Curious entanglements: interactions between mosquitoes, their microbiota, and arboviruses.

Mosquitoes naturally harbor a diverse community of microorganisms that play a crucial role in their biology. Mosquito-microbiota interactions are abundant and complex. They can dramatically alter the mosquito immune response, and impede or enhance a mosquito's ability to transmit medically important arboviral pathogens. Yet critically, given the massive public health impact of arboviral disease, few such interactions have been well characterized. In this review, we describe the current state of knowledge of the role of microorganisms in mosquito biology, how microbial-induced changes to mosquito immunity moderate infection with arboviruses, cases of mosquito-microbial-virus interactions with a defined mechanism, and the molecular interactions that underlie the endosymbiotic bacterium Wolbachia's ability to block virus infection in mosquitoes.

Biological characterization of Aedes albopictus (Diptera: Culicidae) in Argentina: implications for arbovirus transmission.

Aedes albopictus (Diptera: Culicidae) is an invasive mosquito, native to Asia, that has expanded its range worldwide. It is considered to be a public health threat as it is a competent vector of viruses of medical importance, including dengue, chikungunya, and Zika. Despite its medical importance there is almost no information on biologically important traits of Ae. albopictus in Argentina. We studied life cycle traits, demographic parameters and analyzed the competence of this mosquito as a virus vector. In addition, we determined the prevalence of Wolbachia strains in Ae. albopictus as a first approach to investigate the potential role of this bacteria in modulating vector competence for arboviruses. We observed low hatch rates of eggs, which led to a negative growth rate. We found that Ae. albopictus individuals were infected with Wolbachia in the F1 but while standard superinfection with wAlbA and wAlbB types was found in 66.7% of the females, 16.7% of the females and 62.5% of the males were single-infected with the wAlbB strain. Finally, despite high levels of infection and dissemination, particularly for chikungunya virus, Ae. albopictus from subtropical Argentina were found to be relatively inefficient vectors for transmission of both chikungunya and dengue viruses.

Initial experience with imported Zika virus infection in Spain / Experiencia inicial en infección importada por virus Zika en España

Introduction: A considerable increase of imported Zika virus (ZIKV) infection has been reported in Europe in the last year. This is the result of the large outbreak of the disease in the Americas, along with the increase in the numbers of travellers and immigrants arriving from ZIKV endemic areas. Methods: A descriptive study was conducted in the Tropical Medicine Unit of Hospital La Paz-Carlos III in Madrid on travellers returning from an endemic area for ZIKV from January to April 2016. Demographic, clinical and microbiological data were analyzed. Results: A total of 185 patients were screened for ZIKV (59.9% women, median age of 37.7±10.3 years). Main purpose of the travel was tourism to Colombia, Brazil, and México. Just under three-quarters (73%) were symptomatic, mostly with fever and headache. A total of 13 patients (7% of those screened) were diagnosed with ZIKV infections, of which four of them were pregnant. All of them were symptomatic patients, the majority immigrants, and mainly from Colombia. Diagnostic tests were based on positive neutralization antibodies (8 cases, 61.6%) and a positive RT-PCR in different organic fluids (7 cases, 53.8%) The four infected pregnant women underwent a neurosonography every 3 weeks, and no alterations were detected. RT-PCR in amniotic fluid was performed in three of them, with negative results. One of the children has already been born healthy. Conclusions: Our cases series represents the largest cohort of imported ZIKV to Spain described until now. Clinicians must increase awareness about the progression of the ZIKV outbreak and the affected areas so that they can include Zika virus infection in their differential diagnosis for travellers from those areas (AU)

