Ilias G. Bouzalas

Neurotropic Astrovirus in Cattle with Nonsuppurative Encephalitis in Europe

ABSTRACT Encephalitis is a frequently diagnosed condition in cattle with neurological diseases. Many affected animals present with a nonsuppurative inflammatory reaction pattern in the brain. While this pattern supports a viral etiology, the causative pathogen remains unknown in a large proportion of cases. Using viral metagenomics, we identified an astrovirus (bovine astrovirus [BoAstV]-CH13) in the brain of a cow with nonsuppurative encephalitis. Additionally, BoAstV RNA was detected with reverse transcription-PCR and in situ hybridization in about one fourth (5/22 animals) of cattle with nonsuppurative encephalitis of unknown etiology. Viral RNA was found primarily in neurons and at the site of pathology. These findings support the notion that BoAstV infection is a common cause of encephalitis in cattle. Phylogenetically, BoAstV-CH13 was closely related to rare astrovirus isolates from encephalitis cases in animals and a human patient. Future research needs to be directed toward the pathogenic mechanisms, epidemiology, and potential cross-species transmission of these neurotropic astroviruses.

Caprine PRNP polymorphisms at codons 171, 211, 222 and 240 in a Greek herd and their association with classical scrapie

The association between PRNP variation and scrapie incidence was investigated in a highly affected Greek goat herd. Four mutations were identified at codons 171Q/R, 211R/Q, 222Q/K and 240P/S. Lysine at codon 222 was found to be associated with the protection from natural scrapie (P=0.0111). Glutamine at codon 211 was observed in eight animals, all of them being scrapie-negative, indicating a possible protective role of this polymorphism although statistical analysis failed to support it (P=0.1074). A positive association (P=0.0457) between scrapie-affected goats and the wild-type Q(171)R(211)Q(222)S(240) allele is presented for the first time. In addition, a novel R(171)RQS allele, which is identical to the A(136)R(154)R(171) allele that has been associated with resistance to classical scrapie in sheep, was observed in low frequency. Resistant alleles that include K(222) and Q(211) are absent or rare in sheep and can provide the basis for the development of a feasible breeding programme for scrapie eradication in goats.

