Monitoring of Putative Vectors of Bluetongue Virus Serotype 8, Germany

To identify the vectors of bluetongue virus (BTV) in Germany, we monitored Culicoides spp. biting midges during April 2007–May 2008. Molecular characterization of batches of midges that tested positive for BTV suggests C. obsoletus sensu stricto as a relevant vector of bluetongue disease in central Europe.     

Culicoides Latreille (Diptera: Ceratopogonidae) taxonomy: Current challenges and future directions

Culicoides Latreille biting midges (Diptera: Ceratopogonidae) cause a significant biting nuisance to humans, livestock and equines, and are the biological vectors of a range of internationally important pathogens of both veterinary and medical importance. Despite their economic significance, the delimitation and identification of species and evolutionary relationships between species within this genus remains at best problematic. To date no phylogenetic study has attempted to validate the subgeneric classification of the genus and the monophyly of many of the subgenera remains doubtful. Many informal species groupings are also known to exist but few are adequately described, further complicating accurate identification. Recent contributions to Culicoides taxonomy at the species level have revealed a high correlation between morphological and molecular analyses although molecular analyses are revealing the existence of cryptic species. This review considers the methods for studying the systematics of Culicoides using both morphological and genetic techniques, with a view to understanding the factors limiting our current understanding of Culicoides biology and hence arbovirus epidemiology. In addition, we examine the global status of Culicoides identification, highlighting areas that are poorly addressed, including the potential implementation of emerging technologies.     

Dispersal and survival in the field of chemosterilized, irradiated, and cytoplasmically incompatible male Culex pipiens fatigans

Comparative field studies were made on the dispersal and survival of chemosterilized, irradiated, and cytoplasmically incompatible (D3 strain) male Culex pipiens fatigans. There was no significant difference in dispersal patterns and daily survival rates between laboratory colony males and wild males, and between chemosterilized or irradiated males and untreated males. Although there was no difference in dispersal between adults of the D3 strain and the laboratory colony of the Delhi strain, the former had a lower daily survival rate.     

African horse sickness virus (AHSV) with a deletion of 77 amino acids in NS3/NS3a protein is not virulent and a safe promising AHS Disabled Infectious Single Animal (DISA) vaccine platform

African horse sickness virus (AHSV) is a virus species in the genus Orbivirus of the family Reoviridae. Currently, nine serotypes have been defined showing limited cross neutralization. AHSV is transmitted by species of Culicoides biting midges and causes African Horse Sickness (AHS) in equids with a mortality up to 95% in naïve domestic horses. AHS has become a serious threat for countries outside Africa, since endemic Culicoides species in moderate climates are competent vectors of closely related bluetongue virus. AHS outbreaks cause huge economic losses in developing countries. In the developed world, outbreaks will result in losses in the equestrian industry and will have an enormous emotional impact on owners of pet horses. Live-attenuated vaccine viruses (LAVs) have been developed, however, safety of these LAVs are questionable due to residual virulence, reversion to virulence, and risk on virulent variants by reassortment between LAVs or with field AHSV. Research aims vaccines with improved profiles. Reverse genetics has recently being developed for AHSV and has opened endless possibilities including development of AHS vaccine candidates, such as Disabled Infectious Single Animal (DISA) vaccine. Here, virulent AHSV5 was recovered and its high virulence was confirmed by experimental infection of ponies. ‘Synthetically derived’ virulent AHSV5 with an in-frame deletion of 77 amino acids codons in genome segment 10 encoding NS3/NS3a protein resulted in similar in vitro characteristics as published NS3/NS3a knockout mutants of LAV strain AHSV4LP. In contrast to its highly virulent ancestor virus, this deletion AHSV5 mutant (DISA5) was completely safe for ponies. Two vaccinations with DISA5 as well as two vaccinations with DISA vaccine based on LAV strain AHSV4LP showed protection against lethal homologous AHSV. More research is needed to further improve efficacy, to explore the AHS DISA vaccine platform for all nine serotypes, and to study the vaccine profile in more detail.     