Introducción: En el último año se ha registrado un importante aumento de casos de infección por virus Zika (ZIKV) importados en Europa. Este hecho es un reflejo de la epidemia que actualmente se está produciendo en las Américas, así como del aumento del número de viajeros e inmigrantes que proceden de zonas endémicas. Métodos: Estudio descriptivo de los viajeros retornados de área endémica para ZIKV en la Unidad de Medicina Tropical del Hospital La Paz-Carlos III en Madrid, de enero a abril de 2016. Se recogieron y analizaron datos demográficos, clínicos y microbiológicos. Resultados: Se cribaron para ZIKV un total de 185 pacientes (59,9% mujeres, mediana de edad de 37,7±10,3 años). El propósito por el que habían realizado el viaje fue por turismo a Colombia, Brasil y México. El 73% de los inicialmente cribados presentaron síntomas, fundamentalmente fiebre y cefalea. Se diagnosticó infección por ZIKV a 13 pacientes (7% de los cribados); 4 de ellos eran gestantes. Todos los casos con infección confirmada estaban sintomáticos, y la mayoría eran inmigrantes colombianos. El diagnóstico se basó en la presencia de anticuerpos neutralizantes positivos (8 casos, 61,6%) y RT-PCR positiva en diferentes fluidos orgánicos (7 casos, 53,8%). A las 4 gestantes infectadas se les realizó neurosonografía fetal seriada cada 3 semanas, no detectándose alteraciones en ninguna de ellas. En 3 casos se realizó RT-PCR en líquido amniótico, que fue negativo. Uno de los niños ha nacido, y está completamente sano. Conclusiones: Nuestra serie representa la cohorte más grande de infección por ZIKV importada en España hasta la fecha. Los clínicos deben estar alerta sobre la evolución de la epidemia del ZIKV y las zonas a las que afecta, para poder incluir la infección por ZIKV dentro del diagnóstico diferencial de viajeros que regresan de esas áreas (AU)

Detection of arboviruses and other micro-organisms in experimentally infected mosquitoes using massively parallel sequencing.

Human disease incidence attributed to arbovirus infection is increasing throughout the world, with effective control interventions limited by issues of sustainability, insecticide resistance and the lack of effective vaccines. Several promising control strategies are currently under development, such as the release of mosquitoes trans-infected with virus-blocking Wolbachia bacteria. Implementation of any control program is dependent on effective virus surveillance and a thorough understanding of virus-vector interactions. Massively parallel sequencing has enormous potential for providing comprehensive genomic information that can be used to assess many aspects of arbovirus ecology, as well as to evaluate novel control strategies. To demonstrate proof-of-principle, we analyzed Aedes aegypti or Aedes albopictus experimentally infected with dengue, yellow fever or chikungunya viruses. Random amplification was used to prepare sufficient template for sequencing on the Personal Genome Machine. Viral sequences were present in all infected mosquitoes. In addition, in most cases, we were also able to identify the mosquito species and mosquito micro-organisms, including the bacterial endosymbiont Wolbachia. Importantly, naturally occurring Wolbachia strains could be differentiated from strains that had been trans-infected into the mosquito. The method allowed us to assemble near full-length viral genomes and detect other micro-organisms without prior sequence knowledge, in a single reaction. This is a step toward the application of massively parallel sequencing as an arbovirus surveillance tool. It has the potential to provide insight into virus transmission dynamics, and has applicability to the post-release monitoring of Wolbachia in mosquito populations.

Arboviruses causing human disease in the Australasian zoogeographic region.

Over 65 arboviruses have been reported from countries in the Australasian zoogeographic region, but only a few have been implicated in human disease. These include the flaviviruses Murray Valley encephalitis (MVE), Kunjin (KUN), Kokobera (KOK), and dengue, particularly types 1 and 2; the alphaviruses Ross River (RR), Barmah Forest (BF), and Sindbis (SIN); and the bunyaviruses, Gan Gan and Trubanaman. In this paper recent epidemiological and clinical results pertaining to these viruses are reviewed, with major emphasis on MVE and RR viruses. The extensive early studies of Australian arboviruses have been reviewed by Doherty [49, 50], and their ecology and vectors more recently by Kay and Standfast [87]. In addition, the biology of MVE and KUN [113] and RR [87, 114] viruses have been the subjects of more detailed reviews. The Australasian zoogeographic region is defined as countries east of the Wallace and Weber lines, two hypothetical lines in the Indo-Australian archipelago where the fauna of the Australasian and Oriental regions meet. Seroepidemiological studies of human arboviral infections have suggested that the Japanese encephalitis flavivirus and the chikungunya alphavirus occur only in the Oriental region, whereas the related MVE and RR viruses, respectively, are restricted to the Australasian region [85, 148]. Serological results from Wallacea, the zone between the Wallace and Weber lines, are not so clear-cut [85]. This review is therefore restricted to countries east of Wallacea, specifically New Guinea and Australia.

