Perspectives on the Changing Landscape of Epizootic Hemorrhagic Disease Virus Control

Epizootic hemorrhagic disease (EHD) is an insect-transmitted viral disease of wild and domestic ruminants. It was first described following a 1955 epizootic in North American white-tailed deer (Odocoileus virginianus), a species which is highly susceptible to the causative agent of EHD, epizootic hemorrhagic disease virus (EHDV). EHDV has been detected globally across tropical and temperate regions, largely corresponding to the presence of Culicoides spp. biting midges which transmit the virus between ruminant hosts. It regularly causes high morbidity and mortality in wild and captive deer populations in endemic areas during epizootics. Although cattle historically have been less susceptible to EHDV, reports of clinical disease in cattle have increased in the past two decades. There is a pressing need to identify new methods to prevent and mitigate outbreaks and reduce the considerable impacts of EHDV on livestock and wildlife. This review discusses recent research advancements towards the control of EHDV, including the development of new investigative tools and progress in basic and applied research focused on virus detection, disease mitigation, and vector control. The potential impacts and implications of these advancements on EHD management are also discussed.     

Blood-sucking biting midges of the genus Culicoides (Diptera, Ceratopogonidae) from Poland

Further records of Culicoides collected from new localities and regions are presented. C. vidourlensis (Baltic Coasts), C. abchazicus (Ojców National Park, Babia Góra Mts) and C. jurensis (Pieniny and Babia Góra Mts) are for the first time recorded from the country. At present 48 species of the genus are known to occur in Poland.     

基于线粒体COI基因序列进行库蠓二囊亚属近似种的分子鉴定

目的 通过采用线粒体COI基因进行库蠓近似种分子鉴定的方法,来弥补传统形态学在种类鉴定中存在的操作繁琐、难度大等不足,从而实现库蠓近似种的快速、准确鉴别。方法 采用DNA测序的方法获得库蠓属二囊亚属的林岛库蠓(C. gaponus)、标翅库蠓(C. insignipennis)、无害库蠓(C. innoxius)、连斑库蠓(C. jacobsoni)、南山库蠓(C. lansangensis)、新竹库蠓(C. liui)、长喙库蠓(C. longirostris)、异域库蠓(C. peregrinus)等8种库蠓近似种的部分线粒体COI序列,并对其进行分子鉴定;基于Kimura 2-parameter模型分析遗传距离,同时应用MEGA 6.0软件以荒川库蠓C. arakawai为外群构建系统发育树。结果 8种库蠓COI序列长度约650 bp;遗传距离在种内和种间具有统计学差异(t=119.452,P<0.05);系统发育树中不同库蠓种类各自构成单系(群),同种类不同地理种群聚为一支。结论 本研究证实了线粒体COI序列可用于进行库蠓近似种的分子鉴定。     

Characterization of a Novel Reassortant Epizootic Hemorrhagic Disease Virus Serotype 6 Strain Isolated from Diseased White-Tailed Deer (Odocoileus virginianus) on a Florida Farm

We report an outbreak of a novel reassortant epizootic hemorrhagic disease virus serotype 6 (EHDV-6) in white-tailed deer (WTD) on a Florida farm in 2019. At necropsy, most animals exhibited hemorrhagic lesions in the lung and heart, and congestion in the lung, liver, and spleen. Histopathology revealed multi-organ hemorrhage and congestion, and renal tubular necrosis. Tissues were screened by RT-qPCR and all animals tested positive for EHDV. Tissues were processed for virus isolation and next-generation sequencing was performed on cDNA libraries generated from the RNA extracts of cultures displaying cytopathic effects. Six isolates yielded nearly identical complete genome sequences of a novel U.S. EHDV-6 strain. Genetic and phylogenetic analyses revealed the novel strain to be most closely related to a reassortant EHDV-6 strain isolated from cattle in Trinidad and both strains received segment 4 from an Australian EHDV-2 strain. The novel U.S. EHDV-6 strain is unique in that it acquired segment 8 from an Australian EHDV-8 strain. An RNAscope^® in situ hybridization assay was developed against the novel U.S. EHDV-6 strain and labeling was detected within lesions of the heart, kidney, liver, and lung. These data support the novel U.S. reassortant EHDV-6 strain as the cause of disease in the farmed WTD.     

