African swine fever (ASF) is a notifiable transboundary disease of domestic pigs and wild suids that causes severe haemorrhagic fever, with high mortality, and substantial socio-economic losses worldwide. In the absence of effective vaccines or therapeutics, ASF control relies primarily on early diagnosis, strict biosecurity, and stamping-out strategies. In this study, we developed a rapid, field-deployable recombinase polymerase amplification (RPA)-CRISPR/Cas12a assay for the detection of African swine fever virus using both fluorescence- and lateral flow strip (LFS)-based readouts. The assay operates isothermally at 37 °ºC and enables rapid, equipment-free visual detection suitable for on-site testing. To improve diagnostic sensitivity and robustness, a dual-target approach was employed targeting the highly conserved C-terminal region of the p72 (B646L) gene and the early-expressing p22 (KP177R) gene. In the uniplex format, fluorescence-based assays achieved limits of detection of 10.18 and 13.68 copies/reaction for p72 and KP177R, respectively, whereas the LFS format detected 1,018 and 1,368 copies/reaction. Notably, the duplex RPA-CRISPR/Cas12a assay demonstrated enhanced sensitivity with detection limits of 1.02 copies/reaction in the fluorescence format and 102 copies/reaction in the LFS format. The assay showed 100% analytical specificity against other porcine viral pathogens and strong agreement with real-time PCR. Furthermore, lyophilized reagents formulated in a two-tube format retained their analytical performance after storage at 4 °C for one week, highlighting the platform's potential for decentralized ASF surveillance and outbreak response.

Diarrhea is an acute disease of cattle that has been extensively studied in calves; however, it has been far less investigated in adult cattle, despite its significant impact on milk production and animal health. In this study, we investigated an outbreak in a dairy herd in Uruguay for which initial diagnostic testing for the main known pathogens yielded negative results. Therefore, the outbreak was further investigated in depth using shotgun metagenomic sequencing, molecular virology assays and viral isolation. Of the three fecal samples from affected cows that were submitted for diagnostic testing and were negative for the main enteric pathogens, including BCoV, two were positive for bovine torovirus (BToV) by RT-qPCR. Next-generation sequencing-based virome analysis revealed over 3,300 viral operational taxonomic units (vOTUs), predominantly bacteriophages, but also eukaryotic viruses from 11 families. The complete genome of BToV was obtained and a putative novel protoparvovirus species was characterized. In addition, two distinct enteroviruses belonging to the species Enterovirus fitauri and Enterovirus idromi were identified. Notably, Enterovirus idromi had previously been reported exclusively in dromedaries; its detection in cattle expands the known host range of this species. Furthermore, the Enterovirus idromi strain appears to represent an interspecies recombinant enterovirus. These results highlight the importance of emerging approaches such as next-generation sequencing for identifying novel pathogens in cattle diseases and may serve as a basis for evaluating their potential role in disease pathogenesis, as well as for incorporating them into routine initial diagnostic testing.

Many infectious diseases commonly affecting domestic cats may pose potential threats to wild felid populations. Several established contagious feline viruses - including feline leukemia virus (FeLV), feline immunodeficiency virus (FIV), feline coronavirus (FCoV), feline calicivirus (FCV), feline herpesvirus (FHV), and feline parvovirus (FPV) - are well-known drivers of disease epidemiology. In addition, recently identified agents such as feline circovirus (FeCV), feline morbillivirus (FeMV) and other novel paramyxoviruses as well as polyomaviruses may represent emerging risks to wild populations. We conducted a comprehensive molecular epidemiological survey to investigate the occurrence of these viruses in free-ranging European wildcats (Felis s. silvestris) from the Harz Mountains, a low mountain range in central Germany. A total of 42 individuals were sampled between 2010 and 2018. Molecular screening was performed using a comprehensive set of conventional PCR, reverse transcription PCR (RT-PCR), and quantitative RT-PCR (qRT-PCR) assays to detect viral nucleic acids. FeLV DNA was detected in 5/42 individuals (11.90%), and FPV DNA was detected in 2/42 wildcats (4.76%). Overall, viral nucleic acid was identified in seven animals (16.67%). No evidence of the remaining target viruses was found using the respective (q)RT-PCR and conventional PCR assays. However, negative PCR results should be interpreted with caution due to the exclusive use of long-term ethanol-preserved spleen samples, which may have reduced analytical sensitivity. The findings provide molecular evidence of FPV and FeLV, in free-ranging European wildcats in the Harz Mountains. Continued surveillance remains essential to better understand pathogen dynamics, evaluate potential spillover from domestic cats, and inform conservation strategies aimed at safeguarding wild felid populations in Central Europe.

