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.

Culex quinquefasciatus from areas with the highest incidence of microcephaly associated with Zika virus infections in the Northeast Region of Brazil are refractory to the virus

Zika virus (ZIKV) is widely distributed in Brazil and the Northeast Region (NE) is the most affected zone, showing the highest incidence of microcephaly associated with ZIKV congenital infections worldwide. We report attempts to infect three populations of Culex quinquefasciatus from severely affected sites in the NE and Southeast Region (SE) of Brazil with three strains of ZIKV isolated from these localities. An Aedes aegypti population from the SE was used as a positive control. All tested Cx. quinquefasciatus populations were refractory to the ZIKV isolates. For these reasons, we believe Cx. quinquefasciatus should not be considered a potential vector of ZIKV in Brazil.

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.

Saint louis encpphalitis virus in mato grosso, central-western Brazil / Vírus da encefalite de Saint Louis em Mato Grosso, Centro-Oeste, Brasil

The dengue virus (DENV), which is frequently involved in large epidemics, and the yellow fever virus (YFV), which is responsible for sporadic sylvatic outbreaks, are considered the most important flaviviruses circulating in Brazil. Because of that, laboratorial diagnosis of acute undifferentiated febrile illness during epidemic periods is frequently directed towards these viruses, which may eventually hinder the detection of other circulating flaviviruses, including the Saint Louis encephalitis virus (SLEV), which is widely dispersed across the Americas. The aim of this study was to conduct a molecular investigation of 11 flaviviruses using 604 serum samples obtained from patients during a large dengue fever outbreak in the state of Mato Grosso (MT) between 2011 and 2012. Simultaneously, 3,433 female Culex spp. collected with Nasci aspirators in the city of Cuiabá, MT, in 2013, and allocated to 409 pools containing 1-10 mosquitoes, were also tested by multiplex semi-nested reverse transcription PCR for the same flaviviruses. SLEV was detected in three patients co-infected with DENV-4 from the cities of Cuiabá and Várzea Grande. One of them was a triple co-infection with DENV-1. None of them mentioned recent travel or access to sylvatic/rural regions, indicating that transmission might have occurred within the metropolitan area. Regarding mosquito samples, one pool containing one Culex quinquefasciatus female was positive for SLEV, with a minimum infection rate (MIR) of 0.29 per 1000 specimens of this species. Phylogenetic analysis indicates both human and mosquito SLEV cluster, with isolates from genotype V-A obtained from animals in the Amazon region, in the state of Pará. This is the first report of SLEV molecular identification in MT.

O vírus da dengue (DENV), frequentemente envolvido em epidemias de grande proporção, e o vírus da febre amarela (YFV), responsável por surtos silvestres esporádicos, são considerados os flavivírus circulantes mais importantes no Brasil. Por este motivo, o diagnóstico laboratorial de doença febril aguda indiferenciada durante períodos epidêmicos é frequentemente direcionado para dengue e febre amarela no país, dificultando a detecção de outros arbovírus possivelmente circulantes, incluindo o vírus da encefalite de Saint Louis (SLEV), que é amplamente disperso nas Américas. O objetivo deste estudo foi investigar molecularmente a presença de 11 flavivírus no soro de 604 pacientes durante grande epidemia de dengue no estado de Mato Grosso (MT), Centro-Oeste do Brasil, entre 2011- 2012. Concomitantemente, 3.433 fêmeas de Culex spp. capturadas com aspirador de Nasci na cidade de Cuiabá, MT e alocadas em 409 pools com 1-10 mosquitos em 2013 foram testadas por multiplex seminested RT-PCR para os mesmos flavivírus. O SLEV foi detectado em três pacientes co-infectados com o DENV-4 das cidades de Cuiabá e Várzea Grande, MT. Um dos pacientes apresentava tripla co-infecção com DENV-1. Nenhum paciente referiu histórico recente de viagem ou acesso a áreas rurais/silvestres. Um pool contendo uma fêmea de Culex quinquefasciatus foi positivo para o SLEV, apresentando taxa de infecção mínima (MIR) de 0,29 por 1000 espécimes desta espécie. A análise filogenética indica que ambas as amostras formam um cluster com isolados do genótipo V-A do SLEV obtidos de animais na região amazônica do estado do Pará. Este é o primeiro relato de identificação molecular do SLEV no MT.

