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.

Fatal bluetongue virus infection in an alpaca (Vicugna pacos) in California.

In October 2008, a 15-year-old female alpaca (Vicugna pacos) housed at a breeding farm in northern California died after a brief illness characterized by sudden onset of weakness, recumbency, and respiratory distress. Postmortem examination revealed severe hydrothorax and hydropericardium, marked pulmonary edema, and acute superficial myocardial hemorrhage affecting the left ventricle. Bluetongue virus (BTV) was detected in the spleen by quantitative real-time reverse transcription polymerase chain reaction and confirmed by sequence analysis. No antibodies against BTV were detected in the serum using a competitive enzyme-linked immunosorbent assay, confirming acute, fulminant BTV infection.

Pilot immunization of mice infected with an equine strain of Corynebacterium pseudotuberculosis.

This pilot study evaluated protection of an equine autogenous bacterin-toxoid vaccine against Corynebacterium pseudotuberculosis infection. Twenty-four BALB/c mice were inoculated with two doses of bacterin-toxoid vaccine or two injections of a placebo. Clinical, microbiologic, and pathologic outcomes were assessed after intradermal infection with one of two equine-origin C. pseudotuberculosis strains. Mice receiving bacterin-toxoid from fast-growing C. pseudotuberculosis showed significant protection from challenge infection, as evidenced by a higher survival rate, fewer gross and histopathologic lesions, and lower bacterial levels on culture. Successful protection via a vaccine against equine internal abscesses might provide supplementary management options against an important, potentially fatal disease.

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).

Persistent equine arteritis virus infection in HeLa cells.

The horse-adapted virulent Bucyrus (VB) strain of equine arteritis virus (EAV) established persistent infection in high-passage-number human cervix cells (HeLa-H cells; passages 170 to 221) but not in low-passage-number human cervix cells (HeLa-L cells; passages 95 to 115) or in several other cell lines that were evaluated. However, virus recovered from the 80th passage of the persistently infected HeLa-H cells (HeLa-H-EAVP80) readily established persistent infection in HeLa-L cells. Comparative sequence analysis of the entire genomes of the VB and HeLa-H-EAVP80 viruses identified 16 amino acid substitutions, including 4 in the replicase (nsp1, nsp2, nsp7, and nsp9) and 12 in the structural proteins (E, GP2, GP3, GP4, and GP5). Reverse genetic studies clearly showed that substitutions in the structural proteins but not the replicase were responsible for the establishment of persistent infection in HeLa-L cells by the HeLa-H-EAVP80 virus. It was further demonstrated that recombinant viruses with substitutions in the minor structural proteins E and GP2 or GP3 and GP4 were unable to establish persistent infection in HeLa-L cells but that recombinant viruses with combined substitutions in the E (Ser53-->Cys and Val55-->Ala), GP2 (Leu15-->Ser, Trp31-->Arg, Val87-->Leu, and Ala112-->Thr), GP3 (Ser115-->Gly and Leu135-->Pro), and GP4 (Tyr4-->His and Ile109-->Phe) proteins or with a single point mutation in the GP5 protein (Pro98-->Leu) were able to establish persistent infection in HeLa-L cells. In summary, an in vitro model of EAV persistence in cell culture was established for the first time. This system can provide a valuable model for studying virus-host cell interactions, especially virus-receptor interactions.

Evaluation of hepatic disease in mice following intradermal inoculation with Corynebacterium pseudotuberculosis.

OBJECTIVE: To evaluate clinical, microbiologic, and pathologic outcomes in mice after inoculation with 4 equine-origin Corynebacterium pseudotuberculosis strains. ANIMALS: 15 C3H/HeJ mice. PROCEDURES: In a preliminary study, the optimum route of inoculation was determined. In the main study, mice were allocated to 4 treatment groups (3 mice/group). One slow- or rapid-growing equine-origin C pseudotuberculosis strain was inoculated ID into the mice of each treatment group. RESULTS: All 4 strains had distinct tropism for the liver. Histologic lesions associated with rapid-growing strains included focally extensive unencapsulated areas of acute, massive coagulative necrosis of hepatocytes with intralesional colonies of bacteria and variable portal hepatitis characterized by accumulations of mononuclear and polymorphonuclear inflammatory cells. In contrast, the livers of mice inoculated with slow-growing strains had multiple discrete, randomly distributed foci of hepatocellular necrosis and neutrophilic hepatitis that were considerably less severe than the lesions in the mice inoculated with the rapid-growing strains. Significantly more bacterial colonies were recovered from the organs of mice inoculated with rapid-growing than with slow-growing strains of bacteria. Bacteria were isolated from the liver, spleen, lungs, and mesenteric lymph nodes of mice inoculated with rapid-growing strains and from the liver and lymph nodes of mice inoculated with slow-growing strains. CONCLUSIONS AND CLINICAL RELEVANCE: Study of host-bacteria interactions in hosts that are naturally infected with C pseudotuberculosis is difficult because of underlying genetic variability among animals, expense, and requirements for multiple replicates and control animals. The C3H/HeJ mice may provide a useful means for studying virulence mechanisms of C pseudotuberculosis.

