Oral susceptibility of South African stock-associated Culicoides species to bluetongue virus

Field-collected South African Culicoides species (Diptera, Ceratopogonidae) were fed on sheep blood containing bluetongue virus (BTV) represented by 13 low-passage reference serotypes: -1, -2, -4, -6, -7, -8, -9, -10, -11, -12, -13, -16 and -19. After 10 days of extrinsic incubation at 23.5 degrees C, of the 13 serotypes used, seven were recovered from C. (Avaritia) imicola Kieffer and 11 from C. (A.) bolitinos Meiswinkel. Virus recovery rates and the mean titres for most serotypes were significantly higher in C. bolitinos than in C. imicola. In addition, BTV was recovered from three non-Avaritia Culicoides species, namely C. (Remmia) enderleini Cornet & Brunhes (BTV-9), C. (Hoffmania) milnei Austen (BTV-4) and C. (H.) zuluensis de Meillon (BTV-16). No virus could be recovered from 316 individuals representing a further 14 Culicoides species. In Culicoides species fed on blood containing similar or identical virus titres of distinct BTV serotypes, significant differences were found in virus recovery rates. The results of this study confirm the higher vector competence of C. bolitinos compared with C. imicola.     

Recovery rates of bluetongue virus serotypes 1, 2, 4 and 8 Spanish strains from orally infected Culicoides imicola in South Africa

Bluetongue (BT) is an infectious disease of ruminants that has spread northwards in Europe during the last decade. The aetiological agent of the disease is an arbovirus [bluetongue virus (BTV)] that belongs to the genus Orbivirus (family Reoviridae). The virus is transmitted by certain species of biting midge within the genus Culicoides (Diptera: Ceratopogonidae). Information on the vector status of the Culicoides species in a specific area will be essential to predict the risk for BTV incursion. Field-collected Culicoides (Avaritia) imicola Kieffer from South Africa were fed on blood containing several Spanish isolates of BTV. Despite the high virus concentrations in the bloodmeal (5.1-6.4 log(10) TCID(50) /mL of blood), virus was recovered from <1% of midges assayed after incubation. Virus concentrations >2.5 log(10) TCID(50) /midge in individual infected C. imicola suggest virus replication with possible risk for transmission to susceptible vertebrate hosts in the field for at least two of the serotypes assayed (BTV-1 and BTV-2). A third serotype (BTV-4) was very close to the estimated threshold for transmission. The relatively low to near refractory status of C. imicola compared with other vector species such as Culicoides bolitinos supports previous results, indicating that Culicoides species other than C. imicola may play a more important role in the epidemiology of BTV.     

Virus recovery rates for wild-type and live-attenuated vaccine strains of African horse sickness virus serotype 7 in orally infected South African Culicoides species

Previously reported virus recovery rates from Culicoides (Avaritia) imicola Kieffer and Culicoides (Avaritia) bolitinos Meiswinkel (Diptera, Ceratopogonidae) orally infected with vaccine strain of African horse sickness virus serotype 7 (AHSV-7) were compared with results obtained from concurrently conducted oral infections with five recent AHSV-7 isolates from naturally infected horses from various localities in South Africa. Culicoides were fed sheep bloods spiked with 10(7.6) TCID(50)/mL of a live-attenuated vaccine strain AHSV-7, and with five field isolates in which virus titre in the bloodmeals ranged from 10(7.1) to 10(8.2) TCID(50)/mL). After an extrinsic incubation of 10 days at 23.5 degrees C, virus recovery rates were significantly higher in C. imicola (13.3%) and C. bolitinos (4.2%) infected with the live-attenuated virus than in midges infected with any of the field isolates. The virus recovery rates for the latter groups ranged from 0% to 9.5% for C. imicola and from 0% to 1.5% for C. bolitinos. The C. imicola population at Onderstepoort was significantly more susceptible to infection with AHSV-7 isolated at Onderstepoort than to the virus strains isolated from other localities. Results of this study suggest that tissue culture attenuation of AHSV-7 does not reduce its ability to orally infect competent Culicoides species and may even lead to enhanced replication in the vector. Furthermore, oral susceptibility in a midge population appears to vary for geographically distinct isolates of AHSV-7.     

