The first serological detection of West Nile virus infection among residents living in northern Turkey

Background & objectives: West Nile virus (WNV) is transmitted by a mosquito-borne virus whose natural reservoir is birds. Humans and horses are considered accidental hosts. Even if the vast majority of WNV infections in humans have asymptomatic or mild disease settings, serious neurological disorders with lethal outcomes can also be observed in around 1% of the cases. We aimed to serologically investigate the presence of WNV in humans living in Black sea of Turkey, and to obtain epidemiological data that will contribute to the implementation of public health policies to control and prevent potentially other life-threatening arboviral infections. Methods: In the current study, a total of 416 human sera were collected from native patients of Samsun and its boroughs attending Samsun Training and Research Hospital; these sera were tested for WNV with pooling method, using anti-IgM and IgG ELISA commercial kits. All pools that were found positive for both IgM and IgG were individually retested for the detection of positive WNV sera. After that, all positive samples were tested using realtime PCR to detect the presence of WNV-RNA particles. Results: Total seropositivity rates of WNV in terms of IgM and IgG were found as 0.96% and 0.72%, respectively. No presence of WNV-RNA could be detected in positive samples. Interpretation & conclusion: According to the data, further studies should be conducted to better understand the epidemiological dynamics of WNV in Turkey. It is recommended that other antigenically related flaviviruses which can give cross-reaction with WNV should also be investigated.

Seroprevalence of arboviruses in Ecuador: Implications for improved surveillance / Seroprevalencia de arbovirus en Ecuador: implicaciones para mejorar su vigilancia

Abstract | Introduction: Arthropod-borne viruses (arboviruses) cause morbidity and mortality in humans and domestic animals worldwide. The percentage of population immunity or susceptibility to these viruses in Ecuador is unknown. Objectives: To investigate the proportion of Ecuadorian populations with IgG antibodies (Abs) (past exposure/immunity) and IgM Abs (current exposure) against flaviviruses and alphaviruses and to study the activity of these viruses in Ecuador. Materials and methods: During 2009-2011, we conducted a serosurvey for selected arboviruses in humans (n=1,842), equines (n=149), and sentinel hamsters (n=84) at two coastal locations and one in the Amazon basin (Eastern Ecuador) using enzyme-linked immunosorbent assay and hemagglutination inhibition test. Results: From 20.63% to 63.61% of humans showed IgG-antibodies for the flaviviruses: Dengue virus (DENV), yellow fever virus (YFV) Saint Louis encephalitis virus, and West Nile virus (WNV); from 4.67% to 8.63% showed IgG-Abs for the alphaviruses: Venezuelan equine encephalitis virus, eastern equine encephalitis virus, and western equine encephalitis virus. IgM-Abs were found for DENV and WNV. Equines and hamsters showed antibodies to alphaviruses in all locations; two hamsters seroconverted to YFV in the Amazonia. Conclusions: The results show a YFV vaccination history and suggest the activity of arboviruses not included in the current surveillance scheme. Enhanced arbovirus and mosquito surveillance, as well as continued YFV vaccination and evaluation of its coverage/ effectiveness, are recommended.

Resumen | Introducción. Los virus transmitidos por artrópodos (arbovirus) causan morbilidad y mortalidad en humanos y animales domésticos mundialmente. Se desconoce el porcentaje de inmunidad o vulnerabilidad de la población ecuatoriana ante estos virus. Objetivos. Investigar la proporción de poblaciones ecuatorianas con anticuerpos IgG (exposición o inmunidad pasada) y anticuerpos IgM (exposición reciente) contra flavivirus y alfavirus, e investigar su actividad en Ecuador. Materiales y métodos. Entre 2009 y 2011, se llevó a cabo una encuesta serológica para arbovirus en humanos (n=1.842), equinos (n=149) y hámsters centinela (n=84) en dos localidades costeras y en una en la Amazonía, utilizando la prueba ELISA (Enzyme-Linked ImmunoSorbent Assay) y la prueba de inhibición de la hemaglutinación. Resultados. Entre el 20,63 y el 63,61 % de los humanos registraron IgG contra el virus del dengue (DENV), el de la fiebre amarilla (YFV), el de la encefalitis de San Luis y el del Nilo Occidental (WNV); entre 4,67 y 8,63 % tenían IgG para los virus de la encefalitis equina venezolana, de la encefalitis equina del este y de la encefalitis equina del oeste. Se encontró IgM para DENV y WNV. En los equinos y en los hámsters se encontraron anticuerpos contra alfavirus en todas las localidades muestreadas; dos hámsters mostraron seroconversión a YFV en la Amazonía. Conclusiones. Los resultados del estudio evidenciaron los antecedentes de vacunación contra el YFV y sugieren la actividad de arbovirus no incluidos en el esquema de vigilancia actual. Se recomienda ampliar la vigilancia de arbovirus y mosquitos, continuar con la vacunación contra el YFV, y evaluar su cobertura y efectividad.

