Npro Region Research Articles

Increased genetic variation of bovine viral diarrhea virus in dairy cattle in Poland

BackgroundBovine viral diarrhea virus (BVDV) causes severe economic losses and is one of the most important viral pathogens of ruminants worldwide. The infection manifests itself in a variety of clinical symptoms. Phylogenetic studies based mainly on 5’UTR of its genome, identified many different subtypes of BVDV. Previous study indicated the predominance of BVDV-1b and BVDV-1d in Poland. The aim of this study was to genotype BVDV isolates currently circulating in Polish dairy herds.ResultsBVDV was detected in 30 herds. Viral subtypes were identified using sequences of the 5’UTR fragment and they were confirmed within a fragment of the Npro region. Seven subtypes of BVDV-1 species have been identified: 1b, 1 g, 1f, 1d, 1r, 1 s and 1e.ConclusionThe number of subtypes of BVDV in Poland evolves and 2 new subtypes have been identified for the first time. Such studies may have a positive impact on successful eradication of the virus using effective vaccines and diagnostic tests.

Experimental infection of Korean native goats (Capra aegagrus hircus) with bovine viral diarrhea virus 1b

BackgroundBovine viral diarrhea virus (BVDV) infects various ungulates and causes reproductive failure in infected goats. BVDV has been detected among goats in the Republic of Korea, but the route of transmission remains unclear. Here, we aimed to investigate whether BVDV-1b circulating among Korean cattle can be transmitted to Korean native goats (Capra aegagrus hircus) and characterize the outcomes of BVDV infection in these goats.ResultsFour goats were inoculated intranasally with the Korean noncytopathic (ncp) BVDV-1b strain. Two goats exhibited clinical signs of illness, including coughing and nasal discharge. Nasal swabs and blood were collected to screen for viral RNA and BVDV antibodies. Using the 5′-untranslated region (UTR), viral RNA was detected in the nasal swabs of two goats (Goat 1 and 3) on 12 day post-inoculation (dpi) and in the blood sample of one goat (Goat 1) on 7 and 19 dpi. Using the N-terminal protease (Npro) region, viral RNA was detected in the blood sample of Goat 1 on 7 and 12 dpi. Antibodies to BVDV were detected in Goats 1 and 3 on 16–21 dpi using enzyme-linked immunosorbent assay. Sequence analysis of the virus from nasal swabs and blood samples, which was detected via RT-PCR, using the 5′-UTR and Npro regions led to the identification of the strain as ncp BVDV-1b and revealed changes in the nucleotide sequence of these goats.ConclusionsOur results indicate that changes in the nucleotide sequence are associated with the establishment of BVDV infection in Korean native goats; these changes may be owing to a process required for the establishment of infection in a new host reservoir. Broadly, these findings highlight the importance of BVDV surveillance in ungulates other than cattle.

Complete nucleotide sequence of a novel bovine viral diarrhea virus subtype 1 isolate from Italy.

To gain further insight into the genomic features of bovine viral diarrhea virus 1 (BVDV-1) subtypes, we sequenced the complete genome of the BVDV-1 isolate VE/245/12. This is an uncommon subtype that was isolated from a persistently infected animal. Here, we report the complete genome sequence, consisting of 12,295 nucleotides (nt) with an open reading frame of 11,694 nt encoding 3,898 amino acids. Phylogenetic analysis of the full-length genome, 5'-UTR, and Npro region confirmed that the BVDV-1 isolate differed significantly from all of the bovine pestiviruses identified so far, providing evidence for the presence of a distinct novel genetic group.

