Genome sequencing and phylogenetic analysis of Banna virus (genus <Emphasis Type="Italic">Seadornavirus</Emphasis>, family Reoviridae) isolated from Culicoides

In an investigation of blood-sucking insects and arboviruses, a virus (YN12243) was isolated from Culicoides samples collected in the Sino-Burmese border region of Yunnan Province, China. The virus caused cytopathic effect (CPE) in C6/36 cells and passaged stably. Polyacrylamide gel analysis showed that the genome of YN12243 was composed of 12 segments of double-stranded RNA (dsRNA), with a distribution pattern of 6-6. The nucleotide and amino acid sequences of the coding region (1?12 segments) were 17,803 bp and 5,925 amino acids in length, respectively. The phylogenetic analysis of VP1 protein (RdRp) revealed that YN12243 belonged to genus Seadornavirus of family Reoviridae, and further analysis indicated that YN12243 belongs to the Banna virus (BAV) genotype A2. Additionally, YN12243 was located in the same evolutionary cluster as BAV strains isolated from different mosquito species, suggesting that the BAV isolated from Culicoides does not have species barriers. These results indicate that Culicoides can also be a vector for BAV. In view of the hematophagous habits of Culicoides on cattle, horses, deer, and other large animals, as well as the possibility of spreading and causing a variety of animal arboviral diseases, it is important to improve infection detection and monitor the BAV in large livestock.     

Isolation of Tibet orbivirus,TIBOV, from Culicoides Collected in Yunnan,China

We isolated a novel virus strain (YN12246) from Culicoides spp. specimens collected at the China-Laos-Myanmar border in southern Yunnan Province. This virus had a cytopathic effect (CPE) on both insect cells (C6/36) and mammalian cells (BHK-21). Electron microscopy revealed the structure of the virions to be spherical with a diameter of 75 nm. Polyacrylamide gel analysis demonstrated that the viral genome consisted of 10 segments of double-stranded RNA (dsRNA), with a distribution pattern of 3-3-3-1. The coding sequences of 9 genome segments of YN12246 (Seg1, Seg3-Seg10) were obtained by high-throughput sequencing and Sanger sequencing. Comparisons of conserved genome segments 1 and 3 (Seg1 and Seg3), encoding the polymerase-VP1 and sub-core T2 protein, respectively, showed that YN12246 groups with the Culicoides-borne orbiviruses. The highest levels of sequence identity were detected between YN12246 and Tibet orbivirus (TIBOV), indicating that they belong to the same virus species (with amino acid identity of 98.8% and 96.4% for the polymerase and T2 protein, respectively). The data presented here confirm that YN12246 is a member of the TIBOV species, which was first isolated from mosquitoes in 2009. This is the first report of the isolation of TIBOV from Culicoides.     

中国分离巴泰病毒基因组全编码区序列测定和分析

采用RT-PCR和TAIL-PCR方法,首次对我国分离的巴泰病毒(YN92-4株)基因组的全编码区进行序列测定和分析。结果显示,YN92-4株病毒基因组由S、M、L三个片段组成,长度分别为947、4 371、6 860个核苷酸。其中,S片段基因编码由234个氨基酸残基组成的核衣壳蛋白和由102个氨基酸残基组成的非结构蛋白,M片段基因编码由1 435个氨基酸残基组成的前体蛋白,L片段基因编码由2 239个氨基酸残基组成的RNA聚合酶。与国外其它地区的巴泰病毒分离株进行基因组全编码区序列比较后发现,YN92-4株与日本牛血清分离株(ON-7/B/01株)在S、M片段核苷酸(氨基酸)的同源性最高,分别为97.7%(100%)和95.7%(98%);由于本研究首次开展对巴泰病毒L基因片段核苷酸序列的研究,因此国际基因库尚无可参考的序列信息,本研究比较了我国分离的巴泰病毒与同一血清组的代表病毒Bunyamwera病毒L片段的核苷酸和氨基酸序列同源性,分别为73.5%和81.6%。系统进化分析显示,YN92-4株基因组与其它巴泰病毒分离株在各自分支下形成独立分支。本研究提示我国分离的巴泰病毒YN92-4株未发生基因重配(...     

Radioimmunoassay of neuropeptide Y

The development of a radioimmunoassay to the newly isolated peptide, neuropeptide Y is described. Four separate antisera have been developed using different immunisation schedules. Two of these antisera (YNI and YNIO) are directed to the C-terminal region of the peptide and cross-react with the related peptide PYY, whereas YN7 is specific being directed to the N-terminal region of NPY, YN6 is similarly specific for NPY, but is unable to bind the available fragments. These four antisera provide similar results for determination of NPY immunoreactivity within porcine brain extracts, however YN6 consistently undervalues all extracts from the other species examined (human, rat, guinea pig, cat and mouse). Chromatographic analysis by means of reverse phase high pressure liquid chromatography (HPLC) shows that NPY immunoreactivity of human extracts elutes in an earlier position than the porcine standard. It seems likely therefore that human and porcine NPY differ in their amino acid sequences.     