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.

Identification of an additional neutralization determinant of equine arteritis virus.

We recently established an in vitro model of equine arteritis virus (EAV) persistence in HeLa cells. The objective of this study was to determine whether viral variants with novel neutralization phenotypes emerged during persistent EAV infection of HeLa cells, as occurs during viral persistence in carrier stallions. Viruses recovered from persistently infected HeLa cells had different neutralization phenotypes than the virus in the original inoculum, as determined by neutralization assays using EAV-specific monoclonal antibodies and polyclonal equine antisera raised against different strains of EAV. Comparative sequence analyses of the entire structural protein genes (ORFs 2a, 2b, and 3-7) of these viruses, coupled with construction of chimeric viruses utilizing an infectious cDNA clone of EAV, confirmed that the alterations in neutralization phenotype were caused by amino acid changes in the GP5 protein encoded by ORF5. Site-directed mutagenesis studies unequivocally confirmed that amino acid 98 in the GP5 protein was responsible for the altered neutralization phenotype of these viruses. Amino acid 98 in the GP5 protein, which has not previously been identified as a neutralization determinant of EAV, should be included in an expanded neutralization site D (amino acids 98-106).

High subclinical West Nile virus incidence among nonvaccinated horses in northern California associated with low vector abundance and infection.

Although horse cases frequently are reported during West Nile virus (WNV) outbreaks, few investigations have focused on the epidemiology of this transmission. From April to October 2003 to 2005, mosquito abundance and infection were monitored 3 days per week at an equine research facility at the University of California, Davis. Thirty-two nonvaccinated horses enrolled as controls in a vaccine study were bled monthly, and their serum was tested for evidence of WNV infection by plaque reduction neutralization test (PRNT). In 2004, one positive Culex pipiens pool was associated with a single horse that presented with confirmed WNV disease in late September. The annual incidence of clinical and subclinical WNV infection in the nonvaccinated horses was 16%, with an apparent to inapparent ratio of 1:4 among infected horses. In 2005, two Culex tarsalis and two Cx. pipiens WNV-positive pools were associated with an equine infection incidence of 62%, with an apparent to inapparent ratio of 1:17. The majority (79%) of 70 blood-engorged Cx. pipiens fed on birds and the remaining on equines (21%). Conversely, Cx. tarsalis fed primarily on equines (n = 23, 74%), followed by birds (n = 7, 23%) and 1 (3%) fed on a lagomorph. These data indicated that nonvaccinated horses were a sensitive indicator of WNV activity and that their risk of infection was associated with the presence of infection in Cx. pipiens and Cx. tarsalis, which served as both enzootic and bridge vectors amplifying WNV among birds and transmitting WNV to horses.

Isolation of a gammaherpesvirus similar to asinine herpesvirus-2 (AHV-2) from a mule and a survey of mules and donkeys for AHV-2 infection by real-time PCR.

Equids are commonly infected by herpesviruses, but isolation of herpesviruses from mules has apparently not been previously reported. Furthermore, the genomic relationships among the various equid herpesviruses are poorly characterized. We describe the isolation and preliminary characterization of a mule gammaherpesvirus tentatively identified as asinine herpesvirus-2 (AHV-2; also designated equid herpesvirus-7 (EHV-7)) from the nasal secretions (NS) of a healthy mule in northern California. The virus was initially identified by transmission electron microscopic examination of lysates of cell culture inoculated with NS collected from the mule. A 913 nucleotide sequence of the DNA polymerase gene was amplified using degenerate primers, and comparison of this sequence with those of various other herpesviruses showed that the mule herpesvirus was most closely related to EHV-2 (AHV-2 sequences were not available for comparison). The sequence of a shorter portion (166 nucleotides) of the mule herpesvirus DNA polymerase gene was identical to that of the published sequence of an asinine gammaherpesvirus, previously designated as AHV-4-3 (AY054992). AHV-2 was detected by real-time polymerase chain reaction assay in the NS of approximately 8% of a cohort of 114 healthy mules and 13 donkeys.

