Genetic Typing of Bovine Viral Diarrhoea Virus in Cattle on Irish Farms

Transcription

1 Genetic Typing of Bovine Viral Diarrhoea Virus in Cattle on Irish Farms Thesis presented by Eoin O Brien BSc. Submitted to the University of Limerick, November 2015, for the award of Master of Science

2 Genetic Typing of Bovine Viral Diarrhoea Virus in Cattle on Irish Farms Thesis presented by Eoin O Brien BSc. Student: Eoin O'Brien Student ID: Date: July 2015 Supervisors: Prof. Sean Arkins Prof. Ann Cullinane Submitted to the University of Limerick, November 2015, for the award of Master of Science

3 Table of Contents Abstract Bovine viral diarrhoea (BVD) is a pestivirus infection of cattle and other domesticated and free living animals. Two genotypes, bovine viral diarrhoea virus type 1 (BVDV-1) and bovine viral diarrhoea virus type 2 (BVDV-2) are recognised with 17 subgroups of BVDV 1 and 3 subgroups of BVDV 2. However, little is known about the genotypes of BVDV circulating in Irish cattle herds and the aim of this project was to characterise the BVD virus types archived at the Irish Equine Centre. A database of all BVDV positive diagnostic samples archived at the Irish Equine Centre was compiled. This consisted of information relating to in excess of one thousand blood and milk samples submitted for analysis between 2011 and A randomized panel of 375 virus positive samples representative of location, type (beef or dairy), gender, age, breed, year and month of detection was selected for genotyping. Total nucleic acid was extracted from all samples and the RNA concentration was measured using a biophotometer. Forward and reverse primers were synthesised to target two regions consistently conserved among all pestiviruses, a highly conserved 288bp portion of the 5 untranslated region (UTR) and a more variable 428bp segment of the N-terminal autoprotease (N pro ) gene. A one-step reverse transcriptase polymerase chain reaction (RT- PCR) method was specifically optimized to amplify both targets. Amplified products were gel purified, sequenced and aligned with 5 UTR and N pro gene sequences representative of all known BVDV genotypes and sub-genotypes. The genotype and subgenotype was established for 325 field viruses and one commercial vaccine viral strain based on the analysis of at least one genomic region. BVDV-1a was the prominent subgenotype (n=317) with BVDV-1b (n=6), BVDV-1d (n=1) and BVDV-1e (n=1) also observed. A number of nucleotide sequence alignments and phylogenetic trees were constructed to represent the relationship between Irish field viruses and reference strains representative of all known BVDV genotypes and subgenotypes. Evidence of both herd specific clustering and circulation of multiple strains within herds was observed; however no evidence of county specific clustering was observed. A number of completely conserved nucleotide regions were observed and the 5 UTR was found to be more conserved than the N pro fragment analysed. The results of this study provide a detailed and comprehensive insight into the genetic diversity of BVDV in circulation within Irish cattle herds and will act as a baseline reference for future BVDV genotyping studies. i

4 Table of Contents Author s Declaration I hereby declare that this thesis has not been previously submitted to this or any other institution. It is entirely my own original work and where the use or reference has been made of the work of any other persons, it has been fully acknowledged and properly referenced. Singed: Date: Eoin O'Brien ii

5 Table of Contents Acknowledgements I would like to take this opportunity to acknowledge the following; Prof. Ann Cullinane, Head of Virology Department, Irish Equine Centre, for her unfailing support and provision of unlimited resources which enabled me to conduct this research project Prof. Sean Arkins, Head of Life Sciences Department, University of Limerick, for his inspiration and guidance as my academic supervisor Marie Garvey and all the staff within the Virology Department of the Irish Equine Centre who generously shared their vast knowledge and experience of molecular biology and phylogenetic analysis throughout the duration of this research project The Irish Research Council together with their strategic funding partners, Teagasc and the Department of Agriculture, Food and the Marine (DAFM), who provided funding without which this project would not have been possible. iii

6 Table of Contents Table of Contents Abstract... i Author s Declaration...ii Acknowledgements... iii Table of Contents... iv List of Abbreviations... vi List of Figures... viii List of Tables... ix Chapter 1 Literature Review...1 Introduction... 2 Causative Agent... 4 Classification of Pestiviruses... 5 Molecular Characteristics of BVDV... 8 Pathogenesis Clinical Signs Epidemiology Laboratory Diagnosis Prevalence of BVDV Genotypes and Sub-genotypes Control of BVD Irish National BVD Eradication Programme Economic Impact of BVDV Conclusion Thesis Objectives Chapter 2 Materials and Methods Field Viruses RNA Isolation iv

7 Table of Contents RT-PCR Amplification DNA Purification, Quantification and Sequencing Sequence Alignment and Phylogenetic Analysis Serological Analysis The effect of Variable Factors Chapter 3 Results Chapter 4 Discussion and Conclusions Genetic Diversity of BVDV in Ireland Importance of Surveillance Possible Source(s) of Increasing Genetic Diversity in Ireland Genotyping and Identification of Transmission Pathways Genetic Clustering of BVDV on Irish Farms Nucleotide Sequence Analysis Genotyping and Vaccine Efficacy The Benefits of a National/International BVDV Database Conclusion Bibliography Appendices Appendix Appendix Appendix Appendix v

8 Table of Contents List of Abbreviations Bovine viral diarrhoea Bovine viral diarrhoea virus Bovine viral diarrhoea type 1 Bovine viral diarrhoea type 2 Border disease virus Classical swine fever virus BVD BVDV BVDV-1 BVDV-2 BDV CSFV 5 untranslated region 5 UTR N-terminal autoprotease Persistent infection Transient infection Cytopathic Noncytopathic Enzyme-Linked Immunsorbent Assay Polymerase Chain Reaction Reverse Transcription Polymerase Chain Reaction Mucosal disease Ribonucleic acid Deoxyribonucleic acid Virus isolation Animal identification and movement database Irish Cattle Breeders Federation N pro PI TI cp ncp ELISA PCR RT-PCR MD RNA DNA VI AIM ICBF vi