我国2009年新分离乙脑病毒全基因组序列特征研究

本研究对我国2009年新分离的两株乙脑病毒进行全基因组序列测定和分析,以了解病毒全基因组分子特征。通过RT-PCR和核苷酸序列测定方法获得病毒全基因组序列,采用ClustalX、DNASTAR、MEGA等生物学软件完成核苷酸序列及氨基酸序列分析和系统进化分析等。研究结果显示,新分离两株乙脑病毒YN0911和YN0967株基因组全长均为10 965个核苷酸,编码3 432个氨基酸。这2株乙脑病毒之间核苷酸同源性为98.7%,氨基酸同源性为99.8%。与国际乙脑病毒流行株相比,核苷酸同源性为83.5%～98.9%,氨基酸同源性为94.8%～99.7%。与乙脑病毒疫苗株SA14-14-2相比,在E蛋白有13个氨基酸差异位点,但都位于抗原关键位点之外。这2株病毒在3′UTR区域存在11nt缺失。基于C/PrM区段、E基因、全基因组系统进化分析结果均显示新分离2株乙脑病毒为G I乙脑病毒,并且和越南、四川、贵州、广西以往的分离株遗传进化关系较近。本研究提示我国新分离的2株乙脑病毒均为G I乙脑病毒,决定病毒毒力的关键氨基酸位点未见明显变化。     

Occurrence of Culicoides spp. (Diptera: Ceratopogonidae) in Tunisia, with emphasis on the bluetongue vector Culicoides imicola

Following the bluetongue (BT) outbreaks in Tunisia from 1999 to 2002, BTV (bluetongue virus) serotype 2 was isolated; however, no entomological investigation was performed. In the study presented here, we assessed the Culicoides species populations (particularly C. imicola) in proximity to the BT outbreaks locations, both as a retrospective analysis and to update the list of Culicoides species present in Tunisia. The insects were caught using light traps and the species identification was performed according to the standard entomological methods. This study reveaaled the presence of significant numbers of C. imicola in all the tested locations. In addition, we reported a new Culicoides species for the Tunisian fauna C. punctatus.     

Characterization of the Influenza A Virus Gene Pool in Avian Species in Southern China: Was H6N1 a Derivative or a Precursor of H5N1?

In 1997, an H5N1 influenza virus outbreak occurred in chickens in Hong Kong, and the virus was transmitted directly to humans. Because there is limited information about the avian influenza virus reservoir in that region, we genetically characterized virus strains isolated in Hong Kong during the 1997 outbreak. We sequenced the gene segments of a heterogeneous group of viruses of seven different serotypes (H3N8, H4N8, H6N1, H6N9, H11N1, H11N9, and H11N8) isolated from various bird species. The phylogenetic relationships divided these viruses into several subgroups. An H6N1 virus isolated from teal (A/teal/Hong Kong/W312/97 [H6N1]) showed very high (>98%) nucleotide homology to the human influenza virus A/Hong Kong/156/97 (H5N1) in the six internal genes. The N1 neuraminidase sequence showed 97% nucleotide homology to that of the human H5N1 virus, and the N1 protein of both viruses had the same 19-amino-acid deletion in the stalk region. The deduced hemagglutinin amino acid sequence of the H6N1 virus was most similar to that of A/shearwater/Australia/1/72 (H6N5). The H6N1 virus is the first known isolate with seven H5N1-like segments and may have been the donor of the neuraminidase and the internal genes of the H5N1 viruses. The high homology between the internal genes of H9N2, H6N1, and the H5N1 isolates indicates that these subtypes are able to exchange their internal genes and are therefore a potential source of new pathogenic influenza virus strains. Our analysis suggests that surveillance for influenza A viruses should be conducted for wild aquatic birds as well as for poultry, pigs, and humans and that H6 isolates should be further characterized.     

