Sequences Related to Chimay Rhabdovirus Are Widely Distributed in Ixodes ricinus Ticks across England and Wales.

Ticks are the main arthropod vector of pathogens to humans and livestock in the British Isles. Despite their role as a vector of disease, many aspects of tick biology, ecology, and microbial association are poorly understood. To address this, we investigated the composition of the microbiome of adult and nymphal Ixodes ricinus ticks. The ticks were collected on a dairy farm in Southwest England and RNA extracted for whole genome sequencing. Sequences were detected from a range of microorganisms, particularly tick-associated viruses, bacteria, and nematodes. A majority of the viruses were attributed to phlebo-like and nairo-like virus groups, demonstrating a high degree of homology with the sequences present in I. ricinus from mainland Europe. A virus sharing a high sequence identity with Chimay rhabdovirus, previously identified in ticks from Belgium, was detected. Further investigations of I. ricinus ticks collected from additional sites in England and Wales also identified Chimay rhabdovirus viral RNA with varying prevalence in all tick populations. This suggests that Chimay rhabdovirus has a wide distribution and highlights the need for an extended exploration of the tick microbiome in the United Kingdom (UK).

High-Throughput Sequencing Reveals Three Rhabdoviruses Persisting in the IRE/CTVM19 Cell Line.

Cell cultures derived from ticks have become a commonly used tool for the isolation and study of tick-borne pathogens and tick biology. The IRE/CTVM19 cell line, originating from embryos of Ixodes ricinus, is one such line. Previously, reovirus-like particles, as well as sequences with similarity to rhabdoviruses and iflaviruses, were detected in the IRE/CTVM19 cell line, suggesting the presence of multiple persisting viruses. Subsequently, the full genome of an IRE/CTVM19-associated rhabdovirus was recovered from a cell culture during the isolation of the Alongshan virus. In the current work, we used high-throughput sequencing to describe a virome of the IRE/CTVM19 cell line. In addition to the previously detected IRE/CTVM19-associated rhabdovirus, two rhabdoviruses were detected: Chimay rhabdovirus and Norway mononegavirus 1. In the follow-up experiments, we were able to detect both positive and negative RNA strands of the IRE/CTVM19-associated rhabdovirus and Norway mononegavirus 1 in the IRE/CTVM19 cells, suggesting their active replication in the cell line. Passaging attempts in cell lines of mammalian origin failed for all three discovered rhabdoviruses.

Divergent Rhabdovirus Discovered in a Patient with New-Onset Nodding Syndrome.

A divergent rhabdovirus was discovered in the bloodstream of a 15-year-old girl with Nodding syndrome from Mundri West County in South Sudan. Nodding syndrome is a progressive degenerative neuropathy of unknown cause affecting thousands of individuals in Sub-Saharan Africa. The index case was previously healthy until she developed head-nodding seizures four months prior to presentation. Virus discovery by VIDISCA-NGS on the patient's plasma detected multiple sequence reads belonging to a divergent rhabdovirus. The viral load was 3.85 × 103 copies/mL in the patient's plasma and undetectable in her cerebrospinal fluid. Further genome walking allowed for the characterization of full coding sequences of all the viral proteins (N, P, M, U1, U2, G, U3, and L). We tentatively named the virus "Mundri virus" (MUNV) and classified it as a novel virus species based on the high divergence from other known viruses (all proteins had less than 43% amino acid identity). Phylogenetic analysis revealed that MUNV forms a monophyletic clade with several human-infecting tibroviruses prevalent in Central Africa. A bioinformatic machine-learning algorithm predicted MUNV to be an arbovirus (bagged prediction strength (BPS) of 0.9) transmitted by midges (BPS 0.4) with an artiodactyl host reservoir (BPS 0.9). An association between MUNV infection and Nodding syndrome was evaluated in a case-control study of 72 patients with Nodding syndrome (including the index case) matched to 65 healthy households and 48 community controls. No subject, besides the index case, was positive for MUNV RNA in their plasma. A serological assay detecting MUNV anti-nucleocapsid found, respectively, in 28%, 22%, and 16% of cases, household controls and community controls to be seropositive with no significant differences between cases and either control group. This suggests that MUNV commonly infects children in South Sudan yet may not be causally associated with Nodding syndrome.