The susceptibility of Culicoides imicola and other South African livestock-associated Culicoides species to infection with bluetongue virus serotype 8

In 2006, a strain of bluetongue virus serotype 8 (BTV-8) of sub-Saharan origin was responsible for the first outbreaks in recorded history of clinical bluetongue disease (BT) in northern Europe. In this study, we examine the oral susceptibility of Culicoides (Avaritia) imicola Kieffer (Diptera: Ceratopogonidae) and other livestock-associated Culicoides species from southern Africa to infection with several strains of BTV-8. Following feeding using an artificial membrane-based method and incubation, virus was found in <1% of C. imicola individuals tested. Higher rates of susceptibility were found, however, for a variety of other South African species, including Culicoides (Avaritia) bolitinos Meiswinkel. Although these results do not preclude the role of C. imicola as a vector of BTV-8, its low susceptibility to BTV indicates that other less abundant Culicoides species may have the potential to play decisive roles in the epidemiology of this virus and should not be excluded from risk assessment studies.     

African horse sickness epidemiology: vector competence of south african Culicoides species for virus serotypes 3, 5 and 8

The oral susceptibilities of 17 Culicoides species to infection with African horse sickness virus (AHSV) serotypes 3, 5 and 8 were determined by feeding field-collected midges on AHSV infected horse blood. The mean titres of virus in the bloodmeals for the three serotypes of AHSV were between 5.7 and 6.5 log10 TCID50/ml. Virus was detected, after 10 days incubation at 23.5 degrees C, in the Culicoides imicola Kieffer (Diptera: Ceratopogonidae) that had fed on blood containing AHSV 5 (8.5%) and 8 (26.8%), and in the Culicoides bolitinos Meiswinkel that had fed on AHSV 3 (3.8%), 5 (20.6%) and 8 (1.7%). Although 44.4% of the C. imicola were shown to have ingested AHSV 3 immediately after feeding, no virus was detected in 96 C. imicola after incubation. The relatively high titres of virus recorded in individual midges of both species after 10 days incubation suggested a fully disseminated infection. Previously, C. imicola was considered to be the only field vector of AHSV in Africa. Identifying C. bolitinos as a potential vector for AHSV is an important finding, which if proven will have a significant impact on our understanding of the epidemiology of AHS. No AHSVs could be detected in the other 15 species of Culicoides assayed, which suggests that some of the southern African Culicoides species are refractory to AHSV infection. However, further work with larger numbers of each species will be necessary to confirm this observation.     

Vector competence of Culicoides bolitinos and C. imicola for South African bluetongue virus serotypes 1, 3 and 4

Abstract .The susceptibility of field-collected Culicoides bolitinos to infection by oral ingestion of bluetongue virus serotypes 1, 3 and 4 (BLU 1, 3 and 4) was compared with that of field-collected C. imicola and laboratory reared C. variipennis sonorensis . The concentration of the virus per millilitre of bloodmeal was 10^5.0 and 10^6.0TCID₅₀ for BLU 4 and 10^7.2TCID₅₀ for BLU 1 and 3. Of 4927 C. bolitinos and 9585 C. imicola fed, 386 and 287 individual midges survived 10 days extrinsic incubation, respectively. Midges were assayed for the presence of virus using a microtitration assay on BHK-21 cells and/or an antigen capture ELISA. Infection prevalences for the different serotypes as determined by virus isolation ranged from 22.7 to 82.0% in C. bolitinos and from 1.9 to 9.8% in C. imicola; infection prevalences were highest for BLU 1, and lowest for BLU 4 in both species. The mean log₁₀ TCID₅₀ titre of the three BLU viruses per single fly was higher in C. bolitinos than in C. imicola . The results suggested that C. bolitinos populations are capable vectors of the BLU viruses in South Africa. A high correlation was found between virus isolation and ELISA results for the detection of BLU 1, and less for BLU 4; the ELISA failed to detect the presence of BLU 3 in infected flies. The C. v. sonorensis colonies had a significantly lower susceptibility to infection with BLU 1, 3 and 4 than C. bolitinos and C. imicola . However, since infection prevalence of C. v. sonorensis was determined only by ELISA, this finding may merely reflect the insensitivity of this assay at low virus titres, compared to virus isolation.     

