Genetic diversity of ruminant Pestivirus strains collected in Northern Ireland between 1999 and 2011 and the role of live ruminant imports

Two genotypes of bovine viral diarrhoea virus (BVDV) referred to as BVDV types 1 and 2 (BVDV-1, BVDV-2), are currently recognised. BVDV-2 was first detected when virulent strains caused significant losses among cattle in North America in the 1990s [1]. Subsequently, BVDV-2 has been found in many other countries, with the first reported detection in the United Kingdom in 2002 [2]. While BVDV-1 includes at least 18 subtypes (BVDV-1a to 1r) [3–6], BVDV-2 strains are classified into four subtypes (BVDV-2a to 2d) [7–9]. A new species of atypical bovine pestivirus, Hobi virus, (BVDV-3) consisting of viral strains first detected in foetal calf serum (FCS) has also been proposed [10]. The batch of FCS from which Hobi virus was identified was produced in South America and commercialised in European countries [11] and the same viral strain was also subsequently detected in a buffalo from Brazil [12], in a calf in Thailand [13], in calves in Italy [14] and in dairy cattle in India [15].

Other “atypical” pestiviruses of non-bovine origin, including isolates from a giraffe in Kenya, a pronghorn antelope in Africa and Bungowannah virus of pigs in Australia [7, 16, 17] have also been suggested as novel pestiviral species.

The 5’ untranslated region (5’ UTR) of BVDV is highly conserved and has been used as the standard target for diagnostic RT-PCR [18, 19]. The sequence variation of this region is sufficient to enable phylogenetic analysis to assign isolates to genotypes and subtypes [3, 18]. Knowledge of the predominant genotypes in a given region is necessary to ensure that suitable diagnostic tests are used and to assess the protection likely to be afforded by available vaccines, as well as providing surveillance for the emergence of new genotypes or sub-types. Inactivated BVDV vaccines licensed in the United Kingdom and Ireland contain BVDV-1a. It has been estimated than 51 % of dairy herds and 26 % of suckler herds in Northern Ireland vaccinate the adult herd [20]. Vaccines containing BVDV-1 have been shown to provide incomplete protection against type 2 challenge [21–23]. In addition, the ability of vaccines of one BVDV-1 subtype to fully protect against other BVDV-1 subtypes has also been questioned [24, 25]. In 2015, a double deleted modified live vaccine containing two types of a modified live BVDV (BVDV-1 and BVDV-2) has been brought to the market in England, Wales, Northern Ireland and Scotland.

With the exception of a single study comprising 25 sequences [26] and a more recent study confined to the Republic of Ireland [27], there is no published information available on the diversity of ruminant pestiviral strains in Northern Ireland. The current study was undertaken to address this knowledge gap, with emphasis on the genotypes and sub-genotypes present, their relationship to strains present in the rest of the United Kingdom (UK) and in the Republic of Ireland (ROI) and the role of imported cattle and sheep in driving diversity.

In the present study the molecular typing of pestiviral strains from cattle in Northern Ireland, including those imported from other parts of the UK and continental Europe, was performed and the results analysed. This study represents the largest survey of this type undertaken to date in Northern Ireland. The initial phase of the work, using Panel I samples, investigated diversity to the species level. No evidence was found of the presence of BVDV-2 or BDV in Panel I samples. This is in contrast to the situation elsewhere in the UK, where the presence of BVDV-2 and BDV has been reported in cattle, albeit infrequently [2, 33]. The lack of detection of BVDV-2 is encouraging, and taken together with the failure to detect BVDV-2 in two studies in ROI (R O’Neill, personal communication, [27]), supports the hypothesis that the island of Ireland remains free of BVDV-2.

Sequencing of Panel II strains collected between 2008 and 2011, provided a more in-depth analysis of genetic diversity, allowing characterisation to subtype level. A high degree of homogeneity was detected, with BVDV-1a being the predominant subgenotype (86/91 samples; 94.5 %). The remaining 5 strains clustered to BVDV-1b reference strains. This is the first report of BVDV-1b in cattle in Northern Ireland.

Both of the inactivated vaccines currently licenced for use in the UK contain BVDV-1a. These findings indicate that they are well suited, in terms of antigenicity, to provide protection against the predominant challenge strains. Some studies suggest that protection against heterologous BVDV-1 subtypes is inferior to homologous BVDV-1 subtypes [23–25]. Because of this concern, it was important to determine the circulating pestiviral strains. A recently licenced modified live vaccine includes both BVDV-1 and BVDV-2 components. The BVDV-1 strain included (field isolate KE9) is genetically related to several other European virus strains.

