Identification, Isolation, and Molecular Characterization of Betacoronavirus in Oryx leucoryx.

Coronaviruses (CoVs) are enveloped viruses with a large RNA genome (26 to 32 kb) and are classified into four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. CoV infections cause respiratory, enteric, and neurologic disorders in mammalian and avian species. In 2019, Oryx leucoryx animals suffered from severe hemorrhagic diarrhea and high morbidity rates. Upon initial diagnosis, we found that the infected animals were positive for coronavirus by pancoronavirus reverse transcriptase RT-PCR. Next, we detected the presence of CoV particles in these samples by electron microscopy and immunohistochemistry. CoV was isolated and propagated on the HRT-18G cell line, and its full genome was sequenced. Full-genome characterization and amino acid comparisons of this viral agent demonstrated that this virus is an evolutionarily distinct Betacoronavirus belonging to the subgenus Embecovirus and the Betacoronavirus 1 species. Furthermore, we found that it is most similar to the subspecies dromedary camel coronavirus HKU23 by phylogenetic analysis. Here, we present the first report of isolation and characterization of Betacoronavirus associated with enteric disease in Oryx leucoryx. IMPORTANCE CoVs cause enteric and respiratory infections in humans and animal hosts. The ability of CoVs to cross interspecies barriers is well recognized, as emphasized by the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The identification of novel CoV strains and surveillance of CoVs in both humans and animals are relevant and important to global health. In this study, we isolated and characterized a newly identified Betacoronavirus that causes enteric disease in a wild animal, Oryx leucoryx (the Arabian oryx). This work is the first report describing CoV infection in Oryx leucoryx and provides insights into its origin.

Comparative Molecular and Epidemiological Analyses of Israeli Bluetongue Viruses Serotype 1 and 9 Causing Outbreaks in 2018-2020.

Israel is endemic to bluetongue virus (BTV). The introduction of novel-for-the-region arboviruses have been recorded annually in recent years. In 2019, previously non-reported in-the-country BTV-1 and BTV-9 were identified. BTV-1 caused a single-season outbreak, probably linked to mild infection in ruminants. BTV-9 was retrospectively detected in the field samples collected from August 2018 until 2020. It was the dominant serotype in 2019, out of the six serotypes recorded during that calendar year. Clinical manifestation of the disease in cases diagnosed with BTV-9 were compared to those in cases determined to have BTV-1. BLAST and phylogenetic analyses of BTV-1 showed that the nucleotide (nt) sequence coding the viral outer protein 1 (VP2) determining the serotype is closely related to BTV-1 isolated in Sudan in 1987, and the coding sequence of the outer protein 2 (VP5) is related to South African BTV-1 from 2017. A probable common ancestor with Libyan BTV-9 strains isolated in 2008 was seen in an analysis of Israeli BTV-9 nt sequences. Notably, the outbreak-caused BTV-9 strains collected in 2019 exhibited a distinct level of genetic reassortment with local Israeli strains compared to BTV-9 strains registered in 2018 and 2020.

Identification and Genetic Characterization of Viral Pathogens in Ruminant Gestation Abnormalities, Israel, 2015-2019.

Infectious agents including viruses are important abortifacients and can cause fetal abnormalities in livestock animals. Here, samples that had been collected in Israel from aborted or malformed ruminant fetuses between 2015 and 2019 were investigated for the presence of the following viruses: the reoviruses bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV), the flaviviruses bovine viral diarrhea virus (BVDV) and border disease virus (BDV), the peribunyaviruses Shuni virus (SHUV) and Akabane virus (AKAV), bovine herpesvirus type 1 (BoHV-1) and bovine ephemeral fever virus (BEFV). Domestic (cattle, sheep, goat) and wild/zoo ruminants were included in the study. The presence of viral nucleic acid or antigen could be confirmed in 21.8 % of abnormal pregnancies (213 out of 976 investigated cases), with peribunyaviruses, reoviruses and pestiviruses being the most prevalent. At least four different BTV serotypes were involved in abnormal courses of pregnancy in Israel. The subtyping of pestiviruses revealed the presence of two BDV and several distinct BVDV type 1 strains. The peribunyaviruses AKAV and SHUV were identified annually throughout the study period, however, variation in the extent of virus circulation could be observed between the years. In 2018, AKAV even represented the most detected pathogen in cases of small domestic ruminant gestation abnormalities. In conclusion, it was shown that various viruses are involved in abnormal courses of pregnancy in ruminants in Israel.

A high-resolution melting (HRM) assay for the differentiation between Israeli field and Neethling vaccine lumpy skin disease viruses.

Lumpy skin disease (LSD) is a constant threat to the Middle East including the State of Israel. During vaccination programs it is essential for veterinary services and farmers to be able to distinguish between animals affected by the cattle-borne virulent viruses and vaccinated animals, subsequently affected by the vaccine strain. This study describes an improved high resolution-melting (HRM) test that exploits a 27 base pair (bp) fragment of the LSDV126 extracellular enveloped virion (EEV) gene that is present in field viruses but is absent from the Neethling vaccine strain. This difference leads to ∼0.5 °C melting point change in the HRM assay, when testing the quantitative PCR (qPCR) products generated from the virulent field viruses compared to the attenuated vaccine. By exploiting this difference, it could be shown using the newly developed HRM assay that virus isolated from vaccinated cattle that developed disease symptoms behave similarly to vaccine virus control, indicating that the vaccine virus can induce disease symptoms. This assay is not only in full agreement with the previously published PCR gradient and restriction fragment length polymorphism (RFLP) tests but it is faster with, fewer steps, cheaper and dependable.

Development of an assay to differentiate between virulent and vaccine strains of lumpy skin disease virus (LSDV).

Lumpy skin disease (LSD) was and still is a constant threat to the State of Israel, since the first outbreaks in 1989 and in 2006-2007. Recently, another massive outbreak occurred, at the beginning of July 2012, in the northern part of Israel. An intensive vaccination campaign with a sheeppox-based vaccine was initiated, in addition to culling symptomatic animals in the dairy herds. In spite of this, there was a need to apply extra efforts to completely contain and control the spread of the disease by introducing for the first time in Israel a vaccine based on the Neethling vaccine virus strain. However, in case of appearance of LSD symptoms it was essential to be able to distinguish between cattle-carried virulent strain and the vaccine strain. This paper describes the development and utilization of a molecular assay that can differentiate between the virulent isolates from the vaccine strain. The system is based on 3 different tests; it was found that the vaccine strain carries 27 bases less than the virulent virus in the extracellular enveloped virions (EEV) gene. A temperature-gradient PCRs were done using primers which are identical to the vaccine strain but differ at the 3' end nucleotides to the virulent virus. PCR-RFLP was carried out on the presence of an MboI site unique to the vaccine strain. Thus, all three tests presented here are able to differentiate specifically between the two viral appearances.