Vector Competence of Eucampsipoda africana (Diptera: Nycteribiidae) for Marburg Virus Transmission in Rousettus aegyptiacus (Chiroptera: Pteropodidae)

This study aimed to determine the vector competence of bat-associated nycteribiid flies (Eucamsipoda africana) for Marburg virus (MARV) in the Egyptian Rousette Bat (ERB), Rousettus aegyptiacus. In flies fed on subcutaneously infected ERBs and tested from 3 to 43 days post infection (dpi), MARV was detected only in those that took blood during the peak of viremia, 5–7 dpi. Seroconversion did not occur in control bats in contact with MARV-infected bats infested with bat flies up to 43 days post exposure. In flies inoculated intra-coelomically with MARV and tested on days 0–29 post inoculation, only those assayed on day 0 and day 7 after inoculation were positive by q-RT-PCR, but the virus concentration was consistent with that of the inoculum. Bats remained MARV-seronegative up to 38 days after infestation and exposure to inoculated flies. The first filial generation pupae and flies collected at different times during the experiments were all negative by q-RT-PCR. Of 1693 nycteribiid flies collected from a wild ERB colony in Mahune Cave, South Africa where the enzootic transmission of MARV occurs, only one (0.06%) tested positive for the presence of MARV RNA. Our findings seem to demonstrate that bat flies do not play a significant role in the transmission and enzootic maintenance of MARV. However, ERBs eat nycteribiid flies; thus, the mechanical transmission of the virus through the exposure of damaged mucous membranes and/or skin to flies engorged with contaminated blood cannot be ruled out.     

The sexual activity of Culicoides nubeculosus (Mg.) (Diptera, Ceratopogonidae) (author's transl)

The sexual activity of C. nubeculosus has been studied in relation to the age of males and females. The criteria chosen were stoutness of spermatheca and frequency of mating, respectively. A unit N. M. H. (number of matings/male/hour) has been determined. Sexual activity reaches its maximum on 2nd day of imaginal life and is then slowly decreasing. This maximum activity on 2nd day rapidly decreases in the hours following blood meal, down to a rate which then remains nearly constant.     

Modeling Abundance of Culicoides stellifer,a Candidate Orbivirus Vector,Indicates Nonrandom Hemorrhagic Disease Risk for White-Tailed Deer (Odocoileus virginianus)

(1) Background: Hemorrhagic diseases in white-tailed deer (Odocoileus virginianus) are caused by orbiviruses and have significant economic impact on the deer ranching industry in the United States. Culicoides stellifer is a suspected vector of epizootic hemorrhagic disease virus (EHDV), with recent field evidence from Florida, but its natural history is poorly understood. Studying the distribution and abundance of C. stellifer across the landscape can inform our knowledge of how virus transmission can occur locally. We may then target vector management strategies in areas where viral transmission can occur. (2) Methods: Here, we used an occupancy modeling approach to estimate abundance of adult C. stellifer females at various physiological states to determine habitat preferences. We then mapped midge abundance during the orbiviral disease transmission period (May–October) in Florida. (3) Results: We found that overall, midge abundance was positively associated with sites in closer proximity to large-animal feeders. Additionally, midges generally preferred mixed bottomland hardwood and agricultural/sand/water habitats. Female C. stellifer with different physiological states preferred different habitats. (4) Conclusions: The differences in habitat preferences between midges across states indicate that disease risk for deer is heterogeneous across this landscape. This can inform how effective vector management strategies should be implemented.     

