Detection and subtyping of dengue 1–4 and yellow fever viruses by means of a multiplex RT‐nested‐PCR using degenerated primers
María Paz Sánchez‐SecoD. RosarioLourdes Mateos‐HernándezCristina DomingoKatia ValdésMauricio GuzmánAntônio Tenório
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Abstract:
Differential diagnosis of infections that cause similar diseases and may be active simultaneously in the same geographical areas is greatly needed. Dengue and yellow fever viruses (DENV and YFV) are transmitted by the same species of mosquito and both can cause haemorrhagic fever symptoms. These viruses are active mainly in regions where expensive and sophisticated technologies are not available. Our objective was to develop a simple, reliable and easy-to-perform method to detect and identify these viruses.We slightly modified a generic RT-PCR able to detect the mentioned viruses and other members of this genus: specific primers for each one of these viruses were designed and included in the nested reaction instead of one of the generic ones. The reaction was optimized and viruses are amplified giving rise to bands of different sizes distinguishable in agarose gels.This test is able to detect and identify the four DENVs and YFV to a high level of sensitivity and specificity and can be used with clinical samples. This simple, reliable and easy-to-perform method able to detect and identify dengue 1-4 and YFV can be used in poor endemic countries.Keywords:
Subtyping
Multiplex
Flavivirus
Yellow fever vaccine
West Nile virus
Antiviral Therapy
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Zika virus (ZIKV) belongs to the flaviviruses (family Flaviviridae), which includes dengue, yellow fever, West Nile, and Japanese encephalitis viruses. Zika virus was isolated in 1947, in the Zika forest near Kampala, Uganda, from one of the rhesus monkeys used as sentinel animals in a yellow fever research program.
Zika Virus
Flavivirus
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Flavivirus
Endemic diseases
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Zika virus (ZIKV) is a member of the Family Flaviviridae. The virus spreads through mosquitoes, like Aedes aegypti and A. albopictus. The virus was isolated in 1947 and the virus is derived from the Ziika Forest of Uganda. The virus is antigenically related to the dengue, yellow fever, Japanese encephalitis, and West Nile viruses. Zika virus causes Zika fever which causes only mild symptoms as similar to the dengue fever.
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Flavivirus
Aedes albopictus
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West Nile fever is a viral fever caused by the West Nile virus (WNV). In 1937, West Nile virus is initially
determined in the West Nile district of Uganda. The virus is meanly single stranded RNA virus. The virus has
belonged to the family of Flaviviridae, and the genus is Flavivirus, which also carrier of the Zika virus, Yellow
fever virus, Dengue virus, St. Louis encephalitis, Japanese encephalitis. The Mosquito is the main vector of
the West Nile virus which spread the disease of human population. About 80% infected people have no
symptoms, 20% people are suffering from fever, headache, vomiting, and also rushs.1% people are suffering
from serious condition. Specific vaccine is not available for West Nile fever. West Nile fever is controlled by
the use of mosquito net, ware full cloths, various types of mosquito relax spray. The further discussion is
transmission, signs and symptoms, diagnosis, management and control will be carried out below.
Flavivirus
West Nile virus
Viral encephalitis
Veterinary virology
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The Zika virus belongs to the family Flaviviridae, which includes the well-known viruses associated with dengue, yellow fever, Nile fever in addition to the Japanese encephalitis virus. The discovery dates back to 1947 in Uganda, Africa, from an enzootic cycle between primates and wild Aedes aegypti, culminating in infections in humans [1].
Zika Virus
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Flavivirus
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Dengue virus, a mosquito-borne flavivirus, is one of the most formidable public health threats in tropical and subtropical regions. As yet, there is no licensed vaccine to protect against the disease. A chimeric yellow fever (YF) 17D/dengue (DEN) type 1 virus was constructed by replacing the pre-membrane and envelope genes of YF 17D virus with those from DEN 1 VeMir95 virus, a Venezuelan isolate. The chimeric YF 17D/DEN 1 VeMir95 virus was regenerated from full-length infectious clones stably propagated in Escherichia coli by transfection of Vero cells with in vitro transcribed RNA. The chimeric virus proliferated efficiently in Vero cells ( approximately 6.6 log(10) plaque-forming units/ml). The chimeric virus was not neurovirulent to 3-week-old Swiss Webster mice inoculated by the intracerebral route, in contrast to the YF 17DD vaccine strain that was lethal for 90% of the mice. The YF 17D/DEN 1 virus at Passage 6 was more attenuated for rhesus monkeys than the YF 17DD commercial vaccine after intracerebral inoculation according to the standard neurovirulence test. This virus is a potential candidate to be included in a tetravalent DEN vaccine formulation. The availability of the cloned cDNA allows further structure/function studies on the viral envelope.
Flavivirus
Vero cell
Dengue vaccine
Plaque-forming unit
Yellow fever vaccine
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Viral encephalitis
Flavivirus
Chimera (genetics)
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ABSTRACT We previously reported a simple subtyping method, restriction site-specific PCR (RSS-PCR), for dengue virus serotypes 2 and 3; here we describe its application for subtyping dengue virus serotypes 1 and 4. Three major RSS-PCR types were observed for dengue virus serotype 1 and two types were observed for dengue virus serotype 4, in agreement with previous strain classifications based on sequence analysis. Because of its simplicity, this method is amenable to rapid subtyping and application to epidemiological studies of dengue in countries where dengue is endemic.
Subtyping
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