Ariel Vina-Rodrı́guez

Cuban Neuroscience Center

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Martin Eiden

Friedrich-Loeffler-Institut

Martin H. Groschup

Friedrich-Loeffler-Institut

Santiago Dueñas‐Carrera

Centro de Ingeniería Genética y Biotecnología

Nelson Acosta‐Rivero

Centro de Ingeniería Genética y Biotecnología

Juan Morales

Universidad Nacional de Colombia

Liz Alvarez‐Lajonchere

Centro de Ingeniería Genética y Biotecnología

Gillian Martínez

Centro de Ingeniería Genética y Biotecnología

Ute Ziegler

Friedrich-Loeffler-Institut

Josephine Schlosser

Freie Universität Berlin

Julio César Alvarez‐Obregón

Centro de Ingeniería Genética y Biotecnología

Cooperative Institutions

Birds Canada

147

Centro de Ingeniería Genética y Biotecnología

Friedrich-Loeffler-Institut

Cuban Neuroscience Center

Center of Molecular Immunology (Cuba)

National Center for Genetic Engineering and Biotechnology

German Center for Infection Research

Instituto de Gastroenterología

Universidad Nacional Autónoma de México

Centre National de la Recherche Scientifique

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RNA virus detection and identification using techniques based on DNA hybridization

Ariel Vina-Rodrı́guez

Humanity is constantly confronted with the emergence and reemergence of infectious diseases. Many of them produce large or devastating epidemics, like AIDS (HIV) and Ebola. Others have been long neglected, yet pose immediate threats to global public health as evidences the abrupt emergence of Zika virus in South America and its association with microcephaly in babies. The examples illustrate, that many of these diseases are provoked by RNA viruses. One of the first steps in understanding and eliminating those threats is the development of sensitive and rapid diagnostic methods. A general and relatively rapid method is the direct detection and examination of the agent’s genome. However, the nature of (re)emerging RNA viruses poses a series of very specific problems for the design of such methods. Therefore, a systematic approach was proposed for the design of DNA-hybridization-base methods to detect and characterize RNA viruses that will have both a high sensitivity and a specificity sufficiently broad to detect, per reaction, down to a single copy of any of the possible variants of the viral genome. Following this approach a series of assays were designed, developed or adapted and put into use for detection and characterization of important RNA viruses. One of those viruses is West Nile virus (WNV), which after its explosive introduction into USA become the most widespread flavivirus throughout the world and, consequently, many countries began an intensive monitoring. While existing assay detected predominantly the Lineage 1, in Europa Lineage 2 was expected. Two new RT-qPCR for the detection of both lineages were developed, and reportedly used by independent laboratories. Due to more than 50000 associated deaths per year, the Hepatitis E virus also received an increasing attention to elucidate novel routes of transmission. This virus (especially genotype 3) has the zoonotic potential of transmission from pigs and wild boar to humans. RT-qPCR and nested qPCR for detection and characterization of this virus as well as a methodology for subtyping were developed and the first detected case of subtype 3b in a German wild animal was documented. In addition a novel assay for flaviviruses conformed by a RT-qPCR coupled with a low density DNA microarray was developed, which enabled the identification of WNV in mosquitoes from Greece. A RT-qPCR suitable for surveillance and diagnostic of all known variants of Venezuelan equine encephalitis virus was developed too. A causative agent of hemorrhagic infections, the Ngari virus, was detected and characterized in animal samples from Mauritania. These achievements were supported by the development of software applications for selection and visualization of primers and probes from aligned DNA sequences and for modeling of DNA hybridizations using unaligned sequences. In conclusion a general methodology for rapid development of sensitive diagnostic methods based in DNA-hybridization technics (PCR, sequencing and microarray) was stablished and successful applications are reported.