9 Table of Contents Nucleotide Base pair Bovine parainfluenza virus type 3 Bovine respiratory syncytial virus Bovine neonatal pancytopenia Animal Health Ireland Antibody Antigen Friedrich Loeffler Institut Sample Identification Department of Agriculture Food and the Marine Ethylenediaminetetraacetic nt bp bpi3 brsv BNP AHI Ab Ag FLI SID DAFM EDTA vii

10 Table of Contents List of Figures Figure 1.1: Phylogenetic tree representing genetic diversity within the Pestivirus genus Figure 1.2: Structure of the BVDV Genome... 9 Figure 1.3: Phylogenetic tree of a 244 bp portion of the BVDV 5 UTR reference strains used in this study Figure 3.1: Phylogenetic tree representing a 244 bp portion of the BVDV 5 UTR for a selection of viruses sequenced in this study Figure 3.2: Phylogenetic tree representing a 380 bp region of the BVDV Npro for a selection of viruses sequenced in this study Figure 3.3: Phylogenetic tree illustrating the herd specific clustering and diversity among a selection of viruses following alignment of a 244 bp fragment of the BVDV 5 UTR for a selection of viruses sequenced in this study Figure 3.4: Phylogenetic tree illustrating the herd specific clustering and diversity among a selection of viruses following alignment of a 380 bp fragment of the BVDV Npro gene for a selection of viruses sequenced in this study Figure 3.5: Phylogenetic tree illustrating the genetic diversity among a selection of viruses collected in Cork, Galway and Offaly following alignment of a 244 bp fragment of the BVDV 5 UTR Figure 3.6: Pairwise multiple alignment of a 244 bp fragment from the BVDV 5 UTR of a selection of the field viruses analysed in this study Figure 3.7: Pairwise multiple alignment of a 380 bp fragment from the BVDV Npro gene of a selection of the field viruses analysed in this study Appendix 1.1: Phylogenetic tree representing the upper portion of a 244 bp region of all the BVDV 5 UTR nucleotide sequences analysed in this study Appendix 1.2: Phylogenetic tree representing the lower portion of a 244 bp region of all the BVDV 5 UTR nucleotide sequences analysed in this study Appendix 2.1: Phylogenetic tree representing the upper portion of a 380 bp region of all the BVDV Npro nucleotide sequences analysed in this study Appendix 2.2: Phylogenetic tree representing the lower portion of a 380 bp region of all the BVDV Npro nucleotide sequences analysed in this study viii

11 Table of Contents List of Tables Table 1.1: List of commercial BVD ELISA kits approved for use by the Friedrich Loeffler Institut Table 1.2: Worldwide prevalence of BVDV genotypes and sub-genotypes Table 2.1: Summary of the quantity and sample identification (SID) of viruses per sample type analysed in this study Table 2.2: Sequence detection primers used in this study for the partial amplification of the BVDV 5 UTR and the N pro gene Table 2.3: Components of master mix used for RT-PCR amplification Table 2.4: RT-PCR conditions optimised in this study for the partial amplification of the BVDV 5 UTR and the N pro Table 2.5: Description of Pestivirus reference strains used during phylogenetic analysis Table 3.1: Summary of field viruses analysed in this study Table 3.2: Breakdown of the breed groups included in this study Table 3.3: Summary of herds with multiple viruses Table 3.4: Summary of viruses which share 100% nucleotide identity in both a 244 bp fragment of the 5 UTR and a 380 bp fragment of the Npro gene Table 4.1: Summary of live cattle imported into Ireland between 2005 and ix

12 Chapter 1 Literature Review Chapter 1 Literature Review 1

13 Chapter 1 Literature Review Introduction Bovine Viral Diarrhoea virus (BVDV) is associated with serious clinical disease in cattle and significant economic loss (Bachofen et al. 2010; Barrett et al. 2011; Houe 1999; Houe 2003; Lindberg 2002; Sayers 2014). Eradication and control programmes are becoming increasingly common internationally (Stahl et al. 2005; Lindberg 2002; Presi et al. 2011) and will benefit from a greater understanding of the complex epidemiology and pathogenesis of the virus. The International Committee on Taxonomy of viruses classifies BVDV type 1 and 2 as members of the Pestivirus genus, along with classical swine fever and border disease virus, within the Flaviviridae family (International Committee on Taxonomy of viruses 2013). Numerous atypical Pestiviruses have been isolated from various host species internationally; however the genus currently contains four officially classified species: Bovine viral diarrhoea type 1 (BVDV-1), Bovine viral diarrhoea type 2 (BVDV-2), Border disease virus (BDV) and Classical swine fever virus (CSFV). BVD is an infectious disease of both domesticated and free living animals including: cattle, (Mainar-Jaime et al. 2001), pigs (Tao et al. 2013), sheep (Paton et al. 1995), deer (Becher et al. 1999; Harasawa 2000), alpaca (Kim et al. 2009) and camels (Gao et al. 2013). BVDV is endemic in many countries internationally including numerous countries within Europe, North and South America, Oceania, Asia and Africa. An analysis of laboratory submissions in Ireland between 2005 and 2008 concluded that the prevalence of BVDV positive animals was 0.6% while the seroprevalence of BVD was established as 69% (O Neill et al. 2009). Sayers et al. (2015a) identified that 88% of bulk milk samples tested were seropositive while a lower seroprevalence (25%) was identified when younger animals (mean age = 291 days) were analysed. The percentage positivity for BVDV during the first year of the compulsory Irish national BVD eradication programme was 0.77% (Animal Health Ireland 2014c). BVDV can be categorized into two species: the more prevalent BVDV type 1 (BVDV-1) and the more virulent BVDV type 2 (BVDV-2). The sub-division of both species of BVDV has been documented with the more genetically diverse, BVDV-1, containing 17 subgenotypes (Gao et al. 2013). Two subgenotypes of BVDV-2 2