Vaccines against bluetongue in Europe.

After the incursion of bluetongue virus (BTV) into European Mediterranean countries in 1998, vaccination was used in an effort to minimize direct economic losses to animal production, reduce virus circulation and allow safe movements of animals from endemic areas. Vaccination strategies in different countries were developed according to their individual policies, the geographic distribution of the incurring serotypes of BTV and the availability of appropriate vaccines. Four monovalent modified live virus (MLV) vaccines were imported from South Africa and subsequently used extensively in both cattle and sheep. MLVs were found to be immunogenic and capable of generating strong protective immunity in vaccinated ruminants. Adverse side effects were principally evident in sheep. Specifically, some vaccinated sheep developed signs of clinical bluetongue with fever, facial oedema and lameness. Lactating sheep that developed fever also had reduced milk production. More severe clinical signs occurred in large numbers of sheep that were vaccinated with vaccine combinations containing the BTV-16 MLV, and the use of the monovalent BTV-16 MLV was discontinued as a consequence. Abortion occurred in <0.5% of vaccinated animals. The length of viraemia in sheep and cattle that received MLVs did not exceed 35 days, with the single notable exception of a cow vaccinated with a multivalent BTV-2, -4, -9 and -16 vaccine in which viraemia persisted at least 78 days. Viraemia of sufficient titre to infect Culicoides insects was observed transiently in MLV-vaccinated ruminants, and natural transmission of MLV strains has been confirmed. An inactivated vaccine was first developed against BTV-2 and used in the field. An inactivated vaccine against BTV-4 as well as a bivalent vaccine against serotypes 2 and 4 were subsequently developed and used in Corsica, Spain, Portugal and Italy. These inactivated vaccines were generally safe although on few occasions reactions occurred at the site of inoculation. Two doses of these BTV inactivated vaccines provided complete, long-lasting immunity against both clinical signs and viraemia, whereas a single immunization with the BTV-4 inactivated vaccine gave only partial reduction of viraemia in vaccinated cattle when challenged with the homologous BTV serotype. Additional BTV inactivated vaccines are currently under development, as well as new generation vaccines including recombinant vaccines.

Phylogenetic comparison of the S10 genes of recent isolates of bluetongue virus from the United States and French Martinique Island.

The sequences of the S10 genes of 28 recent isolates (1994-2004) of bluetongue virus (BTV) from the United States (US) and French Martinique Island (2006) in the Caribbean Basin were compared in phylogenetic analyses to those of viruses previously isolated in the same regions. Although the analyses segregated the recent virus isolates from the two regions into distinct topotype clusters, the analyses also confirm that viruses from the US and the Caribbean Basin/Central America can share similar S10 genes despite the fact that distinct constellations of BTV serotypes occur in the two regions.

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.

Temporal detection of equine herpesvirus infections of a cohort of mares and their foals.

The objectives of this study were to estimate the prevalence of equine herpesviruses (EHV) 1-5 in the nasal secretions (NS) of a cohort of 12 mares and their foals from birth to 6 months of age, estimate the prevalence of EHV-1-5 infection of peripheral blood mononuclear cells (PBMC) of selected foals, and investigate phylogenetic relationships amongst the various strains of EHV-2 and 5. Virus-specific PCR assays were used to detect EHV-1-5 in NS and PBMC. A homologous portion of the glycoprotein B (gB) gene of the various strains of EHV-2 and 5 was sequenced and compared. EHV-2, 4, and 5 were all detected in NS from the horses, but only EHV-4 was associated with respiratory disease (P=0.005). EHV-2 and 5 infections were both common, but foals shed EHV-2 in their NS earlier in life than EHV-5 (P=0.01). Latent EHV-2 and 5 infections were detected in the PBMC of 75 and 88%, respectively, of the foals at approximately 6 months of age. The strains of EHV-2 shed in the NS of individual horses were more genetically heterogeneous than the strains of EHV-5 (95.5-99.3% versus 98.8-99.3% nucleotide identity, respectively). One-month-old foals typically shed strains of EHV-2 that were identical to those infecting their dams whereas older foals often shed virus strains that were different from those of their dams. Although herpesvirus infections were ubiquitous in this cohort of horses, there were distinct clinical consequences and clear epidemiological differences between infections with the different viruses.

