The first complete genomic characterization of an Amur virus isolate from China.

Amur virus (AMRV) is a member of the genus Hantavirus in the family Bunyaviridae. In this study, we determined for the first time the complete genome sequence of the AMRV H8205 strain, which was isolated from a patient with hemorrhagic fever with renal syndrome (HFRS) in China. The complete nucleotide sequence of the S segment of AMRV H8205 is 1699 nt long, with a 5' noncoding region (5'NC) of 36 nt, followed by a coding sequence of 1290 nt and a 3'NC of 373 nt. The complete sequence of the M segment is 3615 nt long, with a 5'NC of 40 nt, followed by a coding sequence of 3408 nt and a 3'NC of 167 nt. The complete sequence of the L segment is 6536 nt long, with a 5'NC of 37 nt, followed by a coding sequence of 6453 nt and a 3'NC of 40 nt. The major open reading frame (ORF) of each of the three segments (S, nt 37-1326; M, nt 41-3445; L, nt 38-6490) has a coding capacity of 430 aa, 1135 aa, 2151 aa, respectively. Phylogenetic analysis of the nucleotide sequences using the NJ method indicated that H8205 virus, together with the Amur strains isolated from Far-Eastern Russia and Korea, forms a well-supported lineage. Our results will provide insights into the genetic diversity of hantaviruses (HNTV).

The confirmation of three repeated sequence elements in the 3' untranslated region of Chikungunya virus.

In this study, the complete genomic nucleotide sequence of Chikungunya virus (CHIKV) strain S27 African prototype was determined and three 21 nucleotides repeated sequence elements (RSEs) at positions 11398-11418, 11533-11553, and 11620-11640 in the 3' untranslated region (3'UTR) were confirmed. In addition, the 3'UTRs of all CHIKV strains deposited in GenBank were analyzed. The results displayed that the majority of the CHIKV strains consisted of the three 21 nucleotides RSEs in the 3'UTRs, and the third RSE was the most conservative. The conservation of the three RSEs of 21 nucleotides within the 3'UTR of CHIKV genome may play an important role on the virus replication cycle.

Complete genomic characterization of two tick-borne encephalitis viruses isolated from China.

Tick-borne encephalitis (TBEV) is prevalent over a wide area of the Eurasian continent. TBE viruses cause severe encephalitis in humans, with serious sequelae, and have a significant impact on public health in these endemic regions. To gain insight into genetic evolution of tick-borne encephalitis virus (TBEV) in China, the complete genomic sequences of two TBEV strains Senzhang and MDJ01, which were isolated in 1953 and 2001 respectively, were characterized. The complete genome sequences of two strains were all consist 10,784 nucleotides and there are 364 nucleotides deletion in the 3' nontranslated region. Compared with other TBEV strains, homology range from 85.2% (Zausaev) to 99.6% (MDJ02 and MDJ03) on the level of nucleotide. Phylogenetic trees based on the complete genome, open reading frame and E gene nucleotide sequences all showed that the strains Senzhang and MDJ01 belong to Far-Eastern subtype and cluster with other Chinese TBEV strains. All these implied that TBEVs prevalent in China were highly conservative, other measurement should be taken to improve protective efficacy of present vaccine.

Complete genome sequence analysis of tick-borne encephalitis viruses isolated in northeastern China.

Tick-borne encephalitis virus (TBEV) causes lethal encephalitis in humans, posing a growing public-health problem in many European and Asian countries. TBEV is currently endemic in northeastern China, but the complete genome sequences of Chinese TBEV strains have not been reported. During a TBE outbreak in 2010 in Mudanjiang City, Heilongjiang Province, China, two TBEV strains were isolated from serum samples of two patients, and the complete sequences were determined and compared with other known TBEV strains. Both Mudanjiang isolates consisted of 10,774 nucleotides and encoded a single open reading frame coding for a polyprotein of 3414 amino acids, and a unique deletion of 364 nucleotides in the 3' untranslated region (UTR) was recorded. Phylogenetic analysis based on the amino acid sequence of the E protein and the nucleotide sequence of the 3'UTR revealed that the Mudanjiang isolates are closely related to the Senzhang and Sofjin-HO strains and belong to the Far Eastern subtype of TBEV. These findings provide insight into the evolutionary relationships among Chinese TBEV isolates and are useful for laboratory diagnosis and vaccine development for TBEV.