14 Chapter 1 Literature Review (BVDV type 2a and BVDV type 2b) have also been demonstrated in a number of studies (Fulton et al. 2005; Flores et al. 2002). A third subtype was tentatively suggested in 2001 (Tajima et al. 2001) prior to a German outbreak of BVDV in 2012 and 2013 from which BVDV-2c was genotyped (Doll and Holsteg 2013). Numerous studies have been conducted internationally to identify the genotypes and sun-genotypes of BVDV in circulation within individual countries or regions (Booth et al. 2013; Strong et al. 2013; Vilcek et al. 1999; Graham et al. 2001; Arias et al. 2003). Little is known about the genotypes of BVDV circulating in Irish cattle herds. This absence of published literature classifying BVD viruses in Ireland prompted this study which aimed to identify the genotype and subgenotypes of a representative population of viruses collected from Irish herds. BVDV can induce a broad range of disease in cattle including; fever, diarrhoea, ill thrift, reduced milk yield, reduced reproductive performance, immunosuppression and, in a high proportion of cases, subclinical infection which often may go undetected (Peterson 2010; Paton et al. 1989; Grooms 2004; Lanyon et al. 2004; Evermann and Ridpath 2002). BVDV-1 and BVDV-2 infection can result in an acute Transient Infection (TI) and/or a Persistent Infection (PI). Following a 2-5 day incubation period animals transiently infected with BVDV, and shed virus for a period of 4-10 days (Brownlie et al. 1987). During this period a slow immune response is initiated with antibody levels peaking at weeks (Brownlie et al. 1987). In the majority of cases the disease is self-limiting (Peterson 2010). However, animals infected with a more virulent strain of BVDV can experience a more aggressive acute infection which may be fatal (Bachofen et al. 2010). When a pregnant female is transiently infected with a non-cytopathic (ncp) strain of BVDV persistent infection in the foetus may occur (Bachofen et al. 2010). PI animals act as the main reservoir of BVDV as they shed a large amount virus continuously throughout their life time (Brownlie et al. 1984). There are a number of diagnostic tools available to identify the presence of BVDV within a herd and to identify if an individual animal is infected. The circulation of BVDV within a herd can be identified by initially screening a sample population for the presence of BVDV specific antibodies. If animals are diagnosed as antibody positive the source of infection may be determined by virus detection tests. Many 3

15 Chapter 1 Literature Review BVD control and eradication programmes are based on the principle of the identification and removal of PI animals (Dubovi 2013; Barrett et al. 2011). Infection can have a serious negative impact on herd health and welfare within farms (Brülisauer et al. 2010). This can often result in an economic loss due to reduced milk yield, poor fertility, respiratory disease and fatalities (Houe 1999). Animal Health Ireland (Kadir et al. 2008) estimates that BVD causes 102 million in losses to the Irish cattle industry annually (Stott et al. 2012). BVD was identified as a high priority disease among farmers and experts following a Policy Delphi study commissioned by AHI which prompted the implementation of a national BVD eradication programme in Ireland (More et al. 2010). The aim of the present study was to identify the genotypes and subgenotypes of BVDV strains in circulation throughout Ireland between 2011 and An additional aim was to identify whether genetic clustering has occurred within geographic regions and within individual herds in Ireland. Causative Agent BVDV-1 and BVDV-2, together with Border Disease virus (BDV) and Classical Swine Fever virus (CSFV) belong to the genus Pestivirus in the Flaviviridae family. There are three remaining genera within this family of viruses, Flavivirus (53 species), Hepacivirus (1 species) and Pegivirus (2 species). The Flaviviridae family remains unassigned to an order of viruses (International Committee on Taxonomy of viruses 2013). Flaviviridae virus s share a number of common structural traits as all are single stranded, positive sense RNA viruses with non-segmented genomes and one open reading frame (Kronen 2008). The complete nucleotide genome of Flaviviridae viruses is 9,500 to 12,500 nucleotides in length (Virus Pathogen Resource 2014). Some members of the Flaviviridae family such as West Nile virus, Japanese Encephalitis virus and the tick borne encephalitis viruses are of significant global concern as in addition to being veterinary pathogens, they are a major risk to humans. Transmission typically occurs via vectors such as mosquitoes and ticks; however direct contact with infected animals is sufficient for the transmission of 4

