (referred to the complete sequence of BJ01) within an overlapped region composed of BGI-PUP5 (BGI-postulated uncharacterized protein 5) and BGI-PUP6 upstream of the N (nucleocapsid) protein. The discovery of this minor genotype, GD-Ins29, suggests a significant genetic event and differentiates it from the previously reported genotype, the dominant form among all sequenced SARS-CoV isolates. A 17-nt segment of this extra sequence is identical to a segment of the same size in two human mRNA sequences that may interfere with viral replication and transcription in the cytosol of the infected cells. It provides a new avenue for the exploration of the virus-host interaction in viral evolution, host pathogenesis, and vaccine development.

pDVWS501 was a genomic-length cDNA clone of dengue 2 virus, through which infectious virus (MON501) could be rescued. MON501 was neurovirulent in mice, whose E residues 62 and 203 were Lys and Asn, respectively. Two genomic-length cDNA clones (TB62 and TB203) were constructed by pointed mutation of pDVWS501 with OL-PCR, E62 of TB62 and E203 of TB203 were converted to Glu and Asp, respectively. RNA transcripts of pDVWS501, TB62 and TB203 were produced in vitro and electroporated into BHK-21 cells. The cultures were collected after 7 days and used as inoculum to infect C6/36 cells. The existence of rescued dengue viruses in the culture was proved by RT-PCR, and the typical cytopathic effect (CPE) of C6/36 caused by dengue virus emerged after 2-5 days' inoculation. Sequence analysis further confirmed the existence of recovered and recombinant DEN2 viruses, whose 5' termini had an additional non-virus nucleotides "G", while the 3' terminal sequences remained the same as natural. The neurovirulence of three viruses was evaluated in 1-day-old mice by the intracerebral route with 10(5)-10(2) TCID50. Compared with MON501 group, the number of infected mice with the signs of encephalitis in HFT62 and HFT203 groups was less, and the surviving time was longer. The properties of these mutants demonstrated that E62 and E203 are determinants of suckling mice neurovirulence.

The genome sequence of the Severe Acute Respiratory Syndrome (SARS)-associated virus provides essential information for the identification of pathogen(s), exploration of etiology and evolution, interpretation of transmission and pathogenesis, development of diagnostics, prevention by future vaccination, and treatment by developing new drugs. We report the complete genome sequence and comparative analysis of an isolate (BJ01) of the coronavirus that has been recognized as a pathogen for SARS. The genome is 29725 nt in size and has 11 ORFs (Open Reading Frames). It is composed of a stable region encoding an RNA-dependent RNA polymerase (composed of 2 ORFs) and a variable region representing 4 CDSs (coding sequences) for viral structural genes (the S, E, M, N proteins) and 5 PUPs (putative uncharacterized proteins). Its gene order is identical to that of other known coronaviruses. The sequence alignment with all known RNA viruses places this virus as a member in the family of Coronaviridae. Thirty putative substitutions have been identified by comparative analysis of the 5 SARS-associated virus genome sequences in GenBank. Fifteen of them lead to possible amino acid changes (non-synonymous mutations) in the proteins. Three amino acid changes, with predicted alteration of physical and chemical features, have been detected in the S protein that is postulated to be involved in the immunoreactions between the virus and its host. Two amino acid changes have been detected in the M protein, which could be related to viral envelope formation. Phylogenetic analysis suggests the possibility of non-human origin of the SARS-associated viruses but provides no evidence that they are man-made. Further efforts should focus on identifying the etiology of the SARS-associated virus and ruling out conclusively the existence of other possible SARS-related pathogen(s)., (© Science in China Press 2003.)

A large quantity of SARS-CoV virus was proliferated in Vero cells, inactivated with β-propiolactone, then purified by Sepharose 4FF column chromatography to prepare inactivated vaccine. The vaccine was identified by Western blot, mass spectrographic analysis, ELISA and electron microscopy. The vaccine with or without aluminum hydroxide adjuvant was inoculated into female BALB/c mice at different dosages. The result showed that the antibodies to SARS-CoV were induced in the mice. The antibody levels induced by the vaccine with aluminum hydroxide were higher than those without aluminum hydroxide., (© Science in China Press 2003.)

The amplified PrM gene of dengue-2 virus was cloned into the downstream SP6 promoter-pSFV vector and the recombinant plasmid (pSF.rM2) DNA which contained sense- or antisense-PrM gene, was selected. pSF.rM2 DNA and helper DNA linearized by the enzyme SpeI digestion were both transcribed in vitro into recombinant RNAs which contained the capping analog on the 5'-end and contaansfected into BHK cells by electroportation. The the transfected host cells were challenged with dengue-2 virus and the resistant efficiency of recombinant virus RNAs containing sense- or antisense-PrM gene to virus infection were observed, respectively. The recombinant plasmids (pSFV-PrM) containing sense- or antisense-PrM gene were selected with determination of the nucleotide sequence. The recombinant virus particles were obtained with recombinant RNA and helper RNA co-transfected into BHK cells. Host cells transfected with antisense-PrM RNA derived complete resistance to dengue-2 virus replication and the efficiency was higher than that of the recombinant virus RNA containing sense-PrM gene.

