Evaluation of 2012 US EHDV-2 outbreak isolates for genetic determinants of cattle infection.

Following a summer of severe drought and abnormally high temperatures, a major outbreak of EHDV occurred during 2012 in the USA. Although EHDV-1, -2 and -6 were isolated, EHDV-2 was the predominant virus serotype detected during the outbreak. In addition to large losses of white-tailed deer, the Midwest and northern Plains saw a significant amount of clinical disease in cattle. Phylogenetic analyses and sequence comparisons of newly sequenced whole genomes of 2012 EHDV-2 cattle isolates demonstrated that eight of ten EHDV-2 genomic segments show no genetic changes that separate the cattle outbreak sequences from other EHDV-2 isolates. Two segments, VP2 and VP6, did show several unique genetic changes specific to the 2012 cattle outbreak isolates, although the impact of the genetic changes on viral fitness is unknown. The placement of isolates from 2007 and 2011 as sister group to the outbreak isolates, and the similarity between cattle and deer isolates, point to environmental variables as having a greater influence on the severity of the 2012 EHDV outbreak than viral genetic changes.

Assessment of reproducibility of a VP7 Blocking ELISA diagnostic test for African horse sickness.

The laboratory diagnosis of African horse sickness (AHS) is important for: (a) demonstrating freedom from infection in a population, animals or products for trade (b) assessing the efficiency of eradication policies; (c) laboratory confirmation of clinical diagnosis; (d) estimating the prevalence of AHS infection; and (e) assessing postvaccination immune status of individual animals or populations. Although serological techniques play a secondary role in the confirmation of clinical cases, their use is very important for all the other purposes due to their high throughput, ease of use and good cost-benefit ratio. The main objective of this study was to support the validation of AHS VP7 Blocking ELISA up to the Stage 3 of the World Animal Health Organization (OIE) assay validation pathway. To achieve this, a collaborative ring trial, which included all OIE Reference Laboratories and other AHS-specialist diagnostic centres, was conducted in order to assess the diagnostic performance characteristics of the VP7 Blocking ELISA. In this trial, a panel of sera of different epidemiological origin and infection status was used. Through this comprehensive evaluation we can conclude that the VP7 Blocking ELISA satisfies the OIE requirements of reproducibility. The VP7 Blocking ELISA, in its commercial version is ready to enter Stage 4 of the validation pathway (Programme Implementation). Specifically, this will require testing the diagnostic performance of the assay using contemporary serum samples collected during control campaigns in endemic countries.

Recent US bluetongue virus serotype 3 isolates found outside of Florida indicate evidence of reassortment with co-circulating endemic serotypes.

Since 1999, 11 serotypes of bluetongue virus (BTV) similar to Central American or Caribbean strains have been isolated in the southeastern United States, predominantly in Florida. The majority of the incursive serotypes have remained restricted to the southeastern US. In recent years, BTV serotype 3 (BTV-3) has been isolated in areas increasingly distant from Florida. The current study uses whole genome sequencing of recent and historical BTV-3 isolates from the US, Central America and the Caribbean with additional sequences from GenBank to conduct phylogenetic analyses. The individual segments of the BTV genome were analysed to determine if recent BTV-3 isolates are reassortants containing genomic segments from endemic US serotypes or if they retain a majority of Central American/Caribbean genotypes. The analyses indicate that BTV-3 isolates Mississippi 2006, Arkansas 2008 and Mississippi 2009 are closely related reassortants that contain five to six genomic segments that are of US origin and two to three segments of Central American/Caribbean origin. In contrast, the BTV-3 South Dakota 2012 isolate contains seven genomic segments that are more similar to isolates from Central American and the Caribbean. These different evolutionary histories of the BTV-3 isolates suggest that there are at least two different lineages of BTV-3 that are currently circulating in the US.

The First 10 Years (2006-15) of Epizootic Hemorrhagic Disease Virus Serotype 6 in the USA.

Epizootic hemorrhagic disease virus (EHDV) is a Culicoides biting midge-transmitted orbivirus (family Reoviridae) of wild and domestic ruminants and is an important pathogen of white-tailed deer (Odocoileus virginianus). Historically, only two serotypes, EHDV-1 and EHDV-2, have been known to be endemic in the US. However, in 2006, an exotic serotype (EHDV-6) was first detected in the US by a long-term passive surveillance system for EHDV and bluetongue viruses. Here we report EHDV-6 detections made through these passive surveillance efforts by the Southeastern Cooperative Wildlife Disease Study (University of Georgia, Athens, Georgia, USA) and the National Veterinary Services Laboratories (US Department of Agriculture, Ames, Iowa, USA) over a 10-yr period (2006-15). The results demonstrated that EHDV-6 was detected from ruminants every year since 2006 and was widespread in the central and eastern US, providing evidence that EHDV-6 is likely now established in the US.

