Objective To perform quality control in live attenuated yellow fever vaccine(chicken embryo cell)virus seed bank at the genomic level using the new generation Illumina/Solexa sequencing platform.Methods The live attenuated yellow fever vaccine strain YF17D-204 was inoculated into primary chicken embryo cells,and the chicken embryo cell adapted strains of live attenuated yellow fever vaccine were screened to establish YFV17D-CEC tertiary virus seed bank. The genome RNA of virus seeds was extracted,and the RNA library was prepared. The new generation Illumina/Solexa sequencing platform was used for high-throughput RNA sequencing. The whole genome nucleic acid sequence of yellow fever virus was systematically analyzed by using biological softwares such as FastQC,Trimmomatic,SPAdes,GapFiller,PrInSeS-G,Prokka,RepeatMasker,CRT,NCBI Blast~+,KAAS,HMMER3,TMHMM,SignalP,LipoP,ProtCamp and MegAlign.Results The whole genome of YFV17D-CEC tertiary virus seed bank contained 10 862 nucleotides,including an open reading frame(ORF)from 119 to 10 354(10 236 bp),encoding 3 412 amino acids. Sequence alignment analysis showed that the sequence of YF17D-CEC tertiary virus seed bank was 100% identical with YFV17D RKI(JN628279.1),YF/Vaccine/USA/Sanofi-Pasteur-17D-204/UF795AA/YFVax(JX503529.1)and YFV17D-204(KF769015.1),and no mutation occurred in the whole genome of the tertiary virus seed bank. Comparison of the sequences of different live attenuated yellow fever vaccine strains showed that yellow fever virus had multiple polymorphic sites.Conclusion YFV17DCEC has good genetic stability in primary chicken embryo cells. High-throughput RNA sequencing technology can quickly detect the whole genome information of YF17D-CEC virus seed bank,and the sequence analysis data can be used in the gene level quality control of yellow fever vaccine virus seed banks.
High-throughput RNA sequencing ; Live attenuated yellow fever vaccine ; Gene expression ; Virus seed bank ; Quality control

A rapid diagnostic test (RDT) kit was developed to detect non-structural protein 1 (NS1) of yellow fever virus (YFV) using monoclonal antibody. NS1 protein was purified from the cultured YFV and used to immunize mice. Monoclonal antibody to NS1 was selected and conjugated with colloidal gold to produce the YFV NS1 RDT kit. The YFV RDTs were evaluated for sensitivity and specificity using positive and negative samples of monkeys from Brazil and negative human blood samples from Korea. Among monoclonal antibodies, clones 3A11 and 3B7 proved most sensitive, and used for YFV RDT kit. Diagnostic accuracy of YFV RDT was fairly high; Sensitivity was 0.0% and specificity was 100% against Dengue viruses type 2 and 3, Zika, Chikungunya and Mayaro viruses. This YFV RDT kit could be employed as a test of choice for point-of-care diagnosis and large scale surveys of YFV infection under clinical or field conditions in endemic areas and on the globe.
Animals ; Antibodies, Monoclonal ; Brazil ; Clone Cells ; Dengue Virus ; Diagnosis ; Diagnostic Tests, Routine ; Gold Colloid ; Haplorhini ; Humans ; Korea ; Mice ; Point-of-Care Systems ; Sensitivity and Specificity ; Yellow fever virus ; Yellow Fever