Renal tubular necrosis and interstitial hemorrhage ("turkey-egg kidney") in a circovirus-infected Yorkshire cross pig.

A juvenile Yorkshire cross pig with rapidly progressive acute renal failure was submitted for necropsy. There was marked edema and disseminated petechiation of both kidneys, producing the "turkey-egg" appearance that is characteristic of exotic diseases such as African and classical swine fever. Microscopic findings included renal tubular epithelial necrosis with extensive interstitial edema and hemorrhage; lymphoplasmacytic, eosinophilic, and histiocytic tubulointerstitial nephritis; and numerous botryoid intracytoplasmic inclusions within the renal tubular epithelium and interstitial macrophages. Porcine circovirus 2 (PCV2) was readily identified within these lesions by both PCV2-specific immunohistochemistical staining and electron microscopy. Tests for African and classical swine fever viruses, as well as bacterial cultures, were negative. The striking renal lesions in this pig were attributed to PCV2 infection and are distinct from those that are typical of other PCV2-associated diseases.

Isolation of equine herpesvirus-5 from blood mononuclear cells of a gelding.

Horses are commonly infected by herpesviruses, but isolation of equine herpesvirus-5 (EHV-5) has only infrequently been reported. We describe the isolation and characterization of a strain of EHV-5 from the blood mononuclear cells of a healthy adult horse in California. The virus was initially identified by EHV-5 specific polymerase chain reaction (PCR), and it caused lytic infection of cultured rabbit kidney cells only after repeated serial passage. Virions with characteristic herpesvirus morphology were readily demonstrated in cell culture lysate by transmission electron microscopy. A portion of the glycoprotein B gene of this strain of EHV-5 had 99% identity to the published EHV-5 sequence, and it was clearly distinguishable from other EHV (1-4) by virus-specific PCR assays. Prevalence of EHV-5 infection in a group of young racehorses was estimated at 64% using the EHV-5 specific PCR on nasopharyngeal secretions.

Detection of antibodies to West Nile virus in equine sera using microsphere immunoassay.

One hundred and ninety-one sera from horses that recently were exposed to West Nile virus (WNV) by either vaccination or natural infection or that were not vaccinated and remained free of infection were used to evaluate fluorescent microsphere immunoassays (MIAs) incorporating recombinant WNV envelope protein (rE) and recombinant nonstructural proteins (rNS1, rNS3, and rNS5) for detection of equine antibodies to WNV. The rE MIA had a diagnostic sensitivity and specificity, respectively, of 99.3% and 97.4% for detection of WNV antibodies in the serum of horses that were recently vaccinated or naturally infected with WNV, as compared to the plaque reduction neutralization test (PRNT). The positive rE MIA results were assumed to be WNV-specific because of the close agreement between this assay and the PRNT and the fact that unvaccinated control horses included in this study were confirmed to be free of exposure to the related St Louis encephalitis virus. The NS protein-based MIA were all less sensitive than either the rE MIA or PRNT (sensitivity 0-48.0), although the rNSI MIA distinguished horses vaccinated with the recombinant WNV vaccine from those that were immunized with the inactivated WNV vaccine (P < 0.0001) or naturally infected with WNV (P < 0.0001). The rE MIA would appear to provide a rapid, convenient, inexpensive, and accurate test for the screening of equine sera for the presence of antibodies to WNV.

Equine viral arteritis.

EVA is an important if uncommon disease of horses. Potential economic losses attributable to EVA include direct losses from abortion, pneumonia in neonates, and febrile disease in performance horses. Indirect losses are those associated with national and international trade/animal movement regulations, particularly those pertaining to persistently infected carrier stallions and their semen. However, EAV infection and EVA are readily prevented through serological and virological screening of horses, coupled with sound management practices that include appropriate quarantine and strategic vaccination.