Hemagglutination of epizootic hemorrhagic disease virus

Summary Hemagglutination of epizootic hemorrhagic disease virus (EHDV) with a variety of erythrocyte species at 4° C, room temperature and 37° C was dependent on the NaCl molarity and the pH of the diluent. The hemagglutination inhibition test was used to identify EHDV serotypes.     

Characterization of papillomaviruses isolated from cutaneous fibromas of white-tailed deer and mule deer

Deer fibromavirus (DFV), a member of the papillomavirus genus, was isolated from cases of fibromatosis in white-tailed deer (Odocoileus virginianus) and mule deer (Odocoileus hemionus). SDS-polyacrylamide gel electrophoresis analysis of virions indicated no molecular weight differences in the major structural protein. DFV agglutinated mouse erythrocytes and the reaction could be inhibited by both DFV-specific and BPV-1-specific antisera. Although analysis of restriction endonuclease digestion products indicated minor differences in cleavage patterns, the DFV DNAs were indistinguishable by liquid phase hybridization and restriction enzyme cleavage maps indicated most of the sites in common. Comparison of DFV DNA to BPV-1 and BPV-2 DNA under stringent liquid-phase hybridization conditions indicated that 3 to 9% DNA sequence homology could be detected between the genomes of these viruses. Blot-transfer hybridization revealed that DFV DNA reannealed to the same restriction fragments of BPV-2 DNA under stringent conditions that DNA from other papillomaviruses hybridize to under nonstringent conditions.     

Production of a recombinant major inner capsid protein for serological detection of epizootic hemorrhagic disease virus

Constructs of the major core protein, designated VP7, from epizootic hemorrhagic disease virus (EHDV) type 1 were made by amino- or carboxyl-terminal fusion of a six-histidine residue tag to the VP7-1 gene. The resulting fusion proteins were produced in a baculovirus expression system and purified by a rapid, one-step procedure using nickel-nitrilotriacetic acid technology. A high level of VP7-1 protein expression was detected with the N-terminal six-histidine tag fusion construct and was comparable to the level of expression observed with an untagged VP7-1 Bam construct. In contrast, the inclusion of a six-histidine tag at the C terminus adversely affected protein expression. The antigenicity of the N-terminal six-histidine tag EHDV VP7-1 product was identical to that observed with the native virus antigen and untagged EHDV VP7-1 recombinant protein, as determined by reactivity with EHDV specific antibodies in an enzyme-linked immunosorbent assay (ELISA) and Western blot. The high production and purity levels that can be attained for the N-terminal six-histidine tag VP7-1 protein and its reactivity with EHDV-specific sera in a competitive ELISA make it a suitable assay reagent.     

Phylogenetic analyses of the complete nucleotide sequence of the capsid protein (VP3) of Australian epizootic haemorrhagic disease of deer virus (serotype 2) and cognate genes from other orbiviruses

The complete nucleotide sequence of the minor capsid protein (VP3) of epizootic haemorrhagic disease of deer virus (EHDV; Australian serotype 2) was determined using a combination of cloning and sequencing methods. Gene segment 3 that coded for the EHDV VP3 capsid protein was 2768 nucleotides in length with a coding region of 2697 nucleotides flanked by 5' and 3' non-coding regions of 17 and 53 nucleotides, respectively. A protein of 899 amino acids (Mr 103,160) having no overall charge at neutral pH was deduced from the nucleotide sequence. Comparisons with equivalent regions from the other Australian EHDV serotypes showed the VP3 genes and the segments that coded for them were similar, varying by a maximum of 5%. Comparisons with known cognate genes from bluetongue viruses showed that their VP3 genes and the proteins translated from them were remarkably similar to those of EHDV, having approximately 70% to 80% homology at either level, respectively. In an attempt to delineate the evolution of orbiviruses, we have obtained sequence data from the VP3 genes from representative members of all Australian orbiviruses now known. Computer analyses of this data enabled a phylogenetic tree for the orbiviruses to be proposed that incorporated the concept of topotypes.     

