Oropouche virus detection in saliva and urine

Oropouche virus (OROV) is an arthropod-borne virus of the Peribunyaviridae family, transmitted to humans primarily by Culicoides paraensis. It is one of the main arboviruses infecting humans in Brazil, primarily in the Amazon Region. Here, we report the detection of OROV in the saliva and urine of a patient whose samples were collected five days after the onset of symptoms. Nucleotide sequencing and phylogenetic analysis further confirmed the results. To our knowledge, this is the first study reporting the detection of OROV in the saliva and urine of an infected patient. In addition, the results of our study expand the current knowledge pertaining to the natural history of Oropouche fever.

Epidemiological and Clinical Characteristics of Confirmed Cases of Severe Fever With Thrombocytopenia Syndrome in Jeju Province, Korea, 2014-2018 / 예방의학회지

OBJECTIVES: Jeju Province is well known as the region showing the highest incidence of severe fever with thrombocytopenia syndrome (SFTS) in South Korea. The aim of this study was to evaluate the epidemiological and clinical characteristics of SFTS patients in Jeju Province. METHODS: The primary data for this study were obtained from the Integrated Diseases and Health Control System of the Korea Centers for Disease Control and Prevention (KCDCIS). The selection criteria were confirmed cases of SFTS with a residence listed in Jeju Province at the time of diagnosis, reported to the KCDCIS between July 16, 2014 and November 30, 2018. RESULTS: Of 55 confirmed cases of SFTS, the case fatality rate was 10.9% (95% confidence interval [CI], 4.1 to 22.2). The most common presenting symptoms at diagnosis of severe fever, myalgia, and diarrhea had incidences of 83.6% (95% Cl, 71.2 to 92.2), 45.5% (95% Cl, 32.0 to 59.5), and 40.0% (95% CI, 27.0 to 54.1), respectively. CONCLUSIONS: Compared to SFTS patients nationwide in 2013-2015, the subjects of this study exhibited a lower case fatality rate and had a lower incidence of severe fever, myalgia, and confusion.

Multiplexed reverse transcription real-time polymerase chain reaction for simultaneous detection of Mayaro, Oropouche, and Oropouche-like viruses

ABSTRACT We describe a sensitive method for simultaneous detection of Oropouche and Oropouche-like viruses carrying the Oropouche S segment, as well as the Mayaro virus, using a multiplexed one-step reverse transcription real-time polymerase chain reaction (RT-qPCR). A chimeric plasmid containing both Mayaro and Oropouche targets was designed and evaluated for the in vitro production of transcribed RNA, which could be easily used as a non-infectious external control. To track false-negative results due to PCR inhibition or equipment malfunction, the MS2 bacteriophage was also included in the multiplex assay as an internal positive control. The specificity of the multiplex assay was evaluated by Primer-Blast analysis against the entire GenBank database, and further against a panel of 17 RNA arboviruses. The results indicated an accurate and highly sensitive assay with amplification efficiency greater than 98% for both targets, and a limit of detection between two and 20 copies per reaction. We believe that the assay described here will provide a tool for Mayaro and Oropouche virus detection, especially in areas where differential diagnosis of Dengue, Zika and Chikungunya viruses should be performed.

Seroprevalence of Severe Fever with Thrombocytopenia Syndrome in Southeastern Korea, 2015

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease characterized by fever, thrombocytopenia and diarrhea. SFTS was firstly reported in Korea in 2013 but its seroprevalence in the country has yet to be investigated. Here, we investigate the seroprevalence of SFTS in a Korean population. A cross-sectional study was conducted on patients who had their sera tested for various reasons at a tertiary university hospital on particular days in May 2015. This study was conducted in a tertiary hospital in southeastern Korea. Total antibodies including immunoglobulin G (IgG) and immunoglobulin M (IgM), specific to SFTS virus (SFTSV) in serum samples were detected by a double-antigen sandwich enzyme-linked immunosorbent assay (ELISA). A total of 1,069 serum samples were tested. Median age was 59 years (range 12–96 years), and 51.5% were male. Overall, 22 patients (2.1%) were tested positive for anti-SFTSV antibodies. The SFTS seroprevalence increased significantly with age (P = 0.034). The seropositive rate of rural area was higher than that of urban area (7.7% vs. 1.9%, P = 0.040). Seropositive rates were not significantly different among underlying diseases. None of the antibody-positive patients showed typical symptoms or laboratory findings of SFTS at the time of sample collection. Results of real-time reverse transcription polymerase chain reaction (RT-PCR) were negative for all the seropositive patients. Our study shows 2.1% SFTS seroprevalence among the patients visiting a tertiary hospital in Korea. Seroprevalence is higher in older and rural population.

