In 2010, a HoBi-like pestivirus was isolated from clinically affected calves in Italy. This European virus reproduced a milder form of disease under experimental conditions and was genetically related to previously reported HoBi-like strains. Isolation of this novel virus from a clinical outbreak may have implications for cattle health and prophylactic programs.
An outbreak of abortion affecting multiparous cows was associated with Hobi-like pestivirus infection. Viral RNA and antigens were detected in the tissues of two aborted fetuses. Molecular assays for other common abortogenic agents tested negative. At the genetic level, the Hobi-like pestivirus displayed the closest relatedness to Italian, Australian, and South American viruses, whereas it diverged from the prototype Thai isolate. These findings may have important implications for the pestivirus control/ eradication programs in cattle herds. Bovine viral diarrhea viruses (BVDVs) are members of the genus Pestivirus (family Flaviviridae), responsible for a number of clinical signs, including subclinical infections, immunosuppression, acute diarrhea, respiratory disease, reproductive failures, and mucosal disease in persistently infected calves. Reproductive disorders caused by BVDVs vary according to the fetal age and include embryo death, abortion, mummification, congenital abnormalities, or stillbirths (2). Thus far, two different BVDV species have been recognized, BVDV-1 and BVDV-2, that cocirculate in cattle herds worldwide (20). In 2004, an atypical pestivirus, strain D32/00_Hobi, was isolated from a contaminated batch of fetal calf serum (FCS) (19). The virus was distantly related to BVDV-1/BVDV-2, and it was proposed as a prototype of a new pestivirus species (14, 15). Hobi-like sequences have been repeatedly detected in commercial FCS batches (17,22,23), whereas there are few reports on natural infections (4, 5, 21, 23) and clinical outbreaks (5). The virus has been recently detected in aborted bovine fetuses in Brazil, thus suggesting direct clinical implications (4).Here, we report the isolation and genetic characterization of a Hobi-like strain detected from aborted fetuses in southern Italy. The abortion outbreak occurred in June 2011 in a cattle herd where a Hobi-like pestivirus-associated respiratory disease had been recently described (5). Abortion was observed in eight multiparous cows in a group of 270 lactating Holstein cows and occurred between the fourth and sixth months of pregnancy. The animals neither showed prodromal signs nor presented postabortion complications. Two aborted fetuses (280/11-A, 280/11-B) were sent to our laboratory, and tissue samples were collected from lungs, spleens, livers, kidneys, and placentas for diagnostic investigations. Nucleic acids were purified using the DNeasy tissue kit (Qiagen) and QIAamp RNeasy minikit (Qiagen). Reverse transcription (RT)-PCR and PCR assays were performed using SuperScript one-step RT-PCR for long templates (Life Technologies) and LA PCR kit version 2.1 (TaKaRa Bio Inc.), respectively. Positive and negative controls were processed in parallel to the screened samples. The samples tested negative by PCR for Chlamydophila spp. 3, 8). Conversely, the pestivirus genome was detected with two different 24), and the virus was characterized as Hobi-like by a species-specific nested PCR (7) (Fig. 1A). Viral titers, quantified by a TaqMan-based real...
A Hobi-like pestivirus pair consisting of cytopathogenic (cp) and non-cytopathogenic (noncp) strains, Italy 83/10cp and Italy 83/10ncp, was isolated from the lung of a heifer that died of respiratory disease. The noncp and cp viruses were isolated on Madin-Darby bovine kidney cells and separated by plaque purification and end point dilution. Analysis of the nearly full-length genomes revealed that the two viruses were very closely related to each other and to the noncp Hobi-like strain Italy 1/10-1, which had been isolated a few weeks earlier from the same herd. One major difference between noncp and cp viruses concerned the presence of a cellular Jiv sequence in the 39 domain of the NS2-encoding region of the cp strain. This is the first study, to our knowledge, reporting the isolation and molecular characterization of a Hobi-like virus pair. INTRODUCTIONTogether with classical swine fever virus (CSFV), border disease virus (BDV) and other pestiviruses isolated from wild ruminants, bovine viral diarrhea virus (BVDV) belongs to the genus Pestivirus of the family Flaviviridae (Avalos- Ramirez et al., 2001;Becher et al., 2003;Simmonds et al., 2011). BVDV is an enveloped, single-stranded, positivesense RNA virus that is responsible for multiple clinical signs (Lindenbach et al., 2007) and there are two main species, termed BVDV-1 and BVDV-2 (Pellerin et al., 1994;Ridpath et