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.

Identification of two novel ephemeroviruses in pigs infected by classical swine fever virus.

Ephemeroviruses are arthropod-borne rhabdoviruses within Ephemerovirus genus and have been isolated exclusively from cattle and haematophagous arthropods (mosquitoes and biting midges) without any member detected or isolated up to date from pigs, although some serological surveys have indicated that pigs may be a silent host for ephemerovirus infection. Here, many viral reads annotated to, but genetically distinct from, the existing members within the Ephemerovirus genus have been identified in the meta-transcriptomic data of two clinical classical swine fever virus (CSFV)-infected samples (HeN10 and GDMM7). The nearly complete genome sequences of the two novel ephemeroviruses have been obtained through contig assembly, specific RT-PCR and sequencing, therefore named as porcine ephemeroviruses (PoEVs). Genome nucleotide sequence analysis showed that PoEV strains HeN10 and GDMM7 have similar genome organization and 66.5% genomic identity to each other, but both are genetically distant from all members of the Ephemerovirus genus with identity being only 51.1-59.6%. Furthermore, comparison of the most conserved ephemeroviral proteins N and L indicated that PoEV strains HeN10 and GDMM7 share a high sequence identity to each other (N: 78.1%; L: 70.7%), but are diverged from the known ephemeroviruses (N: 43.4-60.7%; L: 47.6-58.5%). The genetic distance is significantly beyond the criteria for demarcation of viruses assigned to different ephemerovirus species. Thereby, two novel viruses named as PoEV1 (strain HeN10) and PoEV2 (strain GDMM7) are identified and these appear to represent two new species within the Ephemerovirus genus. The present study showed the first genome evidence of pig ephemeroviruses, likely expanding the known host range of ephemerovirus.

Hayes Yard virus: a novel ephemerovirus isolated from a bull with severe clinical signs of bovine ephemeral fever is most closely related to Puchong virus.

Bovine ephemeral fever is a vector-borne disease of ruminants that occurs in tropical and sub-tropical regions of Africa, Asia and Australia. The disease is caused by a rhabdovirus, bovine ephemeral fever virus (BEFV), which occurs as a single serotype globally. Although several other closely related ephemeroviruses have been isolated from cattle and/or arthropods, only kotonkan virus from Nigeria and (tentatively) Mavingoni virus from Mayotte Island in the Indian Ocean have been previously associated with febrile disease. Here, we report the isolation of a novel virus (Hayes Yard virus; HYV) from blood collected in February 2000 from a bull (Bos indicus) in the Northern Territory of Australia. The animal was suffering from a severe ephemeral fever-like illness with neurological involvement, including recumbency and paralysis, and was euthanised. Histological examination of spinal cord and lung tissue identified extensive haemorrhage in the dura mata with moderate perineuronal oedema and extensive emphysema. HYV displayed cone-shaped morphology, typical of rhabdoviruses, and was found to be most closely related antigenically to Puchong virus (PUCV), isolated in 1965 from mosquitoes in Malaysia. Analysis of complete genome sequences of HYV (15 025 nt) and PUCV (14 932 nt) indicated that each has a complex organisation (3' N-P-M-G-GNS-α1-α2-ß-γ-L 5') and expression strategy, similar to that of BEFV. Based on an alignment of complete L protein sequences, HYV and PUCV cluster with other rhabdoviruses in the genus Ephemerovirus and appear to represent two new species. Neutralising antibody to HYV was also detected in a retrospective survey of cattle sera collected in the Northern Territory.

Co-circulation and characterization of novel African arboviruses (genus Ephemerovirus) in cattle, Mayotte island, Indian Ocean, 2017.

Mayotte is an island located in the Mozambique Channel, between Mozambique and Madagascar, in the South Western Indian Ocean region. A severe syndrome of unknown aetiology has been observed seasonally since 2009 in cattle (locally named "cattle flu"), associated with anorexia, nasal discharge, hyperthermia and lameness. We sampled blood from a panel of those severely affected animals at the onset of disease signs and analysed these samples by next-generation sequencing. We first identified the presence of ephemeral bovine fever viruses (BEFV), an arbovirus belonging to the genus Ephemerovirus within the family Rhabdoviridae, thus representing the first published sequences of BEFV viruses of African origin. In addition, we also discovered and genetically characterized a potential new species within the genus Ephemerovirus, called Mavingoni virus (MVGV) from one diseased animal. Finally, both MVGV and BEFV have been identified in cattle from the same herd, evidencing a co-circulation of different ephemeroviruses on the island. The clinical, epidemiological and virological information strongly suggests that these viruses represent the etiological agents of the observed "cattle flu" within this region. This study highlights the importance of the strengthening and harmonizing arboviral surveillance in Mayotte and its neighbouring areas, including Africa mainland, given the importance of the diffusion of infectious diseases (such as BEFV) mediated by animal and human movements in the South Western Indian Ocean area.

Three-day fever.

Three-day fever is a viral disease caused by an Ephemerovirus of the family Rhabdoviridae, transmitted by arthropod vectors. It is common in tropical and sub-tropical regions, where it affects mainly domestic cattle and buffaloes, especially in intensive dairy or fattening production systems. It is of economic importance because it reduces milk production and fertility and causes abortion. The disease is generally benign. It manifests in several susceptible subjects simultaneously, with a sudden episode of fever accompanied by muscle involvement with arthritis, stiffness of the limbs, and lameness, followed by rapid recovery. The presence of a serofibrinous exudate in the joints is indicative of the disease. Clinical diagnosis is often difficult in the absence of pathognomonic signs. Epidemiological factors (proliferation of arthropod vectors), associated with a short-lived fever and the presence of many immature neutrophils, point strongly to three-day fever. In the absence of any specific treatment, the symptoms are treated with antibiotics and anti-inflammatories. Medical prophylaxis currently uses live attenuated vaccines, pending the development of recombinant vaccines, which are giving promising results.