In vitro antiviral activity of ribavirin against severe fever with thrombocytopenia syndrome virus

BACKGROUND/AIMS: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by severe fever with thrombocytopenia syndrome virus (SFTSV), a novel bunyavirus. As yet, there is no effective antiviral therapy for SFTS. Ribavirin is a broad-spectrum antiviral agent, which has been tried for treatment of SFTS. In this study, antiviral activity of ribavirin against SFTSV has been investigated. METHODS: Vero cell-grown SFTSV strain Gangwon/Korea/2012 was treated with ribavirin at various concentrations. Antiviral activity of ribavirin was evaluated by inhibition of the SFTSV cytopathic effect in Vero cells and quantification of viral RNA load in culture supernatant using one-step real-time reverse transcription polymerase chain reaction. Cytotoxicity of ribavirin was determined by a tetrazolium-based colorimetric method. RESULTS: Ribavirin reduced SFTSV titers in a dose-dependent manner, with a half-maximal inhibitory concentration ranged from 3.69 to 8.72 μg/mL. Cytopathic effects were reduced as ribavirin concentration increased. No significant cytotoxicity was detected at ribavirin concentrations of ≤ 31.3 μg/mL. CONCLUSIONS: Ribavirin exhibited inhibitory activity against SFTSV replication in vitro, which suggests that ribavirin can be used as a potential antiviral agent for SFTS.

Characterization and Analyses of the Full-length Genome of a Strain of the Akabane Virus Isolated from Mosquitoes in Yunnan Province, China / 病毒学报

We wished to sequence the full-length genomes of the DHL10M110 strain of the Akabane virus (AKV) isolated from mosquitoes in Yunnan Province, China, in 2010. We also wished to analyze the characteristics of these complete nucleotide sequences. The complete genomic sequence of the DHL10M110 strain from Yunnan Province was obtained by reverse transcription-polymerase chain reaction and direct sequencing. We found that the length of the L, M and S gene nucleotide sequences of the DHL10M110 strain were 6 869-bp, 4 309-bp and 856-bp, respectively, including the open reading frame (ORF) nucleotide sequences of 6 756-bp (L), 4 206-bp (M) and 702-bp (S), encoding 2252, 1402 and 234 amino-acid polyproteins, respectively. Phylogenetic analyses based on L-fragment ORF showed that the DHL10M110 strain had a close relationship with the OBE-1 strain of the AKV from Japan and AKVS-7/SKR/2010 strain of the AKV from South Korea. Phylogenetic analyses based on M- and S-fragment ORF showed that the DHL10M110 strain had a close relationship with the epidemic strains of the AKV from Japan, South Korea and Taiwan, but that the DHL10M110 strain had a lone evolutionary branch. In terms of nucleotide (amino acid) homology, the similarity of L-, M- and S-fragment ORFs of the DHL10M110 strain to the OBE-1 strain from Japan was 92.6% (98%), 88.5% (94%) and 96.4% (99.1%), respectively. When comparing the DHL10M110 strain with the OBE-1 strain, we noted 45, 84, and 2 different sites in the amino acids of L, M and S fragments, respectively. Homology and phylogenetic analyses also suggested that the DHL10M110 strain had a distant relationship with the epidemic strains of the AKV from Kenya and Australia. Also, we confirmed by complete genomic sequence analyses that the DHL10M110 strain was clade-Asia of the AKV. However, differences between the DHL10M110 strain compared with strains from Japan and South Korea were also noted. These results suggest that the DHL10M110 strain harbored relatively stable genetic characteristics and distinct regional features. This is the first time that full-length genomic sequences of the DHL10M110 strain of the AKV in mainland China have been obtained.