Evidence for current circulation of an ancient West Nile virus strain (NY99) in Brazil

Abstract INTRODUCTION: In Brazil, West Nile virus (WNV) was first detected, in 2018, in horses with neurological disease. AIM: We report the first case of WNV infection in a horse from Ceará state and the complete genome sequence of an isolate from Espírito Santo state. Both infections occurred in 2019. METHODS: WNV was isolated from the tissues of a horse with neurological signs in Espírito Santo and sequenced by MiSeq. RESULTS: Phylogenetic analysis revealed that the isolate belongs to lineage 1a, clustering with the NY99 strain, a strain that has not circulated in the USA since 2005. CONCLUSIONS: Our findings reinforce the hypothesis that WNV has been silently circulating in Brazil for many years.

Superinfection exclusion studies using West Nile virus and Culex flavivirus strains from Argentina

In Argentina, many Flavivirus were recognised including West Nile virus (WNV). During 2009 several strains of Culex Flavivirus (CxFV), an insect-specific flavivirus, were isolated in the same region where circulation of WNV was detected. Hence, the objective of this study was to analyse the effect of co-infection in vitro assays using CxFV and WNV Argentinean strains in order to evaluate if CxFV could affect WNV replication. Our results showed that WNV replication was suppressed when multiplicity of infection (MOI) for CxFV was 10 or 100 times higher than WNV. Nevertheless, in vivo assays are necessary in order to evaluate the superinfection exclusion potential.

Assessment of Culex pipiens bioforms in the world's southernmost distribution limit

The mosquito Culex pipiens s.s. L. occurs as two bioforms that differ in physiology and behaviour affecting virus transmission cycles. To assess the occurrence of Cx. pipiens bioforms in the southernmost limit of its distribution, specimens were collected aboveground in southern Buenos Aires Province and east Patagonia, Argentina. Ten larvae and 25 adults were individually processed and identified by polymerase chain reaction (PCR) amplification of Ace-2 and CQ11 loci. Culex quinquefasciatus Say (one larva, two adults), Cx. pipiens f. molestus (one larva, one adult) and one adult of hybrid origin were identified in Buenos Aires Province; only Cx. pipiens f. molestus was recorded in Patagonia (eight larvae, 21 adults). The potential absence of bioform pipiens and its implications in arbovirus enzootic cycles is discussed.

Construction of a pseudovirus reporting system for anti-West Nile virus drug screening / 国际药学研究杂志

Objective: To establish a pseudovirus reporting system was established for anti-WNV drug screening in order to by-pass security risk caused by live viruses in West Nile virus(WNV)research. Methods: 293T Cells were co-transfected with WNV replicon recombinant plasmid prWNV-Rluc containing Renilla luciferase reporter gene and recombinant plasmid pcDNA3.1-CME expressing WNV structural proteins C, M and E;and the cell culture supernatant containing pseudovirus was obtained at 72 h post-cotransfection. Results: After pseudovirus infection with the supernatant, BHK21 cells produced fluorescence with the intensity that increased dose-dependently with the supernatant containing pseudovirus. The WNV NS1 Protein expressed in the infected BHK21 cells was identified by Western blot, confirming the presence of WN pseudovirus in the supernatant. Neutralization assay showed that the WNV neutralizing antibody could effectively block the infection of BHK21 cells by the WN pseudovirus in the supernatant. Conclusion: The West Nile pseudovirus reporting system established in this study might be used without the biosafety level 3 laboratory (BSL-3) and could be applied to the screening of anti-WNV drugs.

