Hefer valley virus: a novel ephemerovirus detected in the blood of a cow with severe clinical signs in Israel in 2022

A novel ephemerovirus was identified in a Holstein-Friesian cow in the Hefer Valley, Israel, that showed severe and fatal clinical signs resembling an arboviral infection. A sample taken during the acute phase tested negative for important endemic arboviral infectious cattle diseases. However, sequencing from blood revealed the full genome sequence of Hefer Valley virus, which is likely to represent a new species within the genus Ephemerovirus, family Rhabdoviridae. Archived samples from cattle with comparable clinical signs collected in Israel in 2021 and 2022 tested negative for the novel virus, and therefore, the actual distribution of the virus is unknown. As this is a recently identified new viral infection, the viral vector and the prevalence of the virus in the cattle population are still unknown but will be the subject of future investigations.

from the age 0 to two years old) located in the Hefer Valley in central Israel (Fig. 1a). One of the cows died 14 days after the onset of clinical signs. A blood sample from this cow was collected two days after the onset of clinical signs, which developed during the first seven days after delivery of a healthy female calf. Clinical signs included fever and hypocalcemia, followed by ketonuria, milk reduction, and recumbency. Retrospective collection of data about this animal revealed that it was a six-year-old dairy cow that had been vaccinated several times with ULTRAVAC BEF VAC-CINE (Zoetis) to protect against BEFV. The blood samples were tested by RT-qPCR assays specific for frequently found important arboviral infections of cattle in the region, including bluetongue virus, epizootic hemorrhagic disease virus [9], and BEFV, targeting the G coding region [10] ( Table 1). All of the PCR tests gave negative results. These samples were therefore also tested using a recently developed RT-qPCR assay specific for the N region of BEFV ( Table 1). An AgPath-ID™ One-Step RT-PCR Kit (Applied Biosystems, Warrington, Cheshire, United Kingdom) was used for inhouse RT-qPCR tests ( Table 1, Supplementary Table S1). The real-time RT-PCR program for the thermocycler was performed as described previously [11], and the master mix preparation is shown in Supplementary Table S1. One of the blood samples (that of the animal that died) gave an equivocal result with a recently developed RT-qPCR assay specific for the N region of BEFV. The 100-bp RT-PCR product from that test was purified from a gel using a MEGAquick-spin Total Fragment DNA Purification Kit (iNtRON Biotechnology, Gyeonggi-do, South Korea) and subjected to Sanger sequencing. BLASTn analysis of the sequenced product revealed a high degree of sequence similarity to members of the genus Ephemerovirus. Attempts to isolate the virus from the blood sample on Vero (African green monkey kidney), BHK-BSR (baby hamster kidney-21 clone BSR), and C6/36 (Aedes albopictus) cells failed.
Due to their low melting temperatures, the probes were prepared as alternative MGB probes (https://www.biolegio. com/) Metagenomic RNA sequencing and de novo assembly were performed on the blood sample from the cow that died (the detailed methods are presented in Supplementary Material), and the resulting contigs were matched to protein references of ephemeroviruses using diamond BLASTx (version 2.0.14). A single contig was identified that resembled the viral genome of a potential novel ephemerovirus. We tentatively named it "Hefer Valley virus" (HVV), after the location of sample origin (Fig. 1a).
For phylogenetic classification, individual amino acid sequence alignments of the N and L proteins of HVV and 22 representative viruses of the genera Ephemerovirus and Tibrovirus were made using MUSCLE (version 3.8.425) and then concatenated into a single alignment, and a maximum-likelihood phylogenetic analysis was performed using IQ-TREE2 (see Supplemental Material). The phylogenetic tree based on N and L proteins suggested that HVV is a member of a novel species within the genus Ephemerovirus and is most closely related to HYV, PUCV, and KOV (Fig. 2). The amino acid sequence identity of HVV to HYV, KOV, and PUCV was 87.7-89.4% and 77.3-78.4% for the N and L protein, respectively (Supplementary Fig. S1a and  b).
We submitted the annotated HVV genome sequence to the International Nucleotide Sequence Database Collaboration (https://www.insdc.org) under accession no. OQ679991.
In order to check for the presence of HVV in cases of cattle with comparable clinical signs, we tested 249 available stored samples collected in 2021 and 218 samples collected in 2022 using a pan-ephemerovirus RT-qPCR assay ( Table 1 and Supplementary Table S1). All of the samples were collected from August to January, the usual arbovirus season in the region. No additional positive samples were identified in 2022, while 50 samples collected in 2021 were positive. Identification of the specific ephemeroviruses in these positive samples was performed using a BEFV-specific SYBR Green-based RT-qPCR assay [10] in combination with the generic conventional RT-PCR for the N coding region of ephemeroviruses ( Table 1 and Supplementary Table S1). For the generic conventional RT-PCR, a One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used, and the conditions for thermocycling are shown in Supplementary Materials. All 50 positive samples collected in 2021 were identified as BEFV, and none were HVV (data not shown).
During the last decade, BEFV outbreaks were registered in Israel in 2014, 2015, 2018, and 2021, alternating with periods of "total silence" from the infection. The most serious, long-lasting (July-December) outbreak occurred in 2021, where cases were also reported in arid areas, which had not been observed before. However, due to the absence of systematic screening programs, there is no information available about whether members of the genus Ephemerovirus other than BEFV have circulated in the region and, if so, whether they are of veterinary importance.
In conclusion, we report the identification of HVV, a novel member of the genus Ephemerovirus that is most closely related to HYV, PUCV, and KOV. The blood sample was collected in October 2022 from an adult BEFV-vaccinated milking cow in Israel exhibiting severe illness that subsequently died. Future serological and molecular examinations are expected to reveal the extent of the exposure of cattle to HVV in Israel and to determine its veterinary significance. Also, as the affected cattle had a history of vaccination against BEFV, the efficacy of these vaccines against HVV needs to be evaluated.
As several well-known viruses of the genus Ephemerovirus are transmitted by blood-sucking insects, potential vectors should be collected in regions with affected cattle and investigated for the presence of HVV.