Non-viraemic transmission of tick-borne encephalitis virus: a mechanism for arbovirus survival in nature.

The vectors of arthropod-borne viruses (arboviruses) become infected by feeding on the viraemic blood of an infected animal. This theory is based on transmission studies involving artificial infection of vertebrate hosts by syringe inoculation. To reproduce natural conditions of virus transmission, infected and uninfected vectors (ticks) of tick-borne encephalitis virus, the most important arbovirus in Europe, were allowed to feed together on uninfected wild vertebrate hosts. The greatest numbers of infected ticks were obtained from susceptible host species that had undetectable or very low levels of viraemia. The results suggest that 'nonviremic transmission' is an important mechanism for the survival of certain arboviruses in nature.

[An experimental infection caused by the Issyk-Kul arbovirus]. / Eksperimental'naia infekstiia, vyzvannaia arbovirusom Issyk-Kul'.

Pathogenic properties of Issyk-Kul virus have been experimentally studied in green monkeys, golden hamsters and white mice. The virus was identified in the blood and organs of all the animals. Pathohistological studies have revealed inflammatory and dystrophic changes in the central nervous system, lungs, liver and kidneys. Pronounced immunomorphological changes have been observed in the spleen. The virus possessed pantropic properties and caused generalized infection in all the animals irrespective of the mode of infection. In monkeys asymptomatic infection was accompanied by marked organ damages and virusemia. The presence of specific antibodies in the blood indicated a low efficacy of humoral immunity in this infection.

Estudio preliminar sobre la presencia de arbovirus en poblaciones de Corrientes y Misiones / Preliminary study on the presence of arbovirus in the populations of Corrientes and Misiones

Con la intención de localizar y medir la prevalencia de anticuerpos contra Flavivirus se analizaron por la técnica de inhibición de la hemoaglutinación 479 sueros provenientes de personas que habitan el territorio misionero de la República Argentina y 49 sueros humanos de la provincia de Corrientes. Se estudiaron localidades ribereñas con un tránsito importante con Paraguay y/o Brasil, con el objeto de verificar la posible introducción del Dengue desde estos países. En Ituzaingó (Corrientes) se encontraron dos sueros positivos para Dengue 2. En Misiones se encontraron 3 sueros positivos en Oberá y 2 en Montecarlo para Dengue 2 mientras que en Puerto Iguazú un suero fue reactivo contra Dengue. 1. La seroprevalencia en las poblaciones ribereñas fue significativamente mayor al de las poblaciones de montaña. Las poblaciones sobre el río Uruguay. El 54% de los sueros presentó mayor reactividad para SLE que para FA y Dengue. En los sueros de Misiones se analizaron, a su vez, los anticuerpos contra los alfa-virus EEE y WEE; se observó un patron de distribución complementario con respecto a los flavivirus, siendo mayor la seroprevalencia en las poblaciones de montaña y, dentro de las rivereñas, en las del río Uruguay

Estudio preliminar sobre la presencia de arbovirus en poblaciones de Corrientes y Misiones / Preliminary study on the presence of arbovirus in the populations of Corrientes and Misiones