Identification of a second encephalitis-associated astrovirus in cattle

Dear Editor, Human astroviruses are of particular importance as one of the most common pathogens that cause juvenile gastroenteritis. In recent years, several astrovirus isolates were identified as potential causes of encephalitis in immunocompromised human patients. These discoveries were paralleled by the description of phylogenetically closely related bovine astrovirus isolates (BoAstV) from the nervous tissue of cattle with encephalitis of unknown etiology in the USA (BoAstV NeuroS1) and by our laboratory in Switzerland (BoAstV-CH13). BoAstV-CH13 was retrospectively found in approximately one-quarter (5/22) of the cattle with etiologically unresolved non-suppurative encephalitis in Switzerland; however, most of these cases (17/22) were unrelated to BoAstV-CH13. Here, we report the identification of yet another neuroinvasive astrovirus in this set of cattle with non-suppurative encephalitis, which has less than 65% genetic similarity to currently known astrovirus isolates. In 2006, a 4-year-old Braunvieh cow (case ID 42535) was notified as a clinical bovine spongiform encephalopathy (BSE) suspect to the Swiss authorities. Precise information about the clinical presentation was not available, but the spectrum of signs in BSE-suspect animals usually involves changes in behavior and temperament, hyper-reactivity, and incoordination. Post mortem BSE testing was negative, and histopathological examination led to the diagnosis of severe non-suppurative meningo-encephalomyelitis (Figure 1A). This inflammatory pattern strongly suggested that the animal had a viral infection, but further etiologic investigations were not undertaken. The animal was included in our research on BoAstV infection and encephalitis and was further investigated by unbiased next-generation RNA sequencing (NGS) and a bioinformatics pathogen discovery pipeline. Illumina sequencing of frozen brain tissue RNA extracts (medulla oblongata) from animal 42 535 resulted in 21 443 420 read pairs. After in-silico subtraction of reads that aligned to the bovine reference genome and de novo assembly of the remaining reads, we identified four contiguous sequences (contigs) of 792, 1010, 1103, and 3170 nucleotides that matched with the highest amino acid sequence similarity (64%–83%) to different proteins of a sheep astrovirus isolate entry (accession number NC_002469.1) of the National Center for Biotechnology Information database. Gaps between the contigs were bridged by RT-PCR followed by Sanger sequencing. The 59 and 39 ends of the RNA molecule were determined by rapid amplification of cDNA ends (Supplementary Methods, Supplementary Table S1). This resulted in a sequence of 6287 nucleotides that revealed features of an astrovirus genome with short 59 and 39 untranslated regions, three partially overlapping open reading frames (ORF1a, ORF1b, and ORF2), and a poly-A tail (Figure 1B). RT-PCR targeting a 388-bp fragment in ORF1a confirmed the presence of the viral RNA in frozen tissue samples of the medulla oblongata, cerebellar cortex, midbrain, and cerebral cortex (Supplementary Figure S1). Taken together, these data indicate the presence of a previously unknown astrovirus that we termed BoAstV-CH15 (GenBank accession number KT956903). Other pathogens were not detected using our pipeline. Full genome phylogenetic comparison placed the BoAstV-CH15 in the same cluster of previously described neurotropic astroviruses (Figure 1C, HMO clade) and distant from bovine and human isolates that were derived from feces specimens (Figure 1C, classical clades). BoAstV-CH15 rooted from the same branch as an ovine astrovirus (OvAstV), which was isolated from the feces of a sheep with diarrhea. The same topology was obtained in a maximum-likelihood tree that was based on the full-length capsid protein amino acid sequences (Supplementary Figure S2). A sliding-window, pairwise comparison plot of full genome sequences confirmed the relationship between BoAstV-CH15 and the OvAstV and showed less identity of BoAstVCH15 with BoAstV-CH13 and HuAstV-PS, a human encephalitis isolate, at most positions (Figure 1D). To assess whether other cases of cattle encephalitis are associated with the presence of the newly identified astrovirus, we tested the entire set of frozen tissue samples (n 5 22) from the retrospective study mentioned above using the BoAstV-CH15 RT-PCR protocol. Besides case 42 535, one additional encephalitis case was reactive and showed the specific 388-bp amplicon. This animal was an unrelated neurologically diseased 7-year-old cow that was diagnosed with severe non-suppurative poliomeningoencephalitis and ganglioneuritis in 2007 (ID 42799; Figure 1A). This finding was unexpected in this particular animal because it was previously classified as BoAstV-CH13positive based on RT-PCR results and ISH experiments. However, as the BoAstV-CH15 RT-PCR protocol does not detect BoAstV-CH13 (Seuberlich T, 2015, unpubl. data), these conflicting results could be explained by the presence of either a different type of astrovirus or by

Assessment of bluetongue viraemia in sheep by real-time PCR and correlation with viral infectivity.

Inoculation of embryonated chicken eggs is the standard method for the titration of infectious Bluetongue virus (BTV). Here, six RNA extraction methods coupled with optimised dsRNA denaturation and real-time RT-PCR were evaluated for the quantitation of BTV in blood samples from experimentally infected sheep and results were correlated to infectious virus titres. An exogenous dsRNA internal control (IC) from the closely related Epizootic hemorrhagic disease virus (EHDV) was used to assess the efficiency of BTV genome extraction, dsRNA denaturation, RT, and PCR amplification. Recovery rates of IC and BTV dsRNA copies from extracted blood samples were highly correlated. Adjustment of BTV concentrations according to the IC recovery reduced variation in sample analyses among the different extraction methods and improved the accuracy of BTV quantitation. The EID(50)/ml titre, determined in blood samples from sheep infected experimentally with BTV-1 or BTV-9, correlated highly with the assessed concentration of BTV dsRNA copies. However, this correlation was consistent only during the first 28 days post-infection. The optimised extraction methods and quantitative RT-PCR could be useful for experimental studies of BTV transmission, pathogenesis and vaccine efficacy, or adapted further for the detection and quantitation of EHDV, African horse sickness virus and other dsRNA viruses.