Detección molecular de virus de encefalitis de Saint Louis en mosquitos de Buenos Aires, Argentina

Durante el mes de marzo de 2013 una población de palomas torcazas (Zenaida auriculata) se instaló en una zona céntrica de la ciudad de Buenos Aires. Conociendo el rol que poseen estas aves como hospedadores competentes del virus de la encefalitis de Saint Louis (SLEV), fue colocada en el lugar una trampa de luz tipo CDC, a fin de realizar una vigilancia entomológica. Durante ese mes,fueron capturados 5 grupos de mosquitos (n = 48), 3 correspondieron a la especie Culex pipiens (n = 10) y 2 a Culex spp.(n = 38), no pudiéndose determinar en estos últimos con precisión la especie por encontrarse dañados. En un grupo de mosquitos Culex spp. se detectó el SLEV por técnicas moleculares. Posteriormente fue secuenciado y clasificado como perteneciente al genotipo III.

During March 2013 a population of eared doves (Zenaida auriculata) was established in the center of City of Buenos Aires. Considering the role of these birds as host competent for Saint Louis encephalitis virus (SLEV), a CDC light trap was put in place to perform entomologic surveillance. During this month 5 pools of mosquitoes (n = 48) were collected and taxonomically determined. Three of them were classified as Culex pipiens (n = 10) and the other two were Culex spp. (n = 38). In this case, the mosquitoes species could not be determined due to that individuals were damaged. One of the Culex spp. pool was found to be positive for Saint Louis encephalitis virus by molecular techniques. This was then sequenced and classified as genotype III.

Virulence variation among epidemic and non-epidemic strains of Saint Louis encephalitis virus circulating in Argentina

Saint Louis encephalitis virus caused an outbreak of febrile illness and encephalitis cases in Córdoba, Argentina, in 2005. During this outbreak, the strain CbaAr-4005 was isolated from Culex quinquefasciatus mosquitoes. We hypothesised that this epidemic variant would be more virulent in a mouse model than two other non-epidemic strains (78V-6507 and CorAn-9275) isolated under different epidemiological conditions. To test this hypothesis, we performed a biological characterisation in a murine model, including mortality, morbidity and infection percentages and lethal infection indices using the three strains. Mice were separated into age groups (7, 10 and 21-day-old mice) and analysed after infection. The strain CbaAr-4005 was the most infective and lethal of the three variants, whereas the other two strains exhibited a decreasing mortality percentage with increasing animal age. The strain CbaAr-4005 produced the highest morbidity percentages and no significant differences among age groups were observed. The epidemic strain caused signs of illness in all inoculated animals and showed narrower ranges from the onset of symptoms than the other strains. CbaAr-4005 was the most virulent for Swiss albino mice. Our results highlight the importance of performing biological characterisations of arbovirus strains likely to be responsible for emerging or reemerging human diseases.

Mosquitos (Díptera: Culicidae) vectores potenciales de arbovirus en la región de Urabá, noroccidente de Colombia[

Introducción. Los estudios encaminados a conocer los parámetros ecológicos de las poblaciones de mosquitos selváticos, permiten establecer el riesgo de transmisión de arbovirus y aportar recomendaciones sobre prevención, vigilancia y control a las autoridades de salud. Objetivo. Determinar la diversidad y abundancia de mosquitos nocturnos y crepusculares, potenciales vectores de arbovirus en zonas rurales de Apartadó y Turbo, Antioquia. Materiales y métodos. Se realizaron muestreos trimestrales. Para la recolección de mosquitos se usaron trampas CDC, Shannon y cebo humano protegido, en fragmentos de bosque, entre las 18:00 y las 06:00 horas. Se estimaron los índices de diversidad y abundancia de especies. Resultados. Se capturaron 583 mosquitos de 10 génerosy 27 especies. Las especies más abundantes fueron Coquilletidia venezuelensis (14,6 %), Aedes scapularis (14,08 %), Psorophora ferox (10,82 %) y Culex quinquefasciatus (10,3 %). La riqueza específica y los índices ecológicos calculados fueron mayores en Turbo; el fragmento de bosque estudiado en Turbo se considera de mayor riqueza y uniformidad de especies. El hallazgo de Cx. pedroi, Ae. scapularis, Ae. angustivittatus, Cq. venezuelensis, Cx. nigripalpus, Cx. quinquefasciatus, Cx. declarator, Mansonia titillans, Ma. pseudotitillans, Ps. ferox y Trichiprosopon digitatum reportados como vectores de arbovirus, alerta sobre la posibilidad de transmisión en la zona. Conclusión. La diversidad y abundancia de mosquitos en la zona de estudio son altas. Los análisis ecológicos más los reportes previos de capacidad vectorial de algunas de las especies registradas, permiten concluir que en la zona se pueden presentar brotes de arbovirosis.