Con la intención de localizar y medir la prevalencia de anticuerpos contra Flavivirus se analizaron por la técnica de inhibición de la hemoaglutinación 479 sueros provenientes de personas que habitan el territorio misionero de la República Argentina y 49 sueros humanos de la provincia de Corrientes. Se estudiaron localidades ribereñas con un tránsito importante con Paraguay y/o Brasil, con el objeto de verificar la posible introducción del Dengue desde estos países. En Ituzaingó (Corrientes) se encontraron dos sueros positivos para Dengue 2. En Misiones se encontraron 3 sueros positivos en Oberá y 2 en Montecarlo para Dengue 2 mientras que en Puerto Iguazú un suero fue reactivo contra Dengue. 1. La seroprevalencia en las poblaciones ribereñas fue significativamente mayor al de las poblaciones de montaña. Las poblaciones sobre el río Uruguay. El 54% de los sueros presentó mayor reactividad para SLE que para FA y Dengue. En los sueros de Misiones se analizaron, a su vez, los anticuerpos contra los alfa-virus EEE y WEE; se observó un patron de distribución complementario con respecto a los flavivirus, siendo mayor la seroprevalencia en las poblaciones de montaña y, dentro de las rivereñas, en las del río Uruguay (AU)

Isolation and identification of arboviruses from the Sultanate of Oman.

Sentinel herds and a vector surveillance system were used to identify the presence of arboviruses in Oman. Two strains of bluetongue virus (BTV) serotype 4 and two strains of Akabane virus, were isolated and identified. Both BTV isolates and one Akabane virus isolate came from goats while the second Akabane isolate came from Culicoides imicola. This is the first isolation of an Akabane virus from Culicoides in Arabia. Vector competence studies with the Oman viruses in laboratory reared C. variipennis showed that after oral infection both viruses replicated in Culicoides and were maintained at high titre for at least 10 days post infection.

[Preliminary study on the presence of arbovirus in the populations of Corrientes and Misiones]. / Estudio preliminar sobre la presencia de arbovirus en poblaciones de Corrientes y Misiones.

Human seroprevalence of Flavivirus was determined by hemagglutination inhibition tests on 479 sera from Misiones and 49 from Corrientes provinces. Paraná and Uruguay river bank communities from Argentina and neighbouring countries carry out frequent traffic across the rivers. With the aim of searching for a possible introduction of Dengue virus from Brasil or/and Paraguay, reactivity among people from Paraná and Uruguay river communities was compared with those from mountain communities. Two sera from Ituzaingó (Corrientes Province) were positive for Dengue 2. In Misiones, 3 sera from Oberá and 2 from Montecarlo were reactive for Dengue 2 and 1 serum from Puerto Iguazú was reactive for Dengue 1. Seroprevalence among the river population was significatively higher than among the mountain population. Likewise, populations on Paraná river showed more positive sera than those on Uruguay river; 54% of the samples possessed titers for SLE virus higher than for Dengue or Yellow fever. Anti-alphavirus (EEE and WEE) antibodies tested in sera from Misiones people showed a complementary distribution pattern to flavivirus. Seroprevalence of anti-alphavirus antibodies was higher in the mountain than in the river populations.

[The characteristics of the relationships of arthropods of the refuge complex with the causative agents of transmissible viral infections in bird rookeries]. / Osobennosti sviazei chlenistonogikh ubezhishchnogo kompleksa s vozbuditeliami transmissivnykh virusnykh infektsii v kolonial'nykh poseleniiakh ptits.

The isolation of viruses of tick-borne encephalitis, West Nile fever, and Omsk hemorrhagic fever from arthropods of nests of colonial birds (rook, sand martin, tree sparrow, Laridae) in different zones of West Siberia (from tundra to steppe) has been analyzed. The role of gamasid mites, hematophages and saprophages, characteristic inhabitants of nests of colonial birds, and of the tick Ixodes lividus in connection with their biology, coadaptation with hosts, microclimatic nest conditions, etc. has been evaluated. It has been concluded that the whole nest community of arthropods in a nest rather than one infected individual has to be regarded as an infective unit in colonies.

Geographical distribution of arboviruses in Yugoslavia.