Screening of anti-West Nile virus antibody drugs in vitro / 国际药学研究杂志

Objective: To screen anti-West Nile virus antibody drugs in vitro, using the previously established West Nile pseudovirus reporting system. Methods: At first, the enzyme-linked immunosorbent assay(ELISA)was used to detect the binding of 13 anti-West Nile virus antibody candidate strains previously obtained by the phage display technology to the target protein, West Nile virus E protein domainⅢ(DⅢ). Next, the collected cell culture supernatant containing the West Nile pseudovirus was diluted 102-fold, and the dilutions were incubated with the antibody candidates at relevant concentrations at room temperature for 1 h, respectively. Then, BHK21 or K562 cells were infected with the incubated pseudoviral supernatant+antibody mixture. After 48 hours, the infected cells were lysed to measure luciferase activity(RLU). Results: The tested 13 candidate antibodies could all recognize and bind to the West Nile virus E protein DⅢ. Among them, two antibodies(the antibodies 5 and 7)showed a good neutralizing activity and could effectively block the viral invasion upon target cells at the low concentration 5 mg/L, while two antibodies(the antibodies 3 and 8) showed an antibody-enhancing effect(ADE), which was manifested by enhancing viral infection of target cells. Conclusion: Using the West Nile pseudovirus reporting system that we established previously, we are able to obtain two anti-West Nile virus antibodies with neutralizing activity by the in vitro screening.

Research progress on the West Nile virus inhibitors / 药学学报

West Nile virus is a flavivirus transmitted by culex mosquitoes. People are generally susceptible to it, West Nile virus infection can cause west Nile fever, which can develop West Nile viral encephalitis and even lead to death. There are currently no approved specific antiviral drugs against West Nile virus. Therefore, seeking effective West Nile virus inhibitors is a hot topic in current community of medicinal chemistry. In this article, based on the main targets of West Nile virus, we summarize the new progress research on West Nile virus inhibitors.

Febre do Nilo Ocidental: uma revisão da literatura / West Nile Fever: a literature review / Fiebre del Nilo Occidental: una revisión de la literatura

A partir de seu primeiro isolamento em Uganda, em 1937, até os dias de hoje, o vírus do Nilo Ocidental (WNV) tornou-se um alarmante agente etiológico em humanos e animais. O WNV é mantido e perpetuado na natureza através de um ciclo enzoótico, entre aves e mosquitos, e ocasionalmente causa surtos epizoóticos em razão de uma doença contagiosa em humanos e cavalos. Este vírus é amplamente difundido no mundo e, embora grande parte das infecções humanas causadas por WNV seja assintomática, a doença pode evoluir para um quadro neurológico grave, resultando em sequelas a longo prazo ou óbito do paciente. Este estudo tem por objetivo analisar a literatura específica sobre o WNV para apresentar uma revisão de artigos científicos, buscando explorar os aspectos mais importantes da doença. Foram realizadas buscas nas bases de dados PubMed e SciELO a partir dos seguintes descritores: "West Nile virus", "epidemiology" e "pathogenesi'". A linha temporal pesquisada abrange de 1998 a 2019, o que permitiu a localização de 293 artigos, dos quais, com base na leitura dos resumos, 88 foram selecionados para realização da leitura completa do artigo. Ao final da leitura dos artigos, 33 foram selecionados na análise final, tendo levado à conclusão de que a vigilância epidemiológica e as medidas preventivas são uma necessidade contínua para reduzir os impactos da doença na saúde pública.

From its first isolation in Uganda, in 1937, up to date, the West Nile virus (WNV) has become a major cause of disease in both humans and animals. Maintained in nature through an enzootic cycle involving birds and mosquitoes, the WNV is liable to occasional epizootic outbreaks, causing diseases in humans and horses. This virus is widely spread in the world and, although most human infections with WNV are asymptomatic, the disease may progress into a severe neurological disorder, resulting in long-term sequelae or death. This study comprises a literature review on scientific articles discussing the theme of WNV. For that, a search was conducted in the databases PubMed and Scientific Electronic Library Online (SciELO) for articles published between 1998 and 2019, using the following descriptors: "West Nile virus", "epidemiology", and "pathogenesis". From the 293 articles found, 88 were selected for full-text reading after abstract screening, 33 of which remained in the final analysis. To reduce the impact of the disease on public health, authorities must conduct epidemiological surveillance and develop preventive measures.

