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      Phylogenetic characterization of circulating Dengue and Alkhumra Hemorrhagic Fever viruses in western Saudi Arabia and lack of evidence of Zika virus in the region: A retrospective study, 2010‐2015

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          Abstract

          Flaviviruses represent a global public health concern. They consist of ∼70 viruses with almost half of them causing human diseases with unspecified febrile illnesses. Cities in western Saudi Arabia are endemic for viruses (DENV) with sporadic infections due to Alkhumra hemorrhagic fever virus (AHFV). They also represent a major destination for travelers coming for annual religious pilgrimages (Hajj and Umrah) from all over the world. However, whether other flaviviruses are circulating is not known because of the limited number of surveillance studies. Here, we retrospectively screened 690 samples for flaviviruses in samples from patients with unexplained febrile illnesses between 2010 and 2015 in western Saudi Arabia using a pan‐flaviviruses RT‐PCR assay. Despite Zika virus RNA was not detected, this study confirms circulation and/or sporadic spread of DENV‐2, DENV‐3, and AHFV, higher prevalence of DENV‐2, and a role for visitors from DENV endemic countries in DENV importation into the Kingdom. Further analysis also showed very low genetic diversity of AHFV confirming its slow microevolution. Accordingly, continuous and prospective surveillance for flaviviruses using such assay are warranted in Saudi Arabia which receives millions of Muslims annually to implement effective control measures in light of the global widespread and outbreaks of several flaviviruses.

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          Most cited references38

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          Rapid detection and quantification of RNA of Ebola and Marburg viruses, Lassa virus, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, dengue virus, and yellow fever virus by real-time reverse transcription-PCR.

          Viral hemorrhagic fevers (VHFs) are acute infections with high case fatality rates. Important VHF agents are Ebola and Marburg viruses (MBGV/EBOV), Lassa virus (LASV), Crimean-Congo hemorrhagic fever virus (CCHFV), Rift Valley fever virus (RVFV), dengue virus (DENV), and yellow fever virus (YFV). VHFs are clinically difficult to diagnose and to distinguish; a rapid and reliable laboratory diagnosis is required in suspected cases. We have established six one-step, real-time reverse transcription-PCR assays for these pathogens based on the Superscript reverse transcriptase-Platinum Taq polymerase enzyme mixture. Novel primers and/or 5'-nuclease detection probes were designed for RVFV, DENV, YFV, and CCHFV by using the latest DNA database entries. PCR products were detected in real time on a LightCycler instrument by using 5'-nuclease technology (RVFV, DENV, and YFV) or SybrGreen dye intercalation (MBGV/EBOV, LASV, and CCHFV). The inhibitory effect of SybrGreen on reverse transcription was overcome by initial immobilization of the dye in the reaction capillaries. Universal cycling conditions for SybrGreen and 5'-nuclease probe detection were established. Thus, up to three assays could be performed in parallel, facilitating rapid testing for several pathogens. All assays were thoroughly optimized and validated in terms of analytical sensitivity by using in vitro-transcribed RNA. The >or=95% detection limits as determined by probit regression analysis ranged from 1,545 to 2,835 viral genome equivalents/ml of serum (8.6 to 16 RNA copies per assay). The suitability of the assays was exemplified by detection and quantification of viral RNA in serum samples of VHF patients.
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            Comparison of flavivirus universal primer pairs and development of a rapid, highly sensitive heminested reverse transcription-PCR assay for detection of flaviviruses targeted to a conserved region of the NS5 gene sequences.

            Arthropod-transmitted flaviviruses are responsible for considerable morbidity and mortality, causing severe encephalitic, hemorrhagic, and febrile illnesses in humans. Because there are no specific clinical symptoms for infection by a determined virus and because different arboviruses could be present in the same area, a genus diagnosis by PCR would be a useful first-line diagnostic method. The six published Flavivirus genus primer pairs localized in the NS1, NS3, NS5, and 3' NC regions were evaluated in terms of specificity and sensitivity with flaviviruses (including the main viruses pathogenic for humans) at a titer of 10(5) 50% tissue culture infectious doses (TCID(50)s) ml(-1) with a common identification step by agarose gel electrophoresis. Only one NS5 primer pair allowed the detection of all tested flaviviruses with the sensitivity limit of 10(5) TCID(50)s ml(-1). Using a heminested PCR with new primers designed in the same region after an alignment of 30 different flaviviruses, the sensitivity of reverse transcription-PCR was improved and allowed the detection of about 200 infectious doses ml(-1) with all of the tick- and mosquito-borne flaviviruses tested. It was confirmed that the sequenced amplified products in the NS5 region allowed predictability of flavivirus species by dendrogram, including the New York 99 West Nile strain. This technique was successfully performed with a cerebrospinal fluid sample from a patient hospitalized with West Nile virus encephalitis.
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              A real-time RT-PCR method for the universal detection and identification of flaviviruses.