Prokaryotic expression and purification of HA1 and HA2 polypeptides for serological analysis of the 2009 pandemic H1N1 influenza virus.

Hemagglutinin (HA) is an important influenza virus surface antigen that is highly topical in influenza research. In the present study, the genes encoding the HA1 and HA2 proteins from the 2009 pandemic influenza virus H1N1 (A/California/04/2009(H1N1)) were cloned into a prokaryotic expression plasmid pCold-TF, and soluble fusion proteins containing H1N1 HA1 and HA2 were produced by transformed Escherichia coli. Western blot assays were used to examine the immunoreactivity of the recombinant proteins using polyclonal and monoclonal antibodies derived against the whole virus A/California/04/2009(H1N1). Recombinant protein immunoreactivity was also analyzed qualitatively by ELISA and hemagglutination inhibition using human serum samples. These results will aid future immunological and serological studies of the 2009 pandemic H1N1 virus HA.

Monoclonal antibody induced with inactived EV71-Hn2 virus protects mice against lethal EV71-Hn2 virus infection.

BACKGROUND: Enterovirus 71 (EV71) is a viral pathogen that belongs to the Picornaviridae family, EV71-infected children can develop severe neurological complications leading to rapid clinical deterioration and death. RESULTS: In this study, several monoclonal antibodies (MAbs) were produced by immunizing mice with the inactived EV71 Henan (Hn2) virus strain. The isolated MAbs were characterised by in vitro neutralizing analysis and peptide ELISA. ELISA assay showed that the neutralizing monoclonal antibody 4E8 specifically reacted with synthetic peptides which contain amino acid 240-250 and 250-260 of EV71 VP1. The in vivo protection assay showed that 4E8 can protect two-day-old BALB/c mice against the lethal challenge of EV71 virus. CONCLUSION: The MAb 4E8 could be a promising candidate to be humanized and used for treatment of EV71 infection.

Human neutralizing Fab molecules against severe acute respiratory syndrome coronavirus generated by phage display.

Human recombinant Fab fragments specific for the spike protein of severe acute respiratory syndrome coronavirus (SARS-CoV) were screened from a human Fab library, which was generated from RNAs from peripheral lymphocytes of convalescent SARS patients. Among 50 randomly picked clones, 12 Fabs specially reacted with S protein by an enzyme-linked immunosorbent assay. The microneutralizing test showed that one clone, designated M1A, had neutralizing activity on Vero E6 cells against SARS-CoV. DNA sequence analysis indicated that the light- and heavy-chain genes of M1A Fab belong to the kappa2a and 4f families, respectively. A neutralizing test on purified M1A demonstrated that 0.5 mg/ml of M1A completely inhibited SARS-CoV activity, with an absence of cytopathic effect for 7 days. Real-time fluorescence reverse transcription-PCR also proved the neutralizing capacity of M1A. These data showed that the number of virus copies was significantly reduced in the M1A-treated group, suggesting an important role for M1A in passive immunoprophylaxis against the SARS virus.

Immunogenicity and protective efficacy in monkeys of purified inactivated Vero-cell SARS vaccine.

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.

Antibody responses to individual proteins of SARS coronavirus and their neutralization activities.

A novel coronavirus, the severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), was identified as the causative agent of SARS. The profile of specific antibodies to individual proteins of the virus is critical to the development of vaccine and diagnostic tools. In this study, 13 recombinant proteins associated with four structural proteins (S, E, M and N) and five putative uncharacterized proteins (3a, 3b, 6, 7a and 9b) of the SARS-CoV were prepared and used for screening and monitoring their specific IgG antibodies in SARS patient sera by protein microarray. Antibodies to proteins S, 3a, N and 9b were detected in the sera from convalescent-phase SARS patients, whereas those to proteins E, M, 3b, 6 and 7a were undetected. In the detectable specific antibodies, anti-S and anti-N were dominant and could persist in the sera of SARS patients until week 30. Among the rabbit antisera to recombinant proteins S3, N, 3a and 9b, only anti-S3 serum showed significant neutralizing activity to the SARS-CoV infection in Vero E6 cells. The results suggest (1) that anti-S and anti-N antibodies are diagnostic markers and in particular that S3 is immunogenic and therefore is a good candidate as a subunit vaccine antigen; and (2) that, from a virus structure viewpoint, the presence in some human sera of antibodies reacting with two recombinant polypeptides, 3a and 9b, supports the hypothesis that they are synthesized during the virus cycle.