We developed a Rapid Diagnostic Test (RDT) kit for detecting IgG/IgM antibodies against Zika virus (ZIKV) using monoclonal antibodies to the envelope (E) and non-structural protein 1 (NS1) of ZIKV. These proteins were produced using baculovirus expression vector with Sf9 cells. Monoclonal antibodies J2G7 to NS1 and J5E1 to E protein were selected and conjugated with colloidal gold to produce the Zika IgG/IgM RDT kit (Zika RDT). Comparisons with ELISA, plaque reduction neutralization test (PRNT), and PCR were done to investigate the analytical sensitivity of Zika RDT, which resulted in 100% identical results. Sensitivity and specificity of Zika RDT in a field test was determined using positive and negative samples from Brazil and Korea. The diagnostic accuracy of Zika RDT was fairly high; sensitivity and specificity for IgG was 99.0 and 99.3%, respectively, while for IgM it was 96.7 and 98.7%, respectively. Cross reaction with dengue virus was evaluated using anti-Dengue Mixed Titer Performance Panel (PVD201), in which the Zika RDT showed cross-reactions with DENV in 16.7% and 5.6% in IgG and IgM, respectively. Cross reactions were not observed with West Nile, yellow fever, and hepatitis C virus infected sera. Zika RDT kit is very simple to use, rapid to assay, and very sensitive, and highly specific. Therefore, it would serve as a choice of method for point-of-care diagnosis and large scale surveys of ZIKV infection under clinical or field conditions worldwide in endemic areas.
Antibodies ; Antibodies, Monoclonal ; Baculoviridae ; Brazil ; Cross Reactions ; Dengue Virus ; Diagnosis ; Diagnostic Tests, Routine ; Enzyme-Linked Immunosorbent Assay ; Flavivirus ; Gold Colloid ; Hepacivirus ; Immunoglobulin G ; Immunoglobulin M ; Korea ; Methods ; Neutralization Tests ; Point-of-Care Systems ; Polymerase Chain Reaction ; Reagent Kits, Diagnostic ; Sensitivity and Specificity ; Sf9 Cells ; Yellow Fever ; Zika Virus

We have examined isolation and identification protocols for three virus simulant candidates to biological warfare agents. MS2 phage, a simulant for yellow fever virus and Hantaan virus, was propagated using as a host an E. coli strain with F pilus. MS2 phage genome was examined by reverse transcription and polymerase chain reaction (RT-PCR). Coat protein of the phage preparation was examined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometric analysis. Cydia pomonella granulosis virus (CpGV) is a virus simulant candidate to smallpox virus. CpGV was isolated from a commercialized CpGV pellet. In this study, we developed new isolation and identification protocols for CpGV. One disadvantage of using CpGV is that it is not easy to determine viability of the virus. Here, we have included T4 phage as an alternative. We established a high titer production protocol and developed an easy genome identification protocol that does not require purified phage DNA. Stability of these virus preparations was also examined under various storage conditions. When the virus preparations were not subjected to freeze drying, MS2 phage was most stable when it was stored in liquid nitrogen but unstable at 4℃. In contrast, T4 phage was most stable when it was stored at 4℃. CpGV was stable at −20℃ but not at 4℃. Stability during or after freeze drying was also investigated. The result showed that 70~80% MS2 survived the freeze drying process. In contrast, only about 15% of T4 phage survived during the freeze drying. CpGV was found to be degraded during freeze drying.
Bacteriophage T4 ; Bacteriophages ; Biological Warfare Agents ; DNA ; Electrophoresis ; Freeze Drying ; Genome ; Granulovirus ; Hantaan virus ; Levivirus ; Nitrogen ; Polymerase Chain Reaction ; Reverse Transcription ; Variola virus ; Yellow fever virus

Zika virus (ZIKV) was spread to both eastward and westward from Uganda where the virus was identified approximately in 1947 by a group of arbovirus researchers. In 2015, ZIKV reached Americas with major outbreaks in Brazil. Most countries with mosquito transmitted ZIKV infection are located in tropical and subtropical areas, where ZIKV is endemic with other flaviviruses, including JEV, dengue and yellow fever virus. Approximately 40 countries in Central and South Americas and territories in South Pacific Islands and South East Asia show autochthonous ZIKV endemics. American lineage of ZIKV is known significantly to be mutated in susceptibility to host and in pathogenicity from Asian and Asian lineages approximately since 2014. Early and specific identification of ZIKV infection is very important for the effective management of patients. First of all, optimal collection of specimens for the laboratory diagnosis is required for both nucleic acid testing (NAT) and serological tests. Specimens for NAT tests and serological tests should be determined by the available laboratory resources, work-flow in each laboratory and the geographic areas of specimen collected in addition to days after showing symptoms. Testing strategy for specific differentiation among flaviviruses will vary depending on the prevalence of viruses known to be circulating in the area where the patients were exposed. NAT will be employed for the patients presenting with onset of symptoms less than 7 days. Advanced diagnostic technologies should be continuously developed for the increase of specificity and sensitivity of ZIKV diagnosis.
Americas ; Arboviruses ; Asian Continental Ancestry Group ; Biology* ; Brazil ; Clinical Laboratory Techniques* ; Culicidae ; Dengue ; Diagnosis ; Disease Outbreaks ; Epidemiology* ; Far East ; Flavivirus ; Humans ; Pacific Islands ; Prevalence ; Sensitivity and Specificity ; Serologic Tests ; South America ; Uganda ; Virulence ; Yellow fever virus ; Zika Virus*

