Implicating Culicoides Biting Midges as Vectors of Schmallenberg Virus Using Semi-Quantitative RT-PCR

Background

The recent unprecedented emergence of arboviruses transmitted by Culicoides biting midges in northern Europe has necessitated the development of techniques to differentiate competent vector species. At present these techniques are entirely reliant upon interpretation of semi-quantitative RT-PCR (sqPCR) data in the form of C_q values used to infer the presence of viral RNA in samples.

Methodology/Principal Findings

This study investigates the advantages and limitations of sqPCR in this role by comparing infection and dissemination rates of Schmallenberg virus (SBV) in two colony lines of Culicoides. Through the use of these behaviorally malleable lines we provide tools for demarcating arbovirus infection and dissemination rates in Culicoides which to date have prevented clear implication of primary vector species in northern Europe. The study demonstrates biological transmission of SBV in an arthropod vector, supporting the conclusions from field-caught Culicoides and provides a general framework for future assessment of vector competence of Culicoides for arboviruses using sqPCR.

Conclusions/Significance

When adopting novel diagnostic technologies, correctly implicating vectors of arboviral pathogens requires a coherent laboratory framework to fully understand the implications of results produced in the field. This study illustrates these difficulties and provides a full examination of sqPCR in this role for the Culicoides-arbovirus system.     

Schmallenberg Virus Circulation in Culicoides in Belgium in 2012: Field Validation of a Real Time RT-PCR Approach to Assess Virus Replication and Dissemination in Midges

Indigenous Culicoides biting midges are suggested to be putative vectors for the recently emerged Schmallenberg virus (SBV) based on SBV RNA detection in field-caught midges. Furthermore, SBV replication and dissemination has been evidenced in C. sonorensis under laboratory conditions. After SBV had been detected in Culicoides biting midges from Belgium in August 2011, it spread all over the country by the end of 2011, as evidenced by very high between-herd seroprevalence rates in sheep and cattle. This study investigated if a renewed SBV circulation in midges occurred in 2012 in the context of high seroprevalence in the animal host population and evaluated if a recently proposed realtime RT-PCR approach that is meant to allow assessing the vector competence of Culicoides for SBV and bluetongue virus under laboratory conditions was applicable to field-caught midges. Therefore midges caught with 12 OVI traps in four different regions in Belgium between May and November 2012, were morphologically identified, age graded, pooled and tested for the presence of SBV RNA by realtime RT-PCR. The results demonstrate that although no SBV could be detected in nulliparous midges caught in May 2012, a renewed but short lived circulation of SBV in parous midges belonging to the subgenus Avaritia occured in August 2012 at all four regions. The infection prevalence reached up to 2.86% in the south of Belgium, the region where a lower seroprevalence was found at the end of 2011 than in the rest of the country. Furthermore, a frequency analysis of the Ct values obtained for 31 SBV-S segment positive pools of Avaritia midges showed a clear bimodal distribution with peaks of Ct values between 21–24 and 33–36. This closely resembles the laboratory results obtained for SBV infection of C. sonorensis and implicates indigenous midges belonging to the subgenus Avaritia as competent vectors for SBV.     

RT—PCR检测蓝舌病毒技术的建立

蓝舌病毒 (ＢｌｕｅｔｏｎｇｕｅＶｉｒｕｓ,ＢＴＶ)是呼肠孤病毒科 (Ｒｅｏｖｉｒｉｄａｅ)环状病毒属 (Ｏｒｂｉｖｉｒｕｓ)的代表种 ,含10个节段的双链ＲＮＡ(ｄｓＲＮＡ)作基因组。其中 ,Ｌ2节段编码ＢＴＶ型特异性抗原ＶＰ2 ,Ｌ3节段和Ｓ7节段编码群特异性抗原多肽ＶＰ3和ＶＰ7。其中 ,ＶＰ7是ＢＴＶ粒子的主要结构多肽之一 ,其编码基因序列保守。ＶＰ7具有高度的抗原性 ,能刺激被感机体产生强的群特异性免疫反应[1] 。蓝舌病毒的易感宿主是牛、羊及野生反刍动物 ,死亡率高达 6 0 %～ 70 %以上 ,并且至今仍无有效的防治措施 ,对畜牧业…     