Culicoides in relation to transmission of African Horse Sickness virus in The Gambia

Twelve light trap collections made near overnight shelters of horses and donkeys in four villages in the Central River Division of The Gambia captured fourteen species of biting midge of the genus Culicoides . Five species new to The Gambia were identified. This brought the number of recognized species of Culicoides (after a revision of C. schultzei ) to twenty-nine in The Gambia. Species known or suspected as vectors of African horse sickness virus (AHSV) and bluetongue virus (BTV) comprised 83% of female captures, 65% of captures being C. imicola or its sibling species , C. miombo . Captures of female Culicoides in the late dry season were almost as large as in the early dry season, despite the extreme heat and dryness at this time of the year.
Tests on batches of formalin-preserved female midges, using AHSV or BTV antigen capture ELISAs, did not show the presence of any virus amongst 2286 females in 240 aliquots. Nearly all Gambian equines are reportedly seropositive to AHSV and these results suggest that virus challenge from Culicoides vectors may be a factor in the health of Gambian horses and donkeys.     

Genomic sequences of Australian bluetongue virus prototype serotypes reveal global relationships and possible routes of entry into Australia

Bluetongue virus (BTV) is transmitted by biting midges (Culicoides spp.). It causes disease mainly in sheep and occasionally in cattle and other species. BTV has spread into northern Europe, causing disease in sheep and cattle. The introduction of new serotypes, changes in vector species, and climate change have contributed to these changes. Ten BTV serotypes have been isolated in Australia without apparent associated disease. Simplified methods for preferential isolation of double-stranded RNA (dsRNA) and template preparation enabled high-throughput sequencing of the 10 genome segments of all Australian BTV prototype serotypes. Phylogenetic analysis reinforced the Western and Eastern topotypes previously characterized but revealed unique features of several Australian BTVs. Many of the Australian BTV genome segments (Seg-) were closely related, clustering together within the Eastern topotypes. A novel Australian topotype for Seg-5 (NS1) was identified, with taxa spread across several serotypes and over time. Seg-1, -2, -3, -4, -6, -7, -9, and -10 of BTV_2_AUS_2008 were most closely related to the cognate segments of viruses from Taiwan and Asia and not other Australian viruses, supporting the conclusion that BTV_2 entered Australia recently. The Australian BTV_15_AUS_1982 prototype was revealed to be unusual among the Australian BTV isolates, with Seg-3 and -8 distantly related to other BTV sequences from all serotypes.     

应用单引物差异RT-PCR法鉴定我国分离的甲病毒(YN87448病毒)

甲病毒指披膜病毒科(Togavirdae)甲病毒属(Alphavirus),以蚊虫等吸血昆虫为传播媒介,可引起如辛德毕斯热、基孔肯雅热、东方马脑炎、西方马脑炎等多种人畜共患性传染病[1,2],是医学上重要的虫媒病毒.甲病毒世界性分布,全世界已发现28种甲病毒,其中13种与人畜疾病有关,至少7种甲病毒发生过不同程度流行,造成巨大的经济损失,成为严重的公共卫生问题.张海林等(中国媒介生物学及控制杂志,1992,3(特7):419－421)从云南傣族一位发热病人血中分离到一株病毒,命名为YN87448病毒.经血清学鉴定该病毒符合披膜病毒科甲病毒属病毒特征.由于缺乏标准诊断血清,无法对它作进一步鉴定.该病毒直接分离自发热病人血清,病人主要症状为发热,寒颤,腰疼痛和全身酸痛.由于该病毒直接分离自病人血清,病毒鉴定对解释该病人的发病原因,甚至对于解释我国部分地区流行的"无名热"的病因均具有重要意义.     

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.     

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.     

An investigation on the Culicoides species composition at seven sites in southern Switzerland

In the past decade, there have been regular outbreaks of bluetongue (BT) in many parts of Europe. Owing to the presence of BT disease and its vectors in countries adjacent to Switzerland, an initial entomological survey was conducted in 2003, which established the presence of several midges of the genus Culicoides (Diptera: Ceratopogonidae). Subsequently, a sentinel herd monitoring system was established with the primary entomological aim being the determination and further study of Culicoides population compositions. Insects were collected in 2005 and 2006 at seven sentinel herd sites in the south of Switzerland (canton of Ticino) near the border of Italy, using Onderstepoort-type light traps. This region is botanically and zoologically similar to the Mediterranean and is one of the warmest and most humid areas of the country, hence it is considered a potential access path for BT disease into Switzerland. Collections were made at four cattle farms, two equestrian centres and one goat farm. Sites were sampled four times per month from June to October. Traps were operated from dusk until dawn and samples were collected monthly for analysis through microscopy as well as a Culicoides imicola -specific PCR. Results confirmed the absence of C. imicola (Kieffer) and demonstrated that the potential BT virus vectors are highly abundant, notably: Culicoides obsoletus (Meigen), Culicoides scoticus (Downes & Kettle) and Culicoides dewulfi (Goetghebuer) subgenus Avaritia and Culicoides pulicaris (Linnaeus) subgenus Culicoides . These findings expand the current knowledge of Culicoides population composition in the southern part of the Switzerland. Culicoides cataneii (Clastrier), Culicoides flavipulicaris (Dzhafarov), Culicoides indistinctus (Khalaf), Culicoides nubeculosus (Meigen) and species of the Grisescens complex were reported for the first time in Switzerland.     

