Xue-Dong Yu

A Chimeric Dengue Virus Vaccine using Japanese Encephalitis Virus Vaccine Strain SA14-14-2 as Backbone Is Immunogenic and Protective against Either Parental Virus in Mice and Nonhuman Primates

ABSTRACT The development of a safe and efficient dengue vaccine represents a global challenge in public health. Chimeric dengue viruses (DENV) based on an attenuated flavivirus have been well developed as vaccine candidates by using reverse genetics. In this study, based on the full-length infectious cDNA clone of the well-known Japanese encephalitis virus live vaccine strain SA14-14-2 as a backbone, a novel chimeric dengue virus (named ChinDENV) was rationally designed and constructed by replacement with the premembrane and envelope genes of dengue 2 virus. The recovered chimeric virus showed growth and plaque properties similar to those of the parental DENV in mammalian and mosquito cells. ChinDENV was highly attenuated in mice, and no viremia was induced in rhesus monkeys upon subcutaneous inoculation. ChinDENV retained its genetic stability and attenuation phenotype after serial 15 passages in cultured cells. A single immunization with various doses of ChinDENV elicited strong neutralizing antibodies in a dose-dependent manner. When vaccinated monkeys were challenged with wild-type DENV, all animals except one that received the lower dose were protected against the development of viremia. Furthermore, immunization with ChinDENV conferred efficient cross protection against lethal JEV challenge in mice in association with robust cellular immunity induced by the replicating nonstructural proteins. Taken together, the results of this preclinical study well demonstrate the great potential of ChinDENV for further development as a dengue vaccine candidate, and this kind of chimeric flavivirus based on JE vaccine virus represents a powerful tool to deliver foreign antigens.

Co-circulation of two genotypes of dengue virus serotype 3 in Guangzhou, China, 2009

Dengue is emerging as the most important mosquito borne viral disease in the world. In mainland China, sporadic and large outbreaks of dengue illness caused by the four serotypes of dengue virus (DENV-1 to DENV-4) have been well documented. Guangdong province is the major affected area in China, and DENV-1 has dominantly circulated in Guangdong for a long time. In this study, a family cluster of DENV-3 infection in Guangzhou was described. Three cases were diagnosed as dengue fever based on clinical manifestation, serological and RT-PCR assays. Two DENV-3 strains were isolated in C6/36 cells and the complete genome sequences were determined. Phylogenetic analysis revealed that the new DENV-3 isolates from the family cluster were grouped within genotype III. Considering the fact that several DENV-3 strains within genotype V were also identified in Guangzhou in 2009, at least two genotypes of DENV-3 co-circulated in Guangzhou. Careful investigation and virological analysis should be warranted in the future.

Development of a real-time RT-PCR assay for a novel influenza A (H1N1) virus.

A pandemic caused by a novel influenza A virus (H1N1) poses a serious public health threat. In this study, a real-time reverse transcriptase PCR (RT-PCR) assay based on the hemagglutinin gene was developed that discriminates the novel H1N1 from swine influenza virus, seasonal H1N1/H3N2 virus and the highly pathogenic H5N1 avian influenza virus. The sensitivity of this assay was 0.2 50% tissue culture infective dose of virus and 200 copies of in vitro-transcribed target RNA. Three hundred and forty-eight clinical specimens from suspected H1N1 patients were tested using this assay, and forty-two (12.07%) were found to be positive. Tests using the real-time PCR assay recommended by WHO and virus isolation gave identical results. This sensitive and specific real-time RT-PCR assay will contribute to the early diagnosis and control of the emerging H1N1 influenza pandemic.

Development and characterization of the replicon system of Japanese encephalitis live vaccine virus SA14-14-2

BackgroundViral self-replicating sub-genomic replicons represent a powerful tool for studying viral genome replication, antiviral screening and chimeric vaccine development. Many kinds of flavivirus replicons have been developed with broad applications.FindingsThe replicon system of JEV live vaccine strain SA14-14-2 was successfully developed in this study. Two kinds of replicons that express enhanced green fluorescent protein (EGFP) and Renilla luciferase (R.luc) were constructed under the control of SP6 promoter, respectively. Robust EGFP and R.luc signals could be detected in the replicon-transfected BHK-21 cells. Furthermore, the potential effects of selected amino acids in the C-terminal of envelope protein on replication were characterized using the replicon system.ConclusionsOur results provide a useful platform not only for the study of JEV replication, but also for antiviral screening and chimeric vaccine development.