Tick-borne viruses (TBVs) threaten human and animal health in northern Europe, where Ixodes ricinus and Ixodes persulcatus are expanding their range. In Finland, the genetic diversity, population structure, and tick-associated RNA virome of these species remain insufficiently characterized. Characterizing tick-borne viruses in Finland is essential to detect emerging pathogens at the northern edge of tick expansion, where climate-driven ecological change may facilitate the spread of novel viruses with potential risks to human and animal health. We applied single-tick metatranscriptomic approach to profile and compare the RNA viromes of 92 adult I. ricinus and I. persulcatus collected from two locations in Finland. We identified 42 RNA viruses spanning 12 families, with Nairoviridae, Phenuiviridae, Partitiviridae, Flaviviridae, and Rhabdoviridae being the most represented. Viral community composition differed significantly between tick species, with I. persulcatus exhibiting more constrained but bunyaviridae-dominated viromes, whereas I. ricinus showed greater inter-individual variability and enrichment of partiti-like and iflavirus taxa. Phylogenetic reconstruction of conserved viral proteins supported the presence of several divergent viral lineages and confirmed the first detection of multiple Eurasian tick-associated viruses in Finland, including Nuomin virus, Beiji nairovirus, and Onega tick phlebovirus. Several detected viruses clustered within clades that include known or suspected zoonotic agents, highlighting the need for close monitoring of tick populations, especially in zones of sympatric occurrence where hybridization and associated close contact between the tick species have been observed. These findings reveal substantial, species-structured viral diversity in Finnish Ixodes ticks.

Here, a novel double-stranded RNA (dsRNA) mycovirus, tentatively designated Fusarium oxysporum polymycovirus 1 (FoPmV1), was isolated from Fusarium oxysporum, a phytopathogenic fungus that causes tobacco root rot. The genome of FoPmV1 consists of eight dsRNA segments, referred to as dsRNA1 to dsRNA8, with lengths of 2440 bp, 2276 bp, 2042 bp, 1303 bp, 1144 bp, 1064 bp, 1004 bp, and 913 bp, respectively. Sequence analysis revealed that these dsRNA segments encode 8 open reading frames (ORFs): ORF1 (771 amino acids, aa), ORF2 (699 aa), ORF3 (622 aa), and ORF4 (304 aa) encode an RNA-dependent RNA polymerase (RdRp), scaffold protein, methyltransferase, and coat protein (PASRP), respectively. While ORF5 (304 aa), ORF6 (161 aa), ORF7 (167 aa), and ORF8 (211 aa) encode proteins with unknown functions. BLAST analysis revealed that the RdRp of FoPmV1 shares the maximal sequence identity of 75.75% with the RdRp of Fusarium redolens polymycovirus 1. Phylogenetic analysis based on the RdRp sequence clustered FoPmV1 robustly within the family Polymycoviridae. Overall, this research represents the first report of a polymycovirus infecting F. oxysporum.

Highly pathogenic (HP) H5N1 avian influenza viruses are zoonotic pathogens and are endemic in wild birds and poultry around the world. Pro-inflammatory cytokines are supposed to contribute to the severe clinical symptoms in infected animals and humans. In this study, we determined the infectivity and cytokine induction in primary human lung cells infected with HP H5N1 avian influenza viruses and seasonal H3N2 influenza viruses. MDCK cells were used to measure viral titers in the supernatants of infected human lung cells by log10TCID50/ml. Quantitative real-time PCR was used to detect the induction of cytokine genes using mRNA from the infected primary human lung cells. HP H5N1 avian influenza viruses induced higher viral titers than seasonal H3N2 influenza viruses in supernatants of primary human lung cells. In addition, HP H5N1avian influenza viruses induced the genes of pro-inflammatory cytokines such as IL-23, IL-1α, IL-1β, IL-6, and IL-8 at significantly higher levels than seasonal H3N2 influenza viruses in primary human lung cells. Our data suggest that the high viral replication and the high induction of pro-inflammatory cytokines in the human lung cells infected with HP H5N1 avian influenza viruses may enhance pathogenicity in the infected humans as compared to seasonal H3N2 influenza viruses.

To develop a Vero cell-adapted Japanese encephalitis (JE) SA14-14-2 strain for the production of JE SA14-14-2 live attenuated vaccine (LAV) using Vero cells as the substrate, the SA14-14-2 PHKC strain was adapted to Vero cells and subjected to plaque purification followed by clone selection. A highly attenuated strain, SA14-14-2 VC6, was isolated, whose phenotypic and genotypic characteristics were highly consistent with those of its parental strain SA14-14-2 PHKC. Immunogenicity assays demonstrated that the VC6 strain exhibited comparable immunogenicity to the parental SA14-14-2 strain. Under the experimental conditions used in this study, the VC6 strain induced stronger neutralizing antibody (NAb) responses, protection against intraperitoneal (i.p.) or intracerebral (i.c.) challenge with wild-type JE virus (JEV), viremia inhibition, and cellular immune responses than the chimeric JE-CV vaccine. Collectively, these findings indicate that the SA14-14-2 VC6 retains the immunogenic properties of the parental SA14-14-2 strain after adaptation to Vero cells and warrants further preclinical evaluation before its potential use in Vero cell-based JE vaccine development.