Genetic diversity of bovine diarrhea and mucosal disease virus

Abstract Bovine viral diarrhea virus (BVDV) is classified as a member of the Pestivirus genus of the Flaviviridae family. BVDV is one of the most important viral pathogens of ruminants worldwide, causing severe economic losses. Infection results in a wide range of clinical manifestations, ranging from mild respiratory disease to fetal death and mucosal disease. The virus particles are small and contain a singlestranded, positive-sense RNA molecule of approximately 12.3 kb with one large open reading frame flanked by two untranslated regions (5’UTR and 3’UTR). The polyprotein is proteolytically cleaved by viral and host proteases resulting in the formation of mature viral proteins. It is well established that BVDV strains show considerable genetic diversity. BVD viruses are classified as two species: BVDV-1 and BVDV-2. Quite recently, a new putative species, BVDV-3, was detected. The viruses exist as one of two biotypes: cytopathic or non- cytopathic, based on their activity in cell cultures. The phylogenetic analysis of the 5’UTR and Npro region has revealed at least 21 distinct subtypes of BVDV-1 and 4 subtypes of BVDV-2. Genetic diversity of BVD viruses has serious clinical implications such as immune evasion, increase of virulence, host range alteration and also affects the efficacy of vaccination programmes and diagnostic methods. 1. Introduction. 2. Characteristics of BVD virus. 3. Genetic diversity. 4. The importance of genetic variation. 5. Conclusion

Genomic characterization of a bovine viral diarrhea virus subtype 1i in Brazil.

Bovine viral diarrhea virus 1 (BVDV-1) belongs to the genus Pestivirus within the family Flaviviridae. Based on the 5' untranslated region (UTR) sequence, BVDV-1 can be divided into at least 17 subtypes (1a though 1q). BVDV-1i is an uncommon subtype that has been reported in the United Kingdom and Uruguay. Here, we report the complete genome sequence of the first subtype 1i BVDV-1 (strain ACM/BR/2016) isolated from cattle in southern Brazil. The genome is 12,231 nt in length and contains a single ORF that encodes a polyprotein of 3,896 amino acids, flanked by 5' and 3'UTRs of 325 and 220 nt, respectively. Phylogenetic inferences based on the whole genome, the 5'UTR, and the Npro region showed that strain ACM/BR/2016 is closely related to previously characterized BVDV-1i members. Its 5'UTR shares the highest nucleotide identity (90.5%) with BVDV-1i strains from United Kingdom, and its Npro is most closely related to that of a Uruguayan strain (90.6%). To the best of our knowledge, this is the first BVDV-1i strain from which the whole genome has been completely sequenced and characterized. The complete genome of a BVDV-1i will help future studies on pestivirus evolution and heterogeneity.

Genetic typing of bovine viral diarrhoea virus in cattle on Irish farms

The aim was to carry out a phylogenetic study of bovine viral diarrhoea viruses (BVDV) circulating in Irish cattle herds from 2011 to 2014. Three hundred and twenty five viruses from 267 herds were subtyped by nucleotide sequence analysis of the 5′UTR and/or the Npro regions. All viruses investigated in this study belonged to species BVDV-1 with BVDV-1a as the prominent subtype (97%). Subtypes BVDV-1b, BVDV-1d and BVDV-1e were also identified for the first time in Ireland. Pairwise alignments of 225 viruses with complete sequences for the 5′UTR and the Npro regions were performed to determine a low conflict threshold for virus strain demarcation. One hundred and seventy seven unique virus strains were identified. The study revealed significant levels of herd specific clustering of strains but no geographical or temporal clustering. Similar virus strains were identified in different counties, provinces and years indicating the potential to investigate the epidemiology of the disease by combining sequence analysis with animal movement data.

Evidence for Tunisian-Like Pestiviruses Presence in Small Ruminants in Italy Since 2007.

