A gel-based PCR method to differentiate sheeppox virus field isolates from vaccine strains

Sheeppox (SPP) and goatpox (GTP) are caused by sheeppox virus (SPPV) and goatpox virus (GTPV) of the genus Capripoxvirus of the family Poxviridae [1]. In endemic areas SPP and GTP have a serious economic impact on small ruminant production systems, causing losses in productivity, mortality, damaging skins and hides, as well as inflicting international trade restrictions [2]. They are listed in the group of economically important animal diseases for which outbreaks have to be notified immediately to the World Organization for Animal Health [3].

The main mode of virus transmission is the direct contact between diseased and non-infected animals, but indirect transmission may also occur [4]. Clinical signs of SPPV and GTPV infections are characterized by ocular and nasal discharge and pock-like lesions in the skin and mucosae of the respiratory and gastrointestinal tracts [2, 4, 5]. Most of the isolates are host specific and cause disease mainly in sheep or in goats, whereas some isolates can cause serious disease in both animal species [6].

SPP and GTP are endemic in many African, Middle Eastern and Asian countries and recurrent epidemics have occurred in Greece and Bulgaria in 2013–2014 [7] and in Greece in 2016 and 2017. SPPV is also circulating in the Russian Federation where it causes sporadic outbreaks of disease.

In endemic regions, control of the disease is through effective immunization using killed or live attenuated vaccines derived from SPPV, GTPV or Lumpy skin disease virus (LSDV). In general, live attenuated vaccines are the better choice as compared to inactivated vaccines, as they confer long-lasting immunity. For instance, the Yugoslavian RM65, the Romanian Fanar and KSGP0240 strains, the most commonly used vaccines strains against SPPV, are reported to provide high levels of protection [7]. The Yugoslavian RM65 is widely used in the Middle East, Asia and in the Horn of Africa, while the Romanian Fanar is used in India and Maghreb countries. The Yugoslavian RM65 was attenuated by 30 serial passages on ovine kidney cells, and the Romanian Fanar by 26 serial passages on lamb testis cells [7].

The KSGP 0240 is widely used in several endemic regions in Africa. Nevertheless, KSGP0240 has been shown, by sequencing, to be a LSDV, thus, it can be differentiated from virulent isolates of SPPV using available capripoxvirus genotyping methods [7].

However, when using live attenuated vaccines, the epidemiological investigation of outbreaks can become quite challenging. When outbreaks occur following vaccination, it is essential to identify whether the animals were infected by the field strain because the vaccine did not provide sufficient protection. Alternatively, in some cases the vaccine strain may cause adverse reactions in vaccinated animals or, in rare occasions, re-gain the virulence as suggested by Lee and co-workers for herpesvirus vaccine [8].

Unfortunately, the current live attenuated capripox (CaP) vaccines do not offer the possibility to differentiate vaccinated animals from infected ones. This creates a need to identify a suitable genomic target and develop molecular tools that would enable the differentiation of SPPV field isolates from vaccine strains to rule out the involvement of SPP vaccines during a CaP outbreak in a vaccinated herd. Such a tool will facilitate the management and control of CaP infections in small ruminants.

The present study describes the use of a suitable diagnostic target of the SPPV genome to develop an assay that can discriminate SPP vaccines derived from the Romanian and the Yugoslavian RM/65 strains from SPPV field isolates, to facilitate the diagnosis and surveillance of capripox virus (CaPV) infections in small ruminants.

In this study, we identified a suitable target in CaPV genome and developed a PCR method to discriminate SPP vaccine strains from SPPV field isolates as well as from other CaPVs.

By aligning the full genome sequences of SPPV field isolates with the unpublished full genome of SPPV Morocco vaccine, a Romanian strain, we identified a region of 84 bp nucleotide deletion in the vaccine strains. Primers were designed to amplify this intergenic region located between the DNA ligase gene and the VARV B22R homologue gene of CaPV. The sequencing of this region in SPPV vaccine strains and SPPV field isolates available for this study, showed this deletion to be unique to the SPP vaccines derived from the Romanian and the Yugoslavian RM/65 strains produced in Algeria, Egypt, Morocco and Senegal. However, the sequence of the NISKHI vaccine, whose full genome has been previously published [9] did not carry such a deletion. Nevertheless, the use of the NISHKI strain seems to be confined to Russia and countries of the former Soviet Union such as Kazakhstan [10]. By using the targeted intergenic region, the aim of this study was to develop an assay to discriminate the live attenuated Romanian and Yugoslavian RM/65 SPP vaccines strains from virulent SPPV field isolates and other CaPVs.

