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Identification and characterization of the foot-and-mouth disease virus (FMDV) strains circulating in endemic countries and their dynamics are essential elements of the global FMD control strategy. Characterization of FMDV is usually performed in reference laboratories (RL). However, shipping of FMD samples to RL is a challenge due to the cost and biosafety requirements of transportation, resulting in a lack of knowledge about the strains circulating in some endemic areas. In order to simplify this step and to encourage sample submission to RL, we have previously developed a low-cost protocol for the shipment of FMD samples based on the use of lateral flow devices (LFDs) combined with a simple virus inactivation step using 0.2% citric acid. The present study aimed to evaluate this inactivation protocol in the field. For this purpose, 60 suspected FMD clinical samples were collected in Nigeria, Pakistan, and Turkey, three countries where FMD is endemic. Sample treatment, testing on LFDs, and virus inactivation steps were performed in the field when possible. The effectiveness of the virus inactivation was confirmed at the RL. After RNA extraction from the 60 inactivated LFDs, all were confirmed as FMDV positive by real-time reverse transcription polymerase chain reaction (RT-PCR). The serotype was identified by conventional RT-PCR for 86% of the samples. The topotype and/or lineage was successfully determined for 60% of the samples by Sanger sequencing and sequence analyses. After chemical transfection of RNA extracted from inactivated LFDs, into permissive cells, infectious virus was rescued from 15% of the samples. Implementation of this user-friendly protocol can substantially reduce shipping costs, which should increase the submission of field samples and therefore improve knowledge of the circulating FMDV strains.

West Nile virus (WNV) is amplified in an enzootic cycle involving birds as amplifying hosts. Because they do not develop high levels of viremia, humans and horses are considered to be dead-end hosts. Mosquitoes, especially from the Culex genus, are vectors responsible for transmission between hosts. Consequently, understanding WNV epidemiology and infection requires comparative and integrated analyses in bird, mammalian, and insect hosts. So far, markers of WNV virulence have mainly been determined in mammalian model organisms (essentially mice), while data in avian models are still missing. WNV Israel 1998 (IS98) is a highly virulent strain that is closely genetically related to the strain introduced into North America in 1999, NY99 (genomic sequence homology > 99%). The latter probably entered the continent at New York City, generating the most impactful WNV outbreak ever documented in wild birds, horses, and humans. In contrast, the WNV Italy 2008 strain (IT08) induced only limited mortality in birds and mammals in Europe during the summer of 2008. To test whether genetic polymorphism between IS98 and IT08 could account for differences in disease spread and burden, we generated chimeric viruses between IS98 and IT08, focusing on the 3′ end of the genome (NS4A, NS4B, NS5, and 3′UTR regions) where most of the non-synonymous mutations were detected. In vitro and in vivo comparative analyses of parental and chimeric viruses demonstrated a role for NS4A/NS4B/5′NS5 in the decreased virulence of IT08 in SPF chickens, possibly due to the NS4B-E249D mutation. Additionally, significant differences between the highly virulent strain IS98 and the other three viruses were observed in mice, implying the existence of additional molecular determinants of virulence in mammals, such as the amino acid changes NS5-V258A, NS5-N280K, NS5-A372V, and NS5-R422K. As previously shown, our work also suggests that genetic determinants of WNV virulence can be host-dependent.

The impact of mosquito-borne diseases on human and veterinary health is being exacerbated by rapid environmental changes caused mainly by changing climatic patterns and globalization. To gain insight into mosquito-borne virus circulation from two counties in eastern and southeastern Romania, we have used a combination of sampling methods in natural, urban and peri-urban sites. The presence of 37 mosquito-borne viruses in 16,827 pooled mosquitoes was analyzed using a high-throughput microfluidic real-time PCR assay. West Nile virus (WNV) was detected in 10/365 pools of Culex pipiens (n = 8), Culex modestus (n = 1) and Aedes vexans (n = 1) from both studied counties. We also report the first molecular detection of Sindbis virus (SINV) RNA in the country in one pool of Culex modestus. WNV infection was confirmed by real-time RT-PCR (10/10) and virus isolation on Vero or C6/36 cells (four samples). For the SINV-positive pool, no cytopathic effectwas observed after infection of Vero or C6/36 cells, but no amplification was obtained in conventional SINV RT-PCR. Phylogenetic analysis of WNV partial NS5 sequences revealed that WNV lineage 2 of the Central-Southeast European clade, has a wider circulation in Romania than previously known.

Tick-borne encephalitis virus' (TBEV) geographic range and the human incidence are increasing throughout Europe, putting a number of non-endemic regions and countries at risk of outbreaks. In spring 2020, there was an outbreak of tick-born encephalitis (TBE) in Ain, Eastern France, where the virus had never been detected before. All patients but one had consumed traditional unpasteurised raw goat cheese from a local producer. We conducted an investigation in the suspected farm using an integrative One Health approach. Our methodology included (i) the detection of virus in cheese and milk products, (ii) serological testing of all animals in the suspected farm and surrounding farms, (iii) an analysis of the landscape and localisation of wooded area, (iv) the capture of questing ticks and small mammals for virus detection and estimating enzootic hazard, and (v) virus isolation and genome sequencing. This approach allowed us to confirm the alimentary origin of the TBE outbreak and witness in real-time the seroconversion of recently exposed individuals and excretion of virus in goat milk. In addition, we identified a wooded focus area where and around which there is a risk of TBEV exposure. We provide the first TBEV isolate responsible for the first alimentary-transmitted TBE in France, obtained its full-length genome sequence, and found that it belongs to the European subtype of TBEV. TBEV is now a notifiable human disease in France, which should facilitate surveillance of its incidence and distribution throughout France.