Vector competence of selected South African Culicoides species for the Bryanston serotype of equine encephalosis virus

Equine encephalosis virus (EEV) was recognized and described in the Republic of South Africa in 1967 and subsequent serological studies have shown this orbivirus to be both widespread and prevalent in southern Africa. In the present study it was shown that wild-caught Culicoides (Avaritia) imicola Kieffer (Diptera: Ceratopogonidae) can become infected with and permit the replication of the Bryanston serotype of EEV following membrane-feeding on infective blood containing 5.0 log10 plaque-forming-units (PFU)/ml. The mean prevalence of Bryanston virus infection in C. imicola after 10 days extrinsic incubation at 23.5 degrees C was 22.3% (23/103). The mean infectivity of Bryanston virus in the infected C. imicola increased from 1.3 log10 PFU/midge, in insects assayed immediately after feeding on the blood-virus mixture, to 2.6 log10 PFU/midge in insects assayed after incubation. The virus concentration in individual C. imicola infected with the Bryanston serotype of EEV ranged from 0.7 to 3.6 log10 PFU/midge. Bryanston virus titres higher than 2.5 log10 TCID50, found in individual C. imicola, suggest that this species may be able to transmit this virus to susceptible hosts. Prevalence of virus infection in C. imicola was determined by PFU and microtitration assays on both BHK and Vero cells and confirmation of the Bryanston serotype of EEV was determined by plaque inhibition. No virus replication could be demonstrated in 102 C. nivosus tested after the incubation period, suggesting that not all Culicoides species are equally susceptible to Bryanston virus infection. Other Culicoides species that survived the incubation period and that were negative for the presence of Bryanston virus were C. pycnostictus (42), C. leucostictus (7), C. magnus (2), C. bolitinos (1) and C. bedfordi (1).     

Determination of the oral susceptibility of South African livestock-associated biting midges,Culicoides species,to bovine ephemeral fever virus

A total of 10 607 Culicoides midges (Diptera: Ceratopogonidae) were fed on either sheep or horse blood containing not less than 6.5 log10 TCID50/ml of bovine ephemeral fever virus (BEFV). Insects were collected during two consecutive summers from two distinct climatic areas. Two seed viruses, originating from either South Africa or Australia, were used separately in the feeding trials. Blood-engorged females were incubated at 23.5 degrees C for 10 days and then individually assayed in microplate BHK-21 cell cultures. Of the 4110 Culicoides that survived, 43% were C. (Avaritia) imicola Kieffer and 27% were C. (A.) bolitinos Meiswinkel. The remainder represented 18 other livestock-associated Culicoides species. Although BEFV was detected in 18.9% of midges assayed immediately after feeding, no virus could be detected after incubation. The absence of evidence of either virus maintenance or measurable replication suggests that most of the abundant livestock-associated Culicoides species found in South Africa are refractory to oral infection with BEFV. Future studies should be carried out using species of mosquitoes that are associated with cattle in the BEF endemic areas.     

Spatial distribution of bluetongue virus and its Culicoides vectors in Sicily

During the recent Mediterranean epizootic of bluetongue, an extensive programme of serological and vector (Culicoides biting midges (Diptera: Ceratopogonidae)) surveillance was carried out across Sicily. This paper presents the analysis of 911 light trap catches collected at the times of peak Culicoides abundance (summer to autumn 2000-2002) in 269 sites, in order to produce detailed maps of the spatial distribution of the main European vector, Culicoides imicola Kieffer and that of potential novel vectors. Whereas C. imicola was found at only 12% of sites, potential novel vectors, Culicoides obsoletus group Meigen, Culicoides pulicaris Linnaeus and Culicoides newsteadi Austen were present at over 50% of sites. However, the spatial distribution of C. imicola showed the closest correspondence to that of the 2000 and 2001 bluetongue (BT) outbreaks and its presence and abundance were significant predictors of the probability of an outbreak, suggesting that it was the main vector during these years. Although C. imicola may have played a role in transmission in several sites near Paternó, it was absent from the majority of sites at which outbreaks occurred in 2002 and from all sites in the province of Messina. All three potential novel vectors were widespread across sites at which outbreaks occurred during 2002. Of these, C. newsteadi was an unlikely candidate, as it was significantly less prevalent in outbreak vs. non-outbreak sites in Messina. It is hypothesized that the yearly distribution and intensity of outbreaks is directly attributable to the distribution and abundance of the vectors involved in transmission during each year. When C. imicola operated as the main vector in 2000 and 2001, outbreaks were few in number and were restricted to coastal regions due to low abundance and prevalence of this species. In 2002, it is hypothesized that BTV transmission was handed over to more prevalent and abundant novel vector species, leading to numerous and widespread outbreaks and probably to overwintering of the virus between 2001 and 2002. Based on catch ranges in outbreak vs. non-outbreak sites, it is tentatively suggested that nightly catches of 400 or more C. obsoletus and 150 or more C. pulicaris allow BTV transmission at a site, and provide a strategy for a fuller examination of the relationship between BTV transmission and the abundance and distribution of different vector species.     