RT-PCR检测蓝舌病毒技术的建立

蓝舌病毒(Bluetongue Virus,BTV)是呼肠孤病毒科(Reoviridae)环状病毒属(Orbivirus)的代表种,含10个节段的双链RNA(dsRNA)作基因组.其中,L2节段编码BTV型特异性抗原VP2,L3节段和S7节段编码群特异性抗原多肽VP3和VP7.其中,VP7是BTV粒子的主要结构多肽之一,其编码基因序列保守.VP7具有高度的抗原性,能刺激被感机体产生强的群特异性免疫反应[1].     

Culicoides Midge Bites Modulate the Host Response and Impact on Bluetongue Virus Infection in Sheep

Many haematophagous insects produce factors that help their blood meal and coincidently favor pathogen transmission. However nothing is known about the ability of Culicoides midges to interfere with the infectivity of the viruses they transmit. Among these, Bluetongue Virus (BTV) induces a hemorrhagic fever- type disease and its recent emergence in Europe had a major economical impact. We observed that needle inoculation of BTV8 in the site of uninfected C. nubeculosus feeding reduced viraemia and clinical disease intensity compared to plain needle inoculation. The sheep that developed the highest local inflammatory reaction had the lowest viral load, suggesting that the inflammatory response to midge bites may participate in the individual sensitivity to BTV viraemia development. Conversely compared to needle inoculation, inoculation of BTV8 by infected C. nubeculosus bites promoted viraemia and clinical symptom expression, in association with delayed IFN- induced gene expression and retarded neutralizing antibody responses. The effects of uninfected and infected midge bites on BTV viraemia and on the host response indicate that BTV transmission by infected midges is the most reliable experimental method to study the physio-pathological events relevant to a natural infection and to pertinent vaccine evaluation in the target species. It also leads the way to identify the promoting viral infectivity factors of infected Culicoides in order to possibly develop new control strategies against BTV and other Culicoides transmitted viruses.     

Attraction and Repellent Behaviors of Culicoides Biting Midges toward Cow Dung,Carbon Dioxide,and Essential Oils

Culicoides biting midges (Diptera: Ceratopogonidae) are hematophagous arthropod vectors that transmit epizootic arthropod-borne viruses (arboviruses). Arboviruses are recognized as causes of pregnancy loss, encephalomyelitis, and congenital malformations in ruminants. Therefore, continuous monitoring and control of Culicoides, which causes significant damage to industrial animals are necessary. We performed attraction and repellent tests in Culicoides using various essential oils, cow dung, and carbon dioxide (CO₂). Culicoides tended to move more to cow dung (60.8%, P<0.0001) and CO₂ (63.8%, P<0.01). To the essential oils as repellents, 26.1% (P<0.0001), 18.7% (P<0.001), and 25.5% (P<0.01) of the Culicoides moved to the lavender, lemongrass, and eucalyptus chamber, respectively. The Culicoides that moved to the 3 essential oils chambers showed markedly low activity. Collectively, it was showed that Culicoides tended to be attractive to cow dung and CO₂, and repellent from the 3 essential oils.     

First Record of Culicoides oxystoma Kieffer and Diversity of Species within the Schultzei Group of Culicoides Latreille (Diptera: Ceratopogonidae) Biting Midges in Senegal

The Schultzei group of Culicoides Latreille (Diptera: Ceratopogonidae) is distributed throughout Africa to northern Asia and Australasia and includes several potential vector species of livestock pathogens. The taxonomy of the species belonging to this species group is confounded by the wide geographical distribution and morphological variation exhibited by many species. In this work, morphological and molecular approaches were combined to assess the taxonomic validity of the species and morphological variants of the Schultzei group found in Senegal by comparing their genetic diversity with that of specimens from other geographical regions. The species list for Senegal was updated with four species: Culicoides kingi, C. oxystoma, C. enderleini and C. nevilli being recorded. This is the first record of C. oxystoma from Africa south of Sahara, and its genetic relationship with samples from Israel, Japan and Australia is presented. This work provides a basis for ecological studies of the seasonal and spatial dynamics of species of this species group that will contribute to better understanding of the epidemiology of the viruses they transmit.     

