Wildlife surveillance allowed the monitoring of the zoonotic mosquito-borne Usutu virus (USUV) in birds and bats (Pipistrellus pipistrellus) in southern Belgium in 2017 and 2018. USUV-RNA was detected in 69 birds (of 253) from 15 species, among which 7 species had not previously been reported to be susceptible to the infection. Similarly, 2 bats (of 10) were detected positive by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). USUV-associated lesions were mainly found in Eurasian Blackbirds (Turdus merula), in which USUV antigens were demonstrated by immunohistochemistry in the brain, heart, liver, kidney, intestine, and lung. Partial nonstructural protein 5 gene-based phylogenetic analysis showed several identical or closely related strains from 2016, 2017, and 2018 clustering together within Europe 3 or Africa 3 lineages. Further, one USUV strain detected in a common chaffinch (Fringilla coelebs) manifested a close genetic relationship with the European 1 strains circulating in Hungary and Austria. Our data provide evidence of USUV endemization in southern Belgium in local birds and bats, extension of the host range of the virus and ongoing virus introduction from abroad, likely by migratory birds. Our results highlight the need for vigilance in the forthcoming years toward new virus-associated outbreaks in birds and possible human infections in Belgium.

Usutu virus (USUV) is a neurotropic flavivirus closely related to West Nile virus (WNV). Its enzootic cycle mainly involves mosquitoes and birds. Human infection can occur with occasional, but sometimes severe, neurological complications. Since its emergence and spread in Europe over the last two decades, USUV has been linked to significant avian outbreaks, especially among Passeriformes, including European blackbirds (Turdus merula). Strikingly, no in vivo avian model exists so far to study this arbovirus. The domestic canary (Serinus canaria) is a passerine, which is considered as a highly susceptible model of infection by WNV. Here, we experimentally challenged domestic canaries with two different doses of USUV. All inoculated birds presented detectable amounts of viral RNA in the blood and RNA shedding via feathers and droppings during the early stages of the infection, as determined by RT-qPCR. Mortality occurred in both infected groups (1/5 and 2/5, respectively) and was not necessarily correlated to a pure neurological disease. Subsequent analyses of samples from dead birds showed histopathological changes and virus tropism mimicking those reported in naturally infected birds. A robust seroconversion followed the infection in almost all the surviving canaries. Altogether, these results demonstrate that domestic canaries constitute an interesting experimental model for the study of USUV pathogenesis and transmission.

Usutu virus (USUV) is a mosquito-borne flavivirus, closely related to the West Nile virus (WNV). Similar to WNV, USUV may cause infections in humans, with occasional, but sometimes severe, neurological complications. Further, USUV can be highly pathogenic in wild and captive birds and its circulation in Europe has given rise to substantial avian death. Adequate study models of this virus are still lacking but are critically needed to understand its pathogenesis and virulence spectrum. The chicken embryo is a low-cost, easy-to-manipulate and ethically acceptable model that closely reflects mammalian fetal development and allows immune response investigations, drug screening, and high-throughput virus production for vaccine development. While former studies suggested that this model was refractory to USUV infection, we unexpectedly found that high doses of four phylogenetically distinct USUV strains caused embryonic lethality. By employing immunohistochemistry and quantitative reverse transcriptase-polymerase chain reaction, we demonstrated that USUV was widely distributed in embryonic tissues, including the brain, retina, and feather follicles. We then successfully developed a primary cell line from the chorioallantoic membrane that was permissive to the virus without the need for viral adaptation. We believe the future use of these models would foster a significant understanding of USUV-induced neuropathogenesis and immune response and allow the future development of drugs and vaccines against USUV.

Bovine Viral Diarrhea Virus (BVDV) is one of the main pathogens that affects ruminants worldwide, generating significant economic losses. Like other RNA viruses, BVDV is characterized by a high genetic variability, generating the emergence of new variants, and increasing the risk of new outbreaks. The last report on BVDV genotypes in France was in 2008, since which there have been no new information. The goal of this study is to determine the genetic diversity of BVDV strains currently circulating in France. To this aim, samples of cattle were taken from different departments that are part of the main areas of livestock production during the years 2018 to 2020. Using the partial sequence of the 5'UTR region of the viral genome, we identified and classified 145 samples corresponding to Pestivirus A and one sample corresponding to Pestivirus D. For the Pestivirus A samples, the 1e, 1b, 1d, and 1l genotypes, previously described in France, were identified. Next, the 1r and 1s genotypes, not previously described in the country, were detected. In addition, a new genotype was identified and was tentatively assigned as 1x genotype. These results indicate an increase in the genetic diversity of BVDV in France.

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