Spatial and temporal distribution of bluetongue and its Culicoides vectors in Bulgaria

Surveillance of Culicoides (Diptera: Ceratopogonidae) biting midges was carried out between 2001 and 2003, at 119 sites within a 50 x 50-km grid distributed across Bulgaria, using light trap collections around the time of peak adult midge abundance. Sentinel and ad hoc serum surveillance of hosts susceptible to bluetongue infection was carried out at around 300 sites between 1999 and 2003. Following the initial incursion of bluetongue virus 9 (BTV-9) into Bourgas province in 1999, affecting 85 villages along the southern border, a further 76 villages were affected along the western border in 2001, with outbreaks extending as far north as 43.6 degrees N. The BTV-9 strain in circulation was found to have a low pathogenicity for Bulgarian sheep populations, with less than 2% of susceptible individuals becoming sick and seroconversions detected up to 30 km from recorded outbreaks in the south. The major Old World vector Culicoides imicola Kieffer was not detected among over 70,000 Culicoides identified in summer collections, suggesting that BTV-9 transmission in Bulgaria was primarily carried out by indigenous European vectors. The most likely candidates, the Palaearctic species complexes - the Culicoides obsoletus Meigen and C. pulicaris L. complexes - were widespread and abundant across the whole country. The C. obsoletus complex represented 75% of all individuals trapped in summer and occurred in high catch sizes (up to 15,000 individuals per night) but was not found across all outbreak sites, indicating that both Palearctic complexes probably played a role in transmission. Within the C. pulicaris complex, only C. pulicaris s.s., C. punctatus Meigen and C. newsteadi Austen were sufficiently abundant and prevalent to have been widely involved in transmission, whilst within the C. obsoletus complex most trapped males were C. obsoletus s.s. Adult vectors were found to be largely absent from sites in west Bulgaria for a period of at least 3 months over winter, which, taken along with the spatiotemporal pattern of outbreaks in the region between years, indicates the virus may be overwintering here by an alternative mechanism - either by covert persistence in the vertebrate host or possibly by persistence in larval stages of the vector.     

Modelling the distributions of Culicoides bluetongue virus vectors in Sicily in relation to satellite-derived climate variables

Surveillance data from 268 sites in Sicily are used to develop climatic models for prediction of the distribution of the main European bluetongue virus (BTV) vector Culicoides imicola Kieffer (Diptera: Ceratopogonidae) and of potential novel vectors, Culicoides pulicaris Linnaeus, Culicoides obsoletus group Meigen and Culicoides newsteadi Austen. The models containing the 'best' climatic predictors of distribution for each species, were selected from combinations of 40 temporally Fourier-processed remotely sensed variables and altitude at a 1 km spatial resolution using discriminant analysis. Kappa values of around 0.6 for all species models indicated substantial levels of agreement between model predictions and observed data. Whilst the distributions of C. obsoletus group and C. newsteadi were predicted by temperature variables, those of C. pulicaris and C. imicola were determined mainly by normalized difference vegetation index (NDVI), a variable correlated with soil moisture and vegetation biomass and productivity. These models were used to predict species presence in unsampled pixels across Italy and for C. imicola across Europe and North Africa. The predicted continuous presence of C. pulicaris along the appenine mountains, from north to south Italy, suggests BTV transmission may be possible in a large proportion of this region and that seasonal transhumance (seasonal movement of livestock between upland and lowland pastures) even in C. imicola-free areas should not generally be considered safe. The predicted distribution of C. imicola distribution shows substantial agreement with observed surveillance data from Greece and Iberia (including the Balearics) and parts of mainland Italy (Lazio, Tuscany and areas of the Ionian coast) but is generally much more restricted than the observed distribution (in Sardinia, Corsica and Morocco). The low number of presence sites for C. imicola in Sicily meant that only a restricted range of potential C. imicola habitats were included in the training set and that predictions could only be made within this range. Future modelling exercises will use abundance data collected according to a standardized protocol across the Mediterranean and, for Sicily in particular, should include non-climatic environmental variables that may influence breeding site suitability such as soil type.     