Detection of Oropouche virus segment S in patients and inCulex quinquefasciatus in the state of Mato Grosso, Brazil

This study aimed to investigate the circulation of Orthobunyavirus species in the state of Mato Grosso (MT) Brazil. During a dengue outbreak in 2011/2012, 529 serum samples were collected from patients with acute febrile illness with symptoms for up to five days and 387 pools of female Culex quinquefasciatuscaptured in 2013 were subjected to nested-reverse transcription-polymerase chain reaction for segment S of the Simbu serogroup followed by nucleotide sequencing and virus isolation in Vero cells. Patients (5/529; 0.9%) from Cuiabá (n = 3), Várzea Grande (n = 1) and Nova Mutum (n = 1) municipalities were positive for the S segment of Oropouche virus (OROV). Additionally, eight/387 Cx. quinquefasciatuspools were positive for the segment, with a minimum infection rate of 2.3. Phylogenetic analysis indicated that all the samples belong to the subgenotype Ia, presenting high homology with OROV strains obtained from humans and animals in the Brazilian Amazon. The present paper reports the first detection of an Orthobunyavirus, possibly OROV, in patients and in Cx. quinquefasciatus mosquitoes in MT. This finding reinforces the notion that arboviruses frequently reported in the Amazon Region circulate sporadically in MT during dengue outbreaks.

Infection of the Severe Fever with Thrombocytopenia Syndrome Virus in Balb/C Mice and Hamsters / 病毒学报

The severe fever with thrombocytopenia syndrome virus (SFTSV) is the causative pathogen of an emerging infectious disease severe fever with thrombocytopenia syndrome and a new member in the genus Phlebovirus of family Bunyaviridae. Immune responses and pathological lesions in SFTSV-infected Balb/C mice and hamsters were evaluated by inoculation of SFTSV at 105 TCID50 or 103 TCID50 per animal through four different routes of infection, including intravenous, intramuscular, intraperitoneal, and intracerebral injections. The vehicle control groups were also included. At different time points after the inoculation blood and plasma samples were collected. Blood cell counts, blood viral RNA copies, and plasma antibodies were detected by automatic blood cell counters, real-time PCR, and luminex assays, respectively. At two weeks post inoculation, the animals were sacrificed. Tissues including heart, liver, spleen, lung, kidney, intestine, muscle, and brain, were collected for pathological analyses. Results showed that the SFTSV could infect Balb/C mice and hamsters with SFTSV-specific immunoglobulin (Ig) M and IgG antibodies detected in plasma samples on day 7 post inoculation. The SFTSV-specific IgM levels peaked on day 7 post inoculation and then decreased, whereas the SFTSV-specific IgG levels started to increase on day 7 and then peaked on day 14 post inoculation. Pathological analyses indicated significant pathological lesions in liver and kidney tissues. In conclusion, SFTSV could can infect different strains of rodent animals and cause similar immunological and pathological responses.

Preparation and functional analysis of the monoclonal antibodies against severe fever with thrombocytopenia syndrome bunyavirus structural proteins / 病毒学报

To prepare monoclonal antibodies (mAbs) against structural proteins of severe fever with thrombocytopenia syndrome bunyavirus (SFTSV), BALB/c mice were immunized using purified inactivated SFTSV virions as the antigens. Subsequently, hybridoma cell lines that secreted monoclonal antibodies against nucleoprotein (NP) and glycoproteins (GP) were obtained using a hybridoma technique. The antigen specificities of prepared mAbs were examined by indirect immunofluorescence and immunoprecipitation assays. Functional analyses were then performed,including the detection of IFA antibody titers,the levels of neutralizing activity and antibody affinities. After cell fusion and cloning,13 hybridoma cell lines secreted mAbs specifically against SFTSV-GP and 7 hybridoma cell lines secreted mAbs specifically against SFTSV-NP. Immunofluorescence and immunoprecipitation assays showed that the mAbs had high levels of antigen specificity. Among the 13 anti-SFTSV-GP mAbs,6 recognized Gn,whereas the others reacted with Gc. IFA titers of most anti-SFTSV-GP mAbs were between 1,280 and 20,480, and four anti-SFTSV-Gn mAbs showed neutralizing activity. Seven of the obtained anti-SFTSV-NP mAbs reacted specifically with NP,of which the IFA titers ranged from 5,120 to 20,480 with no observed neutralizing activity. Furthermore, two anti-SFTSV-GP mAbs, 1C8 and 1G8, showed high levels of affinity via a non-competitive ELISA. Our study lays the foundation for the development of further diagnostic assays and basic research into SFTSV.