OIEBTLABNET: the web-based network of the OIE Bluetongue Reference Laboratories.

Bluetongue (BT) is a mild to severe disease of domestic and wild ruminants caused by the Bluetongue virus (BTV) and generally transmitted by Culicoides biting midges. Its occurrence also determines a livestock trade ban in affected countries with severe economic consequences on national and international trade. For this reason, in May 2011, the OIE encouraged the OIE Reference Laboratories to establish and maintain a BT network to provide expertise and training to the OIE and OIE Member Countries for BT diagnosis, surveillance and control. The network is constantly sustained by world leading scientists in the field of virology, epidemiology, serology, entomology and vaccine development. The website, available at http://oiebtnet.izs.it/btlabnet/, hosts an Information System containing data on BTV outbreaks and strains and a WebGIS that distributes maps on BTV occurrence. In this paper we describe the applications and present the benefits derived from the use of the WebGIS in the context of BT international surveillance network.

Combining reverse-transcription multiplex PCR and microfluidic electrophoresis to simultaneously detect seven mosquito-transmitted zoonotic encephalomyelitis viruses.

Several mosquito-transmitted viruses are causative agents for zoonotic encephalomyelitis. Rapid identification of these viruses in mosquito populations is an effective method for surveying these diseases. To detect multiple mosquito-transmitted viral agents, including West Nile virus, Saint Louis encephalitis virus, Venezuelan equine encephalomyelitis virus, Western equine encephalomyelitis virus, Eastern equine encephalomyelitis virus, Highlands J virus and Japanese encephalitis virus, an assay using multiplex reverse-transcription PCR combined with microfluidic electrophoresis was developed and evaluated. Tailed nested primers were used in the assay to amplify specific viral genomic segments, and products with specific length were further analyzed by using a microfluidic electrophoresis chip. The assay exhibited good specificity and analytical sensitivity (10(2) copies/µL). This technology can be helpful in the quarantine and surveillance of exotic encephalomyelitis viruses which are transmitted by mosquitoes.

Diagnostic Tools for Bluetongue and Epizootic Hemorrhagic Disease Viruses Applicable to North American Veterinary Diagnosticians.

This review provides an overview of current and potential new diagnostic tests for bluetongue (BT) and epizootic hemorrhagic disease (EHD) viruses compiled from international participants of the Orbivirus Gap Analysis Workshop, Diagnostic Group. The emphasis of this review is on diagnostic tools available to North American veterinary diagnosticians. Standard diagnostic tests are readily available for BT/EHD viruses, and there are described tests that are published in the World Organization for Animal Health (OIE) Terrestrial Manual. There is however considerable variation in the diagnostic approach to these viruses. Serological assays are well established, and many laboratories are experienced in running these assays. Numerous nucleic acid amplification assays are also available for BT virus (BTV) and EHD virus (EHDV). Although there is considerable experience with BTV reverse-transcriptase PCR (RT-PCR), there are no standards or comparisons of the protocols used by various state and federal veterinary diagnostic laboratories. Methods for genotyping BTV and EHDV isolates are available and are valuable tools for monitoring and analyzing circulating viruses. These methods include RT-PCR panels or arrays, RT-PCR and sequencing of specific genome segments, or the use of next-generation sequencing. In addition to enabling virus characterization, use of advanced molecular detection methods, including DNA microarrays and next-generation sequencing, significantly enhance the ability to detect unique virus strains that may arise through genetic drift, recombination, or viral genome segment reassortment, as well as incursions of new virus strains from other geographical areas.

Whole genome sequence analysis of circulating Bluetongue virus serotype 11 strains from the United States including two domestic canine isolates.