Isolation of arboviruses from mosquitoes, biting midges, sandflies and vertebrates collected in Queensland, 1969 and 1970

30 virus strains were isolated from arthropods or vertebrates taken at 3 centres in Queensland in 1969 and 1970. They included representatives of 6 virus types distinct from any previously isolated in Australia. Prototype strains, suggested names and sources of first or repeatable isolations of the 6 viruses were: Ch9824, Charleville virus, from Phlebotomus spp. and the reptile Gehyra australis; Ch9935, Warrego virus, from Culicoides spp.; MRM10434, Mitchell River virus, from Culicoides spp.; Ch12048, Wallal virus, from Culicoides spp.; MRM13443, Wongorr virus, from Aedes lineatopennis; MRM14556, Ngaingan virus, from Culicoides brevitarsis.Mouse neutralization tests on sera from vertebrates of various species detected serum inhibitors to Warrego, Wallal and Ngaingan viruses in wallabies and kangaroos, and to Warrego, Mitchell River, Wongorr and Ngaingan viruses in cattle.These findings increase to 30 the number of viruses isolated in Australia and tentatively viewed as arboviruses, and underline the importance of biting midges and sandflies as virus vectors.     

Stress dependent infection cost of the human malaria agent Plasmodium falciparum on its natural vector Anopheles coluzzii

Unraveling selective forces that shape vector–parasite interactions has critical implications for malaria control. However, it remains unclear whether Plasmodium infection induces a fitness cost to their natural mosquito vectors. Moreover, environmental conditions are known to affect infection outcome and may impact the effect of infection on mosquito fitness. We investigated in the laboratory the effects of exposition to and infection by field isolates of Plasmodium falciparum on fecundity and survival of a major vector in the field, Anopheles coluzzii under different conditions of access to sugar resources after blood feeding.The results evidenced fitness costs induced by exposition and infection. When sugar was available after blood meal, infected and exposed mosquitoes had either reduced or equal to survival to unexposed mosquitoes while fecundity was either increased or decreased depending on the blood donor. Under strong nutritional stress, survival was reduced for exposed and infected mosquitoes in all assays.We therefore provide here evidence of an environmental-dependant reduced survival in mosquitoes exposed to infection in a natural and one of the most important parasite-mosquito species associations for human malaria transmission.     

Effects of genetic factors and infection status on wing morphology of Triatoma dimidiata species complex in the Yucatán peninsula, Mexico

Triatoma dimidiata is one of the main vectors of Chagas disease, and it has been shown to be a species complex. In the Yucatán peninsula, Mexico, vector populations are non-domiciliated, and the transmission of Trypanosoma cruzi thus critically relies on vector dispersal. This leads us to study the morphologic variations in T. dimidiata wings with respect to genetic factors (sex and genotype at the ITS-2 locus), geographic location, and T. cruzi-infection status. Females were found to have larger and more symmetrical wings than males. Wing shape was influenced by ITS-2 genotypes, although differences are unlikely sufficient to allow taxonomic discrimination of the sibling species. Hybrids were shown to have similar fluctuating asymmetries in wing size and shape as parental species, but the level of asymmetry in shape varied slightly between villages. The two later findings are consistent with a high level of gene flow between parental species, and the high dispersal potential of these non-domiciliated vectors. More surprisingly, individuals infected with T. cruzi were found to have larger wings than non-infected ones. This effect, which was consistently observed across sexes, genotypes and villages, is likely to be due to a direct impact of T. cruzi on insect development. Sex and infection status are thus likely to be key factors influencing vector dispersal with important impacts on disease transmission, since dispersal directly controls the domestic abundance of these vectors. These aspects should be investigated further to fully capture the ecology and evolution of Chagas disease transmission by non-domiciliated vectors.     

Genomic Detection of Schmallenberg Virus,Israel

We discuss genomic detection of Schmallenberg virus in both Culicoides midges and affected ruminants during June 2018–December 2019, demonstrating its circulation in Israel. This region is a geographic bridge between 3 continents and may serve as an epidemiologic bridge for potential Schmallenberg virus spread into Asia.     

