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Arthropod-borne viruses (arboviruses) are an increasingly significant threat to public health in tropical regions. In this study, we investigated the seroprevalence of various arboviruses in two species of sloth (Choloepus hoffmanni and Bradypus variegatus) in rural and peri-urban areas of Western Panama province. Between 2013 and 2018, blood samples from 60 sloths were tested for neutralizing antibodies against ten arboviruses. Significant variation in seroprevalence of different arboviruses was observed: 6.7% of sloths were seropositive for Madariaga virus, 6.7% for Venezuelan equine encephalitis virus, and 4.8% for Oropouche virus, while none were seropositive for dengue type 2, Zika, chikungunya, Una, Mayaro, or Punta Toro viruses. Notably, two Changuinola virus (CGLV) strains, which were previously isolated from Panamanian sloths in the 1970s, showed high seroprevalence: Pansloth 149 (23.3%) and D50 (53.3%). Given the high seroprevalence detected in our study and the lack of genomic characterization of the historical Pansloth 149 isolate, we performed next-generation sequencing of its complete genome using Illumina technology to understand its genetic diversity and evolutionary relationship with other CGLV strains.

Corriparta virus (CORV), an arbovirus within the Orbivirus genus, exhibits a broad vertebrate host range but limited pathogenicity. In this study, we report the first isolation and characterization of a novel orbivirus genetically related to CORV, temporarily designed as novel duck orbivirus (NDORV), from Beijing ducks in Henan province, China, in 2024. Genomic characterization revealed that NDORV possesses a 10‐segment double‐stranded RNA (dsRNA) genome, consistent with the structural hallmarks of the Orbivirus genus, with a high genetic similarity to Parry’s Lagoon virus (PLV) and CORV. To evaluate its pathogenicity, specific pathogen‐free (SPF) ducks were experimentally inoculated with NDORV. Gross pathological examination revealed splenomegaly and blood stasis as primary lesions, with no mortality observed. Histopathological analysis identified tissue damage in the spleen, lungs, heart, liver, and kidneys. The highest viral loads were observed in the spleen and lungs, peaking at 3 days postinoculation (dpi). This study provides the first comprehensive characterization of a novel orbivirus genetically akin to CORV isolated from ducks in China. These findings highlight the potential prevalence of NDORV in domestic duck populations and underscore the urgency of enhanced surveillance and research on CORV‐related arboviruses.

Background Tibet Orbivirus (TIBOV) is a recently discovered Orbivirus known to infect cattle, Asian buffalo and goats in south-western China. It was first isolated from mosquitoes and subsequently from biting midges ( Culicoides spp.) in Yunnan, China, indicating that it is an arbovirus. Little is known of its potential to cause disease, but the economic importance of related viruses promoted an investigation of potential Culicoides spp. vectors of TIBOV. Methods Biting midges were collected approximately once per week between May and December 2020, at a cattle farm in Wulong village, Shizong County, Yunnan Province, China. Approximately 3000 specimens of nine species were subsequently used in attempts to isolate virus, and a further 2000 specimens of six species were tested for the presence of bluetongue virus (BTV) and TIBOV using a RT-qPCR test. Results Virus isolation attempts resulted in the isolation of three viruses. One isolate from a pool of Culicoides jacobsoni was identified as TIBOV, while the other two viruses from C. orientalis and C. tainanus remain unidentified but are not BTV or TIBOV. RT-qPCR analysis did not detect BTV in any specimens, but a single pool containing five specimens of C . jacobsoni and another containing five specimens of C . tainanus produced PCR quantification cycle (Cq) values of around 28 that may indicate infection with TIBOV. Conclusions The isolation of TIBOV from C . jacobsoni satisfies one criterion required to prove its status as a vector of this virus. This isolation is supported by a low Cq value produced from a different pool of this species in the RT-qPCR test. The low Cq value obtained from a pool of C . tainanus suggests that this species may also be able to satisfy this criterion. Both of these species are widespread throughout Asia, with C . jacobsoni extending into the Pacific region, which raises the possibility that TIBOV may be more widespread than is currently known. Graphical abstract

