The genus Ephemerovirus, which belongs to the family Rhabdoviridae comprises viruses that primarily infect ruminants and are transmitted by blood-sucking insects [1, 2], including e.g. Puchong virus (PUCV) from mosquitoes in Malaysia in 1965 and Hayes Yard virus (HYV) from a bull in Australia in 2000 [3,4]. However, several ephemeroviruses were detected in other origins, as porcine ephemeroviruses 1 and 2, which were identified in porcine tissues in China [5], and New Kent County virus was isolated from ticks in Northern America (origin:GenBank).
Bovine ephemeral fever virus (species: Ephemerovirus febris; BEFV) is the type member of the genus and an important pathogen of cattle and water buffalo. It causes a short-lasting disease characterised by a bi‐phasic fever, salivation, ocular and nasal discharge, recumbency, muscle stiffness, lameness and anorexia. The morbidity is high, but the mortality is usually low (<1%) [6]. BEFV is apparently transmitted by two types of arthropod vectors: Culicoides and mosquitoes (culicine and anopheline mosquitoes) [7]. Besides BEFV, other ephemeroviruses have been reported to cause comparable clinical diseases in cattle [7,8].
The genome of ephemeroviruses consists of ssRNA(-) and is about 15 kb in length encoding ten open reading frames. The genes are flanked by conserved transcription initiation and transcription termination/polyadenylation (UGAAAAAAA) sequences, and are separated by intergenic regions [1,2].
In October 2022, a six-year old dairy cow from Ein HaHoresh, a cooperative farm in central Israel, located in the Hefer Valley (Figure 1a) manifested clinical signs resembling an arboviral infection. The cow was previously vaccinated several times with ULTRAVAC BEF VACCINE (Zoetis), to protect against BEFV. During the first seven days after delivery of a healthy female calf, the cow developed fever and hypocalcemia, followed by ketonuria, milk reduction and recumbency. The cow died 14 days after the onset of the clinical signs. A blood sample was collected and sent to the department of Virology, Kimron Veterinary Institute, Israel, for laboratory diagnosis at October 20th, 2022. The sample was tested by RT-qPCRs specific for arboviral viruses such as bluetongue virus, epizootic hemorrhagic disease viruses, and BEFV, targeting the G coding region [10] (Supplementary Table S1). These RT-qPCRs scored negative. The result of an RT-qPCR specific for the N-region of BEFV (Supplementary Table S1) was equivocal and BLASTn analysis of the sequenced BEFV N-region RT-qPCR product revealed highest identity to members of the genus Ephemerovirus. Virus isolation on Vero (African green monkey kidney), BHK-BSR (baby hamster kidney - 21 clone BSR) and C6/36 (Aedes albopictus) cells failed.
Metagenomic RNA sequencing and de novo assembly was performed on a blood sample (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 (Figure 1a).
The genome of the novel HVV was 15,033 nt in length. We predicted and characterized 10 open reading frames (ORF), that followed the typical genome structure of ephemeroviruses (3′-N-P-M-G-GNS-α1-α2-β-γ-L-5′; Figure 1b). Furthermore, we identified nine transcription termination sites (UGAAAAAAA) that were located adjacent to the ORFs.
For phylogenetic classification, individual amino acid sequence alignments from N and L proteins of 22 representative viruses from the genera Ephemerovirus and Tibrovirus along with HVV were generated using MUSCLE (version 3.8.425), then concatenated into a single alignment and a maximum-likelihood phylogenetic analysis was conducted using IQ-TREE2 (see Supplemental Material). The phylogenetic tree based on N and L proteins suggested that HVV as a novel species within the genus Ephemerovirus most closely related to HYV, PUCV and KOV (Figure 2). The amino acid identity of HVV to HYV, KOV and PUCV was between 87.7-89.4% and 77.3-78.4% for N and L proteins, respectively (Supplemental Figure S1a and S1b).
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 (Supplementary Tables S1 and S2). All of the samples were collected from August until January, the usual arboviral season in the region. No additional positive samples were identified in 2022, while fifty samples collected in 2021 were positive. Identification of ephemerovirus species on these positive samples were performed using a BEFV-specific SYBR Green based RT-qPCR [9] in combination with the generic conventional RT-PCR for the N-coding region of ephemeroviruses (Supplemental Table S1 and S2). All of the fifty positive samples collected in 2021 were identified as BEFV only, but not HVV (data not shown).
In conclusion, we report identification of the HVV, a novel species within the genus Ephemerovirus most closely related to HYV, PUCV and KOV. The blood sample was collected in October, 2022 from an adult BEFV-vaccinated milking Israeli cow manifesting severe illness with consequent death. Future serological and molecular examinations are expected to reveal the extent of cattle exposure to HVV in Israel, and to determine its veterinary significance. Also, as the affected cattle had a history of vaccination against BEFV, the efficiency of these vaccines against HVV is up to debate.
As several well-known viruses of the genus Ephemerovirus are transmitted by blood-sucking insects, putative vector species should be collected in regions with affected cattle and investigated for the presence of HVV in order to identify its potential insect vector.