Field experiments on the behaviour of malaria vectors in an unsprayed hut and in a hut sprayed with DDT in Northern Nigeria

Investigations on the behaviour of malaria vectors in DDT-sprayed houses are of special interest in malaria control campaigns. In the Northern Region of Nigeria the behaviour of the local vectors was studied by means of experimental huts fitted with exit window-traps. The results obtained show a marked reduction in the number of mosquitos entering the sprayed hut and a depression of the biting rate. The most striking observation is the large number of A. gambiae and of A. funestus that left the sprayed hut and the considerable proportion that escaped unharmed. Both figures show a very marked increase in subsequent months after spraying, indicating a rapid loss of the lethal action of the DDT residue along with a long-lasting irritant effect. It is concluded that under the particular local conditions the application of 1.9 g/m² DDT twice a year may not be sufficient to interrupt malaria transmission in the Northern Region of Nigeria.     

纤维蛋白原的131I标记

目的 通过研究¹³¹I-纤维蛋白原在体内的留滞时间,提出一种简单高效的蛋白质放射性碘标记的方法。方法 利用Iodogen（四氯二苯基甘尿）为氧化剂对纤维蛋白原进行的¹³¹I标记,取样并测定标记率及生物活性。结果 分别使用5、10、20 μg lodogen氧化剂反应20 min后测定的标记率为89.2%、88.4%、88.7%。使用10 μg的lodogen氧化剂进行氧化标记测得的5、15、30 min的标记率为64.3%、89.6%和88.2%,生物活性率为86.1%、84.7%和85.5%。结论 以Iodogen为氧化剂,一步反应,即可获得纤维蛋白原碘标记物。标记率及生物活性率都接近90%。     

Plasticity in the timing of detachment of an Eurasian-African songbird tick,Ixodes frontalis

In non-permanent parasites, host detachment should take place in an environment that ensures the continuation of their life cycle. Timing of detachment - in combination with the host's space use - affects dispersal and transmission success of the parasites and of the pathogens they vector. Before reaching the adult reproductive stage, ticks need to go through multiple immature developmental stages (larva and nymph), each feeding on host blood. In between the feeding bouts, they often remain in the off-host environment for considerable periods of time. With this study, we aimed to obtain more insight in Ixodes frontalis’ off-host habitat use by comparing its detachment pattern in different life stages with that of two habitat-specialized ticks also found on birds: the endophilic tree-hole tick (Ixodes arboricola) and the exophilic sheep tick (Ixodes ricinus), the latter living in humid understory vegetation of forests. For this, we artificially infested hole-roosting (great tits, Parus major) and open-roosting (blackbirds, Turdus merula) birds with ticks under laboratory conditions, and recorded whether detachment occurred during the day or the night. We hypothesize that nocturnal detachment improves off-host mating opportunities and host localization, whereas diurnal detachment optimizes tick dispersal. Ixodes frontalis nymphs detached during the night, especially when feeding on blackbirds. This behaviour was very similar to that of I. arboricola (larva and nymph) feeding on great tits. In contrast, I. frontalis larvae detached during the day, especially when feeding on great tits, which resembles that of I. ricinus’ feeding behaviour (larva and nymph). Ixodes frontalis left the host within seven days, immediately after completion of the blood meal. This is similar to both developmental stages of I. ricinus but contrasts with the very long (up to 20 days) feeding duration in I. arboricola. Thus I. frontalis shows strong plasticity, switching from dispersal-centered (larvae) to host-centered (nymphs) detachment behaviour. Findings are discussed with regard to the ticks’ habitat use, dispersal, life history and host specificity.     