The complete nucleotide sequence of a pathogenic swine vesicular disease virus

The nucleotide sequence of a swine vesicular disease virus (SVDV) strain that is pathogenic for pigs has been determined and compared with that of a non-pathogenic strain of SVDV, as well as a number of other enteroviruses. It shows only 98 base changes in comparison with a non-pathogenic strain of SVDV (Inoue et al., 1989, J. Gen. Virol. 70, 919-934). Fourteen of these nucleotide differences between the pathogenic and the non-pathogenic SVDV strains occur in the 5' non-coding region which, by analogy with the other picornaviruses, has been implicated in the efficiency with which the RNA is employed as mRNA. Additional differences found throughout the coding regions are largely conservative in nature. A number of residues are discussed as candidates for determinants of pathogenicity. This sequence has been submitted to the PIR database and has accession number A30061.     

The adenovirus that causes hemorrhagic disease of black-tailed deer is closely related to bovine adenovirus-3

Summary.  DNA sequence data was obtained from an adenovirus previously shown to be the cause of a distinctive, fatal hemorrhagic disease of black-tailed deer in California. A 256 base fragment of the viral hexon gene was amplified by PCR from purified adenovirus preparations. The amplicon then was cloned and sequenced. Phylogenetic relationships with other mammalian adenoviruses were also determined. Although sequence analysis of this portion of the hexon gene indicates that the black-tailed deer adenovirus is closely related to bovine adenovirus-3, the biologic properties of the two viruses are clearly distinct. Accepted September 2, 1998 Received July 30, 1998     

Detection of epizootic hemorrhagic disease virus in Culicoides variipennis (Diptera: Ceratopogonidae)

Nucleic acid hybridization was used to detect epizootic hemorrhagic disease (EHD) virus serotype 1 and serotype 2 in Culicoides variipennis (Coquillett). Adult females were inoculated intrathoracically with virus, then were assayed daily for the presence of viral RNA for 2 wk, at which time maximum virus replication is likely to occur. Viral RNA of EHD serotypes 1 and 2 was first detected by hybridization on days 9 and 7 after infection, respectively, and then for up to 14 days after infection. EHD serotype 1 viral RNA was detected by hybridization in infected flies fixed in ethanol at room temperature for 7 d and in unfixed (frozen) infected flies. However, weak false positives diminished prospects for application of this method.     

Analysis of intratypic variation evident in an Ibaraki virus strain and its epizootic hemorrhagic disease virus serogroup

A new strain of Ibaraki virus (IBAV) was isolated from cattle showing atypical symptoms of Ibaraki disease. The isolate was genetically characterized, and the genetic diversity and evolution of the capsid proteins of viruses in the epizootic hemorrhagic disease virus (EHDV) serogroup were investigated. The nucleotide sequences of the isolate's viral RNA segments 2, 3, 6, and 7, which encode the viral structural proteins VP2, VP3, VP5, and VP7, respectively, were determined and were then compared against those of the existing strains of IBAV and EHDV, to which IBAV belongs serologically. The nucleotide sequences of segments 3 and 7 were conserved within the EHDV serogroup, particularly well among the strains of IBAV and Australian EHDV. The similarity of the sequence of segment 6 of the isolate to sequences of corresponding segments of the other strains of IBAV and EHDV was found to be about 93%. The similarity of segment 2 of the isolate to segments 2 of the other strains of IBAV and EHDV was less than 70%. Phylogenetic analysis based on the deduced amino acid sequences of segments 3 and 7 revealed that the viruses differed according to their geographical distributions. However, the new isolate of IBAV was categorized as having a distinct lineage in the phylogenetic tree of VP2. These results suggest that the isolate was modified by a reassortment of segment 2 and that it exhibits unique genetic and antigenic characteristics.     