A Duplex Fluorescent Microsphere Immunoassay for Detection of Bluetongue and Epizootic Hemorrhagic Disease Virus Antibodies in Cattle Sera

Bluetongue virus (BTV) causes internationally reportable hemorrhagic disease in cattle, sheep, and white-tailed deer. The closely related, and often co-circulating, epizootic hemorrhagic disease virus causes a clinically similar devastating disease in white-tailed deer, with increasing levels of disease in cattle in the past 10 years. Transmitted by Culicoides biting midges, together, they constitute constant disease threats to the livelihood of livestock owners. In cattle, serious economic impacts result from decreased animal production, but most significantly from trade regulations. For effective disease surveillance and accurate trade regulation implementation, rapid, sensitive assays that can detect exposure of cattle to BTV and/or EHDV are needed. We describe the development and validation of a duplex fluorescent microsphere immunoassay (FMIA) to simultaneously detect and differentiate antibodies to BTV and EHDV in a single bovine serum sample. Performance of the duplex FMIA for detection and differentiation of BTV and EHDV serogroup antibodies was comparable, with higher sensitivity than commercially available single-plex competitive enzyme-linked immunosorbent assays (cELISA) for detection of each virus antibody separately. The FMIA adds to the currently available diagnostic tools for hemorrhagic orbiviral diseases in cattle as a sensitive, specific assay, with the benefits of serogroup differentiation in a single serum sample, and multiplexing flexibility in a high-throughput platform.     

Full Genome Sequencing of Three Sedoreoviridae Viruses Isolated from Culicoides spp. (Diptera,Ceratopogonidae) in China

Sedoreoviridae is a family of viruses belonging to the order Reovirales and comprises six genera, two of which, Orbivirus and Seadornavirus, contain arboviruses that cause disease in humans and livestock. Areas such as Yunnan Province in southwestern China, have high arboviral activity due in part to warm and wet summers, which support high populations of biting flies such as mosquitoes and Culicoides. Three viral isolates previously obtained from Culicoides collected at cattle farms in Shizong County of Yunnan Province, China, between 2019 and 2020 were completely sequenced and identified as Banna virus (BAV) genotype A of Seadornavirus and serotypes 1 and 7 of epizootic hemorrhagic disease virus (EHDV) of Orbivirus. These results suggest that Culicoides tainanus and C. orientalis are potential vectors of BAV and EHDV, respectively, and represent the first association of a BAV with C. tainanus and of an arbovirus with C. orientalis. Analysis using VP9 generally agreed with the current groupings within this genus based on VP12, although the classification for some strains should be corrected. Furthermore, the placement of Kadipiro virus (KDV) and Liao ning virus (LNV) in Seadornavirus may need confirmation as phylogenetic analysis placed these viruses as sister to other species in the genus.     

Vector Competence of German Aedes punctor (Kirby, 1837) for West Nile Virus Lineages 1 and 2

West Nile virus (WNV) is a zoonotic flavivirus transmitted by mosquitoes as a biological vector. Because of its biting behavior, the widespread snow-melt mosquito Aedes punctor could be a potential bridge vector for WNV to humans and nonhuman mammals. However, little is known on its role in transmission of WNV. The aim of this study was to determine the vector competence of German Ae. punctor for WNV lineages 1 and 2. Field-collected larvae and pupae were reared to adults and offered infectious blood containing either an Italian WNV lineage 1 or a German WNV lineage 2 strain via cotton stick feeding. Engorged females were incubated for 14/15 or 21 days at 18 °C. After incubation; surviving mosquitoes were dissected and forced to salivate. Mosquito bodies with abdomens, thoraces and heads, legs plus wings and saliva samples were investigated for WNV RNA by RT-qPCR. Altogether, 2/70 (2.86%) and 5/85 (5.88%) mosquito bodies were found infected with WNV lineage 1 or 2, respectively. In two mosquitoes, viral RNA was also detected in legs and wings. No saliva sample contained viral RNA. Based on these results, we conclude that Ae. punctor does not play an important role in WNV transmission in Germany.     