Investigations leading to the identification of members of the Anopheles umbrosus group as the probable vectors of mouse deer malaria

Although mosquitos of the Anopheles umbrosus group have long been recognized as important vectors of human malaria in Malaya, there have been doubts about the origin of some of the malaria infections found, especially in A. umbrosus and A. letifer. Investigations have accordingly been carried out in the Malayan swamp-forest, in conjunction with laboratory studies, into the nature of malaria infections in wild-caught mosquitos, the biting behaviour of anophelines and the presence of malaria infection in man and animals. The authors conclude from the results reported in this paper that A. umbrosus is a vector of mouse deer malaria and rarely, if ever, transmits primate malaria; that A. letifer transmits both human and mouse deer malaria; and that A. baezai and A. roperi are probably vectors of mouse deer malaria.     

Bacteria expressed hepatitis E virus capsid proteins maintain virion-like epitopes

The protein encoded by ORF2 in hepatitis E virus (HEV) is the only capsid protein for this single-stranded RNA virus. It was previously shown that 148 aa (aa 459–606) was needed for dimer formation, whereas 239 aa (aa 368–606) was necessary to form virus-like particles (VLPs). The self-assembled VLPs of p239 were characterized with a series of methods including high performance size-exclusion chromatography to demonstrate the particulate nature of purified and properly refolded p239. A neutralizing and protective mouse monoclonal antibody (mAb) 8C11 was previously shown to bind three discontinuous peptide segments in the dimer. In addition to the good binding activity to recombinant dimeric form, E2s or E2, and VLP form p239, we demonstrated that 8C11 was able to capture the authentic HEV virions. The capability of virus capturing was demonstrated with a titration curve from 10⁵ to 10⁷ HEV genome copies, making binding activity to 8C11 a surrogate marker of virion-like epitopes on recombinant VLPs as well as vaccine efficacy in eliciting protective and neutralizing antibodies. Taken together, it was demonstrated that Escherichia coli expressed pORF2 proteins, p239 in particular, maintain the virion-like epitopes on VLP surface. This is consistent with the fact that p239 was demonstrated to be an effective prophylactic vaccine (recently licensed as Hecolin^® in China) against HEV-induced hepatitis in a large scale clinical trial.     

Host-feeding patterns and behaviour of 4 Culex species in an endemic area of Japanese encephalitis

Studies were made on the host-feeding patterns and behaviour of 4 Culex species in China (Province of Taiwan) to further assess their importance as vectors of Japanese encephalitis. A relatively unbiased, non-attractant technique (vacuum sweep-net) was used for collecting mosquitos resting outdoors on rural premises; blood meals from engorged specimens were identified by precipitin tests. Additional information was obtained by analysing data from related studies. Precipitin-test results indicated that C. annulus, C. tritaeniorhynchus, and C. fuscocephalus feed principally on mammals, the first species mainly on swine and the latter two largely on bovine hosts. Mosquito forage ratios suggested that the apparent preference of C. annulus for swine over bovine hosts might be a function of host density. C. annulus was found to be zoophilic, exophilic, and exophagic; however, in view of its abundance during the JE epidemic season, contact with human hosts may reach a significant level, even indoors. The other 3 species of Culex were considered to be of lesser importance as JE vectors during the 1971 epidemic season because of their host-feeding patterns and/or population densities.     

A host-restricted viral vector for antigen-specific immunization against Lyme disease pathogen

Newcastle disease virus (NDV) is an avian virus that is attenuated in primates and is a potential vaccine vector for human use. We evaluated NDV as a vector for expressing selected antigens of the Lyme disease pathogen Borrelia burgdorferi. A series of recombinant NDVs were generated that expressed intracellular or extracellular forms of two B. burgdorferi antigens: namely, the basic membrane protein A (BmpA) and the outer surface protein C (OspC). Expression of the intracellular and extracellular forms of these antigens was confirmed in cultured chicken cells. C3H or Balb/C mice that were immunized intranasally with the NDV vectors mounted vigorous serum antibody responses against the NDV vector, but failed to mount a robust response against either the intracellular or extracellular forms of BmpA or OspC. By contrast, a single immunization of hamsters with the NDV vectors via the intranasal, intramuscular, or intraperitoneal route resulted in rapid and rigorous antibody responses against the intracellular or extracellular forms of BmpA and OspC. When groups of hamsters were separately inoculated with various NDV vectors and challenged with B. burgdorferi (10⁸ cells/animal), immunization with vector expressing either intracellular or extracellular BmpA was associated with a significant reduction of the pathogen load in the joints. Taken together, our studies highlighted the importance of NDV as vaccine vector that can be used for simple yet effective immunization of hosts against bacterial infections including Lyme disease.     