Molecular identification, phylogenetic status, and geographic distribution of Culicoides oxystoma (Diptera: Ceratopogonidae) in Israel

Culicoides oxystoma (Diptera: Ceratopogonidae) is an important vector species, reported mainly from Asia, with high potential to transmit viral diseases affecting livestock. In Japan, many arboviruses have been isolated from C. oxystoma, suggesting it as a key player in the epidemiology of several Culicoides-borne diseases. Over the years, C. oxystoma has also been reported in the Middle East region, including Israel. In this region, however, C. oxystoma cannot be easily distinguished morphologically from its sibling species included in the Culicoides schultzei complex. We therefore used genomic data for species identification and phylogeny resolution. Phylogenetic analyses based on internal transcribed spacer 1 (ITS-1) of ribosomal DNA and the mitochondrial gene encoding cytochrome oxidase subunit I (COI) showed that C. oxystoma from Israel is closely related to C. oxystoma from Japan. Using differential probing PCR, we showed that C. oxystoma is distributed all over the country, especially in Mediterranean climate regions. Culicoides oxystoma is less common or even absent in arid regions, while the other genetic cluster of C. schultzei complex was found only in the east of the country (mostly arid and semiarid regions). The molecular finding of C. oxystoma in wide geographical regions, together with its high proportion in the general Culicoides population and its vectoring potential, imply that it may be an important vector species in the Middle East.     

Saliva proteins of vector Culicoides modify structure and infectivity of bluetongue virus particles

Bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) are related orbiviruses, transmitted between their ruminant hosts primarily by certain haematophagous midge vectors (Culicoides spp.). The larger of the BTV outer-capsid proteins, 'VP2', can be cleaved by proteases (including trypsin or chymotrypsin), forming infectious subviral particles (ISVP) which have enhanced infectivity for adult Culicoides, or KC cells (a cell-line derived from C. sonorensis). We demonstrate that VP2 present on purified virus particles from 3 different BTV strains can also be cleaved by treatment with saliva from adult Culicoides. The saliva proteins from C. sonorensis (a competent BTV vector), cleaved BTV-VP2 more efficiently than those from C. nubeculosus (a less competent/non-vector species). Electrophoresis and mass spectrometry identified a trypsin-like protease in C. sonorensis saliva, which was significantly reduced or absent from C. nubeculosus saliva. Incubating purified BTV-1 with C. sonorensis saliva proteins also increased their infectivity for KC cells ～10 fold, while infectivity for BHK cells was reduced by 2-6 fold. Treatment of an 'eastern' strain of EHDV-2 with saliva proteins of either C. sonorensis or C. nubeculosus cleaved VP2, but a 'western' strain of EHDV-2 remained unmodified. These results indicate that temperature, strain of virus and protein composition of Culicoides saliva (particularly its protease content which is dependent upon vector species), can all play a significant role in the efficiency of VP2 cleavage, influencing virus infectivity. Saliva of several other arthropod species has previously been shown to increase transmission, infectivity and virulence of certain arboviruses, by modulating and/or suppressing the mammalian immune response. The findings presented here, however, demonstrate a novel mechanism by which proteases in Culicoides saliva can also directly modify the orbivirus particle structure, leading to increased infectivity specifically for Culicoides cells and, in turn, efficiency of transmission to the insect vector.     

Heterogeneity of ITS1 sequences in the biting midge Culicoides impunctatus (Goetghebuer) suggests a population in Argyll, Scotland, may be genetically distinct.

Ribosomal DNA (rDNA) internal transcribed spacer 1 (ITS1) is a useful genomic region for understanding evolutionary and genetic relationships. In the current study, variation in ITS1 from eight Culicoides species was analysed by PCR, DNA restriction analysis, cloning, and sequencing. ITS1 variants were essentially homogenized within a species, as sequences were identical or closely related. However, Culicoides impunctatus ITS1 sequences derived from one (Argyll) of five populations contained considerable genomic diversity. The secondary structure of each ITS1 was computed. The structure aided the production of an accurate alignment and the identification of a large indel. A phylogenetic analysis was performed. Some of the sequences from the diverse Argyll C. impunctatus population were more related to Culicoides imicola, a vector of animal pathogens in the Old World, than they were to the other C. impunctatus sequences. Thus, the rDNA ITS1 regions of individuals in the Argyll C. impunctatus population were not conforming to the general theory of rDNA homogenization through molecular drive.     

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。