Study on the resistance of severe acute respiratory syndrome-associated coronavirus.

In this study, the persistence of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) was observed in feces, urine and water. In addition, the inactivation of SARS-CoV in wastewater with sodium hypochlorite and chlorine dioxide was also studied. In vitro experiments demonstrated that the virus could only persist for 2 days in hospital wastewater, domestic sewage and dechlorinated tap water, while 3 days in feces, 14 days in PBS and 17 days in urine at 20 degrees C. However, at 4 degrees C, the SARS-CoV could persist for 14 days in wastewater and at least 17 days in feces or urine. SARS-CoV is more susceptible to disinfectants than Escherichia coli and f2 phage. Free chlorine was found to inactivate SARS-CoV better than chlorine dioxide. Free residue chlorine over 0.5 mg/L for chlorine or 2.19 mg/L for chlorine dioxide in wastewater ensures complete inactivation of SARS-CoV while it does not inactivate completely E. coli and f2 phage.

[Expression of tick-borne encephalitis virus prM-E protein in insect cells and studies on its antigenicity].

BACKGROUND: To express the prM-E protein in Sf9 cells, and lay a basis for further study on the function of the viral proteins and development of specific diagnostic reagents. METHODS: The recombinant prM-E protein of tick-borne encephalitis virus was expressed in insect cell Sf9 by RT-PCR amplification of prM-E gene, construction of donor plasmid of Bac-to-Bac baculovirus expression system, homologous recombination of donor plasmid with bacmid DNA at the site of Tn7 and transfection of insect cell Sf9. RESULTS: Recombinant subviral particles, about 30 nm in diameter, consisting of prM-E were observed by electron microscope in the supernatant of infected cells, which indicated that infected cells released virus-like particles (VLPs) into the culture medium. The results of Western-blot and the indirect immunofluorescence assay (IFA) showed that the recombinant proteins retained antigenic and conformational structures similar to those of native virus proteins. Using the recombinant prM-E as antigens to detect samples of patient sera by ELISA and IFA, all of 16 sera from patients with tick-borne encephalitis were positive and all of 6 sera from other patients were negative. CONCLUSION: The prM-E protein expressed in insect cells retains good antigenicity.

[Comparison and discrimination of the biological characteristics between West Nile virus and Japanese encephalitis virus].

BACKGROUND: To compare the biological characteristics of West Nile virus (WNV) and Japanese encephalitis virus (JEV), including cells sensitivity, pathogenicity, viral morphology, as well as the results of immunological and molecular biological detection. METHODS: Cytopathic effect (CPE) and pathogenicity were observed in C6/36 cells and in suckling mice inoculated intracerebrally with the WNV or JEV, respectively. The sliced tissue samples for electron microscopic examination were prepared for the morphologic observation of the viruses. Serum antibody to WNV or JEV was detected using indirect immunofluorescence assay (IFA), and the viral RNA was analyzed by RT-PCR method. RESULTS: WNV or JEV-caused CPE was characterized by cell fusion and cell shedding, respectively. There was no significant difference in the pathogenicity to suckling mice between WNV and JEV. The morphologic observation showed that the shape and size of the two virions were similar. WNV and JEV were found to have antigenic cross-reactivity. The viral RNA could be detected from both WNV and JEV samples with universal primer set, but only nucleoside fragments of corresponding virus could be amplified when specific primers were used. CONCLUSION: CPE in C6/36 cell and detection of the viral RNA should be useful in discrimination of WNV and JEV, and simultaneously examining the titers of serum antibodies against WNV and JEV may be helpful to diagnosis of infection with these agents.

[Ultrastructural characteristics of SARS associated virus in infected cells].

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.

A complete sequence and comparative analysis of a SARS-associated virus (Isolate BJ01).

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).

A genome sequence of novel SARS-CoV isolates: the genotype, GD-Ins29, leads to a hypothesis of viral transmission in South China.

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.