Bluetongue virus (BTV) is a vector-transmitted pathogen that typically infects and causes disease in domestic and wild ruminants. BTV is also known to infect domestic canines as discovered when dogs were vaccinated with a BTV-contaminated vaccine. Canine BTV infections have been documented through serological surveys, and natural infection by the Culicoides vector has been suggested. The report of isolation of BTV serotype 11 (BTV-11) from 2 separate domestic canine abortion cases in the states of Texas in 2011 and Kansas in 2012, were apparently unrelated to BTV-contaminated vaccination or consumption of BTV-contaminated raw meat as had been previously speculated. To elucidate the origin and relationship of these 2 domestic canine BTV-11 isolates, whole genome sequencing was performed. Six additional BTV-11 field isolates from Texas, Florida, and Washington, submitted for diagnostic investigation during 2011 and 2013, were also fully sequenced and analyzed. The phylogenetic analysis indicates that the BTV-11 domestic canine isolates are virtually identical, and both share high identity with 2 BTV-11 isolates identified from white-tailed deer in Texas in 2011. The results of the current study further support the hypothesis that a BTV-11 strain circulating in the Midwestern states could have been transmitted to the dogs by the infected Culicoides vector. Our study also expands the short list of available BTV-11 sequences, which may aid BTV surveillance and epidemiology.

Molecular evolution of American field strains of bluetongue and epizootic haemorrhagic disease viruses.

Recent Orbivirus occurrences in the Americas have been investigated using whole genome amplification and sequencing followed by phylogenetic analysis. The bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) whole genomes were amplified without prior sequence knowledge and deep sequenced. This technology was applied to evaluate BTV­3 isolates spanning 4 decades from Florida, Arkansas, Mississippi, South Dakota, Central America, and the Caribbean basin. The results of the dataset analysis are consistent with the hypothesis that these viruses were introduced into the United States from Central America and the Caribbean basin. A similar analysis has been performed on a recent BTV­2 isolate from California. It indicates that the BTV­2 strain was likely introduced into Florida and then moved South to the Caribbean and West to California. A historical (1955­2012) molecular characterisation of EHDV strains was also completed, and subsequently used as reference sequence for comparison of genomes from recent 2012 cattle isolates associated with clinical disease. Finally, this analysis was performed on BTV­11 isolated from 2 canine cases and demonstrated that the genome sequences of the virus isolates from these cases were almost identical. These studies indicate the value of this technology in understanding virus epidemiology and ecology.

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.

Coxiella burnetii serology assays in goat abortion storm.

Many commercial antibody detection enzyme-linked immunosorbent assay (ELISA) kits for Q fever utilize the Nine Mile (Montana tick) strain of Coxiella burnetii as antigen. An ELISA kit manufactured in France employs ovine placenta-sourced antigen and has been used in Europe. Sera from goats experiencing a Q fever abortion storm in the United States were used to compare the sensitivity and specificity of these 2 ELISA formats and the Q fever complement fixation test (CFT). Latent class estimates of sensitivity ranged from 97% to 100% with a specificity of 95-100% for the 2 ELISA kits. Estimates for sensitivity and specificity of the CFT were 89% and 82%, respectively. There was not a significant increase in ELISA sensitivity observed with the ovine-sourced antigen kit in this study. Real-time polymerase chain reactions performed on a portion of the sera found that 15 out of 20 sera were congruent across 4 tests for positive and negative sera.

Genetic characterization of feline calicivirus strains associated with varying disease manifestations during an outbreak season in Missouri (1995-1996).

Feline calicivirus (FCV) is a common cause of mild to severe upper respiratory tract disease (URTD) in cats. FCV strain 21223 was isolated from a kitten with severe pneumonia in a disease outbreak with unusually high mortality (35 %) that occurred in a Missouri feline colony in 1995-1996. Phylogenetic analysis of the genome sequence of strain 21223 indicated the emergence of a new FCV strain. Analysis of the full-length genome sequence of a closely related (99.5 % nucleotide identity) strain, 3786, obtained from an asymptomatic animal in the same colony four months later, showed the presence of seven amino acid substitutions, with six of them located in the VP1 capsid sequence encoded by ORF2. Comparative analysis of the E-region sequences (426-521 aa ORF2) presumably involved in virus-host cell receptor interactions did not identify amino acid substitutions unique to the virulent strain. We determined the complete genome sequences of four virus isolates that were collected in regional catteries in the months following the outbreak that were associated with different manifestations of the disease (URTD, chronic stomatitis, and gingivitis). We show that genetically distinct FCV strains were cocirculating in the area, and no apparent correlation could be made between overall sequence and observed disease.

