Experimental Infection of Mid-Gestation Pregnant Female and Intact Male Sheep with Zika Virus.

Zika virus (ZIKV) is an arbovirus that causes birth defects, persistent male infection, and sexual transmission in humans. The purpose of this study was to continue the development of an ovine ZIKV infection model; thus, two experiments were undertaken. In the first experiment, we built on previous pregnant sheep experiments by developing a mid-gestation model of ZIKV infection. Four pregnant sheep were challenged with ZIKV at 57-64 days gestation; two animals served as controls. After 13-15 days (corresponding with 70-79 days of gestation), one control and two infected animals were euthanized; the remaining animals were euthanized at 20-22 days post-infection (corresponding with 77-86 days of gestation). In the second experiment, six sexually mature, intact, male sheep were challenged with ZIKV and two animals served as controls. Infected animals were serially euthanized on days 2-6 and day 9 post-infection with the goal of isolating ZIKV from the male reproductive tract. In the mid-gestation study, virus was detected in maternal placenta and spleen, and in fetal organs, including the brains, spleens/liver, and umbilicus of infected fetuses. Fetuses from infected animals had visibly misshapen heads and morphometrics revealed significantly smaller head sizes in infected fetuses when compared to controls. Placental pathology was evident in infected dams. In the male experiment, ZIKV was detected in the spleen, liver, testes/epididymides, and accessory sex glands of infected animals. Results from both experiments indicate that mid-gestation ewes can be infected with ZIKV with subsequent disruption of fetal development and that intact male sheep are susceptible to ZIKV infection and viral dissemination and replication occurs in highly vascular tissues (including those of the male reproductive tract).

Experimental Infection of Pregnant Female Sheep with Zika Virus During Early Gestation.

Zika virus (ZIKV) is a vertically and sexually transmissible virus resulting in severe congenital malformation. The goal of this study was to develop an ovine model of ZIKV infection. Between 28-35 days gestation (DG), four pregnant animals were infected with two doses of 6 × 106 PFU of ZIKV; four control animals received PBS. Animals were evaluated for 45 days (D) post-infection (PI) and necropsies were performed. Viral RNA was detected in infected ewe peripheral blood mononuclear cells (PBMC) during the first week PI; however, all fluids and tissues were negative upon culture. Anti-ZIKV IgM (1:400) and neutralizing antibodies were detected in all infected animals. Clinical disease, virus, or ZIKV antibodies were not detected in control ewes. After two weeks PI, fetal loss occurred in two infected animals, and at necropsy, three infected animals had placental petechiation and ecchymosis and one had hydramnion. Fetal morphometrics revealed smaller cranial circumference to crown-rump length ratios (p < 0.001) and relative brain weights (p = 0.038) in fetuses of infected animals compared with control fetuses. Immunophenotyping indicated an increase in B cells (p = 0.012) in infected sheep. Additionally, in vitro experiments using both adult and fetal cell lines demonstrated that ovine cells are highly permissive to ZIKV infection. In conclusion, ZIKV infection of pregnant sheep results in a change in fetal growth and gestational outcomes.

Teratogenic bluetongue and related orbivirus infections in pregnant ruminant livestock: timing and pathogen genetics are critical.

Congenital infections of domestic animals with viruses in several families, including Bunyaviridae, Flaviridae, Parvoviridae, and Reoviridae, are the cause of naturally occurring teratogenic central nervous system and/or musculoskeletal defects (arthrogryposis) in domestic animals. Congenital infections of ruminant livestock with bluetongue virus (BTV) and some related members of the genus Orbivirus (family Reoviridae) have clearly shown the critical role of gestational age at infection in determining outcome. Specifically, fetuses infected prior to mid-gestation that survive congenital BTV infection are born with cavitating central nervous system defects that range from severe hydranencephaly to cerebral cysts (porencephaly). Generally, the younger the fetus (in terms of gestational age) at infection, the more severe the teratogenic lesion at birth. Age-dependent virus infection and destruction of neuronal and/or glial cell precursors that populate the developing central nervous system are responsible for these naturally occurring virus-induced congenital defects of animals, thus lesions are most severe when progenitor cells are infected prior to their normal migration during embryogenesis. Whereas congenital infection is characteristic of certain BTV strains, notably live-attenuated (modified-live) vaccine viruses that have been passaged in embryonating eggs, transplacental transmission is not characteristic of many field strains of the virus and much remains to be determined regarding the genetic determinants of transplacental transmission of individual virus strains.