We present the complete genome sequence of a novel alternavirus, tentatively named "Fusarium falciforme alternavirus 1 (FfAV1)", isolated from Fusarium falciforme. The host, F. falciforme strain Fod375, was isolated from a soil sample in Spain in 2012 and was found to be infected with a virus containing a tetra-segmented double-stranded (ds) RNA genome. The genome segments, designated as dsRNA1 (3529 bp), dsRNA2 (2641 bp), dsRNA3 (2459 bp), and dsRNA4 (1471 bp), each possess a single open reading frame (ORF). The protein predicted from dsRNA1 contains the typical domains of an RNA-dependent RNA polymerase (RdRP) homologous to those of previously reported alternaviruses, while the protein predicted from dsRNA3 shows homology to alternavirus capsid proteins. The proteins encoded by dsRNA2 and dsRNA4 are of unknown function. All predicted proteins exhibited the highest sequence identity with their counterparts in Hebei alternavirus and Marquandomyces marquandii alternavirus 1. Phylogenetic analysis supported the placement of this FfAV1 isolate within the genus Alternavirus. Considering these results, we propose that FfAV1, along with the two closely related unassigned alternaviruses, represents a new species within the genus.

In recent years, various bacteriophages that infect Vibrio spp. have been isolated and characterized. However, many characteristics concerning their infection mechanisms remain unknown. Here, we isolated and characterized a novel phage, VipHU7, that infects Vibrio parahaemolyticus. The morphology of VipHU7 was examined using transmission electron microscopy, which demonstrated that it has a short, noncontractile tail characteristic of podoviruses. VipHU7 formed clear plaques with halo zones on a bacterial lawn of V. parahaemolyticus MFS 1101, and host range analysis revealed that it had a limited host range. Analysis of the propagation and one-step growth curve of VipHU7 in liquid medium indicated that its replication rate increases in the presence of divalent cations, which did not affect its adsorption. Genome sequencing revealed that the VipHU7 genome is 76,454 bp long, with a 38.48% GC content and 112 predicted open reading frames. VipHU7 has a genomic structure similar to that of other Varunavirus phages that infect Vibrio spp. VIRIDIC analysis showed that the intergenomic similarity between VipHU7 and Vibrio phage BUCT194 was 83.7%, indicating that VipHU7 is a novel species belonging to the family Schitoviridae and genus Varunavirus.

Hydrangea febrifuga (syn. Dichroa febrifuga) is a traditional medicinal plant distributed in China and Southeast Asia, and febrifugine, one of its principal bioactive constituents, has served as an important lead compound for antimalarial drug development. Viral infections may adversely affect the quality of medicinal plants; however, no emaravirus has previously been reported from H. febrifuga. Here, high-throughput sequencing was performed on H. febrifuga leaves exhibiting mosaic symptoms collected in Yunnan Province, China. Combined with RT-PCR, Sanger sequencing, and 5'/3' rapid amplification of cDNA ends (RACE), five full-length genomic RNA segments of a putative novel emaravirus, tentatively designated Dichroa emaravirus (DEV), were identified and characterized. The five negative-sense single-stranded RNA (-ssRNA) segments have a combined length of 12,971 nt and encode an RNA-dependent RNA polymerase (RdRp), glycoprotein precursor (GP), nucleocapsid protein (NP), movement protein (MP), and an uncharacterized accessory protein, P5. The maximum amino acid sequence identities of DEV P1-P4 with recognized emaraviruses were 73.90%, 51.82%, 65.60%, and 81.30%, respectively, whereas P5 showed a maximum identity of 49.16% with its closest homolog. Thus, three of the four core proteins had maximum identities below 80%, consistent with the current ICTV species demarcation criterion for the genus Emaravirus. Maximum-likelihood phylogenetic analyses based on the four core proteins further supported the placement of DEV within the genus Emaravirus (family Fimoviridae). These results support DEV as a putative novel emaravirus and represent the first report of an emaravirus associated with H. febrifuga.