Development and performance evaluation of a streamlined method for nucleic acid purification, denaturation, and multiplex detection of Bluetongue virus and Epizootic hemorrhagic disease virus.

Bluetongue virus (BTV) and Epizootic hemorrhagic disease virus (EHDV) possess similar structural and molecular features, are transmitted by biting midges (genus Culicoides), and cause similar diseases in some susceptible ruminants. Generally, BTV causes subclinical disease in cattle, characterized by a prolonged viremia. EHDV-associated disease in cattle is less prominent; however, it has emerged as a major economic threat to the white-tailed deer (Odocoileus virginianus) industry in many areas of the United States. The recent emergence of multiple BTV and EHDV serotypes previously undetected in the United States demonstrates the need for robust detection of all known serotypes and differential diagnosis. For this purpose, a streamlined workflow consisting of an automated nucleic acid purification and denaturation method and a multiplex one-step reverse transcription quantitative polymerase chain reaction for the simultaneous detection of BTV serotypes 1-24 and EHDV serotypes 1-7 was developed using previously published BTV and EHDV assays. The denaturation of double-stranded (ds) BTV and EHDV RNA was incorporated into the automated nucleic acid purification process thus eliminating the commonly used separate step of dsRNA denaturation. The performance of this workflow was compared with the World Organization of Animal Health BTV reference laboratory (National Veterinary Services Laboratory, Ames, Iowa) workflow for BTV and EHDV detection, and high agreement was observed. Implementation of the workflow in routine diagnostic testing enables the detection of, and differentiation between, BTV and EHDV, and coinfections in bovine blood and cervine tissues, offering significant benefits in terms of differential disease diagnosis, herd health monitoring, and regulated testing.

Isolation of Bluetongue virus from canine abortions.

Three aborted canine fetuses were submitted to the Animal Health Diagnostic Center at Cornell University in November 2011 and September 2012 for diagnostic workups to determine the causes of the reproductive difficulties. Histological assessments of the sampled tissues were inconclusive due to the autolysis. Tests to detect bacterial causes of the abortions were also negative. Virus isolation testing on pooled tissues from the fetuses identified a cytopathogenic agent in cell cultures. Fluorescent antibody tests on the infected cells gave a positive reaction for Bluetongue virus, and subsequent serotype specific reverse transcription polymerase chain reaction assays identified the isolates as Bluetongue virus serotype 11. The current report describes the isolation of Bluetongue virus from dogs unrelated to contaminated vaccines and in the absence of a raw meat diet.

Isolation of Equine rhinitis A virus from a horse semen sample.

Semen from an apparently healthy 4-year-old American Quarter Horse was submitted to the National Veterinary Services Laboratories for Equine arteritis virus isolation. Visual inspection of the semen sample upon arrival noted it was unusually yellow in color. The semen sample was inoculated onto cell monolayers, and cytopathic effect was observed 5 days postinoculation. The resultant isolate tested negative for Equine arteritis virus, and was subsequently identified as Equine rhinitis A virus. Equine rhinitis A virus has been isolated from horse urine, but has not been described in stallion semen. The present study documents the isolation of Equine rhinitis A virus from stallion semen that was likely contaminated with urine at the time of collection.

Identification and differentiation of the twenty six bluetongue virus serotypes by RT-PCR amplification of the serotype-specific genome segment 2.

Bluetongue (BT) is an arthropod-borne viral disease, which primarily affects ruminants in tropical and temperate regions of the world. Twenty six bluetongue virus (BTV) serotypes have been recognised worldwide, including nine from Europe and fifteen in the United States. Identification of BTV serotype is important for vaccination programmes and for BTV epidemiology studies. Traditional typing methods (virus isolation and serum or virus neutralisation tests (SNT or VNT)) are slow (taking weeks, depend on availability of reference virus-strains or antisera) and can be inconclusive. Nucleotide sequence analyses and phylogenetic comparisons of genome segment 2 (Seg-2) encoding BTV outer-capsid protein VP2 (the primary determinant of virus serotype) were completed for reference strains of BTV-1 to 26, as well as multiple additional isolates from different geographic and temporal origins. The resulting Seg-2 database has been used to develop rapid (within 24 h) and reliable RT-PCR-based typing assays for each BTV type. Multiple primer-pairs (at least three designed for each serotype) were widely tested, providing an initial identification of serotype by amplification of a cDNA product of the expected size. Serotype was confirmed by sequencing of the cDNA amplicons and phylogenetic comparisons to previously characterised reference strains. The results from RT-PCR and sequencing were in perfect agreement with VNT for reference strains of all 26 BTV serotypes, as well as the field isolates tested. The serotype-specific primers showed no cross-amplification with reference strains of the remaining 25 serotypes, or multiple other isolates of the more closely related heterologous BTV types. The primers and RT-PCR assays developed in this study provide a rapid, sensitive and reliable method for the identification and differentiation of the twenty-six BTV serotypes, and will be updated periodically to maintain their relevance to current BTV distribution and epidemiology (http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/rt-pcr-primers.htm).