A new cluster of rhabdovirus detected in field-caught sand flies (Diptera: Psychodidae: Phlebotominae) collected from southern Thailand.

BACKGROUND: The distribution of phlebotomine sand flies is changing rapidly due to climate change. This issue has implications for the epidemiology of sand fly-borne diseases, especially sand fly-associated viruses. Few studies concerning sand fly-associated viruses have been conducted in Thailand. Therefore, this study aimed to perform a molecular survey of groups of pathogenic RNA viruses belonging to the Orbivirus, Phlebovirus, and Flavivirus genera and family Rhabdoviridae in sand fly samples collected from southern Thailand. METHODS: Sand flies were collected at two locations in Trang and Songkhla provinces of southern Thailand, and individual sand fly samples were processed for species identification and virus detection. The Orbivirus, Phlebovirus, and Flavivirus genera and family Rhabdoviridae molecular determination was performed by RT-PCR, and positive samples were identified by cloning and sequencing, cell culture inoculation, and phylogenetic analysis. RESULTS: The results presented in this study were based on the analysis of a total of 331 female sand flies. This molecular study revealed evidence of Rhabdoviridae family virus presence in Phlebotomus papatasi (3/331, 0.9%). The findings demonstrated a new cluster of rhabdovirus that was closely related to Bactrocera dorsalis sigmavirus strain BDSV.abc5 and the lineages of insect-specific Rhabdoviridae. In addition, the Bayesian tree suggested that the common ancestor of this group was the dimarhabdovirus clade. It was assumed that the virus may have switched hosts during its evolution. However, the detection of Orbivirus, Phlebovirus, and Flavivirus genera using specific primers for RT-PCR was negative in the collected sand flies. CONCLUSIONS: There is limited knowledge on the genetic diversity and ecology of Rhabdoviridae in Thailand. This is the first data regarding the circulation of Rhabdoviridae in Ph. papatasi from Thailand. We found a new cluster of rhabdoviruses that was close to the new B. dorsalis sigmavirus. It is possible that there is a great deal of diversity in this family yet to be discovered, and a more extensive survey for new rhabdoviruses may uncover viruses from a wide diversity of host taxa and broaden our understanding of the relationships among the Rhabdoviridae.

High-Throughput Sequencing Indicates Novel Varicosavirus, Emaravirus, and Deltapartitivirus Infections in Vitis coignetiae.

Vitis coignetiae samples were collected from several locations in the northern area of Japan, and virome analysis using a high-throughput sequencing technique was performed. The data indicated that some of the collected samples were in mixed infections by various RNA viruses. Among these viruses, three were identified as newly recognized species with support of sequence identity and phylogenetic analysis. The viruses have been provisionally named the Vitis varicosavirus, Vitis emaravirus, and Vitis crypticvirus, and were assigned to the genus Varicosavirus, Emaravirus, and Deltapartitivirus, respectively.

Joá yellow blotch-associated virus, a new alphanucleorhabdovirus from a wild solanaceous plant in Brazil.

We identified a novel plant rhabdovirus infecting native joá (Solanum aculeatissimum) plants in Brazil. Infected plants showed yellow blotches on the leaves, and typical enveloped bacilliform rhabdovirus particles associated with the nucleus were seen in thin sections by electron microscopy. The virus could be graft-transmitted to healthy joá and tomato plants but was not mechanically transmissible. RT-PCR using degenerate plant rhabdovirus L gene primers yielded an amplicon from extracted total RNA, the sequence of which was similar to those of alphanucleorhabdoviruses. Based on close sequence matches, especially with the type member potato yellow dwarf virus (PYDV), we adopted a degenerate-primer-walking strategy towards both genome ends. The complete genome of joá yellow blotch-associated virus (JYBaV) is comprised of 12,965 nucleotides, is less than 75% identical to that of its closest relative PYDV, and clusters with PYDV and other alphanucleorhabdoviruses in L protein phylogenetic trees, suggesting that it should be taxonomically classified in a new species in the genus Alphanucleorhabdovirus, family Rhabdoviridae. The genome organization of JYBaV is typical of the 'PYDV-like' subgroup of alphanucleorhabdoviruses, with seven genes (N-X-P-Y-M-G-L) separated by conserved intergenic regions and flanked by partly complementary 3' leader and 5' trailer regions.