A novel caulimovirus was identified from diseased leaves of Dregea volubilis exhibiting yellowing and vein-associated chlorosis in Yuanjiang County, Yunnan Province, China. The virus was tentatively named Dregea volubilis caulimovirus 1 (DVCaV1). The complete genome sequence of DVCaV1, determined by de novo assembly of high-throughput sequencing data, comprises 8,160 bp of circular double-stranded DNA containing two intergenic regions and seven open reading frames (ORFs). These ORFs encode (in order) a movement protein (MP), an aphid transmission factor (ATF), a virion-associated protein (VAP), a coat protein (CP), a polymerase polyprotein (Pol, containing protease, reverse transcriptase, and RNase H domains), a transactivator/viroplasmin (TAV) protein, and a hypothetical protein of unknown function. Sequence comparisons revealed the highest nucleotide similarity with strawberry vein banding virus (SVBV; NC_001725). Phylogenetic analysis confirmed DVCaV1 as a member of the genus Caulimovirus, with SVBV as its closest known relative. According to current ICTV species demarcation criteria for the genus Caulimovirus (host range and > 20% nucleotide sequence divergence in the polymerase region), DVCaV1 represents a novel species. This is, to our knowledge, the first report of a caulimovirus detected in naturally symptomatic Dregea volubilis.

Chili leaf curl virus (ChiLCV) is a Begomovirus chillicapsici that is one of the most devastating viruses impacted on the production of chili in the world, especially in South Asia. In the present study, we combined high-throughput computational genomics with experimental analysis of global diversity. A workflow was created using automated Python scripts to download, curate and process ChiLCV genomes from public database. About 410 complete ChiLCV genomes download from public databases. Using a phylogenetic approach, these isolates were subdivided into 34 strains, belonging to 10 major clades, showing significant genetic diversity. Geographic analysis revealed that Pakistan (207 isolates) and India (148 isolates) were the main sources of ChiLCV diversity and the remainder of the isolates were from Oman, Bangladesh, Iran, Saudi Arabia and Sri Lanka. Recombination was observed as a major evolutionary force as more than twenty recombination events were detected. Analysis of cis-regulatory elements showed a complex structure of the viral promoter, including multiple binding sites for transcription factors, hormone-response elements, light-responsive elements, and stress-responsive elements, indicating a high number of interactions between viral regulatory elements and host signaling pathways. Pangenome analysis showed the presence of a highly dynamic open pangenome made up of strain-specific orthologous groups (species-specific orthogroups). Experimental inoculation of chili plants was also carried out to assess the biological effects of infection, along with phytochemical, FTIR, HPLC, and qPCR analyses.

High-throughput sequencing revealed a new emaravirus named clerodendrum yellow blotch virus (ClYBV), infecting Clerodendrum thomsoniae plants in São Paulo State, Brazil. Four genomic RNA segments (RNA1, RNA3, RNA4, and RNA5) were recovered, whereas RNA2 remained undetected. Phylogenetic analysis and pairwise comparisons based on amino acid sequences showed ClYBV is most closely related to redbud yellow ringspot-associated virus (RYRaV, Emaravirus cercidis). No ClYBV particles were observed by transmission electron microscopy in leaf tissues from infected plants. These results support the identification of ClYBV as a new Emaravirus member, though the undetectable RNA2 suggests the isolate may be defective, lacking this segment and unable to produce virus particles. The binomial species name "Emaravirus clerodendri" is proposed.

Emerging outbreaks of the arbovirus chikungunya virus affect millions of individuals globally, causing debilitating arthralgia and fever. A study on the burden and etiologies of acute febrile illnesses (AFI) in Kenya reported an increase in CHIKV cases in Mombasa between December 2017 and December 2019. In January and February 2020, another outbreak of CHIKV occurred in Dadaab-Hagadera. Using next-generation sequencing on the Illumina MiSeq platform, we established molecular characteristics and phylogenetic differences of the CHIKVs collected from the two epidemiologically distinct settings. Sequenced viruses belonged to the Indian Ocean Lineage and clustered according to geographic region, with E1:K211E and E2:V264A mutations present in both settings. Additional mutations previously reported during Kenyan CHIKV outbreaks and suggested to influence CHIKV adaptation to Aedes albopictus, including E1:T82I and E1:V84D, were also identified. These molecular markers provide useful information on CHIKV spread and can help strengthen outbreak preparedness and response. Clinical features were described only for laboratory-confirmed CHIKV cases from the AFI surveillance cohort and compared with AFI cases testing negative for CHIKV. Sore muscles, headache, and convulsions were more frequently reported among CHIKV-positive participants, whereas diarrhea was less common. No differences were observed in the frequency of cough, skin rash, conjunctivitis, or vomiting. Given the limited number of confirmed CHIKV cases and the predominance of young children in the cohort, the reported clinical symptom associations should only be interpreted as exploratory and warrant confirmation in larger and more representative populations.