Development of human antibodies against the Gn protein of severe fever with thrombocytopenia syndrome virus / 病毒学报

To obtain human antibodies against the Gn protein of Severe fever with thrombocytopenia syndrome virus (SFTSV) with phage display technology, this study aimed to screen anti-Gn protein antibodies from an anti-SFTSV Fab human phage display library. Antibody genes were identified by sequence analysis and the specificity of antibodies was confirmed by ELISA. The Fab antibody genes were cloned into the HL51-14 vector and expressed in a mammalian cell expression system. IgG antibodies were then purified by protein A affinity chromatography,and the results were further confirmed by ELISA,IFA,western blotting assays and micro-neutralization tests. The results showed that, after three rounds of panning, there were 390 human Fab antibodies against SFTSV particles, of which 364 were specific for nucleoprotein. Coated with the Gn protein, eight different Fab antibodies specific for Gn protein were obtained after the determination of the subtype and subclass of antibodies by gene sequencing; five of these antibodies were from the Lambda library and three were from the Kappa library. The eight IgG antibodies could specifically bind to Gn protein according to the ELISA, IFA and Western blotting assays. The micro-neutralization test showed that these eight antibodies had no neutralizing activity,but they could still provide a reference for research in human monoclonal antibodies against SFTSV.

Isolation and identification of the Akabane virus from mosquitoes in Yunnan Province, China / 病毒学报

To evaluate the prevalence of mosquito-borne viruses in Manshi and Ruili (Yunnan Province, China), we collected 2 149 mosquitoes (17 species) in August 2010. Virus isolation was undertaken by the cul- ture of baby hamster kidney cells (BHK-21 cells). Two virus-like isolates were obtained: DHL10M117 was isolated from collected in Mangshi; DHL10M110 was obtained from Anopheles vagus collected in Rui- li. Both isolates caused cytopathic effects,illness and death in suckling mice inoculated with these isolates via the intracerebral route. Two positive amplicons, 702-bp from the S segment and 456-bp from the M segment,were obtained using reverse transcription-polymerase chain reaction using primers specific for the Akabane virus (AKV). Phylogenetic analysis suggested that these two virus stains had a distant relation- ship with AKVs from Kenya and Australia,but were genetically close to those from Japan,South Korea, and Taiwan. However,they were separate from other Asian strains and grouped into a small branch. The highest nucleotide and amino-acid sequence identity of the S segment was found with the CY-77 strain from Taiwan (96.6% and 99.6% for DHL10M117 and 96.7% and 100% for DHL10M110,respectively). Com- parison of the M segment showed they shared the highest amino acid identity with CY-77 (99.6% and 100%, respectively), whereas the highest nucleotide identity was found with the Iriki strain from Japan (99.6% and 100%, respectively). Compared with the MP496 strain from Kenya,they displayed lower lev- els of sequence homology, at 69.7% and 70.0% for nucleotide sequences of the two loci,and 91. 0% for a- mino acids. Our results identified that DHL10M117 and DHL10M110 were strains of AKV,and provided molecular biological evidence for the existence of AKV in Yunnan Province. These AKV strains that are circulating in Yunnan Province share a close genetic relationship with strains from the rest of Asia. Culex tritaeniorhynchus and Anopheles vagus may serve as transmission vectors.

Purification and Preliminary Research on the Immunogenicity of Inactivated Severe Fever with Thrombocytopenia Syndrome Bunyavirus / 病毒学报