Measures of diagnostic precision (repeatability and reproducibility) for three test methods designed to detect spring viremia of carp virus.

Spring viremia of carp virus (SVCV) is a rhabdovirus of the Sprivivirus genus and the etiological agent of an internationally regulated aquatic animal disease in several fish species, including koi carp Cyprinus carpio L. The virus has a complex lifecycle with both acute and persistent stages of infection and can cause high mortality in affected populations. In this study, the diagnostic repeatability (within laboratory agreement) and reproducibility (between laboratory agreement) of 3 tests were investigated to assess their fitness as SVCV diagnostic tools. The tests, reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays targeting either the SVCV glycoprotein (Q1G) or nucleoprotein (Q2N) genes and virus isolation by cell culture (VI), were performed in a blinded study with four Canadian laboratories. Test panels consisted of duplicate sets of 100 tissue samples collected from 3 SVCV prevalence populations of koi: a low-prevalence negative reference population (n = 20 fish) as well as moderate- (n = 50 fish) and high-prevalence (n = 30 fish) populations of koi experimentally infected with SVCV. The Q1G and Q2N tests were performed with kidney tissue in 3 laboratories and with brain tissue in 1 laboratory whereas pools of kidney, spleen and gill tissues were tested with the VI assay in 2 laboratories. Agreement of binary results was evaluated using the observed proportion of agreement, Cohen's kappa and Gwet's agreement coefficient (AC1) whereas the concordance correlation coefficient (ccc) and Bland Altman's limit of agreement were used to evaluate agreement of the RT-qPCR continuous data. Gwet's AC1 provided a more stable estimate of agreement than Cohen's kappa. Overall, high repeatability (AC1, 0.78-0.90) and reproducibility (AC1, 0.74-0.89) were observed for the Q1G and Q2N tests when kidney tissue was used. Lower agreement estimates of repeatability (AC1, 0.54-0.77) and reproducibility (AC1, 0.50-0.80) were obtained for the VI test. RT-qPCR reproducibility was low with kidney-brain tissue pairs (AC1, 0.09-0.46) and high with inter-test pairs of brain (AC1, 0.76-0.86) or kidney tissue (0.75-0.86). Tissue-specific differences in virus load affected test precision and informed final tissue selection. Repeatability (ccc, 0.94-0.97) and reproducibility (ccc, 0.91-0.97) estimates of agreement for paired continuous data from the RT-qPCR assays were similarly high with kidney tissue and lower with paired brain (ccc, 0.15-0.83) and kidney-brain tissues (ccc, 0.01-0.55). The high precision of Q1G and Q2N with kidney tissue suggests that the tests are performing similarly and are suitable candidates for assessment of their diagnostic accuracy.

Characterization of Novel Rhabdoviruses in Chinese Bats.

Bats, the second largest order of mammals worldwide, harbor specific characteristics such as sustaining flight, a special immune system, unique habits, and ecological niches. In addition, they are the natural reservoirs of a variety of emerging or re-emerging zoonotic pathogens. Rhabdoviridae is one of the most diverse families of RNA viruses, which consists of 20 ecologically diverse genera, infecting plants, mammals, birds, reptiles, and fish. To date, three bat-related genera are described, named Lyssavirus, Vesiculovirus, and Ledantevirus. However, the prevalence and the distribution of these bat-related rhabdoviruses remain largely unknown, especially in China. To fill this gap, we performed a large molecular retrospective study based on the real-time reverse transcription polymerase chain reaction (RT-qPCR) detection of lyssavirus in bat samples (1044 brain and 3532 saliva samples, from 63 different bat species) originating from 21 provinces of China during 2006-2018. None of them were positive for lyssavirus, but six bat brains (0.6%) of Rhinolophus bat species, originating from Hubei and Hainan provinces, were positive for vesiculoviruses or ledanteviruses. Based on complete genomes, these viruses were phylogenetically classified into three putative new species, tentatively named Yinshui bat virus (YSBV), Taiyi bat virus (TYBV), and Qiongzhong bat virus (QZBV). These results indicate the novel rhabdoviruses circulated in different Chinese bat populations.