Zika Virus

Flavivirus

RNA virus

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The influence of preoperative overnight fasting under insulin resistance in patients to undergo a videolaparoscopic cholecystectomy

A. Martinez Immaculata Xess Indulis Vanags Myung Hee Yoon Pernille Vaabengaard

Preoperative Fasting

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Ngari Virus in Goats during Rift Valley Fever Outbreak, Mauritania, 2010

Emerging infectious diseases (2014)

Martin Eiden Ariel Vina-Rodrı́guez Bezeid O. El Mamy Katia Isselmou Ute Ziegler

To the Editor: Ngari virus (NRIV) is a single-stranded RNA virus belonging to the family Bunyaviridae, genus Orthobunyavirus. The genome comprises 3 segments, the small (S), medium (M), and large (L) segments, which encode the nucleocapsid (N) protein, the 2 glycoproteins Gn and Gc, and the RNA-dependent RNA-polymerase, respectively. Sequence analysis showed that NRIV is a reassortant between Bunyamwera virus (BUNV) and Batai virus (BATV), both from the genus Orthobunyavirus. S and L segments derived from BUNV, and the M segment derived from BATV (1,2). NRIV is more virulent than BUNV and BATV and is associated with hemorrhagic fever. NRIV was first isolated from Aedes simpsoni mosquitoes in 1979 and from humans in 1993, both in Senegal (3). During 1997 and 1998, humans were affected with hemorrhagic fever diseases in Kenya and Somalia that were caused by Rift Valley fever virus (RVFV) and by NRIV (2,4). In 2010, during an ongoing RVFV outbreak in Mauritania, we collected 163 serum samples (62 from camels, 8 from cattle, and 93 from small ruminants) (5). RVFV RNA was isolated from serum samples as described previously (5). Further molecular testing of the samples was conducted by a SYBRGreen–based real-time reverse transcription PCR (RT-PCR) adapted from a conventional RT-PCR and based on generic primers (bun_group_forw 5′-CTGCTAACACCAGCAGTACTTTTGAC-3′ and bun_group_rev 5′-TGGAGGGTAAGACCATCGTCAGGAACTG-3′) that target a 250-nt sequence of the S segment of Bunyamwera serogroup members (6). Real-time RT-PCR was performed in a CFX 96 real-time PCR system (Bio-Rad, Hercules, CA, USA) by using 5 μL RNA with a QuantiTect SYBR Green RT-PCR Kit (QIAGEN, Hilden Germany) in a final volume of 25 μL. Cycling conditions included RT at 50°C for 30 min and 95°C for 15 min, followed by amplification with 44 cycles of 95°C for 15 s, 55°C for 25 s, 72°C for 30 s, and 77°C for 5 s. A melting curve analysis was then performed starting with 95°C for 60 s, and a temperature gradient was conducted from 68°C to 94°C in increments of 0.2°C. Of the 163 serum samples tested, 2 samples from goats resulted in a positive signal with cycle thresholds of 23 (sample 51) and 28 (sample 65), respectively. Both samples showed similar melting peaks at ≈78.2°C and shared the identical partial nucleotide sequence of the S segment. The sequence belongs to the Bunyamwera serogroup, but the short partial sequence was not sufficient for accurate virus determination and identification. For this reason, both serum samples were used to inoculate cell monolayers of Vero E6 cells that were assayed for virus replication. Only sample 51 displayed a cytopathic effect after 72 h and was further analyzed. We isolated the viral RNA from cell culture with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and used it to prepare a sequencing library according to a recently published protocol (7) but using Illumina adaptors (Illumina, San Diego, CA, USA). We sequenced the resulting library using the Illumina MiSeq instrument with v2 chemistry. We recovered full-length genome sequences of the S, M, and L segments of the virus and deposited them in GenBank (accession nos. {type:entrez-nucleotide,attrs:{text:KJ716848,term_id:671775231,term_text:KJ716848}}KJ716848–716850). Phylogenetic analysis of complete genome sequences indicated that the virus belongs to the Ngari virus group and showed high homology to previous NRIV isolates in all 3 segments (Figure). As for all previous NRIV strains, the new isolate was highly similar to BUNV regarding the S and the L segment (Figure, panels A, C); the M segment was highly similar to BATV (Figure, panel B). Figure Phylogenetic tree of Ngari virus–derived A) small (975 bp), B) medium (4,507 bp), and C) large (6,887) segment sequences of Bunyamwera and Batai viruses compared with isolate obtained from a goat in Mauritania in 2010 (arrows). The tree was constructed ... This evidence supports the extension of the range of NRIV infection to goats (complete sequences already had been derived from a human and from mosquitoes [8]) and demonstrates the occurrence of NRIV during the 2010 RVFV outbreak in Mauritania. We are aware of only 1 additional report of NRIV-infected sheep (in 1988), also in Mauritania, although no further characterization or isolation has been conducted (9). Both NRIV-positive samples were negative for RVFV RNA but positive for RVFV-specific IgG. In addition, sample 51 contained IgM against RVFV (5), indicating possible co-infection of RVFV and NRIV. Because both ELISAs rely on detection of antibodies against RVFV N protein, which is highly divergent to the deduced NRIV N sequence, cross-reactivity is highly unlikely but needs to be substantiated. Both samples originated from the Adrar region, which was the center of an unusual RVFV outbreak in Mauritania in 2010 (10). The possible clinical importance to livestock and the circulation of NRIV among mosquitoes, livestock, and humans needs to be clarified. No further information about clinical signs of sampled animals or reports of human NRIV cases is available. Because infection with both RVFV and NRIV induces hemorrhagic fever, affected humans also should be tested for NRIV infection. Further development of specific molecular and serologic diagnostic tools for NRIV should be pursued to obtain more information about NRIV distribution in humans and livestock in Mauritania and other African countries.