库蠓源蓝舌病病毒1型的分离和初步鉴定

为监测云南边境地区虫媒库蠓蓝舌病病毒携带情况,本研究对2013年-2017年从云南6个口岸及周边地区采集到的约5 400只库蠓样本,分180组。采用荧光定量RT-PCR检测、鸡胚和细胞分离、目的基因克隆测序分析和间接免疫荧光试验等进行病毒分离与鉴定。结果显示:采集库蠓样本中有20组检出蓝舌病病毒核酸,检出率为11.11%(20/180);接种后有1份样本能导致鸡胚胚体充血出血和死亡以及BHK-21细胞呈现明显的细胞病变;RT-PCR能从感染细胞样本中扩增出蓝舌病病毒VP7基因特异性片段,且该片段序列与国外BTV-1毒株相应序列的相似性达95%~99%;间接免疫荧光试验显示分离病毒能与BTV-1抗体发生特异性结合。结果表明,云南边境地区库蠓携带有蓝舌病病毒,且为BTV-1,因此应加强对云南边境地区蓝舌病的预防与控制。     

Phylogenetic analysis of Culicoides species from France based on nuclear ITS1-rDNA sequences

Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) play important roles in the transmission of viral diseases affecting wild and domestic ruminants and horses, including Bluetongue (BT) and African horse sickness (AHS) respectively. In southern Europe, BT has been largely transmitted by the classical Afro-Asian vector Culicoides imicola Kieffer. However, other species such as C. obsoletus Meigen, C. scoticus Downs & Kettle and C. pulicaris Linné may also be involved in BTV transmission. As a consequence of the discovery of C. imicola followed by BTV-2 outbreaks on the island of Corsica in October 2000, further studies on these biting midges have been carried out. To better characterize the evolution and phylogenetic relations of Culicoides, molecular analysis in parallel with a morphology-based taxonomic approach were performed. Phylogenetic analyses of French Culicoides species were undertaken using the ribosomal DNA (rDNA) internal transcribed spacer 1 (ITS1) as a molecular target. This region was shown to be useful in understanding evolutionary and genetic relationships between species. Construction of several trees showed that molecular phylogeny within the genus Culicoides correlates not only with morphological-based taxonomy but also with ecological patterns.     

Bluetongue vector species of Culicoides in Switzerland

Switzerland is historically recognized by the Office Internationale des Epizooties as free from bluetongue disease (BT) because of its latitude and climate. With bluetongue virus (BTV) moving north from the Mediterranean, an entomological survey was conducted in Switzerland in 2003 to assess the potential of the BTV vectors present. A total of 39 cattle farms located in three geographical regions, the Ticino region, the Western region and the region of the Grisons, were monitored during the vector season. Farms were located in areas at high risk of vector introduction and establishment based on the following characteristics: annual average temperature > 12.5 degrees C, average annual humidity >or= 60%, cattle farm. Onderstepoort black light traps were operated at the cattle farms generally for one night in July and one night in September. A total of 56 collections of Culicoides (Diptera: Ceratopogonidae) were identified morphologically. Only one single individual of Culicoides (Avaritia) imicola, the major Old World vector of BTV, was found in July 2003 in the Ticino region, one of the southernmost regions of Switzerland. In the absence of further specimens of C. imicola from Switzerland it is suggested that this individual may be a vagrant transported by wind from regions to the south of the country where populations of this species are known to occur. Alternative potential BTV vectors of the Culicoides (Culicoides) pulicaris and Culicoides (Avaritia) obsoletus complexes were abundant in all sampled regions with individual catches exceeding 70 000 midges per trap night.     

