Determination of the sensitivity and specificity of bovine tuberculosis screening tests in dairy herds in Thailand using a Bayesian approach.

BACKGROUND: The objective of this study was to determine the sensitivity (Se) and specificity (Sp) of bovine tuberculosis (bTB) screening tests including a single intradermal tuberculin (SIT) test, interferon gamma (IFN-γ) assay, and a commercial ELISA test (M. bovis Ab) in dairy cattle, under field conditions, using a Bayesian approach. RESULTS: The study population consisted of 128 dairy cows from 25 bTB-infected herds in Chiang Mai and Chiang Rai provinces, Thailand. A single-population Bayesian model was implemented assuming conditional dependence between the SIT test and IFN-γ assays. The 95% posterior probability interval (PPI) of the SIT test (severe interpretation) Se ranged from 75.3 to 95.2% (median = 87.6%), while the Sp was slightly lower (median = 83.6%, PPI = 74.2-92.8%). The IFN-γ assay Se was moderate and the 95% PPI ranged from 38.6 to 74.4% (median = 55.7%) with higher Sp (median = 93.5.4%, PPI = 87.0-98.1%). The M. bovis Ab ELISA Se was low, with 95% PPI ranging between 30.0 and 71.2% (median = 47.4%); however, the Sp was high (median = 90.9%, PPI = 84.5-95.5%). CONCLUSION: The SIT test sensitivity was similar to that demonstrated in other regions and can, therefore, be used effectively as part of control programs in this area. The IFN-γ and M. bovis Ab ELISA assays can be applied as supplementary techniques. The test performance of these tests when used as single tests without confirmation, however, are expected to continue to challenge disease eradication efforts.

The pseudorabies virus vhs protein cleaves RNA containing an IRES sequence.

The virion host shutoff protein (vhs), encoded by the gene UL41, has RNase activity and is the key regulator of the early host shutoff response induced by type 1 herpes simplex virus. Despite low amino acid similarity, the vhs protein of the swine herpesvirus, pseudorabies virus (PrV), also exhibits RNase activity. However, the mechanism underlying the action of vhs remains undefined. Here, we report that the RNA degradation profile of PrV vhs is similar, but not identical, to that of type 1 herpes simplex virus vhs. Notably, the presence of a cap structure enhances both the degradation rate and the preferential targeting of the vhs protein towards the 3'-end of the encephalomyocarditis virus internal ribosome entry site (IRES). Furthermore, type 1 herpes simplex virus vhs produces a simple degradation pattern, but PrV vhs gives rise to multiple intermediates. The results of northern blotting using probes recognizing various regions of the RNA substrate found that PrV vhs also cleaves downstream of the IRES region and this vhs protein overall shows 5' to 3' RNase activity. Moreover, addition of the translation initiation factors eIF4H and eIF4B significantly increased the RNase activity of recombinant PrV vhs against capped RNA. Nonetheless, these proteins did not fully reconstitute the IRES-directed targeting pattern observed for vhs translated in a rabbit reticular lysate system. The interaction between PrV vhs and eIF4H/eIF4B implies that the translation initiation machinery within the cell is able to stimulate the nuclease activity of PrV vhs. However, this process remains inefficient in terms of the IRES-targeting pattern.

Roles of nucleic acid substrates and cofactors in the vhs protein activity of pseudorabies virus.

Pseudorabies virus (PrV) belongs to the α-herpesvirinae of which human simplex virus (HSV) is the prototype virus. One of the hallmarks of HSV infection is shutoff of protein synthesis that is mediated by various viral proteins including vhs (virion host shutoff), which is encoded by the UL41 gene. However, the function of PrV vhs is poorly understood. Due to the low sequence similarity (39.3%) between the HSV and PrV UL41 proteins, vhs might not share the same biochemistry characteristics. The purpose of this study was to characterize the nuclease activity of the PrV vhs protein with respect to substrate specificity, its requirements in terms of cofactors, and the protein regions, as well as key amino acids, which contribute to vhs activity. Our results indicated that, similar to HSV vhs, PrV vhs is able to degrade ssRNA and mRNA. However, PrV vhs also targeted rRNA for degradation, which is novel compared to the HSV-1 vhs. Activity assays indicated that Mg(2+) alone enhances RNA degradation mediated by PrV vhs, while K(+) and ATP are not sufficient to induce activity. Finally, we demonstrated that each of the four highly conserved functional boxes of PrV vhs contributes to RNA degradation and that, in particular, residues 152, 169, 171, 172, 173 343, 345, 352 and 356, which are conserved among α-herpesviruses, are key amino acids needed for PrV vhs ribonuclease activity.

Functional analysis of the short isoform of orf virus protein OV20.0.

UNLABELLED: Orf virus (ORFV) OV20.0L is an ortholog of vaccinia virus (VACV) gene E3L. The function of VACV E3 protein as a virulence factor is well studied, but OV20.0 has received less attention. Here we show that like VACV E3L, OV20.0L encodes two proteins, a full-length protein and a shorter form (sh20). The shorter sh20 is an N-terminally truncated OV20.0 isoform generated when a downstream AUG codon is used for initiating translation. These isoforms differed in cellular localization, with full-length OV20.0 and sh20 found throughout the cell and predominantly in the cytoplasm, respectively. Nonetheless, both OV20.0 isoforms were able to bind double-stranded RNA (dsRNA)-activated protein kinase (PKR) and dsRNA. Moreover, both isoforms strongly inhibited PKR activation as shown by decreased phosphorylation of the translation initiation factor eIF2α subunit and protection of Sindbis virus infection against the activity of interferon (IFN). In spite of this apparent conservation of function in vitro, a recombinant ORFV that was able to express only the sh20 isoform was attenuated in a mouse model. IMPORTANCE: The OV20.0 protein of orf virus (ORFV) has two isoforms and contributes to virulence, but the roles of the two forms are not known. This study shows that the shorter isoform (sh20) arises due to use of a downstream initiation codon and is amino-terminally truncated. The sh20 form also differs in expression kinetics and cellular localization from full-length OV20.0. Similar to the full-length isoform, sh20 is able to bind dsRNA and PKR, inactivate PKR, and thus act as an antagonist of the interferon response in vitro. In vivo, however, wild-type OV20.0 could not be replaced with sh20 alone without a loss of virulence, suggesting that the functions of the isoforms are not simply redundant.