To understand the immunogenicity of purified inactivated severe fever with thrombocytopenia syndrome bunyavirus (SFTSV), concentration by ultrafiltration as well as molecular-sieve chromatography (MSC) were used for purification of inactivated SFTSVs. Inactivated viruses in purified samples were analyzed and identified by western blotting and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the glycoprotein (GP) and nucleoprotein (NP) antigen titers of which were detected using a double-sandwich enzyme-linked immunosorbent assay (ELISA). Purified inactivated SFTSVs were enriched and observed by electron microscopy, and the total protein concentration detected using the bicinchoninic acid assay. Purified inactivated SFTSVs were applied to New Zealand rabbits via two immunization programs to evaluate immunogenicity and to compare the immune effect. After SFTSVs were inactivated and concentrated by ultrafiltration, MSC revealed two typical elution peaks. The sample of one peak was identified as inactivated virions, in which GP and NP were detected by SDS-PAGE, western blotting and ELISA. Main corponent of the other peak was NP. After concentration by ultrafiltration, purified inactivated SFTSVs with purity >90% and total protein concentration of 1. 1 mg/mL were obtained, and the typical electron microscopy of bunyavirus was observed. In the sera of animals immunized with purified inactivated SFTSVs, SFTSV-specific IgG antibody and neutralizing antibody were detected at high titers. However, antibody titers were affected by the immunization program. Effect of immunization on days 0, 14 and 28 was significantly better than that on days 0, 7 and 28. Our work revealed that cultivation of SFTSVs contained intact virus particles and large amounts of free NP. Using MSC, purified inactivated SFTSVs of high purity could be obtained. Purified inactivated SFTSVs induced high titers of neutralizing antibody and virus-specific IgG antibody showing satisfactory immunogenicity, which provides important clues for further study on a vaccine for the inactivated virus.

New Method for Stable Expression of SFTS Virus-like Particles in CHO-K1 Cells / 病毒学报

To explore a new method for stable expression of virus-like particles (VLPs) of the severe fever with thrombocytopenia syndrome (SFTS) virus, an expression plasmid for the membrane glycoprotein (GP) and nucleocapsid protein (NP) of the SFTS virus was constructed by fusion of the two proteins via a serine residue, and a yellow fluorescence protein (YFP) gene was introduced into the plasmid as a reporter. CHO-K1 cells were transfected with this plasmid, and stable cell lines constructed using the limited dilution method. Cellular colonies were hand-picked based on YFP with the help of fluorescence microscopy and expanded without selection pressure. Stability of cell lines was evaluated by monitoring of fluctuation of the intensity of YFP for 40 passages. VLP production was characterized using an indirect fluorescence assay, immunoblotting, and electronic microscopy. We showed that GP and NP fusion proteins could be assembled into VLPs in vivo, and that VLPs had similar morphologies to virus particles. Selected cell lines were stable for YFP expression: no significant fluctuation was detected in 40 passages. These data demonstrated the effectiveness of this new method for expression of structural proteins of the SFTS virus and screening for stable cell lines. Our results could provide new concepts for the production of biopharmaceuticals.

Genome sequencing and the molecular evolutionary analysis of a SFTSV isolated from Zhejiang province / 中华预防医学杂志

<p><b>OBJECTIVE</b>To sequence the whole genome and to analyze the molecular and evolutionary of Severe fever with thrombocytopenia syndrome bunyavirus (SFTSV) isolated from Zhejiang province.</p><p><b>METHODS</b>Viral RNA was extracted from the specimens and detected by quantitative real-time RT-PCR. SFTSV strain was isolated. A total of 17 overlapping fragments covering the whole genome were amplified by RT-PCR. And the entire genomes were formed by sequencing and assembly the fragments. The SFTSV sequence of Zhejiang strain was compared with the sequences of SFTSV that have been published to generate the phylogenetic tree. And the SFTSV sequence of Zhejiang strain was compared with the sequences of strains of the genus phlebovirus in the Bunyaviridae family and analysis of homology.</p><p><b>RESULTS</b>SFTSV strain was isolated from SFTSV infection positive serum successfully. The genomic fragments were amplified by RT-PCR. A total of 3 cDNA sections were formed by sequencing and assembly the fragments. The S segment contained 1 745 nucleotides. The M fragment contained 3 378 nucleotides, and the L segment contained 6 368 nucleotides. Molecular phylogenetic analysis result showed SFTSV Zhejiang strain had the highest similarity with Japan/SPL004A/2013 strain. The similarity of the S segment was 98%. The similarity of the M fragment was 97%. And it was 98% that of the L fragment. Meanwhile, the comparison results also confirmed the Zhejiang strain belonged to the genus phlebovirus.</p><p><b>CONCLUSION</b>SFTSV Zhejiang strain of isolated from SFTSV infection positive serum successfully. And the genome sequencing was complete molecular evolution analysis shows SFTSV Zhejiang strain has the maximum similarity with SFTSV Japan strain.</p>