Identification and characterization of a novel non-structural protein of bluetongue virus

Bluetongue virus (BTV) is the causative agent of a major disease of livestock (bluetongue). For over two decades, it has been widely accepted that the 10 segments of the dsRNA genome of BTV encode for 7 structural and 3 non-structural proteins. The non-structural proteins (NS1, NS2, NS3/NS3a) play different key roles during the viral replication cycle. In this study we show that BTV expresses a fourth non-structural protein (that we designated NS4) encoded by an open reading frame in segment 9 overlapping the open reading frame encoding VP6. NS4 is 77-79 amino acid residues in length and highly conserved among several BTV serotypes/strains. NS4 was expressed early post-infection and localized in the nucleoli of BTV infected cells. By reverse genetics, we showed that NS4 is dispensable for BTV replication in vitro, both in mammalian and insect cells, and does not affect viral virulence in murine models of bluetongue infection. Interestingly, NS4 conferred a replication advantage to BTV-8, but not to BTV-1, in cells in an interferon (IFN)-induced antiviral state. However, the BTV-1 NS4 conferred a replication advantage both to a BTV-8 reassortant containing the entire segment 9 of BTV-1 and to a BTV-8 mutant with the NS4 identical to the homologous BTV-1 protein. Collectively, this study suggests that NS4 plays an important role in virus-host interaction and is one of the mechanisms played, at least by BTV-8, to counteract the antiviral response of the host. In addition, the distinct nucleolar localization of NS4, being expressed by a virus that replicates exclusively in the cytoplasm, offers new avenues to investigate the multiple roles played by the nucleolus in the biology of the cell.     

Identification of the Genome Segments of Bluetongue Virus Serotype 26 (Isolate KUW2010/02) that Restrict Replication in a Culicoides sonorensis Cell Line (KC Cells)

Bluetongue virus (BTV) can infect most ruminant species and is usually transmitted by adult, vector-competent biting midges (Culicoides spp.). Infection with BTV can cause severe clinical signs and can be fatal, particularly in naïve sheep and some deer species. Although 24 distinct BTV serotypes were recognized for several decades, additional ‘types’ have recently been identified, including BTV-25 (from Switzerland), BTV-26 (from Kuwait) and BTV-27 from France (Corsica). Although BTV-25 has failed to grow in either insect or mammalian cell cultures, BTV-26 (isolate KUW2010/02), which can be transmitted horizontally between goats in the absence of vector insects, does not replicate in a Culicoides sonorensis cell line (KC cells) but can be propagated in mammalian cells (BSR cells). The BTV genome consists of ten segments of linear dsRNA. Mono-reassortant viruses were generated by reverse-genetics, each one containing a single BTV-26 genome segment in a BTV-1 genetic-background. However, attempts to recover a mono-reassortant containing genome-segment 2 (Seg-2) of BTV-26 (encoding VP2), were unsuccessful but a triple-reassortant was successfully generated containing Seg-2, Seg-6 and Seg-7 (encoding VP5 and VP7 respectively) of BTV-26. Reassortants were recovered and most replicated well in mammalian cells (BSR cells). However, mono-reassortants containing Seg-1 or Seg-3 of BTV-26 (encoding VP1, or VP3 respectively) and the triple reassortant failed to replicate, while a mono-reassortant containing Seg-7 of BTV-26 only replicated slowly in KC cells.     

Effect of temperature on the transmission of orbiviruses by the biting midge,Culicoides sonorensis