Objective: To analyze the sequences of 5' and 3' terminal region of dengue type 2 virus 04 strain., Methods: Total RNA was isolated from dengue type 2 virus 04 strain (D2-04) infected C6/36 cells. With this RNA as template, the cDNA of both 5' and 3' termini of D2-04 were amplified using RACE method respectively. The cDNAs were separately inserted into pGEM-T vector and the recombinant plasmids containing the 5' terminus 535 bp and 3' terminus 503 bp were obtained. The nucleotide sequences of the inserted cDNA fragment were determined. The nucleotide sequences of the 5' and 3' noncoding regions of D2-04 were compared with other dengue type 2 viruses such as JAM, NGC, S1, 16681 and PD-53., Results: The results showed that they shared 98.96%, 98.96%, 93.75%, 98.95%, 97.92% and 97.72%, 97.80%, 90.65%, 94.26%, 94.22% homology with D2-04 strain respectively., Conclusions: Except SI, the homology between D2-04 and other type 2 viruses was higher than 94%.

Aim: To analyse the humoral immune response in mice to nucleic acid vaccines (VR1012/HG-MSP1-17 for intracellular expression or VR1012/TPA/HG-MSP-17 for secretion) containing Plasmodium falciparum merozoite surface protein 1 (MSP1) 17 block gene and gene fragment of several T cell epitopes from MSA1, MSA2, RESA, IL-1 and TT., Methods: BALB/c or C57BL/6 mice received three times intramuscular immunization with 200 micrograms/100 microliters or 100 micrograms/100 microliters of VR1012/HG-MSP1-17 or VR1012/TPA/HG-MSP1-17 per mouse each time. Anti-HG or anti-MSP1-17 antibodies were monitored by indirect ELISA., Results: BALB/c and C57BL/6 mice immunized with 100 micrograms/100 microliters of VR1012/HG-MSP1-17 per mouse raised significantly anti-HG and anti-MSP1-17 antibodies, but the levels of antibodies were not high. BALB/c mice immunized with 200 micrograms/100 microliters of VR1012/HG-MSP1-17 per mouse raised higher anti-HG antibodies but not anti-MSP1-17 antibodies. BALB/c mice immunized with 200 micrograms/100 microliters of VR1012/TPA/HG-MSP1-17 per mouse raised low level of anti-HG antibodies only., Conclusion: VR1012/HG-MSP1-17 is more immunogenic than VR1012/TPA/HG-MSP1-17.

Hybridoma secreting 3D3 monoclonal antibody against dengue virus type 3 was used. The isolated mRNA was reverse-transcribed into cDNA for amplifying the heavy and light chain variable domains genes. The amplified light and heavy chain variable domains' genes were connected by flexible linker to from a single chain fragment (ScFv) gene of 750 bp, which was cloned into phage pCANTAB5E vector using the recombination phage antibody system. pCANTAB5E vector transformed E coli HB2151. Soluble single chain antibody fragment was expressed both in the bacterial supernatant and cell periplasm under the induction of IPTG. The expressed single chain antibody fragment has a molecular weight of about 28 kD detected by SDS-PAGE and can react with dengue virus type 3 by immunofluorescence test.

Hybridoma 3D3 which secreted monoclonal antibody against Dengue type 3 virus was used to extract mRNA and then was reverse-transcripted into cDNA for amplifying heavy and light chain variable domain genes. The amplified light and heavy chain variable domain genes were connected by flexible linker to form a single chain antibody fragment (ScFv) gene of 750 bp, which was cloned into phage pCANTAB5 vector using the recombinant phage antibody system. The ScFv gene was expressed as fusion protein with phage g3p coat protein and displayed on the phage surface. The phagemid was used to transform competent E. coli TG1 cells and then infected with M13K07 helper phage to rescue the phagemid and antibody ScFv gene. 12 of the 20 randomized clones were shown to react with dengue type 3 virus by immunofluorescence.

In this study, RT-PCR product of the NS1 gene of dengue-2 virus isolated in China was inserted into the DNA of T-vector plasmid. DNAs of the positive clones, which were identified by using the blue/white spot and PCR, were digested with enzymes, indicating that the inserted fragments were the same sizes as those of the amplified NS1 genes. The result of the nucleotide sequence determined by dideoxy chain-termination method indicated that 5'-terminal nucleotide sequence of the amplified NS1 fragment was not changed.