Mosquitoes (Diptera: Culiciadae) as potential vectors of arbovirused in the Urabá region, Northwest of Colombia.

Introduction. Studies directed to investigate ecological parameters of sylvatic mosquitoes populations permit the establishment of risk levels in the transmission of arboviruses and provide the basis for recommendations to health authorities about prevention, surveillance and control. Objective. To establish the diversity and abundance of mosquitoes vectors of arbovirus in rural areas of Apartadó and Turbo, Antioquia. Materials and methods. Quarterly sampling was done. For mosquito collections in forest fragments, CDC traps, Shannon traps and human landing methods were used. Diversity and abundance indices were calculated. Results. Five hundred eighty-three mosquitoes were collected and identified in 10 genera and 27 species. The most abundant species were as follows: Coquilletidia venezuelensis (14.6%), Aedes scapularis (14.1%), Psorophora ferox (10.8%) and Culex quinquefasciatus (10.3%). Species richness and ecological indexes were highest in Turbo municipality; where the forest fragment was considered highest in species richness and uniformity. The identification in the samples of Culex pedroi,Ae. scapularis, Aedes angustivittatus, Cq. venezuelensis, Culex nigripalpus, Cx. quinquefasciatus, Culex declarator, Mansonia titillans, Mansonia pseudotitillans, Ps. ferox andTrichoprosopon. Digitatum--allpreviously reported as arbovirus vectors--warns about the possibility of arbovirus transmission in the zone. Conclusions. Mosquito diversity and abundance in the study area was very high. The ecological analysis, plus previous reports about vector competence of several of the recorded species, permits the conclusion that arbovirus outbreaks can occur in the Urabá region.

Isolation and genetic characterization of Japanese encephalitis virus from equines in India

Japanese encephalitis (JE) is an important vector-borne viral disease of humans and horses in Asia. JE outbreaks occur regularly amongst humans in certain parts of India and sporadic cases occur among horses. In this study, JE seroprevalence and evidence of JE virus (JEV) infection among horses in Haryana (India) is described. Antibodies against JEV were detected in 67 out of 637 (10.5%) horses screened between 2006 and 2010. Two foals exhibiting neurological signs were positive for JEV RNA by RT-PCR; JEV was isolated from the serum of one of the foals collected on the second day of illness. This is the first report of JEV isolation from a horse in India. Furthermore, a pool of mosquitoes collected from the premises housing these foals was positive for JEV RNA by RT-PCR. Three structural genes, capsid (C), premembrane (prM), and envelope (E) of the isolated virus (JE/eq/India/H225/2009) spanning 2,500 nucleotides (from 134 to 2,633) were cloned and sequenced. BLAST results showed that these genes had a greater than 97% nucleotide sequence identity with different human JEV isolates from India. Phylogenetic analysis based on E- and C/prM genes indicated that the equine JEV isolate belonged to genotype III and was closely related to the Vellore group of JEV isolates from India.

Occurrence of Pacora virus (PAC: Bunyaviridae: Bunyavirus - like) in Brazilian Amazonia: new findings

The Pacora virus (PAC: Bunyaviridae: Bunyavirus - like), was known only through some isolates obtained from mosquitoes (culex dunni) in Panama, and two isolates obtained in the Brasilian Amazon region from the sylvatic birds Phlegopsis nigromaculata and Automolus ochrolaemus. In order to add to the knowledge of the arboviruses' cycles, mosquitoes were colected in the Zoobotanical Park of Macapá (Amapá State), from August, 18th to September, 11th, in areas of rain forest and savannah. The collections were done at both ground and canopy levels in the savannah, with human bait and light traps. 5642 mosquitoes have been collected, of which 5580 (or 184 pools) have been inoculated intracerebrally in newborn mice. Two strains of PAC virus have been isolated from unidentified Culex mosquitoes. This result is the first report of PAC virus in Amapá State. The virus has also been isolated for the first time from mosquitoes in Brazil. Thus, the presence of the complete cycle of sylvatic transmission of this agent is confirmed in Brazilian Amazonia. The ecological data available for the host show that the virus circulates at the lower level in the rain forest (0-5 m) with mostly nocturnal mosquitoes as vectors.