Translational enhancing activity in 5' UTR of peste des petits ruminants virus fusion gene.

The fusion gene of peste des petits ruminants virus (PPRV-F), a paramyxovirus, contains an unusual long 5' untranslated region (5' UTR) with a high GC content that is capable of folding into secondary structure proximally to the 5' cap. Sequence analysis further suggested that the proximal end of this UTR contains a nine-nucleotide sequence which could perfectly complement the 18S rRNA and might affect translation through mRNA-rRNA interaction. Based on these features, we examined the functional role of the proximal PPRV-F 5' UTR on translational efficiency compared with two other morbilliviruses. From reporter gene assays, PPRV-F 5' UTR functioned as a strong enhancer of translational efficiency independent of cell and gene specificity. Northern blot analysis of the accumulative RNA levels and mRNA stability suggested that elevated gene expression driven by PPRV-F 5' UTR was accompanied by an increased mRNA level and enhanced mRNA stability. Deletion analysis identified the complementary sequence and distal nucleotides necessary for the enhancing activity, and results suggest RNA structural conformation is important. Taken together, we conclude that the proximal PPRV-F 5' UTR functions as a translational enhancer by promoting translation efficiency and mRNA stability.

Mouse mammary tumor virus-like nucleotide sequences in canine and feline mammary tumors.

Mouse mammary tumor virus (MMTV) has been speculated to be involved in human breast cancer. Companion animals, dogs, and cats with intimate human contacts may contribute to the transmission of MMTV between mouse and human. The aim of this study was to detect MMTV-like nucleotide sequences in canine and feline mammary tumors by nested PCR. Results showed that the presence of MMTV-like env and LTR sequences in canine malignant mammary tumors was 3.49% (3/86) and 18.60% (16/86), respectively. For feline malignant mammary tumors, the presence of both env and LTR sequences was found to be 22.22% (2/9). Nevertheless, the MMTV-like LTR and env sequences also were detected in normal mammary glands of dogs and cats. In comparisons of the MMTV-like DNA sequences of our findings to those of NIH 3T3 (MMTV-positive murine cell line) and human breast cancer cells, the sequence similarities ranged from 94 to 98%. Phylogenetic analysis revealed that intermixing among sequences identified from tissues of different hosts, i.e., mouse, dog, cat, and human, indicated the MMTV-like DNA existing in these hosts. Moreover, the env transcript was detected in 1 of the 19 MMTV-positive samples by reverse transcription-PCR. Taken together, our study provides evidence for the existence and expression of MMTV-like sequences in neoplastic and normal mammary glands of dogs and cats.

Multiplex Amplification Refractory Mutation System Polymerase Chain Reaction (ARMS-PCR) for diagnosis of natural infection with canine distemper virus.

BACKGROUND: Canine distemper virus (CDV) is present worldwide and produces a lethal systemic infection of wild and domestic Canidae. Pre-existing antibodies acquired from vaccination or previous CDV infection might interfere the interpretation of a serologic diagnosis method. In addition, due to the high similarity of nucleic acid sequences between wild-type CDV and the new vaccine strain, current PCR derived methods cannot be applied for the definite confirmation of CD infection. Hence, it is worthy of developing a simple and rapid nucleotide-based assay for differentiation of wild-type CDV which is a cause of disease from attenuated CDVs after vaccination. High frequency variations have been found in the region spanning from the 3'-untranslated region (UTR) of the matrix (M) gene to the fusion (F) gene (designated M-F UTR) in a few CDV strains. To establish a differential diagnosis assay, an amplification refractory mutation analysis was established based on the highly variable region on M-F UTR and F regions. RESULTS: Sequences of frequent polymorphisms were found scattered throughout the M-F UTR region; the identity of nucleic acid between local strains and vaccine strains ranged from 82.5% to 93.8%. A track of AAA residue located 35 nucleotides downstream from F gene start codon highly conserved in three vaccine strains were replaced with TGC in the local strains; that severed as target sequences for deign of discrimination primers. The method established in the present study successfully differentiated seven Taiwanese CDV field isolates, all belonging to the Asia-1 lineage, from vaccine strains. CONCLUSIONS: The method described herein would be useful for several clinical applications, such as confirmation of nature CDV infection, evaluation of vaccination status and verification of the circulating viral genotypes.

Differential diagnosis of orf viruses by a single-step PCR.

The complete nucleotide sequence of the A32L gene (named after vaccinia virus, corresponding with open reading frame 108 of the orf virus and encoding an ATPase) of the orf virus was studied using samples of orf virus from infected goats, which were collected from six outbreaks in central Taiwan. DNA sequence analysis of the A32L genes of these and isolates from other countries showed sequence heterogeneity (base pair variation and deletion) in the 3'-terminal regions. This finding led to the development of a polymerase chain reaction (PCR) method for the rapid differential diagnosis of orf virus infections, and the results demonstrated that this was an easy and reliable method for genotyping of orf viruses.