A review of potential bluetongue virus vaccine strategies.

Bluetongue (BT) is an economically important, non-zoonotic arboviral disease of certain wild and domestic species of cloven-hooved ungulates. Bluetongue virus (BTV) is the causative agent and the occurrence of BTV infection is distinctly seasonal in temperate regions of the world, and dependent on the presence of vector biting midges (e.g. Culicoides sonorensis in much of North America). In recent years, severe outbreaks have occurred throughout Europe and BTV is endemic in most tropical and temperate regions of the world. Several vaccines have been licensed for commercial use, including modified live (live-attenuated) and inactivated products, and this review summarizes recent strategies developed for BTV vaccines with emphasis on technologies suitable for differentiating naturally infected from vaccinated animals. The goal of this review is to evaluate realistic vaccine strategies that might be utilized to control or prevent future outbreaks of BT.

A Deterministic Model to Quantify Risk and Guide Mitigation Strategies to Reduce Bluetongue Virus Transmission in California Dairy Cattle.

The global distribution of bluetongue virus (BTV) has been changing recently, perhaps as a result of climate change. To evaluate the risk of BTV infection and transmission in a BTV-endemic region of California, sentinel dairy cows were evaluated for BTV infection, and populations of Culicoides vectors were collected at different sites using carbon dioxide. A deterministic model was developed to quantify risk and guide future mitigation strategies to reduce BTV infection in California dairy cattle. The greatest risk of BTV transmission was predicted within the warm Central Valley of California that contains the highest density of dairy cattle in the United States. Temperature and parameters associated with Culicoides vectors (transmission probabilities, carrying capacity, and survivorship) had the greatest effect on BTV's basic reproduction number, R0. Based on these analyses, optimal control strategies for reducing BTV infection risk in dairy cattle will be highly reliant upon early efforts to reduce vector abundance during the months prior to peak transmission.

Overwintering of Bluetongue virus in temperate zones.

Within Northern California, Culicoides sonorensis is the major vector of Bluetongue virus (BTV) and annual infection of livestock is distinctly seasonal (typically July­November). Our recent studies compare the population dynamics of C. sonorensis midges with occurrence of BTV infection of C. sonorensis and sentinel dairy cattle throughout both the seasonal and interseasonal ('overwintering') periods of BTV activity. Spring emergence and seasonal abundance of adult C. sonorensis on the sampled farms coincided with rising vernal temperature. Intensive surveillance confirmed widespread infection of both sentinel cattle and vector midges during the August­November period of seasonal BTV transmission. Bluetongue virus infection of parous female midges captured in traps set during daylight hours was also detected during the interseasonal period of virus activity, whereas there was no concurrent active infection of sentinel cattle during the overwintering period. The finding of BTV­infected vector midges during mid­Winter suggests that BTV can overwinter in Northern California by infection of long­lived female C. sonorensis midges that were infected during the prior seasonal period of virus transmission and which, then, entered a quiescence in the fall (Autumn) and re­emerged sporadically during the overwintering period. Notably, vertical transmission of BTV was not detected among progeny of midges infected in the laboratory nor in field­collected larvae. In addition to defining the mechanism of BTV over­wintering in a temperate region, the studies reviewed in this article also provide precise documentation of temporal changes in the annual abundance, dispersal and dynamics of BTV infection of Culicoides midges. Collectively these findings are critical to the creation of accurate predictive models of BTV infection in livestock and to development of sound abatement strategies.

African Horse Sickness Caused by Genome Reassortment and Reversion to Virulence of Live, Attenuated Vaccine Viruses, South Africa, 2004-2014.