Northern Australia has long been a hotbed of arboviral discovery, and collections of viral isolates from Northern Australia represent an invaluable resource for both our knowledge of viral diversity and for disease preparedness and treatment. While discovery of novel viruses via classical virology methods is on the decline, next generation sequencing offers the possibility to speed up viral discovery, albeit at the expense of the collection of valuable life history data. By sequencing unknown isolates from historical viral collections, we may leverage both the rich data collected through classical virology and the power of identification using contemporary sequencing technologies. In the present study, we sequenced 76 historical viral isolates held at the Berrimah Veterinary Laboratory in Darwin, northern Australia, for which serological typing had yielded ambiguous results. We determined that 43 of these isolates belong to the genera Hapavirus, Orbivirus, and Orthobunyavirus. Several of these isolates are putatively novel genotypes or potential taxa, which has significant potential implications for human and animal health. This study demonstrates the utility of historical collections for viral discovery and characterisation and how considerable past efforts to isolate and characterise viruses can be enhanced using next generation sequencing approaches.

The discovery and characterisation of new mosquito-borne viruses provides valuable information on the biodiversity of vector-borne viruses and important insights into their evolution. In this study, a broad-spectrum virus screening system, based on the detection of long double-stranded RNA in inoculated cell cultures, was used to investigate the presence of novel viruses in mosquito populations of northern Australia. We detected and isolated a new virus (tentatively named Parry’s Lagoon virus, PLV) from Culex annulirostris, Culex pullus, Mansonia uniformis and Aedes normanensis mosquitoes that shares genomic sequence similarities to Corriparta virus (CORV), a member of the Orbivirus genus of the family Reoviridae. Despite moderate to high (72.2% to 92.2%) amino acid identity across all proteins when compared to CORV, and demonstration of antigenic relatedness, PLV did not replicate in several vertebrate cell lines that were permissive to CORV. This striking phenotypic difference suggests that PLV has evolved to have a very restricted host range, indicative of a mosquito-only life cycle.

Tibet orbivirus (TIBOV) was initially isolated in Tibet in 2009 and subsequently in Guangdong, Hunan, and Yunnan, China. We document the first isolation of TIBOV outside of China: two TIBOV isolates from Culicoides collected in 2009 and 2010 in Kagoshima, Japan. Their complete genome sequences were also determined. Our results suggest that the two virus isolates are of novel serotypes, evident by variability within genome segment 2 encoding VP2. These new putative TIBOV serotypes will help with future virus surveillance and with the evaluation of its potential to cause disease in domestic ruminants.

Tibet orbivirus (TIBOV) is an orbivirus transmitted by mosquitoes and Culicoides , despite specific neutralizing antibodies being detected in pigs, but the molecular genetic characteristics of TIBOV strains in infected pigs are completely uncharted, and their pathogenicity in piglets is poorly elucidated. This study aimed to investigate the genetic characteristics of TIBOV in infected pigs and evaluate the pathogenicity of TIBOV in weaned piglets. Through viral metagenomic sequencing, seven segments (VP1‐VP4, VP6, NS1, and NS2) of TIBOV were obtained from swine tissues, and the sequences showed high identity with TIBOVs isolated from Culicoides , mosquitos, and cattle. After infection with TIBOV, the body temperature, appetite, and behavior of the piglets were normal, whereas hemorrhage nodes were observed on the hooves of all piglets and on the abdominal skin of one pig. Viremia was first detected at 2 days postinfection (dpi), peaked at 6 dpi, and remained high until 21 dpi. The virus was distributed in multiple organs, and the highest viral load and strongest viral nucleic acid signals were observed in the spleen. The most severe lesion was observed in the spleen with white pulp atrophy, a decreased number of lymphocytes, and widened septa of the medullary cord, indicating that the spleen was the most important target organ of TIBOV infection. The levels of inflammatory cytokines, including interleukin (IL)‐18, tumor necrosis factor‐α (TNF‐α), interferon (IFN)‐α, and IFN‐λ3 in peripheral blood lymphocytes decreased significantly from 2 to 6 dpi, and interferon‐stimulated gene‐15 ( ISG-15 ) and IFN regulatory factor 7 ( IRF-7 ) expression levels declined significantly from 2 to 9 dpi, suggesting that the host immune response was inhibited within 6 dpi. Our findings confirmed that TIBOV elicited long‐term viremia with mild clinical symptoms in piglets, the spleen was the target organ of TIBOV proliferation, and the host immune response may be slightly inhibited in the early stage of viral infection.

