Andrew B. Allison

Detection of a novel reassortant epizootic hemorrhagic disease virus (EHDV) in the USA containing RNA segments derived from both exotic (EHDV-6) and endemic (EHDV-2) serotypes

Epizootic hemorrhagic disease virus (EHDV) is a Culicoides-transmitted orbivirus that infects domestic and wild ruminants and is provisionally thought to be distributed throughout Africa, North America, Australia, East Asia and the Middle East. Historically, of the seven proposed serotypes of EHDV, only EHDV-1 and EHDV-2 have been reported from North America. In 2006, EHDV isolates were recovered from moribund or dead white-tailed deer (Odocoileus virginianus) in Indiana and Illinois that could not be identified as either EHDV-1 or EHDV-2 by virus neutralization tests or by serotype-specific RT-PCR. Additional serological and genetic testing identified the isolates as EHDV-6, a serotype that, although originally described from Australia, has recently been recognized as an emerging pathogen of cattle in Morocco, Algeria and Turkey. In 2007 and 2008, EHDV-6 was isolated again from white-tailed deer, this time in Missouri, Kansas and Texas, suggesting that the virus is capable of overwintering and that it may become, or already is, endemic in a geographically widespread region of the USA. Genetic characterization of the virus indicates that it is a reassortant, such that the outer capsid proteins determining serotype specificity (VP2 and VP5) are derived from exotic EHDV-6, whilst the remaining structural and non-structural proteins are apparently obtained from indigenous EHDV-2 (Alberta).

Frequent Cross-Species Transmission of Parvoviruses among Diverse Carnivore Hosts

ABSTRACT Although parvoviruses are commonly described in domestic carnivores, little is known about their biodiversity in nondomestic species. A phylogenetic analysis of VP2 gene sequences from puma, coyote, gray wolf, bobcat, raccoon, and striped skunk revealed two major groups related to either feline panleukopenia virus (“FPV-like”) or canine parvovirus (“CPV-like”). Cross-species transmission was commonplace, with multiple introductions into each host species but, with the exception of raccoons, relatively little evidence for onward transmission in nondomestic species.

Role of Multiple Hosts in the Cross-Species Transmission and Emergence of a Pandemic Parvovirus

ABSTRACT Understanding the mechanisms of cross-species virus transmission is critical to anticipating emerging infectious diseases. Canine parvovirus type 2 (CPV-2) emerged as a variant of a feline parvovirus when it acquired mutations that allowed binding to the canine transferrin receptor type 1 (TfR). However, CPV-2 was soon replaced by a variant virus (CPV-2a) that differed in antigenicity and receptor binding. Here we show that the emergence of CPV involved an additional host range variant virus that has circulated undetected in raccoons for at least 24 years, with transfers to and from dogs. Raccoon virus capsids showed little binding to the canine TfR, showed little infection of canine cells, and had altered antigenic structures. Remarkably, in capsid protein (VP2) phylogenies, most raccoon viruses fell as evolutionary intermediates between the CPV-2 and CPV-2a strains, suggesting that passage through raccoons assisted in the evolution of CPV-2a. This highlights the potential role of alternative hosts in viral emergence.

Absence of humoral response in flamingos and red-tailed hawks to experimental vaccination with a killed West Nile virus vaccine.

Abstract Sixteen Chilean flamingos, Phoenicopterus chilensis, and 10 red-tailed hawks, Buteo jamacensis, were vaccinated in the pectoral muscle with 0.2 ml of a commercially produced killed West Nile virus vaccine intended for use in horses. Half the birds of each species received a booster vaccination 3 weeks after the first injection. Three weeks after the booster vaccination, none of 13 birds surveyed had detectable antibody to West Nile virus.

Evolutionary Reconstructions of the Transferrin Receptor of Caniforms Supports Canine Parvovirus Being a Re-emerged and Not a Novel Pathogen in Dogs

Parvoviruses exploit transferrin receptor type-1 (TfR) for cellular entry in carnivores, and specific interactions are key to control of host range. We show that several key mutations acquired by TfR during the evolution of Caniforms (dogs and related species) modified the interactions with parvovirus capsids by reducing the level of binding. These data, along with signatures of positive selection in the TFRC gene, are consistent with an evolutionary arms race between the TfR of the Caniform clade and parvoviruses. As well as the modifications of amino acid sequence which modify binding, we found that a glycosylation site mutation in the TfR of dogs which provided resistance to the carnivore parvoviruses which were in circulation prior to about 1975 predates the speciation of coyotes and dogs. Because the closely-related black-backed jackal has a TfR similar to their common ancestor and lacks the glycosylation site, reconstructing this mutation into the jackal TfR shows the potency of that site in blocking binding and infection and explains the resistance of dogs until recent times. This alters our understanding of this well-known example of viral emergence by indicating that canine parvovirus emergence likely resulted from the re-adaptation of a parvovirus to the resistant receptor of a former host.

