Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been the cause of human pandemic infection since late 2019. SARS-CoV-2 infection in animals has also been reported both naturally and experimentally, rendering awareness about a potential source of infection for one health concern.

Canine bocaviruses (CBoVs) have been recognized as pathogens associated with intestinal diseases. Hematogenous spreading caused by CBoV has been documented and may potentiate the virus entry across the blood-brain barrier to initiate a brain infection. This study focused attention on CBoV detection in cases of encepahlopathy and attempted to determine its viral localization. A total of 107 dog brains that histologically exhibited encephalopathy (ED) were investigated for the presence of CBoVs using polymerase chain reaction (PCR). Thirty-three histologically normal brain samples from dogs were used as a control group (CD). CBoV-2 was detected in 15 ED dogs (14.02%) but not in CD dogs (p = 0.02), while no CBoV-1 and -3 were detected. Among the CBoV-2 positive dogs, brain histological changes were characterized by nonsuppurative encephalitis, with inclusion body-like materials in some brains. In situ hybridization (ISH) and transmission electron microscopy (TEM) confirmed the presence of CBoV-2 viral particles in glial cells, supporting neurotropism of this virus. ISH signals were also detected in the intestines, lymphoid organs, and the heart, suggesting both enteral and parenteral infections of this virus. Whole genome characterization and evolutionary analysis revealed genetic diversity of CBoV-2 sequences and it was varying among the different countries where the virus was detected. This study points to a possible association of CBoV-2 with encephalopathy in dogs. It also highlights the genetic diversity and cellular tropism of this virus.

Biting midges (Culicoides) are vectors of many pathogens of medical and veterinary importance, but their viromes are poorly characterized compared to certain other hematophagous arthropods, e.g., mosquitoes and ticks. The goal of this study was to use metagenomics to identify viruses in Culicoides from Mexico. A total of 457 adult midges were collected in Chihuahua, northern Mexico, in 2020 and 2021, and all were identified as female Culicoides reevesi. The midges were sorted into five pools and homogenized. An aliquot of each homogenate was subjected to polyethylene glycol precipitation to enrich for virions, then total RNA was extracted and analyzed by unbiased high-throughput sequencing. We identified six novel viruses that are characteristic of viruses from five families (Nodaviridae, Partitiviridae, Solemoviridae, Tombusviridae, and Totiviridae) and one novel virus that is too divergent from all classified viruses to be assigned to an established family. The newly discovered viruses are phylogenetically distinct from their closest known relatives, and their minimal infection rates in female C. reevesi range from 0.22 to 1.09. No previously known viruses were detected, presumably because viral metagenomics had never before been used to study Culicoides from the Western Hemisphere. To conclude, we discovered multiple novel viruses in C. reevesi from Mexico, expanding our knowledge of arthropod viral diversity and evolution.

Members of the Flaviviridae encode XRN1-resistant RNA structures (xrRNAs) within their 3′ untranslated regions (UTRs) that stall the cellular 5′→3′ exoribonuclease 1 (XRN1), resulting in the accumulation of subgenomic flavivirus RNAs (sfRNAs). Zika virus (ZIKV) contains two such elements, xrRNA1 and xrRNA2, which generate sfRNA1 and sfRNA2, respectively. A third sfRNA species (sfRNA3) has also been described. Building on evidence that sequences within the orthoflavivirus UTRs modulate host range, we replaced the xrRNA1 element in the ZIKV genome with xrRNA1 or xrRNA1-like elements from nine evolutionarily divergent flaviviruses and assessed the in vitro host ranges of the resulting chimeric viruses to determine whether xrRNA1 contributes to host specificity. We selected mosquito-borne, tick-borne, insect-specific, and vertebrate-specific orthoflaviviruses, together with representative viruses from the Pestivirus, Pegivirus, and Hepacivirus genera. All genetic exchanges yielded chimeric viruses that replicated in both vertebrate (Vero) and mosquito (C6/36) cells. Wild-type ZIKV replicated to substantially higher titers than all chimeric viruses in Vero cells but showed no consistent replication advantage in C6/36 cells. All chimeric viruses produced sfRNA1, whereas detection of sfRNA2 and sfRNA3 varied according to the substituted xrRNA1 sequence and host cell type, indicating that heterologous xrRNA1 substitution can alter downstream sfRNA production in a host cell-dependent manner. In summary, replacement of the ZIKV xrRNA1 element with evolutionarily divergent xrRNA1 or xrRNA1-like sequences did not alter in vitro host range or abolish sfRNA1 production, but substantially reduced viral fitness in vertebrate cells.