16 Chapter 1 Literature Review certain Flaviviridae viruses (Kronen 2008). A high proportion of Flaviviridae viruses can persist in a host for considerable periods of time creating a natural reservoir of the virus. The typical clinical consequences of Flaviviridae virus infections include: fever, haemorrhagic fever and encephalitis (Virus Pathogen Resource 2014). Classification of Pestiviruses The International Committee on Taxonomy of viruses recognises four species within the Pestivirus genus: Bovine viral diarrhoea type 1 (BVDV-1), Bovine viral diarrhoea type 2 (BVDV-2), Border disease virus (BDV) and Classical swine fever virus (CSFV) (International Committee on Taxonomy of viruses 2013). Interestingly, a fifth Pestivirus species was proposed due to the genetic diversity of an isolate collected from a Kenyan Giraffe in 1967 (Becher et al. 1997).This species has not yet been accepted in to the genus by the committee. Previously Becher and others (1999) suggested that a pair of German Pestivirus isolates collected from a bison and a reindeer housed in Duisburg Zoo belonged to a novel species. However, when the reindeer virus was subjected to further phylogenetic analysis it was found to cluster towards a subgenotype of Border Disease Virus (Vilcek et al. 2010). In addition, two putative species known as Pronghorn and Bungowannah have also been reported in recent years. The Pronghorn virus was isolated from an apparently blind free ranging pronghorn antelope in the US state of Wyoming (Vilcek et al. 2010). The Bungowannah virus was detected on a pig farm in Australia during an outbreak which resulted in numerous sudden deaths, stillbirths and mummified piglets in 2003 (Kirkland et al. 2007). The classification of field isolates collected from sheep and sheep pox vaccines in use in Tunisia during a severe outbreak of reproductive disease in 1995 revealed another potential member of the Pestivirus genus. The onset of suspected BDV immediately following sheep pox vaccination suggested Pestivirus contamination. Phylogenetic analysis illustrated that the field isolates and contaminants in the vaccines form a unique cluster (Thabti et al. 2005), which later became known as Tunisian Sheep Virus (TSV) (Liu et al. 2009). 5

17 Chapter 1 Literature Review Furthermore, an additional species of Pestivirus tentatively referred to as HoBi-like, BVDV type 3 or atypical viruses has been described in a number of studies. The initial discovery was made when a batch of foetal calf serum (FCS) which originated from Brazil was screened and a virus designated D32/00_ HoBi was isolated (Schirrmeier et al. 2004). The first reported case of natural infection with a HoBilike virus was documented following the isolation of virus TH/04_KhonKaen collected from a dairy herd in Thailand in 2003/2004 (Stahl et al. 2007). This group of viruses is believed to be in circulation in Europe following isolation of a HoBilike virus on an Italian farm in The virus, named Italy-1/10-1, was isolated from a calf which died during an outbreak of severe respiratory disease over a three month period (Decaro et al. 2011). The genetic relationship between all recognised Pestivirus species including the proposed atypical viruses mentioned above are represented in Figure 1.1. Categorization of viruses into new Pestivirus species is determined by a number of factors including; nucleotide sequence comparison, the identification of an N pro coding region, antigenic characterisation and in some cases the host of origin (Kirkland et al. 2007; Liu et al. 2009). Although the putative Pestiviruses described above have been proposed as new species, to-date four officially recognised species occupy the genus. The HoBi-like viruses pose a greater risk to livestock globally than other putative species described above due to the increasing demand for FCS which is routinely used in vaccine formulation and embryo transfer procedures (Bauermann et al. 2013). This hypothesis was confirmed when 33 batches of commercial FCS from 11 countries were screened for the detection of bovine Pestiviruses by RT-PCR. All 33 batches tested positive and further phylogenetic analysis suggested that 13 batches were contaminated with an atypical bovine pestivirus strain. The 13 atypical viruses appear to have originated from Brazil, Australia, Canada, Mexico, USA and South America (Xia et al. 2011). 6

18 Chapter 1 Literature Review Figure 1.1: Phylogenetic tree representing genetic diversity within the Pestivirus genus. The tree was computed by the Neighbour-Joining method and incorporated the Kimura two parameter statistical model using the MEGA6 program. The symbol represents members of the BVDV-1 species, = BVDV-2, = Border Disease virus, = Classical Swine Fever virus and signifies atypical pestiviruses. 7

19 Chapter 1 Literature Review Molecular Characteristics of BVDV The BVDV genome is approximately 12,300 bases in length and consists of a positive sense, single stranded RNA molecule (Neill 2013; Sandvik et al. 2001; Vilcek et al. 2007). However, the length of the genome can vary depending on the virus strain analysed as duplications of viral genes or the insertion of host cell genes may alter the amount of bases, particularly in cytopathogenic genomes (Neill 2013). The BVDV genome is constructed of a single open reading frame approximately 11,700 nucleotides in length which is flanked by two highly conserved untranslated regions (UTR). The UTR at the five prime end (5 UTR) can range from 360 to 400 nucleotides long while the three prime UTR fragment (3 UTR) is much shorter, ranging from 190 to 270 nucleotides (Vilcek et al. 1997a; Vilcek et al. 2007). Both UTR regions of the BVDV genome are involved in transcription and translation as they create secondary structures that interact with each other as well as other proteins regulating RNA replication which is initiated at the 3 UTR (Neill 2013; Vilcek et al. 2007). The 5 UTR is commonly targeted for diagnostic assays and genotyping due to its high level of conservation between strains. Most of the genome consists of a single open reading frame of about 4000 codons. Translation of the genome yields one precursor poly protein, which is cleaved co- and postranslationally by viral and host cell encoded proteases to generate structural and non-structural proteins. These include the non-structural N-terminal autoprotease (N pro ) (Vilcek et al. 2007), the structural capsid protein and three envelope glycoproteins E rns, E1, and the E2 protein (Tajima 2004) and the non-structural proteins p7, NS2, NS3 (NS23), NS4a, NS4b, NS5a with NS5b adjoining the 3 UTR (Neill 2013; Goyal and Ridpath 2005). The organisation of the BVDV genome is illustrated in Figure