Vector Competence of Mosquitoes from Germany for Sindbis Virus

Transmission of arthropod-borne viruses (arboviruses) are an emerging global health threat in the last few decades. One important arbovirus family is the Togaviridae, including the species Sindbis virus within the genus Alphavirus. Sindbis virus (SINV) is transmitted by mosquitoes, but available data about the role of different mosquito species as potent vectors for SINV are scarce. Therefore, we investigated seven mosquito species, collected from the field in Germany (Ae. koreicus, Ae. geniculatus, Ae. sticticus, Cx. torrentium, Cx. pipiens biotype pipiens) as well as lab strains (Ae. albopictus, Cx. pipiens biotype molestus, Cx. quinquefasciatus), for their vector competence for SINV. Analysis was performed via salivation assay and saliva was titrated to calculate the amount of infectious virus particles per saliva sample. All Culex and Aedes species were able to transmit SINV. Transmission could be detected at all four investigated temperature profiles (of 18 ± 5 °C, 21 ± 5 °C, 24 ± 5 °C or 27 ± 5 °C), and no temperature dependency could be observed. The concentration of infectious virus particles per saliva sample was in the same range for all species, which may suggest that all investigated mosquito species are able to transmit SINV in Germany.     

Evaluation of Vector Competence of Ixodes Ticks for Kemerovo Virus

Tick-borne viruses are responsible for various symptoms in humans and animals, ranging from simple fever to neurological disorders or haemorrhagic fevers. The Kemerovo virus (KEMV) is a tick-borne orbivirus, and it has been suspected to be responsible for human encephalitis cases in Russia and central Europe. It has been isolated from Ixodes persulcatus and Ixodes ricinus ticks. In a previous study, we assessed the vector competence of I. ricinus larvae from Slovakia for KEMV, using an artificial feeding system. In the current study, we used the same system to infect different tick population/species, including I. ricinus larvae from France and nymphs from Slovakia, and I. persulcatus larvae from Russia. We successfully confirmed the first two criteria of vector competence, namely, virus acquisition and trans-stadial transmission, for both tick species that we tested. The estimated infection rates of engorged and moulted ticks suggest specificities between viral strains and tick species/developmental stages.     

Infection,Dissemination, and Transmission Potential of North American Culex quinquefasciatus,Culex tarsalis,and Culicoides sonorensis for Oropouche Virus

Oropouche virus (OROV), a vector-borne Orthobunyavirus circulating in South and Central America, causes a febrile illness with high rates of morbidity but with no documented fatalities. Oropouche virus is transmitted by numerous vectors, including multiple genera of mosquitoes and Culicoides biting midges in South America. This study investigated the vector competence of three North American vectors, Culex tarsalis, Culex quinquefasciatus, and Culicoides sonorensis, for OROV. Cohorts of each species were fed an infectious blood meal containing 6.5 log₁₀ PFU/mL OROV and incubated for 10 or 14 days. Culex tarsalis demonstrated infection (3.13%) but not dissemination or transmission potential at 10 days post infection (DPI). At 10 and 14 DPI, Cx. quinquefasciatus demonstrated 9.71% and 19.3% infection, 2.91% and 1.23% dissemination, and 0.97% and 0.82% transmission potential, respectively. Culicoides sonorensis demonstrated 86.63% infection, 83.14% dissemination, and 19.77% transmission potential at 14 DPI. Based on these data, Cx. tarsalis is unlikely to be a competent vector for OROV. Culex quinquefasciatus demonstrated infection, dissemination, and transmission potential, although at relatively low rates. Culicoides sonorensis demonstrated high infection and dissemination but may have a salivary gland barrier to the virus. These data have implications for the spread of OROV in the event of a North American introduction.