A DNA-based West Nile virus replicon elicits humoral and cellular immune responses in mice

While self-replicating, non-infectious subgenomic flavivirus replicons have been described, most of them are RNA transcripts under the control of an Sp6 or T7 promoter. In this study, using West Nile virus (WNV) as a model, a series of DNA-based reporter replicons under the control of a minimal cytomegalovirus (CMV) immediate-early promoter were constructed, and functional analysis showed that these reporter replicons replicate efficiently in mammalian cells. When the DNA-based WNV replicon was used to immunize mice, NS1-specific IgG antibodies and anti-WNV neutralizing antibodies were both induced. Additionally, immunization with this DNA-based WNV replicon induced high levels of lymphocyte proliferation and enhanced the secretion of IFN-γ. These results suggest that this type of DNA-based replicon can induce humoral and cellular immune responses in mice, indicating that this type of DNA-based replicon may serve as a useful platform for vaccine development and protein expression.

Complete Genome Sequence of a Dengue Virus Serotype 4 Strain Isolated in Guangdong, China

ABSTRACT Here we report the first complete genome sequence of a dengue virus serotype 4 genotype II strain, GZ30, isolated in Guangzhou, Guangdong Province, China, in 2010. The sequence information provided herein will help us to understand the molecular epidemiology of dengue virus and predict the risk of severe diseases in mainland China.

Complete Genome Sequence of a Chikungunya Virus Isolated in Guangdong, China

ABSTRACT Chikungunya virus belongs to the genus Alphavirus in the family Togaviridae. Here we report the complete genome sequence of a chikungunya virus strain, GD05/2010, isolated in 2010 from a patient with chikungunya fever in Guangdong, China. The sequence information is important for surveillance of this emerging arboviral infection in China.

Cross protection against lethal West Nile virus challenge in mice immunized with recombinant E protein domain III of Japanese encephalitis virus

Japanese encephalitis virus (JEV) and West Nile virus (WNV) are closely related mosquito-borne flaviviruses that cause severe encephalitic diseases with global impact. Cross protection among JEV and WNV has been previously described, and most cross reactive epitopes were identified within the domain II of E protein (EDII). In this study, the E protein domain III (EDIII) of JEV was successfully expressed in Escherichia coli, purified by a Ni-NTA column and characterized by Western blotting assay. Competitive inhibition assay showed that this recombinant JEV EDIII blocks the entry of JEV into BHK-21 cells. Mice immunized with the recombinant JEV EDIII developed high IgG and neutralizing antibodies titers against JEV. Most importantly, antibodies induced by JEV EDIII could neutralize WNV in vitro and partially protected mice against lethal WNV challenge. These results demonstrate that immunization with JEV EDIII induces cross-protective immunity against WNV infection, indicating a possible role of EDIII for the cross-protection among flavivirus.

Complete Genome Sequence of Dengue Virus Serotype 2 Cosmopolitan Genotype Strain in Guangdong, China

ABSTRACT Here we report the complete genome sequence of a dengue virus serotype 2 (DENV-2) strain, GZ40, isolated in Guangdong, China, in 2010. A phylogenetic analysis classified GZ40 into the Cosmopolitan genotype, while previous Chinese DENV-2 isolates belong to the Asian I genotype. The reemergence of the Cosmopolitan genotype of DENV-2 in China deserves further investigation.

Severe dengue due to secondary DENV-1 infection in Mainland China.

A 68-year-old female patient was referred for admission on 20 ctober 2011, with complaints of fever, severe myalgia and melena. he patient suddenly fell ill three days before with fever up to 9 ◦C, and dark stools were excreted on the morning of admission. cchymosis was observed on the left forearm and skin. Laboraory findings on peripheral blood examination indicated significant eukopenia, thrombocytopenia and decreased hemoglobin. The ourniquet test was positive. On abdominal ultrasonic examinaion, gall bladder thickening was observed without hepatomegaly Fig. 1 a). On the 4th day post onset, the fever disappeared but myalgia ecame more severe. The platelet count dropped to 12 × 109 cells/L n the 6th day post onset. The patient was managed empirically ith fluid therapy and Traditional Chinese Medicine. All the sympoms disappeared and laboratory parameters returned to normal fter 13 days hospitalization. Gall bladder wall returned normal Fig. 1b). Dengue specific IgM and IgG antibodies were both positive in cute phase sera. The IgM/IgG OD ratio is less than 1.2, indicatng secondary infection.1 PCR and DNA sequencing confirmed the ase was caused by DENV-1 (named GZ63). Phylogenetic analysis ased on the complete envelope gene (GenBank accession numer KC136240) revealed that GZ63 fell into genotype I with close elationship with isolates from Southeast Asia (Fig. 2). Dengue is not currently endemic in Mainland China. According o the new WHO/TDR guidelines issued in 2009,2–4 this is the first evere dengue case in Mainland China. The patient has a history f hypertension and diabetes for 10 years, with warning signals for evere disease. Severe bleeding involving continual black stools and apid decrease in hemoglobin led to a clinical diagnosis of severe engue. In this case, severe thrombocytopenia occurred without ncreased hematocrit. Interestingly, the patient presented with ypokalemia, which has been observed in some Chinese dengue ever patients.5 Additionally, the gall bladder thickening was