The influence of temperature on the likelihood of Culicoides sonorensis Wirth & Jones (Diptera: Ceratopogonidae) transmitting African horse sickness virus (AHSV) serotypes 4 and 6, bluetongue virus (BTV) serotypes 10 and 16 and epizootic haemorrhagic disease of deer virus (EHDV) serotype 1 was investigated. Extrinsic incubation periods (EIP), vector competence and vector survival were determined at 15, 20, 25 and 30 degrees C. The effect of humidity on vector survival was also investigated by maintaining adult C. sonorensis at 40, 75 and 85% r.h. at each temperature. Higher temperatures were associated with a shorter EIP for all virus serotypes except AHSV6, to which C. sonorensis was orally refractory, increased vector competence for AHSV4 and EHDV1, but not for BTV10 or BTV16, and a reduction in vector survival. Humidity interacted with temperature in influencing vector survival, such that at low temperatures, lower humidity (40 and 75% r.h.) was detrimental for survival (up to 18% reduction in longevity), whereas at high temperatures, high humidity (85% r.h.) was detrimental (up to 36% reduction in longevity). In general, the transmission potential of C. sonorensis for AHSV4, EHDV1, BTV10 and BTV16 was greater at higher temperatures, because although vector survival was reduced, this was more than compensated for by the accompanying decrease in duration of the EIP.     

Spatial distribution of Culicoides species in Portugal in relation to the transmission of African horse sickness and bluetongue viruses

Surveillance of Culicoides (Diptera: Ceratopogonidae) biting midge vectors was carried out at 87 sites within a 50 x 50 km grid distributed across Portugal, using light trap collections at the time of peak midge abundance. Culicoides imicola (Kieffer) made up 66% of the 55 937 Culicoides in these summer collections. It was highly abundant in the central eastern portion of Portugal, between 37 degrees 5' N and 41 degrees 5' N, and in a band across to the Lisbon peninsula (at around 38 degrees 5' N). Of all the complexes, its distribution was most consistent with that of previous outbreaks of Culicoides-borne disease, suggesting that it may remain the major vector in Portugal. Its distribution was also broadly consistent with that predicted by a recent climate-driven model validating the use of remote sensing datasets for modelling of Culicoides distribution. Adult C. imicola were found to have overwintered at 12 of 20 sites re-surveyed in winter but it did so in very low numbers. Culicoides obsoletus (Meigen) and Culicoides pulicaris (Linnaeus) complex midges were widespread despite their low summer abundance. The observed coincidence of high abundances of C. imicola and high abundances of C. pulicaris in summer lead us to suggest that C. imicola could bring African horse sickness virus or bluetongue virus into contact with C. pulicaris and the latter complex, together with C. obsoletus, could then transmit these viruses across much wider areas of Europe. The fact that adult C. pulicaris are present in high abundances in winter may provide a mechanism by which these viruses can overwinter in these areas.     

蓝舌病病毒基因节段2与6的重配导致病毒血清型与增殖特性的改变

【背景】蓝舌病病毒(Bluetongue Virus,BTV)是一种侵染反刍动物的虫媒病毒,基因重配可引起病毒的快速变异。【目的】通过我国强致病性BTV-16型毒株与弱致病性BTV-4型毒株间Seg-2与Seg-6基因节段的重配,探讨病毒基因重配与表型变异之间的关系。【方法】采用全长cDNA扩增与高通量测序获取BTV-16/V158的全基因组序列,构建病毒的真核表达质粒,通过免疫荧光与WesternBlot检测目的蛋白表达;通过RT-PCR、体外转录与细胞转染等方法建立BTV反向遗传体系并获取基因重配病毒;通过蚀斑分析、增殖曲线分析与血清中和试验,比较亲本毒株与基因重配病毒在生物学特性上的差异。【结果】获取的BTV-16/V158毒株基因组大小为19 186 bp,与中国和印度BTV-16型毒株具有最近的亲缘关系;将表达BTV VP1、VP3与NS2的真核表达质粒转染细胞,检测到目的蛋白的表达;将BTV的7种真核表达质粒与基因组ssRNA共转染BHK-21细胞,成功拯救出与亲本毒株生物学特性一致的病毒;将BTV-16/V158毒株的Seg-2与Seg-6替换为BTV-4/YTS4毒株的对应基因节段,拯救出基因重配病毒BTV-16/V158-RG (BTV-4/S2,S6);与亲本病毒相比较,基因重配病毒在BHK-21细胞上形成的蚀斑变小,增殖能力减弱,血清型由BTV-16型转化为BTV-4型。【结论】建立了我国流行BTV-16型毒株的反向遗传体系,BTVSeg-2与Seg-6的基因重配可引起病毒在细胞上增殖能力的改变与血清型改变。研究结果为BTV基因重配致病毒变异与新型基因工程疫苗的研究提供了基础。