Studies of arboviruses started in Yugoslavia in 1953 following the isolation of TBE virus which caused a severe epidemic that year. Until now the following viruses have been proven to circulate in the country: tick-borne encephalitis (TBE), Crimean-Congo hemorrhagic fever (CCHF), Bhanja (BHA), sandfly fever (SF), Tahyna (TAH), Calovo (CVO), West Nile (WN), dengue (DEN), Jug Bogdanovac (JB), and Hantaviruses. TBE virus is endemic in the north-west part of the country, causing also epidemics in cyclical intervals. Its typical clinical picture is aseptic meningitis, but severe cases with paralysis have also been described. The bite of ticks is confirmed in about 80% of cases. CCF caused a small epidemic with ten clinical cases in Macedonia in 1976. Bhanja virus was isolated on the Dalmatian island of Brac in 1977, the antibody rate there, determined by the HI method, being about 31%. The first human disease in the world was caused by the Yugoslav Bhanja virus strain. Sandfly fever is still active in the country. The Naples type is prevailing and has proved hazardous for newcomers. Hantaviruses have been studied since 1980. They caused severe epidemics (1967, 1980, 1989) and sporadic cases all over the country. Three different strains are in circulation. Further studies are needed for the rest of the above mentioned viruses to learn more about their significance in human pathology.

Viral encephalitis.

Nonherpetic encephalitis outside the newborn period is usually a self-limited disease. The majority of patients will recover without significant sequelae, and require only supportive therapy during the acute illness. Though the underlying viral etiology frequently will escape detection, identification of the infecting agent has considerable prognostic value which can complement clinical measures of severity of disease. The most important initial task of the clinician faced with a case of presumptive viral encephalitis is to eliminate the possibility of a treatable illness. Once this has been done, the diagnosis of viral encephalitis can be supported by documenting the characteristic slow-wave background activity on EEG, and a mild lymphocellular pleocytosis in the CSF. Because viral encephalitis can be caused by such a large number of organisms, the search for an etiology can be daunting. Realizing that all the agents described above can, at times, cause encephalitis without any clue to their identity, one nevertheless may use several pieces of historical information to narrow the possibilities. Travel history, animal exposures, immunization history, and seasonality all may help to steer the search in a particular direction and, indeed, may point to a nonvirologic cause as well. In addition, detection of extraneurological signs and symptoms may strongly indicate a specific virologic diagnosis. Finally, knowledge of concurrent community epidemic patterns, and of surveillance data routinely collected by local and state health departments, can help to increase or decrease the likelihood of a given pathogen. The causative viral agent usually can be identified by serological testing and viral culture. Occasionally, single serological determinations are diagnostic: in rabies (when the patient has not received immune prophylaxis), eastern equine encephalitis, and HIV, since seropositivity is strongly associated with symptomatic illness; and in Epstein-Barr virus, if a panel of antibody determinations which can time the infection is available. In addition, high CSF: serum titers for antibody against any neurotropic agent is usually diagnostic, though the absence of a high central nervous system antibody titer does not eliminate any potential viral pathogen. With these few exceptions, a single serological determination for a given pathogen is almost always impossible to interpret; paired sera (one obtained upon diagnosis, and one obtained 10 to 14 days later, either just prior to hospital discharge or at a follow-up visit) are far more helpful. Many viruses that directly infect the central nervous system are difficult to recover from the CSF; therefore, viral isolation from the nasopharynx and stool also should be sought.(ABSTRACT TRUNCATED AT 400 WORDS)

[The possible role of the salivary gland substrate in ixodid ticks as an adjuvant enhancing arbovirus transmission]. / Vozmozhnaia rol' substrata sliunnykh zhelez iksodid v kachestve ad''iuvanta, usilivaiushchego peredachu arbovirusov.

