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As proven early on in the pandemic, SARS-CoV-2 is mainly transmitted by aerosols. This urged us to develop a silicon impactor that collects the virus particles directly from breath. Performing PCR on these breath samples proved equally sensitive as nasopharyngeal swabs during the first week of an infection [Stakenborg et al., 2022], yet it remained a mostly manual process and PCR turn-around-time was still long. To overcome these drawbacks, we developed a fast and sensitive, fully integrated point-of-need breath test, comprising a novel breath sampler device and PCR instrument. The breath sampler combines virus collection and in-situ RNA amplification. The PCR instrument performs very fast amplification of the released viral RNA. Sample-to-result time was reduced to

Bovine viral diarrhea virus (BVDV) is a (+) ssRNA virus that belongs to the family Flaviviridae. BVDV is a significant animal pathogen causing substantial economic losses to the cattle industry worldwide through respiratory and gastrointestinal infections and abortion or birth of persistently infected calves. While the immunogenic profile of some of the BVDV proteins (i.e., Erns, E2 and NS3) is well established during viral pathogenesis, very little information is available about most of BVDV’s non-structural proteins in this regard. In recent times, the NS4B protein has emerged as an interesting target of diagnostic, vaccination and therapeutic value in viral infections of other members of the family Flaviviridae due to its key scaffold-like contribution in the viral replication complex. Although, BVDV-NS4B has a membrane topology alongside its role in induction of autophagosomes in vitro. However, information on its immunogenicity during BVDV pathogenesis and vaccination is scarce. To characterize the immunogenic profile of the NS4B, five cows were vaccinated with the live attenuated BVDV vaccine Bovela® and blood samples were taken pre- and post-immunization for serum isolation. Virus neutralization assay (VNA) confirmed the presence of anti-BVDV antibodies in the sera of vaccinated cows. VNA also revealed pre-existing antibodies against BVDV in the pre-immunization sera of two cows. To identify BVDV-NS4B specific antibodies, the NS4B protein was expressed in mammalian cells by using the pCI-neo vector system. The sera from BVDV vaccinated cows were evaluated for the presence of BVDV-NS4B specific antibodies through western blot and indirect ELISA. Interestingly, t sera from cows with pre-existing immunity against BVDV were able to detect NS4B in western blot and ELISA, suggesting the presence of NS4B-specific antibodies. The obtained results provide the first indication of the immunogenic nature of BVDV-NS4B protein in sero-converted animals. These findings are consistent with the observation made for NS4B in other Flaviviridae members and confirm this protein as an interesting target with diagnostic, vaccination and therapeutic value.