The first survey to determine the strain type of pestiviruses in Ireland was carried out in 2001 [26], using 25 field strains collected between 1968 and 1999. All were found to be BVDV-1a. Similar findings were reported in England and Wales in a contemporaneous study [34]. However, more recent studies have shown an increased phylogenetic diversity in England and Wales [31]. Although BVDV-1a was still the predominant subtype detected (77.2 %), an increased frequency of detection of BVDV-1b was found, along with three additional circulating subtypes, BVDV-1e, BVDV-1f and BVDV-Ii. Booth et al. [35] reported similar findings in a recent study of 104 samples from GB. The predominant sub-genotype found was again BVDV-1a (88/104 samples; 85 %). However BVDV-1b, 1e and 1i were also identified and the first BVDV -1d in the UK was reported.

In the ROI a more diverse antigenic variability has also been recently reported [27]. BVDV-1a was still the predominant subgenotype, accounting for 97.5 % (n = 317) of the viral isolates. Other subgenotypes detected were BVDV-1b (n = 6), BVDV-1d (n = 1) BVDV-1e (n = 1).

The predominance of BVDV-1a in the UK and Ireland [26, 31, 35] is in contrast to other European countries including Poland [36], the Netherlands [37], Portugal [38], Italy [39] and Spain [40] where BVDV-1b is the most prevalent subtype.

Panel III sera were tested to examine the hypothesis that importation of infected ruminants was driving the detected increase in genetic diversity. Overall, the findings support this hypothesis with 2.1 % (18/839) of samples from imported cattle giving a positive result and a further 8 (0.95 %), an inconclusive result. It was not possible to establish in all cases whether the positive and inconclusive samples were from persistently or transiently infected animals. However, two animals (INT 12 and INT 13, Table 1) tested virus positive on two separate occasions which is consistent with them being PI.

As the BVDV-1b strains in Panel II samples were detected throughout or after the period during which the Panel III BVDV-1b-positive animals were introduced, the possibility of a link between those herds by cattle movement was explored. Observing the clustering in Fig. 2, it was logical to investigate a relation between Herd D (VSD-78-11) and Herds 1, 2 and 3. Although all the herds in Panel II where BVDV-1b was detected had each added animals within the previous 2 years, analysis of movement data failed to show links between the Panel III and Panel II herds that would explain the BVDV-1b strains detected in this study. Nevertheless, a combination between phylogenetic and movement analysis may be used as an epidemiological tool to determine the origin of infection.

None of the pools from the 4,437 samples from sheep imported between September 2010 and November 2011 were positive for the presence of pestivirus RNA. This could reflect a lower risk of introduction of pestiviruses from sheep or a lack of sensitivity of the method used (including the impact of pooling). Our in house validation records for the RT-PCR assay used showed that the analytical sensitivity for the detection of several BDV species subtypes was lower than for the detection of BVDV-1 and BVDV-2 (data not shown). The rise in detection of different BVD strains, including atypical strains [11–13] and the increase in reports of cattle affected by BDV [33, 41] highlights the need to monitor circulating strains and to review accordingly the suitability of the diagnostic methods used.

Molecular epidemiology has been used to trace sources and routes of BVDV infections within the Swedish BVD eradication programme [42] and to link animal movements with viral isolates in the UK [35]. The sequence information collated in this study may provide a useful tool to trace the origin of new outbreaks. Since the level of movement of animals between Northern Ireland and other regions of the UK and Ireland is very high, a common database where all the sequence data could be stored would be a useful tool for this purpose.

This study suggests that in terms of BVDV the island of Ireland retains a distinct pattern of strain diversity from continental Europe. The reasons behind this are unclear, but some possibilities include the fact that the vast majority of transboundary cattle movements are outward, the existence of strict importation regulations prior to 1992 the system of cattle production is largely grass-based and the obvious absence of a land-link with other cattle populations.

In conclusion, this study found strains of BVDV in the cattle population in Northern Ireland to be predominantly BVDV-1a. No evidence of BVDV-2 was found, but the emergence of BVDV-1b in recent years was detected. Testing of samples from imported cattle highlighted this as a route for introduction of new strains leading to increasing viral heterogeneity, emphasising the need to address imports in the context of current eradication programmes in Northern Ireland and the Republic of Ireland.

AFBI, Agri-Food and Biosciences Institute; APHIS, Animal and Public Information System; BDV, Border disease virus; BTV, Bluetongue virus; BVDV, Bovine viral diarrhoea virus; DAERANI, Department of Agriculture, Environment and Rural Affairs of Northern Ireland; DAFM, Department of Agriculture, Food and Marine; ELISA, Enzyme-linked immunosorbent assay; FCS, Foetal calf serum; NI, Northern Ireland; NTC, No template control; PI, Persistently infected; RLOD, Reproducible level of detection; ROI, Republic of Ireland; SE, South eastern; UK, United Kingdom; UTR, Untranslated region; VSD, Veterinary Sciences Division.