The complete nucleotide sequence of a severe stem pitting isolate of Citrus tristeza virus from Spain: comparison with isolates from different origins

Summary. The genomic RNA of the severe stem pitting Citrus tristeza virus (CTV) isolate T318A from Spain (19252 nt) was completely sequenced. It showed strong sequence similarities with the severe isolates SY568 from California and NUagA from Japan, and distant relationships with mild non-stem pitting isolates T385 from Spain and T30 from Florida. Contrasting with other severe CTV isolates, T318A had a predominant sequence variant even in the highly variable 5′-terminal untranslated region, in which a unique sequence variant (type II) previously associated with severe stem pitting isolates was detected. The high homogeneity of the T318A population suggests that the sequence obtained is probably responsible for the symptoms induced and makes it a useful tool to delimit pathogenicity determinants.     

Analysis of nucleotide sequence of Iranian maize mosaic virus confirms its identity as a distinct nucleorhabdovirus

The nucleotide sequence of the Iranian maize mosaic rhabdovirus (IMMV) was obtained using a random-PCR method (rPCR) followed by PCR with specific primers. Analysis of the complete nucleotide sequence of the IMMV genes and intergenic regions comprising a total of 12,381 nucleotides (including the partial sequences of leader and trailer regions) revealed six open reading frames (ORF) on the viral complementary RNA (vcRNA). On the basis of its similarities to other rhabdovirus sequences, the IMMV genome consists of 3'-leader-N-P-3-M-G-L-5'-trailer. The intergenic regions contained a characteristic consensus sequence, 3'-AAUUCUUUUUGGGUUU/G-5'. The IMMV gene products showed a high similarity to those of maize mosaic virus and taro vein chlorosis virus and a more distant relationship to other rhabdoviruses. Together with the biological, serological and morphological features described earlier, our molecular data provide evidence that IMMV is a distinct member of the genus Nucleorhabdovirus in the family Rhabdoviridae.     

Transmission of bacterial agents from lone star ticks to white-tailed deer

Amblyomma americanum (L.), the lone star tick, is an aggressive ixodid tick that has been implicated as a vector for several bacteria. Among these bacteria are the disease agents Ehrlichia chaffeensis and Ehrlichia ewingii, and the putative disease agent "Borrelia lonestari." The hypothesis in this study was that wild lone star ticks from northeastern Georgia are capable of transmitting all three agents to white-tailed deer, Odocoileus virginianus, a known reservoir host for E. chaffeensis. In this study, transmission of all three agents from wild caught lone star ticks to captive reared white-tailed deer was demonstrated by polymerase chain reaction (PCR), culture, or serology. Two of three deer showed evidence of E. chaffeensis and E. ewingii infection by polymerase chain reaction assay; all three deer showed evidence of B. lonestari by PCR assay. E. chaffeensis was isolated in culture from both PCR-positive deer on multiple days. All three deer seroconverted to E. chaffeensis, whereas one deer seroconverted to B. lonestari. This study supports the role of lone star ticks and white-tailed deer as a vector and reservoir host for E. chaffeensis and E. ewingii and suggests for the first time, transmission of B. lonestari from lone star ticks to white-tailed deer.     

Complete nucleotide sequence of segment 5 of epizootic haemorrhagic disease virus; the outer capsid protein VP5 is homologous to the VP5 protein of bluetongue virus

The complete nucleotide sequence of a cDNA clone representing the segment 5 RNA of epizootic haemorrhagic disease virus (EHDV) United States serotype 1 was determined. The 5' and 3' termini of the RNA are complementary and are capable of forming secondary structures. The comparison of the predicted amino acid sequence of the encoded outer capsid protein (VP5) with the sequences of VP5 from four serotypes of bluetongue virus, the prototype orbivirus, revealed that the protein shares 59% to 62% homologies with various BTV serotypes, including a single conserved glycine residue at the amino terminus. The sequence has been submitted to the Genebank databox (X55782).     