African horse sickness (AHS) is a hemorrhagic viral fever of horses. It is the only equine disease for which the World Organization for Animal Health has introduced specific guidelines for member countries seeking official recognition of disease-free status. Since 1997, South Africa has maintained an AHS controlled area; however, sporadic outbreaks of AHS have occurred in this area. We compared the whole genome sequences of 39 AHS viruses (AHSVs) from field AHS cases to determine the source of 3 such outbreaks. Our analysis confirmed that individual outbreaks were caused by virulent revertants of AHSV type 1 live, attenuated vaccine (LAV) and reassortants with genome segments derived from AHSV types 1, 3, and 4 from a LAV used in South Africa. These findings show that despite effective protection of vaccinated horses, polyvalent LAV may, paradoxically, place susceptible horses at risk for AHS.

Complete Genome Sequences of Four African Horse Sickness Virus Strains from a Commercial Tetravalent Live Attenuated Vaccine.

This is a report of the complete genome sequences of plaque-selected isolates of each of the four virus strains included in a South African commercial tetravalent African horse sickness attenuated live virus vaccine.

Bluetongue virus infection creates light averse Culicoides vectors and serious errors in transmission risk estimates.

BACKGROUND: Pathogen manipulation of host behavior can greatly impact vector-borne disease transmission, but almost no attention has been paid to how it affects disease surveillance. Bluetongue virus (BTV), transmitted by Culicoides biting midges, is a serious disease of ruminant livestock that can cause high morbidity and mortality and significant economic losses. Worldwide, the majority of surveillance for Culicoides to assess BTV transmission risk is done using UV-light traps. Here we show that field infection rates of BTV are significantly lower in midge vectors collected using traps baited with UV light versus a host cue (CO2). METHODS: We collected Culicoides sonorensis midges in suction traps baited with CO2, UV-light, or CO2 + UV on three dairies in southern California to assess differences in the resulting estimated infection rates from these collections. Pools of midges were tested for BTV by qRT-PCR, and maximum likelihood estimates of infection rate were calculated by trap. Infection rate estimates were also calculated by trapping site within a dairy. Colonized C. sonorensis were orally infected with BTV, and infection of the structures of the compound eye was examined using structured illumination microscopy. RESULTS: UV traps failed entirely to detect virus both early and late in the transmission season, and underestimated virus prevalence by as much as 8.5-fold. CO2 + UV traps also had significantly lower infection rates than CO2-only traps, suggesting that light may repel infected vectors. We found very high virus levels in the eyes of infected midges, possibly causing altered vision or light perception. Collecting location also greatly impacts our perception of virus activity. CONCLUSIONS: Because the majority of global vector surveillance for bluetongue uses only light-trapping, transmission risk estimates based on these collections are likely severely understated. Where national surveillance programs exist, alternatives to light-trapping should be considered. More broadly, disseminated infections of many arboviruses include infections in vectors' eyes and nervous tissues, and this may be causing unanticipated behavioral effects. Field demonstrations of pathogen-induced changes in vector behavior are quite rare, but should be studied in more systems to accurately predict vector-borne disease transmission.

Complete Genome Sequences of the Three African Horse Sickness Virus Strains from a Commercial Trivalent Live Attenuated Vaccine.

This is a report of the complete genome sequences of plaque-selected isolates of each of the three virus strains included in a South African commercial trivalent African horse sickness attenuated live virus vaccine.

Development of three triplex real-time reverse transcription PCR assays for the qualitative molecular typing of the nine serotypes of African horse sickness virus.