A review of research on Schmallenberg virus / 病毒学报

Schmallenberg virus (SBV), a novel orthobunyavirus, was first isolated in 2011. SBV preferentially infects the central nervous system of cattle and sheep and causes fever, diarrhea, a drop in milk yields, congenital malformations and stillbirths. Until June 2014, more than 200 scientific publications regarding SBV have been published. Although more than 20 articles on SVB were published in China, most of these articles provided only a brief introduction of the disease without fully discussing the associated disease characteristics. As a new disease, it has been made a focus of the National Research Center for Exotic Animal Diseases at the China Animal Health and Epidemiology Center. In this review, in order to provide a reference for research into SBV in China, we have reviewed the state of current research progress on the etiology, diagnosis and epidemiology of SBV, and vaccine development.

Prevalence and homology analysis on human and animals severe fever with thrombocytopenia syndrome virus infection in Yantai of Shandong province / 中华流行病学杂志

<p><b>OBJECTIVE</b>To learn the prevalence of infection of human and animals severe fever with thrombocytopenia syndrome bunyavirus (SFTSV) in Yantai, Shandong province, and to analyze the pathogenic features of SFTSV as well as its relationship between human and animal hosts.</p><p><b>METHODS</b>From April to November in 2011, 3 576 serum samples were collected from domesticated animals, including sheep, cattle, pigs, dogs, chickens, in Laizhou and Penglai areas where fever with thrombocytopenia syndrome frequently occurred among local residents. Total SFTSV antibodies and virus-specific nucleic acids of the serum were tested by ELISA and Real time RT-PCR, respectively. SFTSV infection on each animal was observed in different months. 2 590 human serum samples were also collected in Laizhou and Penglai areas, with IgG antibodies tested by ELISA. Virus was isolated with Vero cells from the serum which SFTSV viral nucleic acids were positive. S fragments were amplified by RT-PCR and sequenced, with homology analysis conducted on these sequences.</p><p><b>RESULTS</b>The overall positive rate of serum samples from animals on the total SFTSV antibodies was 40.24% (1 439/3 576) while the positive rate for specific nucleic acids was 4.56% (163/3 576). The positive rates for SFTSV antibodies were 62.78%, 52.97%, 45.56%, 28.73%, 1.45% and the positive rates for specific nucleic acids were 5.72%, 4.63%, 3.02%, 5.25% and 3.73%, in sheep, cattle, chickens, dogs, pigs, respectively. The antigens/antibodies for SFTSV in animals changed seasonally. The overall positive rate for SFTSV IgG antibody from 2 590 human samples was 5.41%. Thirteen virus strains were isolated from these serum samples (10 strains from human and 3 strains from animals). The nucleotide homology of 13S fragments' sequences ranged from 95.23% to 100.00% and the nucleotide homology with the isolates from other provinces were between 94.72% and 99.13%. The homology was considered to be high.</p><p><b>CONCLUSION</b>High prevalence of SFTSV infections occurred both in human and domestic animals in Yantai city. The nucleotide sequences of SFTSV were highly homologous among human and domestic animals. The findings suggested that domesticated animals might serve as SFTSV proliferation and the hosts for transmission thus should be attached great importance.</p>

Severe fever with thrombocytopenia syndrome virus nucleoprotein specifically binds to 60kD SSA/Ro protein in host cells / 病毒学报

This study aims to investigate whether the nucleoprotein (NP) of severe fever with thrombocytopenia syndrome virus (SFTSV) can impact the cellular immunity of host cells. Gene segments that encode the NP and non-structural protein (NSs) of SFTSV were inserted into eukaryotic expression vector VR1012. Host proteins that interact with NP and affect immunity were identified with co-immunoprecipitation (IP), SDS-PAGE, mass spectrometry (MS), and Western blot. Co-localization of NP and the identified host proteins was confirmed by confocal microscopy. A 60kD SSA/Ro, a protein related to immunity, interacted with NP, as found by IP and MS. Confocal microscopy showed that NP and SSA/Ro were co-localized in cytoplasm. These results indicated that SFTSV NP may specifically bind to 60kD SSA/Ro and cause a series of immune responses and clinical symptoms.