20 Chapter 1 Literature Review Figure 1.2: Structure of the BVDV Genome Bovine viral diarrhoea virus is classified into two genotypes, BVDV-1 and BVDV-2, both of which contain a number of sub genotypes. The number of BVDV-1 subgenotypes is continuously increasing as more isolates are analysed internationally. Vilcek et al. (2001) identified that BVDV-1 contains eleven subtypes; however following a more recent study Xue et al. (2010) suggested there are at least sixteen subtypes; BVDV-1a to BVDV-1p. In 2013 a BVDV-1q subgroup was isolated from a Bactrian camel in China (Gao et al. 2013). There is less diversity between BVDV-2 isolates, with two subtypes, 2a and 2b, identified (Fulton et al. 2005; Flores et al. 2002), although, a third subtype, 2c has been proposed (Tajima et al. 2001). This subtype was found to be the cause of a severely virulent BVD outbreak in Germany during 2012 and 2013 (Doll and Holsteg 2013). Figure 1.3 illustrates the relatedness between viruses representative of all know BVDV genotypes and subgenotypes. 9

21 Chapter 1 Literature Review Figure 1.3: Phylogenetic tree of a 244 bp portion of the BVDV 5 UTR reference strains used in this study. Each reference strain is labelled with the name of the strain followed by the subgenotype it belongs to. Additional information relating to reference strains used in this study can be found in Table 2.5. The symbol included in the reference strain name represents the region from which the virus was detected; = Ireland, = UK, = Europe, = USA and represents the other regions. The tree was computed by the Neighbour-Joining method and incorporated the Kimura two parameter statistical model using the MEGA6 program (Tamura et al. 2013; Saitou and Nei 1987; Kimura 1980). 10

22 Chapter 1 Literature Review The BVDV structural proteins are those that are incorporated into the viral capsid or envelope. The E rns differs from the E1 and E2 proteins as it is not embedded directly in to the membrane but bound by a C-terminal domain. This results in the protein being secreted (hence the name rns) in a soluble form by infected cells having a weak neutralizing activity (Neill 2013). The continuous secretion of the E rns protein plays an important role in the diagnosis of BVDV as its detection in a clinical sample is indicative of BVDV infection (Tews and Meyers 2007). The gene encoding the E2 protein is noted to be the least conserved region of the BVDV genome. The E2 protein contains antigenic determinants (epitopes) that are recognised by the host s immune system initiating the production of neutralizing antibodies. This characteristic can be exploited when designing vaccines to provide immunity against BVDV infection (Wang et al. 2015). To date, little is known about the function of E1 structural protein but both E1 and E2 are necessary for infectivity. The C- terminus of the E2 ectodomain, i.e. the portion of the protein outside the virus, is believed to possess the receptor binding function (Liang et al. 2003). The N pro is unique to Pestiviruses and while it is believed not to be required for virus replication, it does however provide the important function of inhibiting interferon production by the host cells (Seago et al. 2010). This inhibition aids the virus in penetrating cells as it suppresses the host s ability to defend cells from infection (Seago et al. 2010). The N pro gene is useful for genotyping as it does not appear to have any function influenced by evolutionary selective pressure and is suitable for typing closely related strains as it is a more genetically diverse region than the 5 UTR (Sandvik et al. 2001). It therefore provides better resolution of the branches on a phylogenetic tree (Vilcek et al. 2005). The p7 non-structural protein follows the E2 protein from which it is ineffectively cleaved resulting in either two separate proteins (e2 and p7) or a combined structure (E2-p7) (Neill 2013; Goyal and Ridpath 2005). The production of infectious virus particles is believed to be the main role of p7 as it appears to have no role in BVDV replication (Goyal and Ridpath 2005). A role in cell-to-cell spread of virus has been proposed (Griffin et al. 2004). The two non-structural proteins, NS2 and NS3 are encoded following the p7 protein. A noncytopathic (ncp) infection results in a single uncleaved unprocessed protein, 11

23 Chapter 1 Literature Review NS2/3. A cytopathic (cp) infection is the result of the cleavage between the two proteins forming NS2 and NS3 due to genomic insertions, duplications or mutations (Kummerer and Meyers 2000; Qi et al. 1992) The cleavage of NS2 and NS3 is also regulated by a protease activity of the NS2 protein and cleavage between NS2/NS3 is believed to be required for replication in the early stages of an ncp infection before cleavage reduces considerably as the infection progresses (Neill 2013). NS2 is also essential for the migration of the protein to the endoplasmic reticulum. The NS3 protein is essential for the viability of the virus as it contains a serine protease domain which cleaves peptide bonds and also contains an RNA helicase domain which assists with replication and transcription of the virus (Neill 2013). The NS4a and NS4b proteins are both hydrophobic and do not appear to induce an immune response by the host (Weiskircher et al. 2009). NS4a is believed to be a cofactor for the NS3 serine protease, while NS4b is involved in the replication of RNA and also appears to influence the cytopathogenicity of the virus. NS5a is a hydrophilic phosphoprotein with apparent RNA binding activity and has a role in RNA replication (Isken et al. 2014). NS5b functions as the dominant protein in genomic RNA replication due to its RNA-dependent RNA polymerase activity (Neill 2013). Phosphorylation of both NS5a and NS5b proteins by cellular kinases has been observed in all Flaviviridae genera including both BVDV species (Reed et al. 1998). Pathogenesis Postnatal animals with no BVDV immunity develop a transient infection when infected with the virus, typically with a mild fever as the initial clinical sign (Corapi et al. 1989; Pedrera at al. 2012). Experimental infection of calves has identified that BVDV RNA was detectable between 3 and 12 days post infection (Pedrera at al. 2012), however virus has been detected up to 40 days post infection (Corapi et al. 1989). Leukopenia is frequently observed in acutely infected animals coupled to suboptimal B and T-cell response which results in the host becoming susceptible to a range of other pathogens (Howard 1990). The ability of BVDV to bind to CD46, a 12