Comparison of Q fever serology methods in cattle, goats, and sheep.

Coxiella burnetii is an obligate intracellular bacterium that is responsible for the zoonotic disease Q fever. The distribution of this agent is worldwide except for New Zealand, and infection can be asymptomatic in both human beings and animals. Chronic exposures can produce abortions, stillbirths, and infertility issues in animals and endocarditis in human beings. A commercial enzyme-linked immunosorbent assay (ELISA) kit marketed in the European Union was purchased to compare C. burnetii antibody detection methods. The current study examined the agreement of ELISA and complement fixation results in over 668 diagnostic ruminant sera submitted to the National Veterinary Services Laboratories for Q fever serologic testing. The majority of combined sera (548) were negative on both tests. Fifty-seven of the combined sera were positive on both tests. There were 45 combined sera with low complement fixation titers at 1:10 and negative ELISA results. The results were surprising given the expectations that ELISA methods, by nature, amplify detection of antibody-antigen interactions leading to higher sensitivity. Potential mechanisms for these discrepant results are discussed.

Serologic evidence of West Nile Virus in wild ducks captured in major inland resting sites for migratory waterfowl in South Korea.

The rapid global expansion of West Nile virus (WNV) has recently raised concerns regarding its possible spread into South Korea. To date, WNV infection in wild birds in South Korea has not been identified. Bird migration is thought to be involved in spreading WNV, and wild birds are the possible routes of introduction of WNV infection. To assess the risk of WNV infection in South Korea, we conducted a nationwide WNV surveillance of wild birds, with an emphasis on migratory ducks from WNV-affected areas. Our chief aim was to determine whether birds with the potential to introduce WNV are present in South Korea by testing migrating and resident wild birds for WNV antibodies. We collected blood samples from 1531 wild birds representing 57 bird species at several major inland resting sites for migratory waterfowl in South Korea. A seroepidemiological analysis of WNV and Japanese encephalitis virus (JEV) infections was conducted using plaque-reduction neutralization tests (PRNTs) for each virus. To search for recent WNV infections, sera were also evaluated by IgM antibody capture ELISA. Of the 1531 serum samples, 5 (0.3%) tested positive for WNV-specific antibodies, and 70 (4.6%) tested positive for JEV-specific antibodies. A total of 9 (0.6%) samples were positive for both WNV and JEV antibodies; these samples were interpreted as having a flavivirus exposure. All birds that had neutralizing antibodies specific to WNV were negative for IgM, which indicates the likelihood of a relatively old infection. Along with the recognized distribution of flaviviruses along several duck species' migratory routes, our findings strongly suggest that some of the birds captured in this study had been exposed to WNV or JEV.

Concentration of acrylamide in a polyacrylamide gel affects VP4 gene coding assignment of group A equine rotavirus strains with P[12] specificity.

BACKGROUND: It is universally acknowledged that genome segment 4 of group A rotavirus, the major etiologic agent of severe diarrhea in infants and neonatal farm animals, encodes outer capsid neutralization and protective antigen VP4. RESULTS: To determine which genome segment of three group A equine rotavirus strains (H-2, FI-14 and FI-23) with P[12] specificity encodes the VP4, we analyzed dsRNAs of strains H-2, FI-14 and FI-23 as well as their reassortants by polyacrylamide gel electrophoresis (PAGE) at varying concentrations of acrylamide. The relative position of the VP4 gene of the three equine P[12] strains varied (either genome segment 3 or 4) depending upon the concentration of acrylamide. The VP4 gene bearing P[3], P[4], P[6], P[7], P[8] or P[18] specificity did not exhibit this phenomenon when the PAGE running conditions were varied. CONCLUSIONS: The concentration of acrylamide in a PAGE gel affected VP4 gene coding assignment of equine rotavirus strains bearing P[12] specificity.