Desde su primer aislamiento en Uganda en 1937 hasta la actualidad, el virus del Nilo Occidental (WNV) se ha convertido en un importante agente etiológico en humanos y animales. El WNV es un virus mantenido y perpetuado en la naturaleza a través de un ciclo enzoótico, entre aves y mosquitos, y ocasionalmente ocurren brotes epizoóticos, causando enfermedad en humanos y caballos. Es un virus ampliamente difundido en el mundo, que causa infecciones asintomáticas en humanos en la mayoría de los casos, sin embargo, la enfermedad puede evolucionar a un cuadro neurológico grave, ocasionando secuelas a largo plazo o el óbito del paciente. Este estudio tiene por objetivo analizar la literatura específica sobre el WNV para presentar una revisión de artículos científicos referentes al tema. Se realizaron búsquedas en las bases de datos PubMed y SciELO a partir de los siguientes descriptores: "West Nilo virus", "epidemiology" y "pathogenesi". La línea temporal de estudio abarcaba de 1998 a 2019, en la cual se encontraron 293 artículos y con base en la lectura de los resúmenes se seleccionaron 88 para realizar la lectura completa. Al final de la lectura de los artículos, 33 artículos fueron seleccionados en el análisis final, lo que se concluye que la vigilancia epidemiológica y las medidas preventivas son una necesidad continua a fin de reducir los impactos de esa enfermedad en la salud pública.

First Evidence of West Nile Virus in Hodeidah, Yemen: Clinical and Epidemiological Characteristics

ntroduction:West Nile Virus (WNV) infection is an important arthropod-borne zoonosis viral disease. This virus is neglected in Yemen especially in Hodeidah.Aim of the Study:The purpose of this study was to detect WNV infection,determinethe epidemiological and clinical characteristics within febrile patients in Hodeidah city and to determine some risk factors associatedwith WNVinfection.Materials and Methods: 136 febrile patients in a hospital base study were diagnosed in Center of Tropical Medicine and Infectious Diseases (CTMID), Authority of General Al-Thawara Hospital Hodeidah, Yemen from January of 2017 to December of 2017. WNV infection was detected by enzyme linkage immune sorbent assay (ELISA) on serum samples. Results and Discussion:The results showed that 5 cases (3.67%) were WNV –positive namely IgM thatwas detected inwinter and spring seasons,the most prevalent antibodies of WNVwere IgG namely 75 cases (55.14%). Most common symptoms were fever, headache, fatigue, weakness, arthralgia, myalgiaand photophobia. The treatment based on the intravenoustherapy (IV)with anti-pyritic,plasma in some cases and all cases were recovered whilemortality rate was 00%. Conclusion:WNVwas detected in Hodeidah which placed in Tehama "western Yemen", as first time by our preliminary study that confirmed the evidence of WNV IgM and IG antibodies presence on 2017, in order to increase safety of diagnosis of febrile diseases, it is essential to continue surveillance of this emerging infection, suggesting that this emergence has been transported by migratory birds from winteringareas to Tehama region.

Cloning, expression & evaluation of potential immunogenic recombinant capsid premembrane protein of West Nile virus

Background & objectives: West Nile virus (WNV) is a neurotropic flavivirus that has emerged globally as a significant cause of viral encephalitis. The early confirmatory diagnosis of WNV infections is important for timely clinical management and in areas where multiple flaviviruses are endemic. Diagnosis of WNV infection is primarily based on serodiagnosis, followed by virus isolation and identification. The aim of this study was to develop and evaluate a highly sensitive and specific immunoglobulin M (IgM) ELISA using the recombinant CprM protein (rWNV-CprM) for rapid, early and accurate diagnosis of WNV. Methods: The gene coding for the CprM protein of WNV was cloned and expressed in pET 28a vector followed by purification. An indirect IgM microplate ELISA using purified rWNV-CprM protein was optimized having no cross-reactivity with healthy human serum and serum samples obtained from patients with dengue and Japanese encephalitis viruses infection. Results: The comparative evaluation of this rWNV-CprM protein-specific IgM ELISA with plaque reduction neutralization test using 105 blood samples collected from patients suspected to have acute WNV infection revealed 98 per cent concordance with sensitivity and specificity of 100 and 97 per cent, respectively. Interpretation & conclusions: The recombinant CprM protein-based WNV-specific ELISA reported in this study may be useful for rapid screening of large numbers of blood samples in endemic areas during outbreaks.