24 Chapter 1 Literature Review complement receptor which expresses itself on a range of cell types within the host, also promotes the susceptibility of the host to a range of secondary pathogens (Cattaneo 2004). Many transient infections are subclinical. In some cases, however the animals may experience anorexia, reduced milk yield or diarrhoea before an immune response slowly develops at approximately 2 weeks post infection reaching peak levels at 10 to 12 weeks (Brownlie et al. 1987). The clinical significance of a BVDV infection can be exacerbated by secondary infection due to an impaired immune system which may explain why some field outbreaks have a greater clinical severity than experimental infections (Brownlie et al. 1987). Evidence of reduced reproductive performance has also been observed with pregnancy rates of 33% (group 1) and 39% (group 2) recorded for heifers experimentally infected 4 days post insemination and 9 day prior to insemination respectively, compared to 79% within the control group (McGowan et al. 1993). Conception failure was prominent in group 2, while there was a higher level of embryonic death in the group infected post insemination (McGowan et al. 1993). The virus may occupy and replicate within the placenta during pregnancy resulting in transmission to the foetus. However there has also been evidence of maternal vascular endothelium damage post infection which may result in abortion due to the onset of placentitis (Brownlie et al. 1998). All genotypes of BVDV can be classified as one of two biotypes, cytopathic (cp) or noncytopathic (ncp), based on their behaviour when grown in cell culture. Infection with a cytopathic strain of BVDV, in vitro, leads to the disruption of cellular structure while noncytopathic infection causes no change to cell structure (Bendfeldt et al. 2007; Goyal and Ridpath 2005). NCP viruses have the potential to develop into CP biotypes when subjected to genomic insertions, duplications or mutations (Kummerer and Meyers 2000; Qi et al. 1992). The co-circulation of both biotypes has been associated with virus-induced apotosis which has the potential to result in severe clinical consequences for the host (Ammari et al The ability of BVDV to persist is due to a transplacental infection of the foetus by a ncp BVDV strain during the middle stage of gestation (Bachofen et al. 2010; Corapi et al. 1988; Bendfeldt et al. 2007). A PI calf is the progeny of a TI dam infected between 18 and 125 days of pregnancy. Alternatively a PI dam will always yield a PI 13

25 Chapter 1 Literature Review calf (Bolin 2002). It is believed that while the embryo is unattached during the first 18 days of pregnancy persistence is unlikely (Moenning and Leiss 1995). However following implantation the foetus is susceptible to persistent infection up to 125 days of pregnancy. During this period the foetal immune system has not developed and cannot protect the foetus against the invading virus (Niewiesk 2014). This failure to recognise BVDV as foreign results in immunotolerance, allowing the virus to persist throughout the host s lifetime as the viral proteins are recognised as self-antigens (Brownlie et al. 1998). After day 125 of gestation a foetus typically has adequate immunocompetence to mount an effective immune response against BVDV infection and are born seropositive (Grooms 2004; Radostits and Littlejohns 1988). Teratogenesis by BVDV may result in a number of outcomes depending on the severity of lesions and the stage of pregnancy (Brownlie et al. 1998) resulting in the development of congenital defects (Van Oirschot 1983). Congenital defects are highly variable with central nervous system (CNS) defects most frequently observed (Grooms 2004). Congenital defects develop in the foetus typically when infected during the mid-gestation stage (Radostits and Littlejohns 1988). CNS defects include cerebellar hypoplasia (Ward et al. 1969), microencephalopathy, hydrocephalus and hydranencephaly (Badman et al. 1981), while incidences of optic neuritis, alopecia (Yeruham et al. 2001) and growth retardation have also been observed (Done et al. 1980). Experimental BVDV infection identified the presence of antigen-bearing cells in multiple lymphoid organs and some squamous epithelial cells of acutely infected animals. Viral antigen was located in the cerebral cortex and within multiple tissues of non-lymphoid organs of persistently infected animals (Marshall et al. 1996). This distribution of BVDV within multiple organs results in the highly variable clinical consequences. Cytopathic BVDV is the causative agent of mucosal disease which occurs as a result of spontaneous mutation of the original ncp BVDV strain persistent in the animal (Lindberg 2002; Bachofen et al. 2010). A cp virus is distinctive due to its behaviour in cell culture and it is this characteristic which caused cell destruction to the host typical of an animal infected with MD (Neill 2013). Evidence suggests that clinical MD effects animals which were infected with BVDV in early pregnancy and have a 14

26 Chapter 1 Literature Review specific immunotolerance (Radostits and Littlejohns 1988). Interestingly, mucosal disease was found to be more prevalent in older animals and has been suggested that this may be due to a longer time period for genetic mutations to occur as a result of viral replication (Bachofen et al. 2010). There is also evidence to suggest that MD may develop due to the super infection of an animal persistently infected with ncp BVDV becoming infected with a secondary cp BVDV (Bachofen et al. 2010). This process has been demonstrated experimentally with both ncp and cp BVDV isolated from MD infected animals (Brownlie et al. 1984; Bolin et al. 1985). The cp virus is unchallenged by the host immune system as the host is BVDV immunotolerent due to the ncp persistent infection (Brownlie et al. 1998). Post-mortem analysis of MD infected animals typically reveals mucosal lesions and cytopathicity of the lymphoid tissue in the gastrointestinal tract (Liebler et al. 1995; Brownlie et al. 1984). Both biotypes have been isolated from chronically infected animals which presented with mucosal disease. The secondary cp BVDV infection can be derived from a number of sources including the PI animal becoming infected with a new field strain of cp BVDV or due to administering a vaccination containing a modified live cp strain of BVDV which in some cases can result in the development of mucosal disease (Goyal and Ridpath 2005). There are a number of clinical consequences of a BVDV infection which are highly variable due to the genetic and antigenic diversity of the virus (Bachofen et al. 2010). BVDV can also induce immunosuppression leaving the animal susceptible to other viral or bacterial diseases (Zhang et al. 2014). Seronegative (i.e. immunologically) naïve females are a high risk population within herds. If this population of females become infected with BVDV the outcome is dependent on the age and immunological maturity of the foetus at the time of infection (Altamiranda et al. 2013). Numerous studies suggest varying timelines of gestation as to when immunotollerance is initiated, ranging from 18 to 125 days of gestation indicating that case to case variation exists (Bolin 2002; Grooms 2004; Lanyon et al. 2004; Evermann and Ridpath 2002). 15