Blood samples collected as part of routine diagnostic investigations from South African horses with clinical signs suggestive of African horse sickness (AHS) were subjected to analysis with an AHS virus (AHSV) group specific reverse transcription quantitative polymerase chain reaction (AHSV RT-qPCR) assay and virus isolation (VI) with subsequent serotyping by plaque inhibition (PI) assays using AHSV serotype-specific antisera. Blood samples that tested positive by AHSV RT-qPCR were then selected for analysis using AHSV type specific RT-qPCR (AHSV TS RT-qPCR) assays. The TS RT-qPCR assays were evaluated using both historic stocks of the South African reference strains of each of the 9 AHSV serotypes, as well as recently derived stocks of these same viruses. Of the 503 horse blood samples tested, 156 were positive by both AHSV RT-qPCR and VI assays, whereas 135 samples that were VI negative were positive by AHSV RT-qPCR assay. The virus isolates made from the various blood samples included all 9 AHSV serotypes, and there was 100% agreement between the results of conventional serotyping of individual virus isolates by PI assay and AHSV TS RT-qPCR typing results. Results of the current study confirm that the AHSV TS RT-qPCR assays for the identification of individual AHSV serotypes are applicable and practicable and therefore are potentially highly useful and appropriate for virus typing in AHS outbreak situations in endemic or sporadic incursion areas, which can be crucial in determining appropriate and timely vaccination and control strategies.

The changing global face of Bluetongue: from the beginning.

Bluetongue (BT) was first recognized and described in Southern Africa, and only later elsewhere. It is now known that the causative agent of BT [BT virus (BTV)] occurs throughout temperate and tropical regions of the world. Previous OIE symposia in 1984, 1991 and 2003 clarified the global distribution of BTV and its epidemiology, pathogenesis, and diagnosis. Since the 3rd symposium in 2003, however, there have been significant developments. Notably, BT has emerged in Northern Europe and novel BTV serotypes have appeared in other endemic areas raising substantial questions regarding the environmental and anthropogenic drivers of emergence of BTV, including the invasion and spread of laboratory propagated viruses. Additional BTV serotypes with novel properties have recently been identified in Europe and the Middle East. Recent studies also confirm the importance of the Culicoides vector as the essential overwintering reservoir of BTV in temperate regions such as California and not ruminant livestock, proving wrong the prevailing thesis circulated prior to the 1st symposium in 1984. The challenge for participants of this 4th symposium is to predict collectively what the future might hold in terms of emergence of BTV globally, and what strategies are likely to be most feasible, justified, and effective for its control.

Orbiviruses: A North American Perspective.

Orbiviruses are members of the Reoviridae family and include bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV). These viruses are the cause of significant regional disease outbreaks among livestock and wildlife in the United States, some of which have been characterized by significant morbidity and mortality. Competent vectors are clearly present in most regions of the globe; therefore, all segments of production livestock are at risk for serious disease outbreaks. Animals with subclinical infections also serve as reservoirs of infection and often result in significant trade restrictions. The economic and explicit impacts of BTV and EHDV infections are difficult to measure, but infections are a cause of economic loss for producers and loss of natural resources (wildlife). In response to United States Animal Health Association (USAHA) Resolution 16, the US Department of Agriculture (USDA), in collaboration with the Department of the Interior (DOI), organized a gap analysis workshop composed of international experts on Orbiviruses. The workshop participants met at the Arthropod-Borne Animal Diseases Research Unit in Manhattan, KS, May 14-16, 2013, to assess the available scientific information and status of currently available countermeasures to effectively control and mitigate the impact of an outbreak of an emerging Orbivirus with epizootic potential, with special emphasis given to BTV and EHDV. In assessing the threats, workshop participants determined that available countermeasures are somewhat effective, but several weaknesses were identified that affect their ability to prevent and control disease outbreaks effectively.

Vaccines for Prevention of Bluetongue and Epizootic Hemorrhagic Disease in Livestock: A North American Perspective.