The genus Pestivirus, which belongs to the Flaviviridae family, includes ssRNA+ viruses responsible for infectious diseases in pigs, cattle, sheep, goats and other domestic and wild ruminants. Like most of the RNA viruses, pestivirus has high genome variability with practical consequences on disease epidemiology, diagnosis and control. In addition to the officially recognized species in the genus Pestivirus, such as BVDV-1, BVDV-2, BDV and CSFV, other pestiviruses have been detected. Furthermore, most of the ruminant pestiviruses show low or absent species specificity observed in serological tests and are able to infect multiple species. Particularly, small ruminants are receptive hosts of the most heterogeneous group of pestiviruses. The aim of this study was to carry out the molecular characterization of pestiviruses isolated from sheep and goats in Sicily, Italy. Phylogenetic analysis of two viral genomic regions (a fragment of 5'-UTR and the whole Npro regions) revealed the presence of different pestivirus genotypes in the analysed goat and sheep herds. Two of five viral isolates were clustered with BVDV-1d viruses, a strain widespread in Italy, but never reported in Sicily. The other three isolates formed a distinct cluster with high similarity to Tunisian isolates, recently proposed as a new pestivirus species. This represents the first evidence for Tunisian-like pestivirus presence in small ruminants in Italy. Furthermore, one of the isolates was collected from a goat, representing the first isolation of Tunisian-like pestivirus from this species.

First Evidence of Bovine Viral Diarrhea Virus Infection in Wild Boars

Background: The farming of wild boars has growing due to the interest of the human consumption of this exotic meat. Such a development may pose an increased risk of disease transmission between boars and domestic animals. The wild boar population has increased in South America in the last years due the absence of predator causing economic losses due to direct damage to crops and risk of disease transmission. The genus Pestivirus within the family Flaviviridae are composed by four recognized species by the International Committee on the Taxonomy of Viruses (ICTV): classical swine fever virus (CSFV), border disease virus (BDV), bovine viral diarrhea virus type 1 (BVDV-1) and 2 (BVDV-2). Other putative species denoted as atypical pesitiviruses have been reported as ‘HoBi’-like virus, giraffe pestivirus, Bungowannah pestivirus, Pronghorn antelope virus, atypical porcine pestivirus (APPV), Norwegian rat pestivirus (NrPV) and Rhinolophus affinis bat pestivirus (RaPestV-1). CSFV is commonly detected in wild boars, but despite positive serology, bovine viral diarrhea virus (BVDV) was never detected in this animal species. Thereby, the present communication describes the first detection of BVDV in the lungs of captive boars using RT-PCR and DNA sequencing.Materials, Methods & Results: Forty lung samples from farmed wild boars were collected after slaughter in a commercial abattoir. The organs were crushed separately, centrifuged, and the supernatant was stored for further analysis. The total RNA was isolated using a phenol-based protocol and RT-PCR protocol that amplified 118 bp of 5’ untranslated region (5’UTR) was carried out. One out 40 samples resulted positive. The positive sample had partial fragments of 5’UTR and N terminal autoprotease (Npro) sequenced and analyzed. The strain LV Java/2012 presented 99% of identity in 5’UTR and 98% in Npro region with a BVDV-2 previously reported in bovines in Southern Brazil. In both 5’UTR and Npro phylogenetic analysis, the strain LV Java/2015 clustered with BVDV-2 strains and was most closely related to subtype 2b identified in bovines in Southern Brazil grouping in the same terminal node.Discussion: Wild boars are commonly associated to pathogen transmission to domestic animals. This animal species is considered a reservoir of the pestivirus CSFV and important keys in CSFV control and eradication programs in Europe. Despite indirect presence of BVDV was reported in wild boars by serology tests, the direct detection of the viral agent was never reported. The present study showed the presence of BVDV-2 genomic segments obtained by RT-PCR followed by DNA sequencing in captive wild boars. The reported data suggests a possible importance of this animal species in the epidemiology of ruminant pestiviruses which could interfere in control and eradication programs of these important pathogens for cattle worldwide. The strain LV Java/2012 was closely related to BVDV-2b and presented highest identity with a strain detected in cattle from Southern Brazil. This data suggests that wild boars and bovines could be sharing this pathogen due the similarity of the strains and that both were reported in the same region. It can lead to need of inclusion of wild swines in BVDV control programs since boars can circulate between different regions and carry this pathogen to different cattle herds. The present study reported the first molecular evidence of BVDV in wild boars in the literature. The data generated herein suggests a possible importance of boars in the epidemiology of ruminant pestiviruses.