Fructose in wild-caught Phlebotomus ariasi and the possible relevance of sugars taken by sandflies to the transmission of leishmaniasis

Cold, acidified anthrone was used to test 200 wild-caught sandflies (Phlebotomus ariasi ♀♀) for the presence of fructose. This sugar, or others with a fructose moiety, were demonstrable in 157 (79%) of the flies; proportions of flies positive in different tests ranged from 44 to 91%. Higher proportions of flies gave positive results when tested less than one hour after capture than when kept in the laboratory for more than 12 hours before testing. Similarly high percentages of flies were positive in samples collected coming to engorge on dogs and in those caught in CDC miniature light traps. These results, together with published reports of the demonstration of sugars in 25 individually tested sandflies of other species, confirm that, like other biting flies, sugar is an important part of the diet of sandflies. Natural sugars may influence the development of leishmaniae in sandflies and their subsequent transmission. The anthrone test was found to be simple, rapid and suitable for field use; it is a valuable addition to indirect techniques for the study of the behaviour of sandflies.     

Thirty years of tick population genetics: A comprehensive review

Population genetic studies provide insights into the basic biology of arthropod disease vectors by estimating dispersal patterns and their potential to spread pathogens. In wingless vectors, such as ticks, gene flow will be defined in large part by the mobility of their hosts. However, tick behaviors and life cycle strategies can limit their dispersal even on highly mobile hosts and lead to an increase in genetic structure. In this review we synthesize the published literature from three decades of tick population genetic studies. Based on studies from 22 tick species (including representatives from Amblyomma, Bothriocroton, Dermacentor, Ixodes, Ornithodoros, and Rhipicephalus), observed levels of population genetic structure in ticks varied from no structure to very high levels. In about half of the species (including representatives from Amblyomma, Bothriocroton, Dermacentor, and Ornithodoros), tick genetic structure appeared to be determined primarily by the movement capacity of hosts, with low gene flow observed in ticks that use smaller bodied less mobile hosts and high gene flow in ticks using highly mobile hosts. In a number of other species (primarily from Ixodes, Ornithodoros, and Rhipicephalus), behavioral limitations to gene flow appeared to result in greater genetic structure than expected based upon host movement capability alone. We also discuss the strengths and limitations of genetic markers and their applicability to ticks and suggest possible analyses when planning population genetic studies for ticks.     

Genetic comparison of Glossina tachinoides populations in three river basins of the Upper West Region of Ghana and implications for tsetse control

Tsetse flies are the cyclical vectors of African animal trypanosomosis (AAT) and human African trypanosomosis (HAT). In March 2010, the Government of Ghana initiated a large scale integrated tsetse eradication campaign in the Upper West Region (UWR) (≈18,000 km²) under the umbrella of the Pan-African Tsetse and Trypanosomosis Eradication Campaign (PATTEC).We investigated the structuring of Glossina tachinoides populations within and between the three main river basins of the target area in the UWR. Out of a total sample of 884 flies, a sub-sample of 266 was genotyped at nine microsatellite loci. The significance of the different hierarchical levels was tested using Yang’s parameters estimated with Weir and Cockerham’s method. A significant effect of traps within groups (pooling traps no more than 3 km distant from each other), of groups within river basins and of river basins within the whole target area was observed. Isolation by distance between traps was highly significant. A local density of 0.48–0.61 flies/m² was estimated and a dispersal distance that approximated 11 m per generation [CI 9, 17]. No significant sex-biased dispersal was detected.Dispersal distances of G. tachinoides in the UWR were relatively low, possibly as a result of the fragmentation of the habitat and the seasonality of the Kulpawn and Sissili rivers. Moreover, very high fly population densities were observed in the sample sites, which potentially reduces dispersal at constant habitat saturation, because the probability that migrants can established is reduced (density dependent dispersal). However, the observed spatial dispersal was deemed sufficient for a G. tachinoides-cleared area to be reinvaded from neighboring populations in adjacent river basins. These data corroborate results from other population genetics studies in West Africa, which indicate that G. tachinoides populations from different river basins cannot be considered isolated.     