First isolation of West Nile virus in Brazil

BACKGROUND Serological evidence of West Nile virus (WNV) infection has been reported in different regions of Brazil from equine and human hosts but the virus had never been isolated in the country. OBJECTIVES We sought to identify the viral etiology of equine encephalitis in Espírito Santo state. METHODS We performed viral culture in C6/36 cells, molecular detection of WNV genome, histopathology and immunohistochemistry from horse cerebral tissue. We also carried out sequencing, phylogenetic analysis and molecular clock. FINDINGS Histopathologic analysis from horse cerebral tissue showed injury related to encephalitis and WNV infection was confirmed by immunohistochemistry. The virus was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) from brain tissue and subsequently isolated in C6/36 cells. WNV full-length genome was sequenced showing the isolated strain belongs to lineage 1a. The molecular clock indicated that Brazilian WNV strain share the same common ancestor that were circulating in US during 2002-2005. MAIN CONCLUSIONS Here we report the first isolation of WNV in Brazil from a horse with neurologic disease, which was clustered into lineage 1a with others US WNV strains isolated in beginning of 2000's decade.

West nile virus infections are here! are we prepared to face another flavivirus epidemic?

Abstract Emerging arthropod-borne viruses (arboviruses), such as chikungunya and Zika viruses, are a major threat to public health in countries like Brazil where biodiversity is high and medical care is sometimes precarious. West Nile fever is a disease caused by the West Nile Virus (WNV), an RNA virus belonging to the Flaviviridae family. It is transmitted by infected mosquitoes to numerous animals like birds, reptiles and mammals, including human and non-human primates. In the last decade, the number of reported cases of WNV infection in humans and animals has increased in the Americas. Circulation of WNV in forests and rural areas in Brazil has been detected based on serological surveys and, in 2014, the first case of West Nile fever was confirmed in a patient from Piauí State. In 2018, the virus was isolated for the first time from a horse from a rural area in the state of Espírito Santo presenting with a neurological disorder; this raises the possibility that other cases of WNV encephalitis may have occurred without clinical recognition and without laboratory diagnosis by specific assays. The imminent WNV outbreak poses a challenge for Brazilian clinicians and researchers. In this review, we summarize the basic biological and ecological characteristics of this virus and the clinical presentation and treatment of febrile illnesses caused by WNV. We also discuss the epidemiological aspects, prophylaxis of WNV infections, and monitoring strategies that could be applied in the possibility of a WNV outbreak in Brazil.

Regulation of p38 mitogen-activated protein kinase pathway by West Nile virus in human neuroblastoma cells / 第二军医大学学报

Objective: To explore the regulation of p38 mitogen-activated protein kinase (MAPK) pathway by West Nile virus (WNV) in human neuroblastoma SH-SY5Y cells and the contributions of p38 MAPK to WNV replication as well as stress and inflammatory response related molecule expression. Methods: Total and phosphorylated p38 MAPK levels were analyzed in SH-SY5Y cells incubated with WNV for short (5, 15, 30 and 60 min) and long (12, 24, 48 and 60 h) durations by Western blotting. Dynamic changes of CCAAT/enhancer-binding protein homologous protein (CHOP), interleukin 6 (IL-6), activating transcription factor 6α (ATF6α) and interferon-stimulated gene 15 (ISG15) mRNA expression in WNV infected cells were detected by qRT-PCR. In response to WNV infection, WNV RNA level and CHOP, IL-6, ATF6α and ISG15 mRNA levels were assessed in SH-SY5Y cells transfected with p38 MAPK siRNA. Results: Incubation with WNV for short durations enhanced p38 MAPK phosphorylation compared to the untreated control. The p38 MAPK signaling pathway was activated at 12 h and 24 h in WNV-infected SH-SY5Y cells, but down-regulated at 48 h and 60 h. WNV infection led to increased mRNA expression of CHOP, IL-6 and ISG15 and reduced ATF6α mRNA. In comparison with control siRNA transfection, the levels of WNV RNA (P<0.05) and ATF6α mRNA (P<0.01) were increased and CHOP mRNA level was decreased (P<0.05) in WNV-infected SH-SY5Y cells with the p38 MAPK siRNA transfection. Conclusion: The p38 MAPK pathway is activated during early stage of WNV infection and such activation may negatively regulate WNV replication. WNV-induced stress response molecules CHOP and ATF6α and proinflammatory cytokine IL-6 production by SH-SY5Y cells are coupled with the regulation of p38 MAPK pathway.