Diagnosis of Barmah Forest Virus Infection by a Nested Real-Time SYBR Green RT-PCR Assay

Barmah Forest virus (BFV) is a mosquito borne (+) ssRNA alphavirus found only in Australia. It causes rash, myalgia and arthralgia in humans and is usually diagnosed serologically. We developed a real-time PCR assay to detect BFV in an effort to improve diagnosis early in the course of infection. The limit of detection was 16 genome equivalents with a specificity of 100%. Fifty five serum samples from BFV-infected patients were tested by the PCR. 52 of 53 antibody-positive samples were PCR negative. Two culture-positive (neutralizing antibody negative) samples were positive on first round PCR, while one sample (IgM and neutralizing antibody strongly positive, IgG negative) was positive on second round PCR, suggesting that viral RNA is detectable and transiently present in early infection. PCR can provide results faster than culture, is capable of high throughput and by sequencing the PCR product strain variants can be characterized.     

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。     

禽流感和新城疫病毒二重荧光定量RT-PCR检测方法的建立

目的：建立二重荧光定量RT-PCR方法,用于禽流感病毒（AIV）和新城疫病毒（NDV）的检测。方法：根据AIV和NDV的基因保守序列,设计了AIV和NDV的2对特异性引物和2条用不同荧光基团标记的TaqMan探针;对反应条件和试剂浓度进行优化,建立了能够同时检测AIV和NDV的Z-重荧光定量RT-PCR方法。结果：所建方法特异性好,对AIV和NDV的检测敏感性均达到2000个模板拷贝数,比常规RT-PCR敏感性高100倍;抗干扰能力强,对AIV和NDV不同模板浓度进行组合,仍可有效地同时检测2种病毒。对保存的AIV或NDV鸡胚尿囊液及临床病料进行二重荧光定量RT-PCR检测,结果尿囊液检测的拷贝数均达到10^10/μL以上,临床病料的拷贝数为2．13x10^8-6．52x10^4／μL。结论：建立了用于检测AIV和NDV的二重荧光定量RT-PCR法,该方法特异、敏感、快速、可定量,对AIV和NDV的防制有重要意义。     

Population Genetic Structure and Potential Incursion Pathways of the Bluetongue Virus Vector Culicoides brevitarsis (Diptera: Ceratopogonidae) in Australia

Culicoides brevitarsis is a vector of the bluetongue virus (BTV), which infects sheep and cattle. It is an invasive species in Australia with an assumed Asian/South East Asian origin. Using one mitochondrial marker (i.e., part of the cytochrome oxidase subunit I gene) and six nuclear markers, we inferred population genetic structure and possible incursion pathways for Australian C. brevitarsis. Nine mitochondrial haplotypes, with low nucleotide sequence diversity (0.0–0.7%) among these, were identified in a sample of 70 individuals from seven sites. Both sets of markers revealed a homogeneous population structure, albeit with evidence of isolation by distance and two genetically distinct clusters distributed along a north-to-south cline. No evidence of a cryptic species complex was found. The geographical distribution of the mitochondrial haplotypes is consistent with at least two incursion pathways into Australia since the arrival of suitable livestock hosts. By contrast, 15 mitochondrial haplotypes, with up to four times greater nucleotide sequence diversity (0.0–2.9%) among these, were identified in a sample of 16 individuals of the endemic C. marksi (sampled from a site in South Australia and another in New South Wales). A phylogenetic tree inferred using the mitochondrial marker revealed that the Australian and Japanese samples of C. brevitarsis are as evolutionarily different from one another as some of the other Australian species (e.g., C. marksi, C. henryi, C. pallidothorax) are. The phylogenetic tree placed four of the species endemic to Australia (C. pallidothorax, C. bundyensis, C. marksi, C. henryi) in a clade, with a fifth such species (C. bunrooensis) sharing a common ancestor with that clade and a clade comprising two Japanese species (C. verbosus, C. kibunensis).     