Increased genetic diversity of BVDV-1: recent findings and implications thereof

Sequence-based genotyping was recently used to distinguish between the BVDV-1 and BVDV-2 species of the bovine viral diarrhoea virus (BVDV). Quite recently, a new putative species, BVDV-3, was also detected. The phylogenetic analysis of the 5'-untranslated region (UTR) and Npro region has revealed at least 17 distinct subtypes for BVDV-1 to date. The aim of this study was to further investigate the genetic heterogeneity of BVDV-1 in Italy, by analysing 173 virus sequences from isolates collected over an 18-year period (1997-2014). Viral RNA was extracted from the original biological samples identified as BVDV-1-positive. Reverse transcription (RT) and polymerase chain reaction (PCR) assays targeting a 288-base pair (bp) region of the 5'-UTR and a 428-bp region encoding the autoprotease Npro were performed, and the RT-PCR products were sequenced. The phylogenetic analysis of the 5'-UTR and Npro sequences re-confirmed the circulation of ten out of eleven subtypes previously discovered in Italy. Interestingly, four isolates differed significantly from all of the bovine pestiviruses identified so far, thereby providing evidence for the circulation of three novel subtypes that have not been documented so far. The growing number of reports on BVDV-1 heterogeneity, including the recent findings reported herein, raises concern related to the emergence and spread of new BVDV variants, with possible implications for animal health and disease control. This global issue needs to be addressed with the highest priority.

Identification and molecular characterization of novel and divergent HoBi-like pestiviruses from naturally infected cattle in India

HoBi-like pestiviruses have been sporadically reported from naturally infected cattle in South America, Asia and Europe. While the closely related bovine viral diarrhoea virus 1 (BVDV-1) and BVDV-2 have been reported from cattle in India, the prevalence and diversity of HoBi-like viruses have not yet been studied. Here we report the genetic diversity and molecular characteristics of HoBi-like viruses, through systematic surveillance in cattle (n=1049) from 21 dairy farms across India during 2012–2013. On the basis of real-time RT-PCR, virus isolation and nucleotide sequencing results, of the 20 pestivirus positive cattle, HoBi-like viruses were identified in 19 cattle from four farms in three states and BVDV-1b in one cattle. Phylogenetic analysis of 5′-UTR and Npro region identified the circulation of two lineages of HoBi-like viruses in India, that were distinct to those circulating globally, highlighting the independent evolution of at least three lineages of HoBi-like viruses globally. Antigenic differences were also evident between the two Indian lineages. In addition to revealing that HoBi-like virus may be more widespread in Indian cattle than previously reported, this study shows greater genetic divergence of HoBi-like viruses indicating a need for continued pestivirus surveillance in cattle.

Molecular investigation of bovine viral diarrhea virus infection in yaks (Bos gruniens) from Qinghai, China

BackgroundBovine viral diarrhea virus (BVDV) is a pestivirus which infects both domestic animals and wildlife species worldwide. In China, cattle are often infected with BVDV of different genotypes, but there is very limited knowledge regarding BVDV infection in Chinese yaks and the genetic diversity of the virus. The objectives of this study were to detect viral infection in yaks in Qinghai, China and to determine the genotypes of BVDV based on analysis of the 5′untranslated region (5′UTR) and N-terminal protease (Npro) region.ResultsBetween 2010 and 2012, 407 blood samples were collected from yaks with or without clinical signs in six counties of Qinghai Province. Ninety-eight samples (24%) were found to be positive by reverse transcription polymerase chain reaction (RT-PCR) targeting a conserved region of BVDV-1 and BVDV-2. The nucleotide sequences of the 5′UTR and complete Npro region were determined for 16 positive samples. Phylogenetic reconstructions demonstrated that all 16 samples belong to subgenotypes BVDV-1b, BVDV-1d and BVDV-1q.ConclusionsThis study provides, for the first time, molecular evidence for BVDV infection in yaks in Qinghai involving multiple subgenotypes of BVDV-1. This may have occurred under three possible scenarios: interspecies transmission, natural infection, and the use of vaccines contaminated with BVDV. The results have important implications for yak production and management in China, and specifically indicate that unscientific vaccination practices should be stopped and bio-security increased.