Généralités sur arbovirus et arboviroses

Arbovirus diseases are mainly tropical affections caused by a heterogenic group of viruses with common epidemiological properties. About 130 known arboviruses belonging to about 12 different virus families are of human or veterinarian medical importance. Many others are not pathogenic but can be interesting models for fundamental virological studies. Arboviruses survive in nature by biological transmission between vertebrate hosts and hematophage arthropoda. Arboviruses reproduce on these without change to their life or fecundity. They are transmitted through bite or sting of infected arthropoda to receptive vertebrates inducing an early and transitional viremia. A more or less complex cycle usually follows: virus, arthropoda vector, vector, vertebrate host. According to the vector’s ecology, each arbovirus is present in a specific area and is always likely to invade a non-endemic zone. The arthropoda in question are ticks, mosquitoes, phlebotomes, or Culicoides (Ceratopogonidae). Vertebrates either spread or amplify the viremia or they are accidental hosts or epidemiological dead-ends. Three types of clinical presentations are described: febrile polyalgia (“dengue-like” syndromes); hemorrhagic fever (frequently severe evolution); meningo-encephalitis (frequently severe evolution ending by death or recovery with severe neurological sequels). Various arboviruses are responsible for the same clinical presentations; but a same virus can induce several types of syndromes which stress the importance of laboratory diagnosis, based on the virus identification and specific serological tests. Prophylaxis relies on vaccination: yellow fever, tick borne encephalitis, Japanese encephalitis and fighting against vectors. New vaccines are being studied but not on the market yet.     

VP2-serotyped live-attenuated bluetongue virus without NS3/NS3a expression provides serotype-specific protection and enables DIVA

Bluetongue virus (BTV) causes Bluetongue in ruminants and is transmitted by Culicoides biting midges. Vaccination is the most effective measure to control vector borne diseases; however, there are 26 known BTV serotypes showing little cross protection. The BTV serotype is mainly determined by genome segment 2 encoding the VP2 protein. Currently, inactivated and live-attenuated Bluetongue vaccines are available for a limited number of serotypes, but each of these have their specific disadvantages, including the inability to differentiate infected from vaccinated animals (DIVA).BTV non-structural proteins NS3 and NS3a are not essential for virus replication in vitro, but are important for cytopathogenic effect in mammalian cells and for virus release from insect cells in vitro. Recently, we have shown that virulent BTV8 without NS3/NS3a is non-virulent and viremia in sheep is strongly reduced, whereas local in vivo replication leads to seroconversion. Live-attenuated BTV6 without NS3/NS3a expression protected sheep against BTV challenge. Altogether, NS3/NS3a knockout BTV6 is a promising vaccine candidate and has been named Disabled Infectious Single Animal (DISA) vaccine.Here, we show serotype-specific protection in sheep by DISA vaccine in which only genome segment 2 of serotype 8 was exchanged. Similarly, DISA vaccines against other serotypes could be developed, by exchange of only segment 2, and could therefore safely be combined in multi-serotype cocktail vaccines with respect to reassortment between vaccine viruses.Additionally, NS3 antibody responses are raised after natural BTV infection and NS3-based ELISAs are therefore appropriate tools for DIVA testing accompanying the DISA vaccine. To enable DIVA, we developed an experimental NS3 ELISA. Indeed, vaccinated sheep remained negative for NS3 antibodies, whereas seroconversion for NS3 antibodies was associated with viremia after heterologous BTV challenge.