Isolation and Characterization of a cDNA Clone Coding for a Glutathione S-Transferase Class Delta Enzyme from the Biting Midge Culicoides variipennis sonorensis Wirth and Jones

Culicoides variipennis sonorensis is the primary vector of bluetongue viruses in North America. Glutathione S-transferases (GSTs) are enzymes that catalyze nucleophilic substitutions, converting reactive lipophilic molecules into soluble conjugates. Increased GST activity is associated with development of insecticide resistance. Described here is the isolation of the first cDNA encoding a C. variipennis GST. The clone consists of 720 translated bases encoding a protein with a M(r) of approximately 24,800 composed of 219 amino acids. The deduced amino acid sequence is similar (64%-74%) to class Delta (previously named Theta) GSTs from the dipteran genera Musca, Drosophila, Lucilia and Anopheles. The cDNA was subcloned into pET-11b, expressed in Epicurian coli BL21 (DE3) and has a specific activity of approximately 28,000 units/mg for the substrate 1-chloro-2,4-dinitrobenzene.     

Isolation and Characterization of Bluetongue Virus Recovered from Blood Samples by Immunoaffinity Purification

An immunoaffinity chromatography (IAC) method was optimized for the selective capture of bluetongue virus (BTV) from blood samples and isolation of the virus in cell culture. The antibody against BTV core particles (lacking the outer capsid proteins VP2 and VP5) was used for the optimization of IAC technique. The antibody against BTV core particle was conjugated with Protein A-virus complex and the complex was dissociated using elution buffer (4 M MgCl₂ with 75 mM HEPES, pH 6.5). The optimized IAC method specifically purified the BTV without capturing other commonly infecting small ruminant’s viruses like gaotpox virus (GTPV), sheeppox virus (SPPV), Peste des petits ruminants virus (PPRV) and Foot and mouth disease virus (FMDV). The blood samples (n?=?22), positive for BTV antigen in sandwich-ELISA were attempted for virus isolation in the BHK-21 cell using the optimized IAC method. A total of seven BTV were isolated by selective capturing of the virion particles. The isolated viruses were characterized by RNA-PAGE, sequence analysis and serum neutralization test (SNT). Electropherotypic analysis of viral dsRNA in the RNA-PAGE revealed the presence of ten dsRNA segments characteristic of BTV. Out of seven isolates, four isolates were identified as BTV-1 and three isolates were identified as BTV-16 based on nucleotide sequences of segment-2. Phylogenetic analysis of segment-2 nucleotide sequence segregated BTV-1 and BTV-16 isolates to monophyletic cluster at close proximity to other eastern topotype. In SNT, hyperimmune serum (HIS) against BTV-1 neutralized the four BTV-1 isolates up to a titer?>?256 and HIS against BTV-16 neutralized the three BTV-16 isolates up to a titer?>?128. The IAC technique will be useful for the selective capture of BTV from mixed infection (BTV with other small ruminant’s viruses) and isolation from blood sample having low viral load by enrichment.     

中国柱蠓属一新种及阿蠓属一新记录（双翅目：蠓科）

报道采自陕西省宝鸡地区的柱蠓属（Stilobezzia)1新种,即宝鸡柱蠓Stilobezzia(Stilobezzia)baojia,sp.nov,和阿蠓属（Alluaudomyla)1新记录,即梯泊阿蠓Allu-audomyia tiberghieni Neveu,1978。模式标本保存在北京市丰台区北京军事医学科学院医学昆虫标本馆。