Koolpinyah and Yata viruses: two newly recognised ephemeroviruses from tropical regions of Australia and Africa.

Koolpinyah virus (KOOLV) isolated from healthy Australian cattle and Yata virus (YATV) isolated from a pool of Mansonia uniformis mosquitoes in the Central African Republic have been tentatively identified as rhabdoviruses. KOOLV was shown previously to be related antigenically to kotonkon virus, an ephemerovirus that has caused an ephemeral fever-like illness in cattle in Nigeria, but YATV failed to react antigenically with any other virus tested. Here we report the complete genome sequences of KOOLV (16,133 nt) and YATV (14,479 nt). Each has a complex genome organisation, with multiple genes, including a second non-structural glycoprotein (GNS) gene and a viroporin (α1) gene, between the G and L genes as is characteristic of ephemeroviruses. Based on an analysis of genome organisation, sequence identity and cross-neutralisation, we demonstrate that both KOOLV and YATV should be classified as two new species in the genus Ephemerovirus.

A reverse-transcription PCR method for detecting all known ephemeroviruses in clinical samples.

Bovine ephemeral fever virus (BEFV) is an economically important vector-borne pathogen of cattle in tropical and sub-tropical regions of Australia, Asia, Africa and the Middle East. Although clinical cases of bovine ephemeral fever are usually attributed to BEFV, definitive diagnosis is rarely performed and at least two other related viruses, kotonkon virus (KOTV; an ephemerovirus) and Fukuoka virus (FUKAV; an unassigned rhabdovirus), can cause similar clinical signs. As vaccines have been developed against BEFV but not against KOTV or FUKAV, a test capable of detecting and differentiating these pathogens would be useful. In the present study, an RT-PCR method using degenerate primers designed to a region of block III of the polymerase (L) gene was developed and optimised for primer annealing temperature and MgCl2 concentration. The RT-PCR detected all known ephemeroviruses and several other closely related insect-transmitted rhabdoviruses, including FUKAV. Viruses could be identified by subsequent sequencing and phylogenetic analysis of the amplicons. BEFV could be detected using tissue culture isolates or cattle blood to a sensitivity of 500 RNA copies per reaction. This test will be useful for establishing the identity of the causative agent of bovine ephemeral fever from field samples and cultured isolates.

Malakal virus from Africa and Kimberley virus from Australia are geographic variants of a widely distributed ephemerovirus.

Kimberley virus (KIMV) is an arthropod-borne rhabdovirus that was isolated in 1973 and on several subsequent occasions from healthy cattle, mosquitoes (Culex annulirostris) and biting midges (Culicoides brevitarsis) in Australia. Malakal virus (MALV) is an antigenically related rhabdovirus isolated in 1963 from mosquitoes (Mansonia uniformis) in Sudan. We report here the complete genome sequences of KIMV (15442 nt) and MALV (15444 nt). The genomes have a similar organisation (3'-l-N-P-M-G-G(NS)-α1-α2-ß-γ-L-t-5') to that of bovine ephemeral fever virus (BEFV). High levels of amino acid identity in each gene, similar gene expression profiles, clustering in phylogenetic analyses of the N, P, G and L proteins, and strong cross-neutralisation indicate that KIMV and MALV are geographic variants of the same ephemerovirus that, like BEFV, occurs in Africa, Asia and Australia.

Development and application of G1-ELISA for detection of antibodies against bovine ephemeral fever virus.

The gene encoding antigenic site G(1) of bovine ephemeral fever virus (BEFV) was highly expressed in the host cell Escherichia coli. An indirect G(1)-ELISA with the recombinant protein as the coating antigen was established to detect antibodies against BEFV. The result revealed that the optimal concentration of the coated antigen was 0.5 microg/well and the dilution of serum was 1:20. It was optimal that sera with P/N >or= 2.2 were considered positive, P/N

Adelaide River virus nucleoprotein gene: analysis of phylogenetic relationships of ephemeroviruses and other rhabdoviruses.

The nucleotide sequence of the Adelaide River virus (ARV) genome was determined from the 3' terminus to the end of the nucleoprotein (N) gene. The 3' leader sequence comprises 50 nucleotides and shares a common terminal trinucleotide (3' UGC-), a conserved U-rich domain and a variable AU-rich domain with other animal rhabdoviruses. The N gene comprises 1355 nucleotides from the transcription start sequence (AACAGG) to the poly(A) sequence [CATG(A)7] and encodes a polypeptide of 429 amino acids. The N protein has a calculated molecular mass of 49429 Da and a pI of 5.4 and, like the bovine ephemeral fever virus (BEFV) N protein, features a highly acidic C-terminal domain. Analysis of amino acid sequence relationships between all available rhabdovirus N proteins indicated that ARV and BEFV are closely related viruses (48.3% similarity) which share higher sequence similarity to vesiculoviruses than to lyssaviruses. Phylogenetic trees based on a multiple sequence alignment of all available rhabdovirus N protein sequences demonstrated clustering of viruses according to genome organization, host range and established taxonomic relationships.