              Here we describe an optimized molecular protocol for the universal detection and identification of flaviviruses. It combines the convenient real-time polymerase chain reaction (PCR) format with a broad spectrum of flavivirus detection. This assay, based on the amplification of a 269-272 nt (depending on the flavivirus tested) region at the N terminal end of the NS5 gene, enabled the amplification of 51 flavivirus species and 3 tentative species. Sequencing of the amplicons produced by reverse transcriptase (RT)-PCR permitted the reliable taxonomic identification of flavivirus species by comparison with reference sequences available in databases, using either the BLASTN algorithm or a simple phylogenetic reconstruction. The limit of detection of the assay (2-20,500 copies/reaction depending on the virus tested) allowed the detection of different flaviviruses from a series of human sera or veterinary samples. Altogether, the characteristics of this technique make it a good candidate for the identification of previously identified flaviviruses in cell culture and the investigation of field samples, and also a promising tool for the discovery and identification of new species, including viruses distantly related to "classical" arthropod-borne flaviviruses.
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                Author and article information

                Contributors
                eazhar@kau.edu.sa
                amhashem@kau.edu.sa
                Journal
                J Med Virol
                J. Med. Virol
                10.1002/(ISSN)1096-9071
                JMV
                Journal of Medical Virology
                John Wiley and Sons Inc. (Hoboken )
                0146-6615
                1096-9071
                08 March 2017
                August 2017
                : 89
                : 8 ( doiID: 10.1002/jmv.v89.8 )
                : 1339-1346
                Affiliations
                [ 1 ] Special Infectious Agents Unit, King Fahd Medical Research Center King Abdulaziz University Jeddah Saudi Arabia
                [ 2 ] UMR “Emergence des Pathologies Virales“ (EPV: Aix‐Marseille university—IRD 190—Inserm 1207—EHESP) Marseille France
                [ 3 ] Institut hospitalo‐universitaire Méditerranée infection APHM Public Hospitals of Marseille Marseille France
                [ 4 ] Faculty of Applied Medical Sciences, Department of Medical Laboratory Technology King Abdulaziz University Jeddah Saudi Arabia
                [ 5 ] Faculty of Medicine, Department of Medical Microbiology and Parasitology King Abdulaziz University Jeddah Saudi Arabia
                Author notes
                [*] [* ] Correspondence

                Esam I. Azhar and Anwar M. Hashem, Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia.

                Email: amhashem@ 123456kau.edu.sa (AMH); eazhar@ 123456kau.edu.sa (EIA)

                Author information
                http://orcid.org/0000-0002-8471-7011
                Article
                JMV24785
                10.1002/jmv.24785
                7167144
                28198548
                0952f988-ccd8-4555-9b2c-99ba8a8af3d4
                © 2017 Wiley Periodicals, Inc.

                This article is being made freely available through PubMed Central as part of the COVID-19 public health emergency response. It can be used for unrestricted research re-use and analysis in any form or by any means with acknowledgement of the original source, for the duration of the public health emergency.

                History
                : 15 October 2016
                : 05 January 2017
                : 24 January 2017
                Page count
                Figures: 3, Tables: 3, Pages: 8, Words: 4649
                Funding
                Funded by: Deanship of Scientific Research (DSR) , open-funder-registry 10.13039/501100004686;
                Funded by: King Abdulaziz University, Jeddah, Saudi Arabia , open-funder-registry 10.13039/501100004054;
                Award ID: G‐145‐140‐36
                Categories
                Research Article
                Research Articles
                Custom metadata
                2.0
                August 2017
                Converter:WILEY_ML3GV2_TO_JATSPMC version:5.8.0 mode:remove_FC converted:15.04.2020

                Microbiology & Virology
                alkhumra hemorrhagic fever,dengue,flaviviruses,saudi arabia
                Microbiology & Virology
                alkhumra hemorrhagic fever, dengue, flaviviruses, saudi arabia

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