Using a model: salivary glands of Dermacentor ticks--tick-borne encephalitis virus--guinea pig--D. marginatus ticks, it became possible to confirm the data of Jones et al. (1989) on the role of a substrate of Rhipicephalus appendiculatus glands as a strong enhancer of orthomyxovirus Togoto transmission during subcutaneous administration of a moderate virus dose to virus-resistant guinea pig. A tendency was only noticed towards better infectivity of ticks with the administration of sub- or supraoptimal virus doses together with the adjuvant (salivary gland substrate), as well as enhanced sensitivity of male individuals to a combination of virus with adjuvant. The latter fact can be explained by a transptyal way of infection typical for male individuals, which was noted earlier during joint nutrition with infected female individuals. A lower level of virus reproducibility in ticks who got it together with the adjuvant, as compared to the control, has been established. Low titer in female individuals after nutrition reduces the likelihood of transovarial transmission of virus with adjuvant.

[Research on arbovirus infections]. / Recherches sur les infections à arbovirus.

The author presents the results of researches done at the "Stefan S. Nicolau" Institute of Virology in Bucharest on the infections induced by arboviruses. The characteristics of three tick encephalitis virus strains (two strains isolated from Ixodes ricinus and Ixodes persulcatus ticks and one from a patient with encephalitis symptoms) are given. Most of the report is devoted to the results of serological survey conducted in a human population, in several domestic birds and mammals from some districts of Romania, as well as in migratory birds from the Danube delta, with regards to the incidence of some Toga-, Bunya- and Reoviruses.

[A seroepidemiologic and virological study of the presence of arboviruses in Moldavia in 1961-1982]. / Studiul seroepidemiologic si virologic al prezentei unor arbovirusuri pe teritoriul Moldovei, 1961-1982.

In Romania, St. Draganescu et al., Miszkolczy et al., based on clinical and epidemiological observations, affirmed the possible presence of "tick-borne encephalitis (TBE)". However, critical retrospective evaluation of certain others' papers published through 1958-1971 reveal some uncommon features. By way of example: a very high incidence of CF- and HI-antibodies in acute and chronic CNS illnesses as well as in general population; replication in nucleus and maturation in mitochondria of a strain of "TBE virus" isolated from a patient CSF; isolation of "group B arbovirus" from Ixodes persulcatus on Romanian territory. As known, this tick was not identified in Romania, "no tick-borne Flavivirus isolated outside the U.S.S.R. proved to be a strain of RSSE virus" and "no arbovirus (Alpha- and Flavivirus or Bunyavirus) was ever detected in nucleus". Based on the HI reaction with West Nile (WN) virus, strain Egypt 101, human, domestic and wild vertebrates sera, treated with acetone for removal of nonspecific inhibitors, were tested as part of a control study, in Moldavia in 1961. WN virus was selected for its capacity to crossreact in the HI test with many members of the family Flaviviridae, genus Flavivirus (formerly Group B arboviruses), including the agents of Central European encephalitis. In order to avoid the interference of nonspecific inhibitors, a new single radial haemolysis test for the assay of antibodies to haemagglutinating arboviruses was calibrated in 1979. The seroepidemiological study performed on all 19.853 sera (10.698 human, 6.452 domestic mammals, 2.477 wild small rodents and 226 migrating aquatic birds) pointed out a very low circulation of Flaviviruses in the investigated area. The attempts to isolate such viruses from Ixodes ricinus on suckling mice yielded a single strain of Kemerovo (genus Orbivirus) virus. The need of extending the study to other viruses transmitted by arthropods and rodents, and mainly on the haemorrhagic fever with renal syndrome, an illness already clinically identified in Moldavia, is emphasized. Final consideration of viability (specificity, sensitivity, accuracy and precision) of the tests used and the need for continuous quality control is also presented.

Investigations of bluetongue and other arboviruses in the blood and semen of naturally infected bulls.

Small groups of bulls were exposed to natural infection with arboviruses. The bulls were bled and ejaculated regularly and the blood and semen were processed for virus isolation. Over a 5-year observation period, virus isolation and serology indicated that the 29 exposed bulls had experienced 79 viraemic episodes with the viruses of the bluetongue, epizootic haemorrhagic disease, Palyam and Simbu serogroups and an incompletely characterised rhabdovirus. In no instance was there unequivocal evidence of bluetongue virus contamination of semen, despite 18 infections in the study period.