Brote de fiebre de Oropuche en dos localidades de la región Cajamarca, Perú, 2011 / Oropuche fever outbreak in two villages of the region Cajamarca, Peru, 2011

La fiebre de Oropuche es una arbovirosis transmitida por especies de Culicoides, tiene un ciclo selvático y otro urbano, comparte las mismas características clínicas con el dengue, pero con escaso compromiso cutáneo y mayor tendencia a la recurrencia. Desde su primer aislamiento en humanos hasta el año 2011 se han reportado más de 30 brotes epidémicos y por lo menos 500 000 personas infectadas. Objetivo: Describir un brote de fiebre de Oropuche en la región Cajamarca en el año 2011. Métodos: Estudio transversal. El ámbito geográfico del brote es eminentemente rural, su población muestra un fluido intercambio migratorio con zonas endémicas de dengue. La investigación se inició luego de detectar el incremento de pacientes febriles a través de la vigilancia sindrómica, se realizaron búsquedas activas institucional y comunitaria mediante las definiciones de caso sospechoso y probable de la directiva sanitaria de dengue. Resultados: Se investigaron un total de 33 pacientes que cumplieron la definición de caso probable de dengue, todos con evolución favorable, 31 casos fueron autóctonos. El laboratorio del Instituto Nacional de Salud (INS) confirmó, por primera vez en la provincia de Cutervo de la región Cajamarca, la presencia de anticuerpos para el virus de Oropuche en 17 muestras de las 26 enviadas. El manejo inicial del brote fue orientado según el contexto clínico y epidemiológico para dengue, lo cual generó demoras en el periodo de confirmación diagnóstica y por ende en la implementación de las medidas de intervención. Conclusiones: La fiebre de Oropuche es uno de los diagnósticos diferenciales de dengue, en tal sentido, los sistemas de vigilancia y la red de laboratorios del país deben ser fortalecidos en su detección temprana y control.

Oropuche fever is an arbovirus transmitted by Culicoides species. It has a jungle and an urban cycle, and shares the same clinical features with dengue, but with limited cutaneous involvement and a greater tendency to recur. Since its first isolation in humans until 2011, more than 30 outbreaks have been reported and at least 500 000 people infected. Objective: To describe an outbreak of Oropuche fever in Cajamarca region in 2011. Methods: Cross-sectional study. The geographical scope of the outbreak was largely rural. The population shows a fluid migration exchanges with dengue endemic areas. The investigation began after detecting an increase of febrile patients by syndromic surveillance. An institutional and community active search of suspect and probable cases of dengue according to health directives was performed. Results:We investigated a total of 33 patients who met the definition for a probable case of dengue, all with favorable course, 31 cases were autochthonous. The laboratory of the National Institute of Health (NIH) confirmed for the first time in the province of Cutervo, Cajamarca region, the presence of antibodies to the virus Oropuche in 17 of 26 samples. Initial management of the outbreak was oriented along the clinical and epidemiological context for dengue, which caused delays in the diagnostic confirmation period and therefore in the implementation of intervention measures. Conclusions: Oropuche fever is one of the differential diagnosis of dengue, as such, surveillance systems and laboratory network in the country should be strengthened in early detection and control.

Investigation of RNA viral genome amplification by multiple displacement amplification technique / 病毒学报

In order to facilitate the detection of newly emerging or rare viral infectious diseases, a negative-strand RNA virus-severe fever with thrombocytopenia syndrome bunyavirus, and a positive-strand RNA virus-dengue virus, were used to investigate RNA viral genome unspecific amplification by multiple displacement amplification technique from clinical samples. Series of 10-fold diluted purified viral RNA were utilized as analog samples with different pathogen loads, after a series of reactions were sequentially processed, single-strand cDNA, double-strand cDNA, double-strand cDNA treated with ligation without or with supplemental RNA were generated, then a Phi29 DNA polymerase depended isothermal amplification was employed, and finally the target gene copies were detected by real time PCR assays to evaluate the amplification efficiencies of various methods. The results showed that multiple displacement amplification effects of single-strand or double-strand cDNA templates were limited, while the fold increases of double-strand cDNA templates treated with ligation could be up to 6 X 10(3), even 2 X 10(5) when supplemental RNA existed, and better results were obtained when viral RNA loads were lower. A RNA viral genome amplification system using multiple displacement amplification technique was established in this study and effective amplification of RNA viral genome with low load was achieved, which could provide a tool to synthesize adequate viral genome for multiplex pathogens detection.