RT-PCR Assays for Seven Serotypes of Epizootic Haemorrhagic Disease Virus & Their Use to Type Strains from the Mediterranean Region and North America

Epizootic haemorrhagic disease virus (EHDV) infects wild ruminants, causing a frequently fatal haemorrhagic disease. However, it can also cause bluetongue-like disease in cattle, involving significant levels of morbidity and mortality, highlighting a need for more rapid and reliable diagnostic assays. EHDV outer-capsid protein VP2 (encoded by genome-segment 2 [Seg-2]) is highly variable and represents the primary target for neutralising antibodies generated by the mammalian host. Consequently VP2 is also the primary determinant of virus “serotype”, as identified in virus neutralisation tests (VNT). Although previous reports have indicated eight to ten EHDV serotypes, recent serological comparisons and molecular analyses of Seg-2 indicate only seven EHDV “types”. Oligonucleotide primers were developed targeting Seg-2, for use in conventional RT-PCR assays to detect and identify these seven types. These assays, which are more rapid and sensitive, still show complete agreement with VNT and were used to identify recent EHDV isolates from the Mediterranean region and North America.

Host-Specific Parvovirus Evolution in Nature Is Recapitulated by In Vitro Adaptation to Different Carnivore Species

Canine parvovirus (CPV) emerged as a new pandemic pathogen of dogs in the 1970s and is closely related to feline panleukopenia virus (FPV), a parvovirus of cats and related carnivores. Although both viruses have wide host ranges, analysis of viral sequences recovered from different wild carnivore species, as shown here, demonstrated that >95% were derived from CPV-like viruses, suggesting that CPV is dominant in sylvatic cycles. Many viral sequences showed host-specific mutations in their capsid proteins, which were often close to sites known to control binding to the transferrin receptor (TfR), the host receptor for these carnivore parvoviruses, and which exhibited frequent parallel evolution. To further examine the process of host adaptation, we passaged parvoviruses with alternative backgrounds in cells from different carnivore hosts. Specific mutations were selected in several viruses and these differed depending on both the background of the virus and the host cells in which they were passaged. Strikingly, these in vitro mutations recapitulated many specific changes seen in viruses from natural populations, strongly suggesting they are host adaptive, and which were shown to result in fitness advantages over their parental virus. Comparison of the sequences of the transferrin receptors of the different carnivore species demonstrated that many mutations occurred in and around the apical domain where the virus binds, indicating that viral variants were likely selected through their fit to receptor structures. Some of the viruses accumulated high levels of variation upon passage in alternative hosts, while others could infect multiple different hosts with no or only a few additional mutations. Overall, these studies demonstrate that the evolutionary history of a virus, including how long it has been circulating and in which hosts, as well as its phylogenetic background, has a profound effect on determining viral host range.

West Nile virus detection in the organs of naturally infected blue jays (Cyanocitta cristata).

Blue jays (Cyanocitta cristata) are an effective indicator species for West Nile virus (WNV) and may be regionally important in surveillance efforts. The sites of WNV replication and sensitivity of virus detection techniques are undefined for blue jays. The objectives of this study were to describe the gross and microscopic pathology associated with natural WNV infection in blue jays, as well as determine the most appropriate tissues to be used for virus isolation, reverse transcription–nested polymerase chain reaction, and immunohistochemistry (IHC) techniques. Blue jays were collected in Georgia, USA, between May and September 2001. Initial screening by virus isolation indicated that 36 of 59 blue jays chosen for evaluation were WNV positive. From this group, 20 positive and five negative birds were chosen to compare virus detection techniques. Six positive and five negative birds were selected for histopathology examination. Splenomegaly and poor body condition were the most consistent gross findings among positive birds. The most consistent histopathologic findings in the tissues of WNV-positive blue jays were mononuclear leukocytosis and epicarditis/myocarditis. Brain, heart, and lung had the highest viral titers, and WNV antigen was most often detected by IHC in heart, kidney, liver, and lung. Reverse transcription–nested polymerase chain reaction proved to be the most sensitive diagnostic test applied in this study irrespective of the tissue type. Brain tissue could be used effectively for both virus isolation and RT-nPCR, and this tissue is simple to remove and process. The success of IHC is highly dependent on tissue selection, and the use of multiple tissues including heart, kidney, liver, or lung is recommended.

West Nile Virus Viremia in Wild Rock Pigeons

Feral rock pigeons were screened for neutralizing antibodies to West Nile virus (WNV) during late winter/spring and summer of 2002 and 2003. Additionally, virus isolation from serum was attempted from 269 birds collected during peak transmission periods. The observed viremia levels and seroprevalence indicate that this species could be involved in amplifying WNV in urban settings.

Eastern Equine Encephalitis in a Free-ranging White-tailed Deer (Odocoileus virginianus)

Eastern equine encephalitis (EEE) was diagnosed in a free-ranging, adult, male white-tailed deer (Odocoileus virginianus) from Houston County, Georgia, USA, in July 2001. The yearling buck had neurologic disease and died during transport to our diagnostic facility. Eastern equine encephalitis virus (EEEV) was isolated in Vero cell culture and identified by reverse-transcriptase polymerase chain reaction; as well, EEEV antigen was detected in brain by immunohistochemistry. This is the first report of fatal EEEV infection in a white-tailed deer. Antibodies to EEEV were demonstrated by microtiter neutralization in 14 of 99 (14%) of the white-tailed deer from Georgia sampled in fall 2001. Most antibody-positive deer originated from the Coastal Plain physiographic region. Eastern equine encephalitis virus should be considered a possible cause of neurologic disease in white-tailed deer where it may occur.