Zika virus was first isolated in from nonhuman primate in 1947. It is in the genus Flavivirus, closely related to other flavivirus like Dengue, West Nile, Yellow fever and Japanese encephalitis virus. Since 2007 epidemic in Yap island, zika virus infections had spread to the countries in Micronesia and South Pacific. In 2015, Zika virus outbreak occurred in Brazil and now more than 40 countries in American continents reported autochthonous infection. The virus is transmitted mainly by Ae. aegypti mosquito with many other Aedes mosquito species known as vector. Recently, Zika virus infection is known to cause severe neurological complications and congenital malformation. In this paper, we will review current knowledge on Zika virus history, biology, clinical characteristics and preventive method.
Aedes ; Biology ; Brazil ; Culicidae ; Dengue ; Encephalitis Virus, Japanese ; Flavivirus ; Methods ; Microcephaly ; Micronesia ; Primates ; Yellow Fever ; Zika Virus Infection* ; Zika Virus*

Zika virus (ZIKV) is an arthropod-borne member of the genus Flavivirus, closely related to the dengue, West Nile, Japanese encephalitis, and yellow fever viruses and is transmitted by Aedes spp. mosquitoes. It has emerged explosively since 2007 to cause a series of epidemics in Micronesia, the South Pacific, and most recently the Americas. Following the first detection of ZIKV on the American continent, autochthonous ZIKV transmission has been confirmed throughout Central and South America. The unprecedented numbers of people infected during recent outbreaks in the South Pacific and the Americas may have resulted in enough ZIKV infections to notice patterns of the associated incidence of congenital microcephaly, Gillain-Barre symdrome, and acute diffuse encephalomyelitis. Here we review the history, emergence, biology, transmission, and control strategies for the ongoing outbreak through vector-centric approaches on ZIKV to date.
Aedes ; Americas ; Arboviruses ; Biology ; Culicidae ; Dengue ; Disease Outbreaks ; Encephalitis, Japanese ; Encephalomyelitis ; Flavivirus ; Incidence ; Microcephaly ; Micronesia ; South America ; Yellow fever virus ; Zika Virus*

Antibodies, Viral ; blood ; China ; epidemiology ; Humans ; Yellow Fever ; epidemiology ; Yellow fever virus ; immunology

Animals ; Genome, Viral ; Humans ; Viral Proteins ; genetics ; metabolism ; Yellow Fever ; virology ; Yellow fever virus ; genetics ; metabolism

This report describes a case of yellow fever vaccine-associated viscerotropic disease (YEL-AVD) that occurred after vaccination in a 23-year-old male. Seven days after vaccination, our patient presented with fever, myalgia, and nausea. The IgM enzyme-linked immunosorbent assay (ELISA) for yellow fever virus was positive. After a 24 day hospitalization, he recovered and was discharged. Yellow fever is a viral hemorrhagic febrile illness caused by a flavivirus and transmitted by mosquitoes. The clinical presentation ranges from a mild febrile illness to a serious infection, leading to hepatic and renal failure, myocardial injury, hemorrhage, and shock, with a case fatality rate of 20-30%. Because yellow fever is a potentially fatal disease, vaccination is encouraged for people traveling to high-risk areas. Although considered a safe vaccine, severe adverse reactions have been reported. In 2001, rare, but severe, acute viscerotropic disease following vaccination was first described. We report the case of a 23-year-old male with fever and hepatitis following vaccination with 17D yellow fever vaccine.
Culicidae ; Enzyme-Linked Immunosorbent Assay ; Fever ; Flavivirus ; Hemorrhage ; Hepatitis ; Hospitalization ; Humans ; Immunoglobulin M ; Male ; Nausea ; Renal Insufficiency ; Shock ; Vaccination ; Yellow Fever ; Yellow Fever Vaccine ; Yellow fever virus ; Young Adult