27 Chapter 1 Literature Review Clinical Signs An animal exposed and subsequently infected with BVDV may develop an acute or subclinical infection (Bolin and Grooms 2004). An acute infection may result in fever, diarrhoea, reduced milk yield, weight loss, anorexia, depression and coughing (Peterson 2010). Bulls may suffer a reduction in sperm motility and an increase in sperm abnormalities (Paton et al. 1989). Subclinical infections are often undetected by the herd owner. BVDV infections may result in poor conception rates, early embryonic death, abortions and the development of PI calves (Bachofen et al. 2010). Reduced reproductive performance, including reduced conception rates and early embryonic death are likely to occur if infection occurs early in gestation. Transplacental BVDV infection of foetuses can result in congenital defects and immunotolerance to the virus, resulting in the birth of a PI calf (Grooms 2004; Lanyon et al. 2004; Evermann and Ridpath 2002). Infection during late gestation, (>125 days), can result in weak or under developed calves or the foetus may mount an effective immune response and be normal at birth (Evermann and Ridpath 2002). PI animals may be clinically normal but often suffer chronic ill thrift. Mortality often reaches 50% in the first year of life. Mucosal Disease (MD) may develop usually between 6 months and 2 years of age (Brownlie et al. 1987). Clinical signs include pyrexia, anorexia, erosions in the mouth and hoof, nasal discharge, diarrhoea and increased salivation. MD is fatal and the animal usually dies 3 to 10 days following the development of the clinical signs (Brownlie et al. 1987). However a minority may survive for several months. PI animals infected with a highly virulent strain of BVDV-2 may lead to the development of haemorrhagic syndrome which like, MD, has a high mortality rate within herds (Jackova et al. 2008). 16

28 Chapter 1 Literature Review Epidemiology BVDV spreads primarily by direct contact between cattle. PI cattle play a major role in the maintenance and transmission of virus (Houe 1999; Lindberg 2002; Mainar- Jaime et al. 2001).. The most efficient method of transmission for BVDV is direct transmission between PI animals and seronegative animals as PI (Houe 1999; Lindberg 2002; Mainar-Jaime et al. 2001). The source of infection also plays a role in the rate of transmission as a TI animal will shed considerable lower levels of virus in comparison to a PI animal leading to a slower progression within the herd. Another contrasting fact is that a TI animal will only shed virus for a few days (Brownlie et al. 1987), whereas a PI animal will continuously shed large amounts of virus for the duration of their lifetime. However, during an outbreak of BVDV-2c in Germany a transiently infected cow continued to shed virus for almost three months until euthanized (Doll and Holsteg 2013). The virus is also spread from TI animals which typically shed lower levels of the virus between 4 and 10 days post infection (Brownlie et al. 1987), although BVDV can be detected by PCR in the lungs and semen of TI animal s for longer periods (Newcomer et al. 2014). A viraemic animal will excrete BVDV in all bodily secretions and it has been reported that one hour of direct nose-to-nose contact between an infected and a susceptible animal is sufficient for transmission of the virus to occur (Houe 1999). The rate of transmission of BVDV within a herd is dependent on a number of factors including stocking densities, age and gender profile of animals within groups and the source of infection. If an infected animal is introduced into a low density holding the rate of transmission may be lower than in a high density holding as the likelihood of susceptible animals being exposed to direct contact with the PI or TI animal will be decreased. The age and gender profile of the group will be an important factor as the presence of an infected animal in a group of pregnant females may lead to the development of PI calves. Thus, maintaining a constant source of BVDV within the herd (Houe 1999; Lindberg 2002; Mainar-Jaime et al. 2001). Strain variation can also be a contributing factor to the rate and extent of transmission. Finally, animals developing severe nasal discharge and/or coughing can lead to the contamination of feed, forage, water sources, feeding equipment, 17

29 Chapter 1 Literature Review bedding material and the general environment with virus laden secretions which promotes the spread of the virus. Coughing can also result in the virus becoming airborne contributing to the transmission into adjoining holding areas or paddocks (Lindberg 2002; Mainar-Jaime et al. 2001). Introducing animals from other herds without establishing their BVD status and without a sufficient quarantine procedure can be detrimental to a herd s health. Various risk factors including nose to nose contact at farm boundaries mixed grazing and cattle shows can promote herd to herd transmission of BVDV (Houe 1999). PI animals play a major role in transmission of BVDV between herds. The acquisition of a pregnant female carrying a PI calf (Trojan cow) has the potential to transfer a new strain of the virus to the recipient herd (Lanyon et al. 2014). The virus can also be transmitted indirectly through infected semen, embryo transfer or through the use of contaminated veterinary instruments or equipment (Manimar-Jamie et al. 2001). When an animal becomes infected after birth with the virus, an immune response is initiated. Antibodies clear the virus and provide immunity against potential future infection (Brownlie et al. 1987). There is no evidence to suggest that BVDV can become latent in TI animals or that they are a risk to naïve animals within the herd once virus shedding has ceased. Historically Pestiviruses were classified based on the host, i.e. ovine BDV, porcine CSFV and bovine BVDV. However genetic analysis of Pestiviruses has revealed the potential for interspecies transmission. Interestingly CSFV is the only Pestivirus which has only been isolated from its host of origin. Paton et al. (1995) identified that both sheep and cattle located on the same farm were infected with similar strains of BVDV and suggested that the virus originated in cattle before being transmitted to the ovine population. This may have significant implications for BVDV control and eradication programmes as many Irish farms contain multiple species. 18