Novel and Diverse Non-Rabies Rhabdoviruses Identified in Bats with Human Exposure, South Dakota, USA.

Bats are a host and reservoir for a large number of viruses, many of which are zoonotic. In North America, the big brown bat (Eptesicus fuscus) is widely distributed and common. Big brown bats are a known reservoir for rabies virus, which, combined with their propensity to roost in human structures, necessitates testing for rabies virus following human exposure. The current pandemic caused by severe acute respiratory syndrome coronavirus 2, likely of bat origin, illustrates the need for continued surveillance of wildlife and bats for potentially emerging zoonotic viruses. Viral metagenomic sequencing was performed on 39 big brown bats and one hoary bat submitted for rabies testing due to human exposure in South Dakota. A new genotype of American bat vesiculovirus was identified in seven of 17 (41%) heart and lung homogenates at high levels in addition to two of 23 viscera pools. A second rhabdovirus, Sodak rhabdovirus 1 (SDRV1), was identified in four of 23 (17%) viscera pools. Phylogenetic analysis placed SDRV1 in the genus Alphanemrhavirus, which includes two recognized species that were identified in nematodes. Finally, a highly divergent rhabdovirus, Sodak rhabdovirus 2 (SDRV2), was identified in two of 23 (8.7%) big brown bats. Phylogenetic analysis placed SDRV2 as ancestral to the dimarhabdovirus supergroup and Lyssavirus. Intracranial inoculation of mouse pups with rhabdovirus-positive tissue homogenates failed to elicit clinical disease. Further research is needed to determine the zoonotic potential of these non-rabies rhabdoviruses.

Transmission of the Bean-Associated Cytorhabdovirus by the Whitefly Bemisia tabaci MEAM1.

The knowledge of genomic data of new plant viruses is increasing exponentially; however, some aspects of their biology, such as vectors and host range, remain mostly unknown. This information is crucial for the understanding of virus-plant interactions, control strategies, and mechanisms to prevent outbreaks. Typically, rhabdoviruses infect monocot and dicot plants and are vectored in nature by hemipteran sap-sucking insects, including aphids, leafhoppers, and planthoppers. However, several strains of a potentially whitefly-transmitted virus, papaya cytorhabdovirus, were recently described: (i) bean-associated cytorhabdovirus (BaCV) in Brazil, (ii) papaya virus E (PpVE) in Ecuador, and (iii) citrus-associated rhabdovirus (CiaRV) in China. Here, we examine the potential of the Bemisia tabaci Middle East-Asia Minor 1 (MEAM1) to transmit BaCV, its morphological and cytopathological characteristics, and assess the incidence of BaCV across bean producing areas in Brazil. Our results show that BaCV is efficiently transmitted, in experimental conditions, by B. tabaci MEAM1 to bean cultivars, and with lower efficiency to cowpea and soybean. Moreover, we detected BaCV RNA in viruliferous whiteflies but we were unable to visualize viral particles or viroplasm in the whitefly tissues. BaCV could not be singly isolated for pathogenicity tests, identification of the induced symptoms, and the transmission assay. BaCV was detected in five out of the seven states in Brazil included in our study, suggesting that it is widely distributed throughout bean producing areas in the country. This is the first report of a whitefly-transmitted rhabdovirus.

Molecular characterization of a novel cytorhabdovirus associated with paper mulberry mosaic disease.

A novel cytorhabdovirus, tentatively named "paper mulberry mosaic-associated virus" (PMuMaV), was discovered and identified by transcriptome sequencing (RNA-seq), small RNA sequencing (sRNA-seq), and RT-PCR amplification. The whole-genome sequence of PMuMaV is 13,736 nucleotides (nt) in length and contains six open reading frames (ORFs) encoding a nucleocapsid protein (N), a phosphoprotein (P), a putative movement protein (P3), a matrix protein (M), a glycoprotein (G), and an RNA-dependent RNA polymerase (L). The coding sequences are flanked by a 194-nt leader and a 370-nt trailer sequence at the 3' terminus and 5' terminus, respectively. Pairwise sequence comparisons showed that PMuMaV is related to northern cereal mosaic virus (NCMV, 38.97%), barley yellow striate mosaic virus (BYSMV, 38.86%), and maize yellow striate virus (MYSV, 38.76%), and phylogenetic analysis also placed these viruses together into the same branch, thus suggesting that PMuMaV is a member of a new species in the genus Cytorhabdovirus.