Bluetongue (BT) and epizootic hemorrhagic disease (EHD) are noncontagious, insect-transmitted diseases of domestic and wild ruminants caused by related but distinct viruses. There are significant gaps in our scientific knowledge and available countermeasures to control an outbreak of orbivirus-induced disease, whether BT or EHD. Both BT virus (BTV) and EHD virus (EHDV) cause hemorrhagic fevers in susceptible ruminants; however, BT is principally a disease of domestic livestock whereas EHD is principally a disease of certain species of wild, non-African ungulates, notably white-tailed deer. The live-attenuated (modified live virus [MLV]) vaccines available in the United States for use in small ruminant livestock do provide good protection against clinical disease following infection with the homologous virus serotype. Although there is increasing justification that the use of MLV vaccines should be avoided if possible, these are the only vaccines currently available in the United States. Specifically, MLVs are used in California to protect sheep against infection with BTV serotypes 10, 11, and 17, and a MLV to BTV serotype 10 is licensed for use in sheep throughout the United States. These MLV vaccines may need to continue to be used in the immediate future for protective immunization of sheep and goats against BT. There are currently no licensed vaccines available for EHD in the United States other than autogenous vaccines. If there is a need to rapidly develop a vaccine to meet an emerging crisis associated with either BTV or EHDV infections, development of an inactivated virus vaccine in a conventional adjuvanted formulation will likely be required. With two doses of vaccine (and in some instances just one dose), inactivated vaccines can provide substantial immunity to the epizootic serotype of either BTV or EHDV. This strategy is similar to that used in the 2006-2008 BTV serotype 8 outbreaks in northern Europe that provided vaccine to the field within 2 years of the initial incursion (by 2008). Further research and development are warranted to provide more efficacious and effective vaccines for control of BTV and EHDV infections.

Seasonal and interseasonal dynamics of bluetongue virus infection of dairy cattle and Culicoides sonorensis midges in northern California--implications for virus overwintering in temperate zones.

Bluetongue virus (BTV) is the cause of an economically important arboviral disease of domestic and wild ruminants. The occurrence of BTV infection of livestock is distinctly seasonal in temperate regions of the world, thus we determined the dynamics of BTV infection (using BTV-specific real time reverse transcriptase polymerase chain reaction) among sentinel cattle and vector Culicoides sonorensis (C. sonorensis) midges on a dairy farm in northern California throughout both the seasonal and interseasonal (overwintering) periods of BTV activity from August 2012 until March 2014. The data confirmed widespread infection of both sentinel cattle and vector midges during the August-November period of seasonal BTV transmission, however BTV infection of parous female midges captured in traps set during daylight hours also was detected in February of both 2013 and 2014, during the interseasonal period. The finding of BTV-infected vector midges during mid-winter suggests that BTV may overwinter in northern California by infection of long-lived female C. sonorensis midges that were infected during the prior seasonal period of virus transmission, and reemerged sporadically during the overwintering period; however the data do not definitively preclude other potential mechanisms of BTV overwintering that are also discussed.

Whole genome sequencing and phylogenetic analysis of Bluetongue virus serotype 2 strains isolated in the Americas including a novel strain from the western United States.

Bluetongue is a potentially fatal arboviral disease of domestic and wild ruminants that is characterized by widespread edema and tissue necrosis. Bluetongue virus (BTV) serotypes 10, 11, 13, and 17 occur throughout much of the United States, whereas serotype 2 (BTV-2) was previously only detected in the southeastern United States. Since 1998, 10 other BTV serotypes have also been isolated from ruminants in the southeastern United States. In 2010, BTV-2 was identified in California for the first time, and preliminary sequence analysis indicated that the virus isolate was closely related to BTV strains circulating in the southeastern United States. In the current study, the whole genome sequence of the California strain of BTV-2 was compared with those of other BTV-2 strains in the Americas. The results of the analysis suggest co-circulation of genetically distinct viruses in the southeastern United States, and further suggest that the 2010 western isolate is closely related to southeastern strains of BTV. Although it remains uncertain as to how this novel virus was translocated to California, the findings of the current study underscore the need for ongoing surveillance of this economically important livestock disease.

Experiences with infectious cDNA clones of equine arteritis virus: lessons learned and insights gained.