Orthobunyavirus

Rift Valley Fever

Phlebovirus

10.3201/eid2012.140787

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Citations (24)

CETP polymorphism adds to the characterisation of premature coronary heart disease and multiple vessel disease

M. Irurita Latasa J. Irurita Lidia López M. N. Saez Milene T. Saavedra

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A Quantitative Real-Time RT-PCR Assay for the Detection ofVenezuelan equine encephalitis virusUtilizing a Universal Alphavirus Control RNA

BioMed Research International (2016)

Ariel Vina-Rodrı́guez Martin Eiden Markus Keller Winfried Hinrichs Martin H. Groschup

Venezuelan equine encephalitis virus (VEEV) is an Alphavirus from the family Togaviridae that causes epizootic outbreaks in equids and humans in Central and South America. So far, most studies use conventional reverse transcriptase PCR assays for the detection of the different VEEV subtypes. Here we describe the development of a TaqMan quantitative real-time reverse transcriptase PCR assay for the specific detection and quantitation of all VEEV subtypes which uses in parallel a universal equine encephalitis virus control RNA carrying target sequences of the three equine encephalitis viruses. The control RNA was used to generate standard curves for the calculation of copy numbers of viral genome of Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV), and VEEV. The new assay provides a reliable high-throughput method for the detection and quantitation of VEEV RNA in clinical and field samples and allows a rapid differentiation from potentially cocirculating EEEV and WEEV strains. The capability to detect all known VEEV variants was experimentally demonstrated and makes this assay suitable especially for the surveillance of VEEV.

Togaviridae

TaqMan

Alphavirus infection

10.1155/2016/8543204

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Detection of HCV-related Structures in the Mitochondria of Apoptotic Hepatocytes in Liver Biopsies from Chronically HCV-infected Patients

Microscopy and Microanalysis (2003)

Viviana Falcón Nelson Acosta‐Rivero Glay Chinea Jorge Gavilondo Marı́a-C de la Rosa

