Usutu Virus Infection of Embryonated Chicken Eggs and a Chicken Embryo-Derived Primary Cell Line.

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.

New Insights into the Susceptibility of Immunocompetent Mice to Usutu Virus.

Usutu virus (USUV) is a mosquito-borne flavivirus that shares many similarities with the closely related West Nile virus (WNV) in terms of ecology and clinical manifestations. Initially distributed in Africa, USUV emerged in Italy in 1996 and managed to co-circulate with WNV in many European countries in a similar mosquito-bird life cycle. The rapid geographic spread of USUV, the seasonal mass mortalities it causes in the European avifauna, and the increasing number of infections with neurological disease both in healthy and immunocompromised humans has stimulated interest in infection studies to delineate USUV pathogenesis. Here, we assessed the pathogenicity of two USUV isolates from a recent Belgian outbreak in immunocompetent mice. The intradermal injection of USUV gave rise to disorientation and paraplegia and was associated with neuronal death in the brain and spinal cord in a single mouse. Intranasal inoculation of USUV could also establish the infection; viral RNA was detected in the brain 15 days post-infection. Overall, this pilot study probes the suitability of this murine model for the study of USUV neuroinvasiveness and the possibility of direct transmission in mammals.

Experimental Usutu Virus Infection in Domestic Canaries Serinus canaria.

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 Epizootic in Belgium in 2017 and 2018: Evidence of Virus Endemization and Ongoing Introduction Events.

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.

First Evidence of Fatal Usutu Virus Natural Infections in an Anatidae, the Common Scoter (Melanitta nigra).

While fatal infections caused by the Usutu virus appeared to concern only passerines (especially the blackbird) and Strigiformes (especially the great gray owl), we report herein that the virus also naturally causes a fatal disease in an anseriforme species, the common scoter (Melanitta nigra).

Moku virus detection in honey bees, Belgium, 2018.

We report the detection of Moku virus in honey bees (Apis mellifera) collected in 2017 from hives with a history of attacks by invasive Asian hornets (Vespa velutina nigrithorax) in Belgium. End 2016, Moku virus was reported in Asian hornets from the same area. In addition, the Moku virus was already present in historical samples of bees collected in 2013, that is, 2 years after the official first detection of Asian hornets in the same area of Belgium. This study suggests a spread of Moku virus to honey bees with possible consequences.

Cell cycle S phase markers are expressed in cerebral neuron nuclei of cats infected by the Feline Panleukopenia Virus.

The cell cycle-associated neuronal death hypothesis, which has been proposed as a common mechanism for most neurodegenerative diseases, is notably supported by evidencing cell cycle effectors in neurons. However, in naturally occurring nervous system diseases, these markers are not expressed in neuron nuclei but in cytoplasmic compartments. In other respects, the Feline Panleukopenia Virus (FPV) is able to complete its cycle in mature brain neurons in the feline species. As a parvovirus, the FPV is strictly dependent on its host cell reaching the cell cycle S phase to start its multiplication. In this retrospective study on the whole brain of 12 cats with naturally-occurring, FPV-associated cerebellar atrophy, VP2 capsid protein expression was detected by immunostaining not only in some brain neuronal nuclei but also in neuronal cytoplasm in 2 cats, suggesting that viral mRNA translation was still occurring. In these cats, double immunostainings demonstrated the expression of cell cycle S phase markers cyclin A, cdk2 and PCNA in neuronal nuclei. Parvoviruses are able to maintain their host cells in S phase by triggering the DNA damage response. S139 phospho H2A1, a key player in the cell cycle arrest, was detected in some neuronal nuclei, supporting that infected neurons were also blocked into the S phase. PCR studies did not support a co-infection with an adeno or herpes virus. ERK1/2 nuclear accumulation was observed in some neurons suggesting that the ERK signaling pathway might be involved as a mechanism driving these neurons far into the cell cycle.