30 Chapter 1 Literature Review Laboratory Diagnosis BVDV infection can result in a broad range of clinical signs in affected animals, many of which may also be consistent with numerous other viral or bacterial diseases. These non-specific clinical signs along with sub-clinical acute and persistent infection may lead to BVDV being relatively difficult to diagnose within a herd. Similar to many other infectious diseases, routine herd health screening and the analysis of clinically ill animals using accurate diagnostic laboratory techniques is the most efficient method of BVDV diagnosis (Dubovi 2013). There are a number of laboratory techniques available to veterinary practitioners and herd owners for the detection of BVDV including: virus isolation (VI), immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) (Zhang et al. 2014). Virus isolation is commonly referred to as the gold standard for accurate BVDV diagnosis. This specialised technique is relatively expensive however, and may take up to 14 days to achieve a definitive result. BVDV grows particularly well in bovine turbinate cells (BT-), bovine testicle cells (Btest-) and Madin Darby bovine kidney cells (MDBK) (Dubovi 2013; Fulton et al. 2005). Certain strains of BVDV can be identified in a cell culture by their cytopathic effect on cells (cp biotype) but in the majority of isolates (ncp biotype) fluoresced labelled or enzyme-marked monoclonal or polyclonal antibodies against viral antigens must be used to detect virus replication (Sandvik, 1999). Successful isolation of a virus is dependent on a number of factors including: type of sample collected (although in PI animals virus can typically be isolated from any sample type), time of sampling, transport and how the sample is processed in the laboratory (Dubovi 2013). Immunohistochemistry is a technique whereby tissue samples are stained and examined using a microscope to identify whether chromophores have bound to BVDV viral particles which are present in infected animals (Brodersen 2004). This method demonstrated 100% sensitivity in the detection of PI animals when used on ear notch tissue samples (Cornish et al. 2005). 19

31 Chapter 1 Literature Review ELISA tests are used to detect the presence specific antigens or antibodies in biological samples in order to diagnose a pathogen within a host (Lanyon et al. 2014). There are a number or commercial ELISA kits available for the rapid and accurate detection of BVDV antigens and antibodies. Table 1.1 lists the commercial BVD ELISA kits approved for use by the Friedrich Loeffler Institut (FLI). All BVD antigen and antibody testing conducted in Ireland must use a FLI approved test kit batch on order to be compliant with the Irish national BVD eradication programme. Table 1.1: List of commercial BVD ELISA kits approved for use by the Friedrich Loeffler Institut. Kit Name Manufacturer Analyte SVANOVIR BVD-Ab, Biphasisch (Confirmation) SVANOVIR BVDV p80 Boehringer Ingelheim Svanova Boehringer Ingelheim Svanova BVD Ab BVD Ab IDEXX BVDV Total Ab IDEXX Europe B.V. BVD Ab IDEXX BVDV Ag / Serum Plus IDEXX Europe B.V. BVD Ag IDEXX BVDV p80 Ab IDEXX Europe B.V. BVD Ab ID Screen BVD p80 Antibody Competition ID VET BVD Ab PrioCHECK BVDV Ab Therno Fisher Scientific BVD Ab SERELISA BVD / MD Antikörper Symbiotics BVD Ab 20

32 Chapter 1 Literature Review ELISA kits designed for the detection of BVDV antigens utilise target antigens that are highly conserved and do not vary between different BVDV genotypes. The most commonly targeted antigens are the E rns and the NS3 proteins (Sandvik 1999; Dubovi 2013). A detectable concentration of both antigens is readily identified in serum, individual milk and some tissue samples, typically ear notch samples (Dubovi 2013). In the laboratory, a defined volume of the clinical sample from the subject animal is added to the well of a microtitre plate which has been coated with a capture antibody. Viral antigens available in the sample will bind to the antibody coated plate before an enzyme-marker antibody specific to BVDV is added, which in turn attaches to the antigen. Following the addition of a substrate solution, the optical density within the well will determine the result (Kramps et al. 1999; Howard et al. 1985). It has been demonstrated that the antigen ELISA tests may be limited to exclusively detecting PI animals due to the high levels of virus present (Sandvik and Krogsrud 1995) although a portion of TI individuals have been detected by antigen ELISA in the Irish eradication scheme. However this may be sufficient for herd health and BVD control programmes that focus on the identification of PI animals only. Furthermore, Cornish et al. (2005) revealed that the antigen capture ELISA method identified 100% of PI animals in the sample population analysed when compared to a number of other diagnostic techniques. Hilbe et al. (2007) reported varying antigen ELISA sensitivity and specificity values in a diagnostic method comparison. The initial ELISA achieved 100% sensitivity and specificity values while analysis of the second assay s performance revealed 93.5% and 99% respectively as this method failed to detect two infected animals (Hilbe et al. 2007). ELISA s that detect antibodies against BVDV proteins, such as the p80 and E 0 proteins, can be a useful screening tool as the status of a herd can be economically established through testing a bulk milk sample (Sayers et al. 2015b) or pooled serum samples (Cowley et al. 2012) samples to reduce cost (Dubovi 2013). PCR is a molecular biological method where a specific sequence of nucleic acid is targeted using specifically designed primers. Primers are designed to complement the forward and reverse sequence of a specific conserved region on the target genome. There are three stages of the PCR process: denaturation, annealing and extension/elongation. The denaturation stage of PCR involves the slow heating of 21