ABSTRACT
Objective To construct the pseudovirus containing nucleic acid(NA)fragments of Marburg virus,Zaire Ebola virus,Sudan Ebola virus,Lassa fever virus and Yellow fever virus by using a lentiviral vector system in order to provide a reference standard for the detection of the five viruses.Methods The gene fragments of the above five viruses were synthesized in vitro,connected into a single gene by fusion PCR technique,and cloned into lentiviral vectors with its auxiliary vector.After co-transfecting into 293T cells,the supernatants were collected on 48 h and 72 h post transfection. The naked NA was cleaned from the supernatants using DNase and RNase digestion before pseudotype virus was purified and concentrated.After the NA of the pseudotype virus,were extracted normal PCR and real-time PCR were conducted. Results Sequence analysis showed that the five target genes in vitro synthesis were properly connected and inserted into lentivirus vectors.Using the NA of the pseudotype virus as the template,both normal PCR and real-time PCR could sensitively amplify the target gene with the primers and probes of the above five,viruses respectively.The result indicated that the pseudovirus particles containing the five kinds of hemorrhagic fever virus target genes were successfully packaged. Conclusion The pseudovirus particles containing gene fragments of five viruses are constructed,which can be used as a common reference standard for NA detection.
ABSTRACT
Objective: To develop specific, sensitive and rapid TaqMan real-time quantification PCR assays for the detection of Zaire Ebola virus (ZEBOV) and Sudan Ebola virus (SEBOV). Methods: We selected primer and probe used by WHO reference laboratory, targeted the NP region of the viral genome, evaluated the specificity, sensitivity and reproducibility of each assay and compared it with original polymerase chain reation. Results: The Zaire assay could detect 34 copies of the artificial plasmid which contains ZEBOV target sequence, while the Sudan assay could detect 24 copies of the artificial plasmid which contains SEBOV target sequence. In addition, the assay was highly specific and reduplicated for Ebola virus, and more rapid and sensitive than the original one. Conclusion: We developed and evaluated TaqMan reverse transcription real time quantification PCR (RT-qPCR) assays for the rapid detection of ZEBOV and SEBOV. It seems that the assays are potentially suitable for diagnosis in the laboratories or field.
ABSTRACT
Zaire Ebola virus (EBOV) is a fatal human pathogen, with a high case fatality rate (CFR) averaging up to 78%. In March 2014, the World Health Organization (WHO) was made aware of a ZEBOV outbreak in rural Guinea, West Africa. Epidemiologic investigation linked the clinical and laboratory confirmed cases with the presumed first fatality of the outbreak in December 2013. EBOV from Guinea is a separate clade from other ZEBOV strains reported from the Democratic Republic of Congo (DRC) and Gabon. Since the outbreak in March, ZEBOV was also reported in Conakry, Guinea's capital and spread to other neighboring countries. In its largest outbreak, ZEBOV disease expanded through Guinea, Liberia, Sierra Leone, and Nigeria and to Spain, the USA, and the UK. The WHO declared the 2013-2015 West African Ebola epidemic a public health emergency of international concern considering its presumable capacity for further international spread. Early manifestations of EVD (Ebola virus disease) include a high fever, body aches, malaise, and fatigue. Severe diarrhea and other gastrointestinal manifestations such as vomiting were common, while bleeding was a more sporadic finding. The fatality rate was 43% and highest in patients aged > or = 45 years and the overall fitted mean incubation period was 10.3 days (95% CI 9.9~10.7). We present a review of the literature on the emergence of Ebola, and the epidemiologic, clinical, and laboratory records of patients in whom EVD was diagnosed in Sierra Leone, Guinea, Liberia, Mali, the USA, and Spain, its zoonotic origin, and the transmission of ZEBOV, as well as presenting original literature on the current Ebola outbreak.