al., 1994;Simmonds et al., 2011), with several subtypes within each species (Baule et al., 1997;Wolfmeyer et al., 1997;Becher et al., 1999; Vilcek et al., 1999a, b, c;Couvreur et al., 2002;Flores et al., 2002;Tajima, 2004). The BVDV genomic RNA, approximately 12.3 kb in size, encodes a polyprotein (NH 2 -N pro -C-E rns -E1-E2-p7-NS2-3-NS4A-NS4B-NS5A-NS5B-COOH) that is processed by viral and cellular proteases, thereby generating structural and non-structural proteins. The single large ORF is flanked by the 59 and 39 UTRs (Lindenbach et al., 2007).An atypical pestivirus, named D32/00_Hobi, was isolated from a contaminated batch of FCS originating from Brazil (Schirrmeier et al., 2004). Hobi-like pestiviruses contaminating FCS of South American origin were later detected in Switzerland (Stalder et al., 2005), Sweden (Liu et al., 2009a), Italy (Peletto et al., 2012), USA, Canada, Mexico and Australia (Xia et al., 2011). Strains responsible for natural infections were recovered in South America and Thailand from bubaline blood (Stalder et al., 2005) and bovine serum (Ståhl et al., 2007), but there was no evidence of associated clinical signs. All these viruses were proposed to belong to a new pestivirus species, tentatively termed BVDV-3 (Liu et al., 2009b). However, there is no agreement among pestivirologists about this proposal, considering the genetic and antigenic distance of the new viruses from other BVD viruses (J. Ridpath, personal communication). The first European Hobi-like virus strain Italy 1/10-1 was isolated from calves with severe respiratory disease in southern Italy (Decaro et al., 2011). This is also the first report, to our knowl...
A TaqMan-based real-time PCR assay targeting the glycoprotein B-encoding gene was developed for diagnosis of canid herpesvirus 1 (CHV-1) infection. The established assay was highly specific, since no cross-reactions were observed with other canine DNA viruses, including canine parvovirus type 2, canine minute virus, or canine adenovirus types 1 and 2. The detection limit was 10(1) and 1.20 x 10(1) DNA copies per 10 microl(-1) of template for standard DNA and a CHV-1-positive kidney sample, respectively: about 1-log higher than a gel-based PCR assay targeting the thymidine kinase gene. The assay was also reproducible, as shown by satisfactory low intra-assay and inter-assay coefficients of variation. CHV-1 isolates of different geographical origins were recognised by the TaqMan assay. Tissues and clinical samples collected from three pups which died of CHV-1 neonatal infection were also tested, displaying a wide distribution of CHV-l DNA in their organs. Unlike other CHV-1-specific diagnostic methods, this quantitative assay permits simultaneous detection and quantitation of CHV-1 DNA in a wide range of canine tissues and body fluids, thus providing a useful tool for confirmation of a clinical diagnosis, for the study of viral pathogenesis and for evaluation of the efficacy of vaccines and antiviral drugs.
An atypical pestivirus ('Hobi'-like pestivirus, putative bovine viral diarrhoea 3, BVDV-3) was identified firstly in contaminated foetal calf serum batches and isolated subsequently from an outbreak of respiratory disease in a cattle herd in Italy. The isolation of the novel pestivirus from animals affected clinically posed concerns about the validity of BVDV eradication programs, considering that 'Hobi'-like pestivirus (BVDV-3) is undetected or mistyped by the molecular diagnostic tools currently employed. In this paper, the development of a nested PCR (nPCR) assay for unambiguous typing of all bovine pestiviruses is reported. The assay consisted of a first-round amplification using an oligonucleotide pair which binds to conserved sequences located in the 5' untranslated region and capsid gene, followed by a heminested PCR using virus-specific forward primers. The assay performances were evaluated analytically, showing good sensitivity and specificity. By analysis of 100 BVDV-positive samples typed using a nPCR assay discriminating ruminant pestiviruses, five samples recognised previously as BVDV-2 were not typed when submitted to the new assay (n=2) or reacted as 'Hobi'-like pestivirus BVDV-3 (n=3). Sequence analysis of the first-round amplification products showed that the untyped strains were border disease viruses, whereas the other three strains were true 'Hobi'-like viruses. The development of a molecular assay able to identify simultaneously all bovine pestiviruses known currently will help warrant biosafety of live vaccines and other biological products and assess the molecular epidemiology of 'Hobi'-like pestivirus, thus leading to the improvement of the eradication programs through unambiguous typing of pestiviruses infecting cattle.
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