Neutralizing monoclonal antibodies against the West Nile virus: research advances / 国际药学研究杂志

West Nile virus(WNV)is a member of the Flavivirus genus, which can cause zoonotic diseases or even death. With the gradual globalization of WNV infection epidemic, there exists an input risk in China. Now there are no approved vaccines and drugs for the prevention and treatment of human infection with WNV. Hence, research and de- velopment of drugs against WNV is necessary. WNV envelope(E)protein, the main antigen of neutralizing antibody, is involving in the infection of the target cells by the virus. The E protein contains three structural domains(D, DⅡ and DIII). This review briefly describes the recent advances in neutralizing antibodies targeting these three domains.

Detection and surveillance of west nile virus infection in southern Xinjiang, China, 2012 / 中华实验和临床病毒学杂志

Objective@#To investigate the west nile virus (WNV) infection in Xinjiang, China.@*Methods@#Serum samples were collected from patients with fever and chicken in southern Xinjiang, 2012. The presence of WNV-specific immunoglobulin M (IgM) antibodies and neutralizing antibodies was examined through enzyme-linked immunosorbent assay (ELISA) and plaque reduction neutraization test (PRNT90).@*Results@#A total of 1 712 serum samples of outpatients and inpatients were collected in 8 counties in southern Xinjiang. As a result , 22 samples were positive for WNV IgM antibody and 48 samples were positive for WNV neutralization antibody, among which 21 WNV IgM antibody positive samples and 42 WNV neutralization antibody positive samples were from Jiashi county. Of 383 chicken serum samples collected in 4 counties in southern Xinjiang, only 28 samples were positive for WNV neutralizing antibody, interestingly, all positive chicken serum samples were collected from Jiashi county.@*Conclusions@#This study revealed that WNV infection occurred in human and poultry in southern Xinjiang, 2012, mainly in Jiashi county.

Diagnostic significance of immunoglobulin G avidity in symptomatic and asymptomatic West Nile virus infection

Abstract INTRODUCTION West Nile virus (WNV) immunoglobulin M (IgM) antibodies have been shown to persist for up to 500 days in certain patients. To evaluate the usefulness of immunoglobulin G (IgG) avidity assessment in the diagnosis of WNV infection, we analyzed 54 WNV IgM- and/or IgG-positive serum samples from 39 patients with neuroinvasive disease and 15 asymptomatic cases tested during a seroprevalence investigation. METHODS Serological tests (WNV IgM/IgG antibody detection, IgG avidity) were performed using commercially available enzyme-linked immunosorbent assays. RESULTS WNV IgM antibodies were detected in 47 (87%) samples. Acute/recent WNV infection was confirmed based on low/borderline avidity index (AI) in 44 IgM-positive samples (93.6%). In three IgM-positive samples (6.4%), high IgG AIs were detected, thus indicating persisting IgM antibodies from previous infections. All IgM-negative samples showed high AIs. Patients with WNV neuroinvasive disease tested within 30 days showed low AIs. In six patients tested 34-50 days after disease onset, AI was borderline (42%-60%), suggesting earlier WNV IgG maturation. Samples with the highest IgM values were associated with the lowest AIs (Spearman's rho coefficient -0.767, p < 0.001). CONCLUSIONS Our results indicate that IgG avidity differentiates current/recent WNV infection from persistent IgM seropositivity from the previous WNV transmission season both in patients with WNV neuroinvasive disease and in asymptomatic persons. A strong negative correlation between IgM antibody levels and AI indicates that in cases with very high IgM levels, determination of IgG avidity may not be necessary. As many patients showed rapid avidity maturation, low IgG avidity is indicative of WNV infection within the previous month.