In this study, we provide a genomic description of the first isolation of the Umattila virus (UMAV) in Brazil. The virus was obtained from the blood of a bird (Turdus fumigatus) and isolated in a C6/36 cell culture. The viral genome contains ten segments, and its organization is characteristic of viruses of the genus Orbivirus (family Sedoreoviridae). The coding region of each segment was sequenced, demonstrating the nucleotide identity with UMAV. The phylogenetic inference results were in line with these findings and demonstrated the formation of two distinct monophyletic clades containing strains isolated around the world, where our isolate, belonging to the same clade as the prototype strain, was allocated to a different subclade, highlighting the genetic divergence between them. This work reports the first isolation of UMAV in Brazil, and due to the scarcity of information on this viral agent in the scientific literature, it is essential to carry out further studies to better understand its epidemiology, dispersion, and, in particular, its interactions with vertebrate hosts, vectors, and the environment.

Bioinformatic analyses have predicted that orbiviruses encode an additional, small non-structural protein (NS5) from a secondary open reading frame on genome segment 10. However, this protein has not previously been detected in infected mammalian or insect cells. NS5-specific antibodies were generated in mice and were used to identify NS5 synthesised in orbivirus-infected BSR cells or cells transfected with NS5 expression plasmids. Confocal microscopy shows that although NS5 accumulates in the nucleus, particularly in the nucleolus, which becomes disrupted, it also appears in the cell cytoplasm, co-localising with mitochondria. NS5 helps to prevent the degradation of ribosomal RNAs during infection and reduces host-cell protein synthesis However, it helps to extend cell viability by supporting viral protein synthesis and virus replication. Pulldown studies showed that NS5 binds to ssRNAs and supercoiled DNAs and demonstrates interactions with ZBP1, suggesting that it modulates host-cell responses.

We isolated a novel virus strain (YN12246) from Culicoides spp. specimens collected at the China-Laos-Myanmar border in southern Yunnan Province. This virus had a cytopathic effect (CPE) on both insect cells (C6/36) and mammalian cells (BHK-21). Electron microscopy revealed the structure of the virions to be spherical with a diameter of 75 nm. Polyacrylamide gel analysis demonstrated that the viral genome consisted of 10 segments of double-stranded RNA (dsRNA), with a distribution pattern of 3-3-3-1. The coding sequences of 9 genome segments of YN12246 (Seg1, Seg3-Seg10) were obtained by high-throughput sequencing and Sanger sequencing. Comparisons of conserved genome segments 1 and 3 (Seg1 and Seg3), encoding the polymerase-VP1 and sub-core T2 protein, respectively, showed that YN12246 groups with the Culicoides-borne orbiviruses. The highest levels of sequence identity were detected between YN12246 and Tibet orbivirus (TIBOV), indicating that they belong to the same virus species (with amino acid identity of 98.8% and 96.4% for the polymerase and T2 protein, respectively). The data presented here confirm that YN12246 is a member of the TIBOV species, which was first isolated from mosquitoes in 2009. This is the first report of the isolation of TIBOV from Culicoides.