English

The genetic diversity of bovine viral diarrhea viruses (BVDV) from Beijing region, China between 2009-2010, was investigated using a phylogenetic analysis of partial 5’untranslated region (5’UTR) nucleotide sequences from 61 BVDV positive samples, and for 13 selected samples, the Npro region was also investigated. All the sixty-one blood samples were collected from cattle that were BVDV persistently infected (PI) within a BVD eradication program on Beijing farms during 2009 and 2010. Phylogenetic analysis based on 5’UTR sequences indicated that all BVDV positive samples were of the BVDV-1 genotype, but were clustered into four different subtypes: sub-genotype 1b (n = 31), 1m (n = 24), 1c (n = 4) and 1d (n = 2). BVDV-1b and 1 m are the most prevalent sub-genotypes in Beijing. The results also demonstrate that in most herds, certain BVDV-1 sub-genotype predominates. It is the first time that BVDV-1d is reported in China with unclear origin. The result of this study will be useful in constructing an effective vaccination plan to further control BVDV in Beijing region.   Key words: bovine viral diarrhea viruses 1 (BVDV-1), 5’untranslated region (5’UTR), Npro, phylogenetic analysis.

A phylogenetic analysis of Bovine Viral Diarrhoea Virus (BVDV) isolates from six different regions of the UK and links to animal movement data

Bovine Viral Diarrhoea Virus (BVDV) is a pestivirus which infects cattle populations worldwide and is recognised as a significant source of economic loss through its impact on health and productivity. Studies investigating the molecular epidemiology of BVDV can give invaluable information about the diversity of viral strains present in a population and this, in turn, can inform control programs, drive vaccine development and determine likely infection sources. The current study investigated 104 viral isolates from forty farms across the UK. Through phylogenetic and nucleotide sequence analysis of the 5′UTR and Npro regions of the isolates investigated, it was determined that BVDV 1a was the predominant sub-genotype. However, BVDV 1b, 1e and 1i were also identified and, for the first time in the UK, BVDV 1d. Through analysis of animal movement data alongside the phylogenetic analysis of these BVD isolates, it was possible to link animal movements to the viral isolates present on several premises and, for the first time, begin to elucidate the routes of viral transmission. With further work, this type of analysis would enable accurate determination and quantification of the true biosecurity risk factors associated with BVDV transmission.

Increased phylogenetic diversity of bovine viral diarrhoea virus type 1 isolates in England and Wales since 2001

Currently, there are two recognised genotypes of Bovine viral diarrhoea virus (BVDV), type 1 and type 2. These genotypes are divided into subtypes based on phylogenetic analysis, namely a–p for BVDV-1 and a–c for BVDV-2. Within this study, the genetic heterogeneity of BVDV-1 in England and Wales was investigated and compared to the situation in 1996/1997. Viral RNA was extracted from 316 blood samples collected between 2004 and 2009 that were previously identified as BVDV-1 positive. A region of the 5′ untranslated region (UTR) was amplified by RT-PCR and the PCR products were sequenced. Phylogenetic analysis of the 5′UTR demonstrated the existence of five subtypes of BVDV-1 circulating in England and Wales, namely BVDV-1a (244 samples), BVDV-1b (50), BVDV-1e (3), BVDV-1f (1) and BVDV-1i (18). Phylogenetic analysis of the nucleotide sequence for the Npro region of the viral genome supported the classification obtained with the 5′UTR. Given the fact that only three subtypes were detected in 1999 this report supports the notion that the restocking of cattle from continental Europe, after the mass culling during the Foot-and-Mouth outbreak in 2001 and slaughter of cattle due to bovine tuberculosis infection, has increased the genetic diversity of BVDV-1 subtypes in England and Wales in the past 10years.