Evaluation of real-time reverse-transcription loop-mediated isothermal amplification assay for clinical diagnosis of West Nile virus in patients

Background & objectives: West Nile virus (WNV) is a mosquito-borne flavivirus. The disease can be diagnosed by isolation followed by fluorescent antibody tests, enzyme-linked immunosorbent assay and polymerase chain reaction (PCR) assay. These diagnostic methods are laborious and time-consuming. The present study was aimed to evaluate the real-time reverse-transcription loop-mediated isothermal amplification (RT-LAMP) method for rapid, early and accurate diagnosis of WNV. Methods: A one-step single tube accelerated quantitative RT-LAMP assay was evaluated by targeting the Env gene of WNV. The gene amplification was accomplished by incubating the reaction mixture at 63°C for 60 min in both real time turbidimeter as well as routine laboratory water bath/dry heating bath. To rule out contamination issues, proper negative controls, including no template, no primer; and no enzyme, were always kept alongside each run. The RT-LAMP assay was evaluated on 105 clinical samples from individuals having ocular infection. Results: Of the 105 samples tested, 27 were positive for WNV by RT-LAMP assay. The comparative evaluation with conventional RT-PCR revealed 100 per cent accordance with sensitivity and specificity of 100 and 95 per cent, respectively. The specificity of this assay was confirmed with serum samples obtained from patients with dengue and chikungunya. Interpretation & conclusions: The RT-LAMP test seemed to be a sensitive and specific method for rapid detection of WNV infection and would be useful for rapid screening of a large number of clinical samples in endemic areas during outbreaks.

Improved reverse transcription-polymerase chain reaction assay for the detection of flaviviruses with semi-nested primers for discrimination between dengue virus serotypes and Zika virus

BACKGROUND The genus Flavivirus includes a variety of medically important viruses, including dengue virus (DENV) and Zika virus (ZIKV), which are most prevalent in Brazil. Because the clinical profile of patients affected by different DENV serotypes or ZIKV may be similar, the development of new methods that facilitate a rapid and accurate diagnosis is crucial. OBJECTIVES The current study aimed to develop an improved reverse transcription-polymerase chain reaction (RT-PCR) protocol for universal detection of flaviviruses by using semi-nested primers that discriminate between DENV serotypes and ZIKV. METHODS The bioinformatics workflow adopted for primer design included: (1) alignment of 1,442 flavivirus genome sequences, (2) characterisation of 27 conserved regions, (3) generation of a primer set comprising 77 universal primers, and (4) selection of primer pairs with greatest coverage and specificity. Following primer design, the reaction was validated in vitro. The same approach was applied to the design of primers specific for DENV and ZIKV, using a species-specific sequence database. FINDINGS The new assay amplified an 800-806 nt variable region of the NS5 gene and allowed discrimination of virtually all flavivirus species using reference-sequence comparison. The 800-806 nt fragment was validated as a template for a semi-nested multiplex PCR using five additional primers for the detection of DENV and ZIKV. These primers were designed to generate amplicons of different sizes, allowing differentiation of the four serotypes of DENV, and ZIKV using agarose gel electrophoresis. MAIN CONCLUSIONS The bioinformatics pipeline allowed efficient primer design, making it possible to identify the best targets within the coding region of the NS5 protein. The multiplex system proved effective in differentiation of DENV1-4 and ZIKV on a 2% agarose gel. The possibility of discriminating DENV serotypes and ZIKV in the same reaction provided a faster result consuming less sample. In addition, this simplified approach ensured the reduction of the cost per analysis.

Survey of pathogens in threatened wild red-tailed Amazon parrot (Amazona brasiliensis) nestlings in Rasa Island, Brazil

ABSTRACT The red-tailed Amazon parrot (Amazona brasiliensis) is a threatened species of psittacine bird that inhabit coastal regions of Brazil. In view of the threat of this species, the aim of this study was to perform a health evaluation in wild nestlings in Rasa Island, determining the prevalence of enterobacteria and infectious agents according to type of nest. Blood samples were collected from 64 birds and evaluated for antibodies of Chlamydia psittaci by commercial dot-blot ELISA. Cloacal and oropharyngeal swabs samples were collected from 23 birds from artificial wooden nests, 15 birds from PVC nests and 2 birds from natural nests for microbiological analysis. Swab samples were collected from 58 parrots for C. psittaci detection by PCR and from 50 nestlings for Avian Influenza, Newcastle Disease and West Nile viruses' detection analysis by real-time RT-PCR. Ten bacterial genera and 17 species were identified, and the most prevalent were Escherichia coli and Klebsiella oxytoca. There was no influence of the type of nest in the nestlings' microbiota. All samples tested by ELISA and PCR were negative. There is currently insufficient information available about the health of A. brasiliensis and data of this study provide a reference point for future evaluations and aid in conservation plans.