Exploring the virome of cattle with non-suppurative encephalitis of unknown etiology by metagenomics

Non-suppurative encephalitis is one of the most frequent pathological diagnosis in cattle with neurological disease, but there is a gap in the knowledge on disease-associated pathogens. In order to identify viruses that are associated with non-suppurative encephalitis in cattle, we used a viral metagenomics approach on a sample set of 16 neurologically-diseased cows. We detected six virus candidates: parainfluenza virus 5 (PIV-5), bovine astrovirus CH13/NeuroS1 (BoAstV-CH13/NeuroS1), bovine polyomavirus 2 (BPyV-2 SF), ovine herpesvirus 2 (OvHV-2), bovine herpesvirus 6 (BHV-6) and a novel bovine betaretrovirus termed BoRV-CH15. In a case-control study using PCR, BoAstV-CH13 (p=0.046), BoPV-2 SF (p=0.005) and BoHV-6 (p=4.3E-05) were statistically associated with the disease. These data expand our knowledge on encephalitis-associated pathogens in cattle and point to the value of NGS in resolving complex infection scenarios in a clinical disease setting.

Isolation of Mycobacterium avium subspecies paratuberculosis from Ugandan cattle and strain differentiation using optimised DNA typing techniques

BackgroundThe occurrence of paratuberculosis in Ugandan cattle has recently been reported but there is no information on the strains of Mycobacterium avium subspecies paratuberculosis (MAP) responsible for the disease. The aim of this study was to isolate and characterise MAP from seropositive cattle and paratuberculosis lesions in tissues obtained from slaughtered cattle in Uganda.ResultsTwenty one isolates of MAP were differentiated into 11 genotype profiles using seven genotyping loci consisting of Insertion Sequence 1311(IS1311), Mycobacterial interspersed repeat units (MIRU) (loci 2, 3), Variable number tandem repeats (VNTR) locus 32 and Short sequence repeats (SSR) (loci 1, 2 and 8). Three different IS1311 types and three MIRU 2 profiles (7, 9, 15 repeats) were observed. Two allelic variants were found based on MIRU 3 (1, 5 repeats), while VNTR 32 showed no polymorphism in any of the isolates from which it was successfully amplified. SSR Locus 1 revealed 6 and 7 G1 repeats among the isolates whereas SSR locus 2 revealed 10, 11 and 12 G2 repeats. SSR locus 8 was the most polymorphic locus. Phylogenetic analysis of SSR locus 8 sequences based on their single nucleotide polymorphisms separated the isolates into 8 genotypes. We found that the use of Ethylene glycol as a PCR additive improved the efficiency of the PCR reactions for MIRUs (2, 3), VNTR 32 and SSR (loci 1 and 2).ConclusionsThere is a high strain diversity of MAP in Uganda since 21 isolates could be classified into 11 genotypes. The combination of the seven loci used in this study results into a very precise discrimination of isolates. However analysis of SNPs on locus alone 8 is very close to this combination. Most of the genotypes in this study are novel since they differed in one or more loci from other isolates of cattle origin in different studies. The large number of MAP strains within a relatively small area of the country implies that the epidemiology of paratuberculosis in Uganda may be complicated and needs further investigation. Finally, the use of Ethylene glycol as a PCR additive increases the efficiency of PCR amplification of difficult templates.

Detection of Astrovirus in Historical Cases of European Sporadic Bovine Encephalitis, Switzerland 1958–1976

European sporadic bovine encephalitis is a frequent diagnosis in neurologically diseased cattle, but its etiology remained unresolved. Using in situ hybridization, we have detected a recently discovered neurotropic bovine astrovirus in historical tissues in a high proportion of brain samples of affected cattle. Our results suggest that astroviruses were already involved in the pathogenesis of the disease several decades ago, but have gone undetected.

Development and validation of an immunohistochemistry procedure for the detection of a neurotropic bovine astrovirus