Journal Article Detection of HCV-related Structures in the Mitochondria of Apoptotic Hepatocytes in Liver Biopsies from Chronically HCV-infected Patients Get access Viviana Falcón, Viviana Falcón Biomedicine Division, Center for Genetic Engineering and Biotechnology., P.O. Box 6162, C.P. 10600, Havana. Email: viviana.falcon@cigb.edu.cu Search for other works by this author on: Oxford Academic Google Scholar Nelson Acosta-Rivero, Nelson Acosta-Rivero Biomedicine Division, Center for Genetic Engineering and Biotechnology., P.O. Box 6162, C.P. 10600, Havana. Email: viviana.falcon@cigb.edu.cu Search for other works by this author on: Oxford Academic Google Scholar Glay Chinea, Glay Chinea Biomedicine Division, Center for Genetic Engineering and Biotechnology., P.O. Box 6162, C.P. 10600, Havana. Email: viviana.falcon@cigb.edu.cu Search for other works by this author on: Oxford Academic Google Scholar Jorge Gavilondo, Jorge Gavilondo Biomedicine Division, Center for Genetic Engineering and Biotechnology., P.O. Box 6162, C.P. 10600, Havana. Email: viviana.falcon@cigb.edu.cu Search for other works by this author on: Oxford Academic Google Scholar María-C de la Rosa, María-C de la Rosa Biomedicine Division, Center for Genetic Engineering and Biotechnology., P.O. Box 6162, C.P. 10600, Havana. Email: viviana.falcon@cigb.edu.cu Search for other works by this author on: Oxford Academic Google Scholar Ivón Menéndez, Ivón Menéndez Biomedicine Division, Center for Genetic Engineering and Biotechnology., P.O. Box 6162, C.P. 10600, Havana. Email: viviana.falcon@cigb.edu.cu Search for other works by this author on: Oxford Academic Google Scholar Santiago Dueñas-Carrera, Santiago Dueñas-Carrera Biomedicine Division, Center for Genetic Engineering and Biotechnology., P.O. Box 6162, C.P. 10600, Havana. Email: viviana.falcon@cigb.edu.cu Search for other works by this author on: Oxford Academic Google Scholar Ariel Viña, Ariel Viña Biomedicine Division, Center for Genetic Engineering and Biotechnology., P.O. Box 6162, C.P. 10600, Havana. Email: viviana.falcon@cigb.edu.cu Search for other works by this author on: Oxford Academic Google Scholar Bienvenido Gra, Bienvenido Gra Institute of Gastroenterology, C.P. 10400, Havana; C.P. 10400, Havana Search for other works by this author on: Oxford Academic Google Scholar Miriam Noa, Miriam Noa National Center for Scientific Research, P.O. Box 6990, Havana Search for other works by this author on: Oxford Academic Google Scholar ... Show more Felix Alvarez, Felix Alvarez Biomedicine Division, Center for Genetic Engineering and Biotechnology., P.O. Box 6162, C.P. 10600, Havana. Email: viviana.falcon@cigb.edu.cu Search for other works by this author on: Oxford Academic Google Scholar Josefina Rodríguez, Josefina Rodríguez Biomedicine Division, Center for Genetic Engineering and Biotechnology., P.O. Box 6162, C.P. 10600, Havana. Email: viviana.falcon@cigb.edu.cu Search for other works by this author on: Oxford Academic Google Scholar Juan Morales Juan Morales Biomedicine Division, Center for Genetic Engineering and Biotechnology., P.O. Box 6162, C.P. 10600, Havana. Email: viviana.falcon@cigb.edu.cu Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 9, Issue S02, 1 August 2003, Pages 1424–1425, https://doi.org/10.1017/S1431927603447120 Published: 19 July 2003

10.1017/s1431927603447120

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HCV core protein localizes in the nuclei of nonparenchymal liver cells from chronically HCV-infected patients

Biochemical and Biophysical Research Communications (2005)

Viviana Falcón Nelson Acosta‐Rivero Mineko Shibayama Glay Chinea Jorge Gavilondo

Hepatic stellate cell

Pathogenesis

Immunostaining

10.1016/j.bbrc.2005.02.107

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Citations (22)

Two New Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction Assays with Unique Target Sites for the Specific and Sensitive Detection of Lineages 1 and 2 West Nile Virus Strains

Journal of Veterinary Diagnostic Investigation (2010)

Martin Eiden Ariel Vina-Rodrı́guez Bernd Hoffmann Ute Ziegler Martin H. Groschup

Two novel 1-step real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays for the simultaneous detection of West Nile virus (WNV) lineage 1 and 2 strains were developed. Primers and the probe of assay 1 target the 5′-untranslated region (UTR), whereas the amplicon of assay 2 is located in the nonstructural region NS2A, which enables an unambiguous and independent WNV diagnosis based on 2 different amplicons. Both assays allow the detection of as few as 2–4 genome copies of WNV strains NY99, Uganda B956, Kunjin, and Sarafend (all cultured on Vero cells). A new synthetic RNA mutant of the 5′-UTR amplicon, which contains 6 twist inverted base-pair changes at the probe attachment site, was used as external calibrator control.

Amplicon

Vero cell

Flavivirus

10.1177/104063871002200515

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Citations (109)

Ultrastructural evidences of HCV infection in hepatocytes of chronically HCV-infected patients

Biochemical and Biophysical Research Communications (2003)

Viviana Falcón Nelson Acosta‐Rivero Glay Chinea Jorge Gavilondo Marı́a-C de la Rosa

Immunoelectron microscopy

10.1016/s0006-291x(03)00884-2

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