Interestingly, Romanian and Yugoslavian RM/65 SPPV vaccines produced amplicons of shorter lengths, as compared to field isolates of SPPV and other CaPVs, and thus could be easily discriminated from them. The assay was found to be very specific and sensitive, and the accuracy and reliability was confirmed by sequencing the corresponding amplicons of SPP vaccine strains and SPPV, GTPV and LSDV field isolates available for this study.

The high sensitivity of this assay for SPPV vaccine strain, as compared to the field isolates, is likely due to the shortest amplicon size in the vaccine strains. No amplification was detected for any non-CaPV samples by the assay. However, the assay could not differentiate SPPV field isolates from LSDVs and GTPVs. Although an insertion of 34–36 nucleotides was observed in all LSDVs, making them longer than GTPVs and SPPV field isolates, it was not possible to resolve these differences on the agarose gel. Owing to the availability of species-specific PCR [11‐13] for CaPV genotype determination, we suggest that the current assay could be used once the genotype of CaPV is established. Alternatively, the exact capripoxvirus genotype can be determined by sequencing the region targeted in this study. Within the SPPV genotype, the test, undoubtedly discriminates between the virulent field isolates and the vaccines derived from the Romanian and the Yugoslavian RM/65 strains.

The availability of an easy-to-use molecular method is needed for the identification of SPPV, to rule out the involvement of SPP vaccines following a CaP outbreak in previously vaccinated flock of small ruminants. In Middle Eastern and Asian countries, small ruminants are protected from CaPV infections using various SPPV, GTPV or LSDV derived vaccines, however, SPP vaccines derived from Romanian and the Yugoslavian RM/65 strains are predominant [14]. In Africa, small ruminants are protected against CaPV using either KSGP O-240 and O-180 vaccines or SPP vaccines derived from the Romanian and the Yugoslavian RM/65 strains. Since KSGP O-240 and O-180 strains are of LSDV genotype, the use of a CaPV species-specific assay [11‐13] can allow for determination of their involvement when the disease occurs in a previously vaccinated herd. However, if a herd is vaccinated with a SPP vaccine from the Romanian and the Yugoslavian RM/65 strains, the full genome sequencing of the viral isolate, collected during an outbreak, would be required to rule out vaccine involvement when an outbreak occurs. This is time-consuming and cost prohibitive for most laboratories in limited resourced countries. Thus, the identification of suitable target in the viral genome to differentiate SPPV vaccine strains from SPPV field isolates greatly reduces the costs, by allowing the sequencing of small specific genome fragments. Furthermore, this region can be targeted in a simple molecular method such as the PCR approach presented in the work, thereby avoiding the use of sequencing. The current assay is intended to be used by all veterinary laboratories, including those with limited resources. It can also be used as a front-line tool for the direct screening of pathological samples collected during CaPV outbreaks, especially those occurring in previously vaccinated small ruminant populations. A study was recently conducted in Morocco to rule out the involvement of the vaccine strain in 2010 SPP outbreaks using a PCR based approach [15]. When compared to the assay developed by Haegeman and co-workers [15], our method presents the advantage of using only one primer pair to target both viruses, and thus, is much simpler to conduct and interpret. In addition, more vaccine strains and field isolates, from various geographical locations were included in this study, which broadened the scope of the applicability to all countries where SPP vaccines derived from the Romanian or the Yugoslavian RM/65 strains are used.

The molecular assay described herein is a reliable and rapid method that can easily be implemented for the differentiation of SPP vaccine derived from the Romanian or the Yugoslavian RM/65 strains from virulent SPPV field isolates. The method is applicable as a routine tool for outbreak investigations and disease surveillance in both SPP and GTP enzootic and disease-free countries. It is expected that its adoption by veterinary laboratories in CaPV affected countries, will help facilitate the control and management of CaP disease in small ruminants.