Identification, molecular characterization and prevalence of a novel cytorhabdovirus infecting zucchini crops in Greece.

A new cytorhabdovirus was identified in zucchini (Cucurbita pepo) in Greece with the aid of high-throughput sequencing technology. The negative-sense, single-stranded genomic RNA of the new virus was determined and includes seven open reading frames in the order 3'-N-P-P3-P4-M-G-L-5' in the antigenomic orientation. Typical rhabdovirus-like particles were observed in infected leaf material. Comparative sequence analysis and phylogenetic reconstructions suggested that the described virus is a new member of the genus Cytorhabdovirus, and it was tentatively named cucurbit cytorhabdovirus 1 (CuCV1). To our knowledge CuCV1 is the first cytorhabdovirus infecting cucurbits in nature. Our surveys indicated that it occurs in a percentage of 36.7 % in zucchini crops in Greece.

Revisiting the Classification of Percid Perhabdoviruses Using New Full-Length Genomes.

Perhabdoviruses are a threat to some freshwater fish species raised in aquaculture farms in Europe. Although the genetic diversity of these viruses is suspected to be high, the classification of isolates is still in its infancy, with just one full-length genome available and only partial sequences for a limited number of others. Here, we characterized a series of viruses isolated from percids in France from 1999 to 2009 by sequencing the nucleoprotein (N) gene. Four main clusters were distinguished, all related at varying levels of similarity to one of the two already-recognized species, namely Perch perhabdovirus and Sea trout perhabdovirus. Furthermore, we obtained the complete genome of five isolates, including one belonging to Sea trout rhabdovirus. The analysis of the complete L genes and the concatenated open reading frames confirmed the existence of four main genetic clusters, sharing 69 to 74% similarity. We propose the assignation of all these viral isolates into four species, including two new ones: Perch perhabdovirus 1, Perch perhabdovirus 2, Sea trout perhabdovirus 1 and Sea trout perhabdovirus 2. In addition, we developed new primers to readily amplify specific portions of the N gene of any isolate of each species by conventional PCR. The presence of such genetically diverse viruses in France is likely due to divergent viral populations maintained in the wild and then introduced to experimental facilities or farms, as well as via trade between farms across the European continent. It is now urgent to improve the identification tools for this large group of viruses to prevent their unchecked dissemination.

Identification of Trichosanthes associated rhabdovirus 1, a novel member of the genus Cytorhabdovirus of the family Rhabdoviridae, in the Trichosanthes kirilowii transcriptome.

The genome sequence of a novel RNA virus, Trichosanthes associated rhabdovirus 1 (TrARV1), was identified in a transcriptome dataset isolated from a root sample of Trichosanthes kirilowii, which is a flowering plant belonging to the family Cucurbitaceae. The fruits, seeds, and root tubers of T. kirilowii have been used clinically in traditional Chinese medicine. The TrARV1 genome sequence was predicted to have six open reading frames (ORFs) encoding five canonical structural proteins of the family Rhabdoviridae (N ORF for nucleocapsid, P ORF for phosphoprotein, M ORF for matrix protein, G ORF for glycoprotein, and L ORF for polymerase), and an accessory protein. Sequence comparisons and phylogenetic analyses based on L and N proteins confirmed that TrARV1 is a novel member of the genus Cytorhabdovirus of the family Rhabdoviridae. TrARV1 is most closely related to Wuhan insect virus 5 and persimmon virus A. The putative cis-regulatory elements involved in transcription termination and polyadenylation, commonly found in the gene junction regions of rhabdoviruses, were also identified in the TrARV1 genome having the consensus sequence 3'- ACUAAAUUAUUUUGAUCUUU-5'. The genome sequence of TrARV1 may be useful to study the evolution and molecular biology of cytorhabdoviruses. Keywords: Trichosanthes associated rhabdovirus 1; Cytorhabdovirus; Rhabdoviridae; Trichosanthes kirilowii.