The advent of recombinant DNA technology, development of infectious cDNA clones of RNA viruses, and reverse genetic technologies have revolutionized how viruses are studied. Genetic manipulation of full-length cDNA clones has become an especially important and widely used tool to study the biology, pathogenesis, and virulence determinants of both positive and negative stranded RNA viruses. The first full-length infectious cDNA clone of equine arteritis virus (EAV) was developed in 1996 and was also the first full-length infectious cDNA clone constructed from a member of the order Nidovirales. This clone was extensively used to characterize the molecular biology of EAV and other Nidoviruses. The objective of this review is to summarize the characterization of the virulence (or attenuation) phenotype of the recombinant viruses derived from several infectious cDNA clones of EAV in horses, as well as their application for characterization of the molecular basis of viral neutralization, persistence, and cellular tropism.

Seasonal variation and impact of waste-water lagoons as larval habitat on the population dynamics of Culicoides sonorensis (Diptera:Ceratpogonidae) at two dairy farms in northern California.

The Sacramento (northern Central) Valley of California (CA) has a hot Mediterranean climate and a diverse ecological landscape that is impacted extensively by human activities, which include the intensive farming of crops and livestock. Waste-water ponds, marshes, and irrigated fields associated with these agricultural activities provide abundant larval habitats for C. sonorensis midges, in addition to those sites that exist in the natural environment. Within this region, C. sonorensis is an important vector of bluetongue (BTV) and related viruses that adversely affect the international trade and movement of livestock, the economics of livestock production, and animal welfare. To characterize the seasonal dynamics of immature and adult C. sonorensis populations, abundance was monitored intensively on two dairy farms in the Sacramento Valley from August 2012- to July 2013. Adults were sampled every two weeks for 52 weeks by trapping (CDC style traps without light and baited with dry-ice) along N-S and E-W transects on each farm. One farm had large operational waste-water lagoons, whereas the lagoon on the other farm was drained and remained dry during the study. Spring emergence and seasonal abundance of adult C. sonorensis on both farms coincided with rising vernal temperature. Paradoxically, the abundance of midges on the farm without a functioning waste-water lagoon was increased as compared to abundance on the farm with a waste-water lagoon system, indicating that this infrastructure may not serve as the sole, or even the primary larval habitat. Adult midges disappeared from both farms from late November until May; however, low numbers of parous female midges were detected in traps set during daylight in the inter-seasonal winter period. This latter finding is especially critical as it provides a potential mechanism for the "overwintering" of BTV in temperate regions such as northern CA. Precise documentation of temporal changes in the annual abundance and dispersal of Culicoides midges is essential for the creation of models to predict BTV infection of livestock and to develop sound abatement strategies.

The immune response of ruminant livestock to bluetongue virus: from type I interferon to antibody.

Infection of ruminants with most (but not all) serotypes of bluetongue virus (BTV) leads to a highly blood cell-associated viremia that may be prolonged but not persistent. Furthermore, recovered animals are resistant to reinfection with the homologous virus serotype, which is the basis for vaccination strategies to prevent BTV infection and the clinical disease (bluetongue) that it causes in domestic livestock. BTV infection is initiated at the site of virus inoculation and the associated draining lymph node, from where the virus is then spread in lymph cells to the systemic circulation and secondary sites of replication. Replication of BTV in target cells, notably mononuclear phagocytic cells (dendritic cells and macrophages) and endothelium, leads to the generation of the innate and adaptive immune responses that mediate both initial virus clearance and subsequent resistance to infection with the homologous virus serotype. The goal of this review is to summarize current understanding of these innate and adaptive immune responses of animals to BTV infection.

Equine arteritis virus.

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory and reproductive disease of equids. There has been significant recent progress in understanding the molecular biology of EAV and the pathogenesis of its infection in horses. In particular, the use of contemporary genomic techniques, along with the development and reverse genetic manipulation of infectious cDNA clones of several strains of EAV, has generated significant novel information regarding the basic molecular biology of the virus. Therefore, the objective of this review is to summarize current understanding of EAV virion architecture, replication, evolution, molecular epidemiology and genetic variation, pathogenesis including the influence of host genetics on disease susceptibility, host immune response, and potential vaccination and treatment strategies.