The S7 gene and VP7 protein are highly conserved among temporally and geographically distinct American isolates of epizootic hemorrhagic disease virus

Complete sequences of genome segment 7 (S7) from six isolates of epizootic hemorrhagic disease virus serotype 1 (EHDV-1) and 37 isolates of serotype 2 (EHDV-2) were determined. These isolates were made between 1978 and 2001 from the southeast, mid-Atlantic, Midwest and intermountain United States. Analysis of the S7 sequence similarities showed 98.1% identity among the EHDV-1 isolates and 91.0% identity among the EHDV-2 isolates. Comparison of the deduced amino acid similarities showed an even greater degree of similarity among the isolates (100% among the EHDV-1 isolates and 98.9% identity among the EHDV-2 isolates). There was only 75.8% identity between the EHDV-1 and EHDV-2 isolates at the nucleic acid level; however, there was 93.7% identity between the two groups at the amino acid level. The ratio of non-synonymous to synonymous nucleotide indicates a strong selection for silent substitutions. There was no evidence for reassortment between EHDV-1 and EHDV-2 isolates. The high degree of conservation of S7 gene codons and the VP7 protein, suggests that little variation is allowed in preserving the function of this protein. The high degree of conservation also validates the use of diagnostic tests for EHDV based on S7 and VP7.     

Development of polymerase chain reaction for specific identification of epizootic hemorrhagic disease virus serotype 1

Summary The diagnostic potential of the polymerase chain reaction (PCR) for specific identification of epizootic hemorrhagic disease virus serotype 1 (EHDV-1) in cell culture and clinical specimens was evaluated. Using oligonucleotide primers, selected from genome segment 2 of EHDV-1 (New Jersey strain), the PCR-based assay resulted in a 862 base pair (bp) PCR product. EHDV-1 RNA from United States prototype serotype 1 and a number of EHDV-1 field isolates, propagated in cell cultures, were detected by this PCR based assay. The specific 862 bp PCR products were visualized on ethidium bromide-stained agarose gel. Identity of the PCR product was confirmed by chemiluminescent hybridization with non radiolabelled internal probe. Using chemiluminescent hybridization, the sensitivity of the PCR assay was 1.0 fg of virus RNA (equivalent to 60 virus particles). Amplification product was not detected when the PCR-based assay was applied to RNA from EHDV serotype 2 (EHDV-2); the United States bluetongue virus (BLU) prototypes serotypes 2, 10, 11, 13, and 17; total nucleic acid extracts from uninfected BHK-21 cell; or blood cells from calves and deer that were EHDV-seronegative and virus isolation negative. Application of this EHDV-1 PCR-based assay to clinical samples resulted in detection of EHDV-1 RNA from blood samples, collected from a calf experimentally infected with EHDV-1. The described PCR-based assay provides a simple, rapid, sensitive, specific and inexpensive method for specific identification of EHDV-1 infection in susceptible ruminants.     

Preliminary observations on the experimental transmission of chronic wasting disease (CWD) from elk and white-tailed deer to fallow deer

To determine the transmissibility of chronic wasting disease (CWD) to fallow deer (Dama dama) and to provide information about clinical course, lesions and suitability of currently used diagnostic procedures for detection of CWD in this species, 13 fawns were inoculated intracerebrally with CWD brain suspension from elk (n=6) or white-tailed deer (n=7). Three other fawns were kept as uninfected controls. Three CWD-inoculated deer were killed 7.6 months post-inoculation (mpi). None had abnormal prion protein (PrPd) in their tissues. One sick deer died at 24 mpi and one deer without clinical signs was killed at 26 mpi. Both animals had a small focal accumulation of PrPd in the midbrain. Between 29 and 37 mpi, three other deer became sick and were killed. All had shown gradual decrease in appetite and some loss of body weight. Microscopical lesions of spongiform encephalopathy were not observed, but PrPd was detected in tissues of the central nervous system (CNS) by immunohistochemistry, western blot and by two commercially available rapid diagnostic tests. This study demonstrates that intracerebrally inoculated fallow deer amplified CWD PrPd from white-tailed deer and elk in the absence of lesions of spongiform encephalopathy. Four years after CWD inoculation, the remaining five inoculated and two control deer are alive and apparently healthy.