Members of the Astroviridae family are best known to cause diarrhea in different mammalian species. Lately, some strains have been associated with encephalitis in humans, minks and cattle. In this study, we developed an immunohistochemistry (IHC) procedure for the detection of a neurotropic bovine astrovirus (BoAstV-CH13/NeuroS1), which is associated with non-suppurative encephalitis in cattle. We expressed five recombinant antigens corresponding to different putative viral proteins of BoAstV-CH13/NeuroS1. Antigens were then used for the production of hyperimmune sera in rabbits. Out of the five hyperimmune sera, the one directed against the conserved N-terminus of the viral capsid protein, termed ORF2-con, clearly surpassed the others in the detection of viral antigens in IHC in terms of strong signal intensity and low background staining. The accuracy of the ORF2-con IHC protocol was then evaluated using different sets of brain tissue samples: 30 samples from 9 animals with confirmed BoAstV-CH13/NeuroS1 infection, 30 samples from 8 animals with non-suppurative encephalitis of another etiology and 30 samples from apparently healthy slaughtered animals. The IHC was positive only with tissue samples from animals with a known positive BoAstV-CH13/NeuroS1 status, but not with those from negative ones, indicating a good diagnostic sensitivity and specificity of the assay. The ORF2-con IHC procedure is therefore an adequate tool for the detection of BoAstV-CH13/NeuroS1 infections in cattle.Members of the Astroviridae family are best known to cause diarrhea in different mammalian species. Lately, some strains have been associated with encephalitis in humans, minks and cattle. In this study, we developed an immunohistochemistry (IHC) procedure for the detection of a neurotropic bovine astrovirus (BoAstV-CH13/NeuroS1), which is associated with non-suppurative encephalitis in cattle. We expressed five recombinant antigens corresponding to different putative viral proteins of BoAstV-CH13/NeuroS1. Antigens were then used for the production of hyperimmune sera in rabbits. Out of the five hyperimmune sera, the one directed against the conserved N-terminus of the viral capsid protein, termed ORF2-con, clearly surpassed the others in the detection of viral antigens in IHC in terms of strong signal intensity and low background staining. The accuracy of the ORF2-con IHC protocol was then evaluated using different sets of brain tissue samples: 30 samples from 9 animals with confirmed BoAstV-CH13/NeuroS1 infection, 30 samples from 8 animals with non-suppurative encephalitis of another etiology and 30 samples from apparently healthy slaughtered animals. The IHC was positive only with tissue samples from animals with a known positive BoAstV-CH13/NeuroS1 status, but not with those from negative ones, indicating a good diagnostic sensitivity and specificity of the assay. The ORF2-con IHC procedure is therefore an adequate tool for the detection of BoAstV-CH13/NeuroS1 infections in cattle.

Distinct Proteinase K-Resistant Prion Protein Fragment in Goats with No Signs of Disease in a Classical Scrapie Outbreak

ABSTRACT Considerable efforts have been directed toward the identification of small-ruminant prion diseases, i.e., classical and atypical scrapie as well as bovine spongiform encephalopathy (BSE). Here we report the in-depth molecular analysis of the proteinase K-resistant prion protein core fragment (PrPres) in a highly scrapie-affected goat flock in Greece. The PrPres profile by Western immunoblotting in most animals was that of classical scrapie in sheep. However, in a series of clinically healthy goats we identified a unique C- and N-terminally truncated PrPres fragment, which is akin but not identical to that observed for atypical scrapie. These findings reveal novel aspects of the nature and diversity of the molecular PrPres phenotypes in goats and suggest that these animals display a previously unrecognized prion protein disorder.

Accurate and precise real-time RT-PCR assays for the identification of astrovirus associated encephalitis in cattle

A novel bovine astrovirus genotype species (BoAstV-CH13/NeuroS1) was recently identified in brain tissues of cattle as a plausible cause of encephalitis. The purpose of the present study was to develop and validate real time RT-PCR assays for the detection of BoAstV-CH13/NeuroS1 in brain tissues of cattle. Three different primer-probe combinations were designed based on BoAstV-CH13/NeuroS1 full-genome sequences of 11 different strains identified in cattle, and established in three distinct one-step real time RT-PCR protocols. These protocols were compared regarding their diagnostic performance using brain tissues of cattle with and without astrovirus associated encephalitis. The limit of detection (LOD) of all three assays was between 1.34 × 101 and 1.34 × 102 RNA copies, leading to an analytical sensitivity two orders of magnitude superior compared to a conventional pan-astrovirus RT-PCR protocol (LOD 1.31 × 104 RNA copies). Amplification efficiency was in the range of 97.3% to 107.5% with linearity (R2) > 0.99. The diagnostic sensitivity and specificity of the assays was determined as 100%, and all three revealed good intra- and inter-test repeatability. In conclusion, the newly developed RT-qPCRs are sensitive, specific, and reliable test formats that will facilitate BoAstV-CH13/NeuroS1 detection in routine diagnostics as well as in research settings.