Identification and characterization of a novel rhabdovirus infecting peach in China.

A novel negative-sense, single-stranded (ss) RNA virus was identified in peach trees by high-throughput sequencing, and named peach virus 1 (PeV1). The genome of PeV1 consists of 13,949 nucleotides (nt), and its organization is typical of rhabdoviruses with six open reading frames (ORFs) encoding deduced proteins N-P-P3-M-G-L on the antisense strand. These ORFs are separated by highly conserved intergenic sequences and flanked by complementary 3'-leader and 5'-trailer sequences. PeV1 shared highest complete genome (41.9%), N amino acid (43.6%), G amino acid (41.0%), and L amino acid (42.7%) identities with viruses which belong to the genus Alphanucleorhabdovirus, suggesting it may belong to a new species. This was further supported by phylogenetic analyses using amino acid sequences of N, G, and L proteins, in which this virus is always clustered with alphanucleorhabdoviruses. Collectively, results suggest that PeV1 is a member of a new alphanucleorhabdovirus species. Moreover, bioassays revealed that it could be transmitted through grafting. The findings expand our knowledge of peach-infecting viruses and alphanucleorhabdoviruses.

Molecular and Biological Characterization of a New Strawberry Cytorhabdovirus.

Virus diseases of strawberry present several complex problems. More than 25 viruses have been described in the genus Fragaria thus far. Here, we describe a novel rhabdovirus, tentatively named strawberry virus 1 (StrV-1), that infects F.ananassa and F.vesca plants. Genomic sequences of three distinct StrV-1 genotypes co-infecting a single F.ananassa host were obtained using combined Illumina and Ion Proton high-throughput sequencing. StrV-1 was transmitted to herbaceous plants via Aphisfabae and A.ruborum, further mechanically transmitted to Nicotianaoccidentalis 37B and sub-inoculated to N.benthamiana, N.benthamiana DCL2/4i, N.occidentalis 37B, and Physalisfloridana plants. Irregular chlorotic sectors on leaf blades and the multiplication of calyx leaves seem to be the diagnostic symptoms for StrV-1 on indexed F.vesca clones. StrV-1 was detected in asymptomatic grafted plants and in 49 out of 159 field strawberry samples via RT-PCR followed by Sanger sequencing. The bacilliform shape of the virions, which have a cytoplasm-limited distribution, their size, and phylogenetic relationships support the assignment of StrV-1 to a distinct species of the genus Cytorhabdovirus. Acyrthosiphonmalvae, A.fabae, and A.ruborum were shown to transmit StrV-1 under experimental conditions.

The complete genome sequence of a novel cytorhabdovirus identified in strawberry (Fragaria ananassa Duch.).

A cytorhabdovirus, tentatively named "strawberry-associated virus 1" (SaV1), was identified in strawberry (Fragaria ananassa Duch.), and its complete genome sequence was determined. Its negative-sense single-stranded RNA genome is composed of 14,159 nucleotides and contains eight open reading frames (ORFs) in the canonical order 3'-N-P-P3-M-G-P6-P7-L-5. The ORFs are separated by conserved intergenic sequences, and the genome coding region is flanked by 3' and 5' untranslated regions of 179 and 856 nt, respectively. SaV1 N and L genes shares 32-57% and 38-64% amino acid sequence identity with those of nine reported cytorhabdoviruses, respectively. Phylogenetic analysis showed that SaV1 clustered with high confidence with representative cytorhabdoviruses and is most closely related to tomato yellow mottle-associated virus. There are two additional small genes of unknown function between the G and L genes. We propose that SaV1 should be considered a member of a novel species in the genus Cytorhabdovirus, family Rhabdoviridae.

Characterization of a novel rhabdovirus isolated from a stranded harbour porpoise (Phocoena phocoena).

An adult male harbour porpoise (Phocoena phocoena) stranded off the coast of Alaska displaying poor body condition, scattered mild ulcerative dermatitis, and necrotizing balanoposthitis. Necropsy findings included severe verminous panniculitis, pneumonia, hepatitis, and enteritis. Histopathological examination of skin lesions revealed a pustular epidermitis and dermatitis, with ballooning degeneration of keratinocytes and occasional amphophilic intracytoplasmic inclusion bodies. A swab sample collected from the ulcerative penile lesions was processed for virus isolation resulting in cytopathic effects observed in primary beluga whale kidney (BWK) cells. Transmission electron microscopy revealed bullet-shaped virions budding from the cell surface of infected BWK cells consistent with a rhabdovirus. A cDNA library was prepared using RNA extracted from infected cell culture supernatant and sequenced on an Illumina MiSeq sequencer. The near-complete genome of a novel rhabdovirus was recovered. Genetic and phylogenetic analyses based on the complete L gene supported the harbour porpoise rhabdovirus (HPRV) as a new species. HPRV clustered phylogenetically with dolphin rhabdovirus (DRV) and this cetacean rhabdovirus clade was found to be the sister group to members of the genus Perhabdovirus that infect fish. A specific nested RT-PCR assay detected HPRV RNA in the epaxial musculature of the harbour porpoise. Our results are consistent with a previous hypothesis that cetacean rhabdoviruses may have arisen following a host jump from fish and suggest that DRV and HPRV represent separate species belonging in a new genus within the family Rhabdoviridae. Further research is needed to determine the health impact of HPRV in harbour porpoise populations, its prevalence, and route of transmission.

The Spodoptera frugiperda Sf9 cell line is a heterogeneous population of rhabdovirus-infected and virus-negative cells: Isolation and characterization of cell clones containing rhabdovirus X-gene variants and virus-negative cell clones.

The Sf9 cell line is broadly used for manufacturing baculovirus-expressed viral vaccines. We previously reported the presence of a novel, rhabdovirus in the Sf9 cell line, which contained a unique X gene (Sf-rhabdovirus; designated as X+ in this paper). These results were extended by other reports describing an Sf-rhabdovirus variant in Sf9 cells, which lacked 320 nucleotides encompassing the X-gene and adjacent intergenic region (designated as X- in this paper), and the development of an Sf-rhabdovirus negative cell line. Here, we report that the Sf9 cell line is a mixed-cell population, based upon isolation of cell clones with distinct phenotypes: Sf-rhabdovirus-negative, X+, and X-. We also show that Sf-rhabdovirus X+ and X- variants replicate independently in Sf-rhabdovirus-negative cells. These results shed light on the detection of different rhabdovirus variants by different laboratories using Sf9-derived cell clones and confirm that both X+ and X- viruses are infectious in rhabdovirus-negative Sf9 cells.

Discovery of Four Novel Viruses Associated with Flower Yellowing Disease of Green Sichuan Pepper (Zanthoxylum Armatum) by Virome Analysis.

An emerging virus-like flower yellowing disease (FYD) of green Sichuan pepper (Zanthoxylum armatum v. novemfolius) has been recently reported. Four new RNA viruses were discovered in the FYD-affected plant by the virome analysis using high-throughput sequencing of transcriptome and small RNAs. The complete genomes were determined, and based on the sequence and phylogenetic analysis, they are considered to be new members of the genera Nepovirus (Secoviridae), Idaeovirus (unassigned), Enamovirus (Luteoviridae), and Nucleorhabdovirus (Rhabdoviridae), respectively. Therefore, the tentative names corresponding to these viruses are green Sichuan pepper-nepovirus (GSPNeV), -idaeovirus (GSPIV), -enamovirus (GSPEV), and -nucleorhabdovirus (GSPNuV). The viral population analysis showed that GSPNeV and GSPIV were dominant in the virome. The small RNA profiles of these viruses are in accordance with the typical virus-plant interaction model for Arabidopsis thaliana. Rapid and sensitive RT-PCR assays were developed for viral detection, and used to access the geographical distributions. The results revealed a correlation between GSPNeV and the FYD. The viruses pose potential threats to the normal production of green Sichuan pepper in the affected areas due to their natural transmission and wide spread in fields. Collectively, our results provide useful information regarding taxonomy, transmission and pathogenicity of the viruses as well as management of the FYD.