The mutual dependence of human and animal health is central to the One Health initiative as an integrated strategy for infectious disease control and management. A crucial element of the One Health includes preparation and response to influenza A virus (IAV) threats at the human-animal interface. The IAVs are characterized by extensive genetic variability, they circulate among different hosts and can establish host-specific lineages. The four main hosts are: avian, swine, human and equine, with occasional transmission to other mammalian species. The host diversity is mirrored in the range of the RT-qPCR assays for IAV detection. Different assays are recommended by the responsible health authorities for generic IAV detection in birds, swine or humans. In order to unify IAV monitoring in different hosts and apply the One Health approach, we developed a single RT-qPCR assay for universal detection of all IAVs of all subtypes, species origin and global distribution. The assay design was centred on a highly conserved region of the IAV matrix protein (MP)-segment identified by a comprehensive analysis of 99,353 sequences. The reaction parameters were effectively optimised with efficiency of 93–97% and LOD95% of approximately ten IAV templates per reaction. The assay showed high repeatability, reproducibility and robustness. The extensive in silico evaluation demonstrated high inclusivity, i.e. perfect sequence match in the primers and probe binding regions, established as 94.6% for swine, 98.2% for avian and 100% for human H3N2, pandemic H1N1, as well as other IAV strains, resulting in an overall predicted detection rate of 99% on the analysed dataset. The theoretical predictions were confirmed and extensively validated by collaboration between six veterinary or human diagnostic laboratories on a total of 1970 specimens, of which 1455 were clinical and included a diverse panel of IAV strains.

Woody perennial plants like grapevine and fruit trees can be infected by several viruses even as multiple infections. Since they are propagated vegetatively, the phytosanitary status of the propagation material (both the rootstock and the variety) can have a profound effect on the lifetime and health of the new plantations. The fast evolution of sequencing techniques provides a new opportunity for metagenomics-based viral diagnostics. Viral derived small RNAs produced by the host immune system during viral infection can be sequenced by next-generation techniques and analyzed for the presence of viruses, revealing the presence of all known viral pathogens in the sample. This method is based on Illumina sequencing of short RNAs and bioinformatics analysis of virus-derived small RNAs in the host. Here we describe a protocol for this challenging technique step by step with notes, in order to ensure success for every user.

Meristem culture and somatic embryogenesis are effective tools for virus elimination of vegetatively propagated crops including grapevine (Vitis vinifera L.). While both have been shown to be useful to eliminate the main grapevine viruses, their efficiency differs depending on the virus and grapevine variety. In our work, we investigated the efficiency of these two virus elimination methods using small RNA high-throughput sequencing (HTS) and RT-PCR as virus diagnostics. Field grown mother plants of four clones representing three cultivars, infected with different viruses and viroids, were selected for elimination via somatic embryogenesis (SE) and meristem culture (ME). Our results show for the first time that using SE, elimination in mother plants was effective for all viruses, i.e., grapevine rupestris vein feathering virus (GRVFV), grapevine Syrah virus 1 (GSyV-1), Grapevine virus T (GVT) and grapevine Pinot gris virus (GPGV). This study also confirms previous studies showing that SE is a possible strategy for the elimination of GFkV, GRSPaV, HSVd, and GYSVd-1. Our results demonstrate that the efficacy of virus elimination via SE is relatively high while the purging of viroids is lower. Our work provides evidence that the efficiency of SE is comparable to that of the technically difficult ME technique, and that SE will offer a more effective strategy for the production of virus-free grapevine in the future.

Porcine epidemic diarrhoea virus (PEDV) is an emerging enteropathogen, causing great economic losses in the pig industry. After many years of quiescence, PEDV was detected in Hungary in 2016 with a recombination in its S gene. In order to determine the extent of this change, an attempt was made to isolate the recombinant PEDV. This study was extended with a variety of samples collected from three separate farms with newly identified PEDV in 2018. The recombinant PEDV from 2016 was isolated successfully along with three viruses from 2018, and one isolate from the new cases was used for whole genome determination. Whole genome sequence alignment revealed the highest identity with recombinant Hungarian and Slovenian PEDV within the low-pathogenic European viruses. This suggests that these recombinant PEDV are circulating in this area and may spread to other parts of the continent.

The dietary supplement Citicoline free-base (choline cytidine 5'-pyrophosphate) was toxicologically evaluated in Sprague-Dawley rats using oral gavage. In an acute 14-day study, 2000 mg/kg was well tolerated. In a 90-day study, 100, 350, and 1000 mg/kg/day doses resulted in no mortality. In males, slight significant increases in serum creatinine (350 and 1000 mg/kg/day), and decreases in urine volume (all treated groups) were observed. In females, slight significant increases in total white blood cell and absolute lymphocyte counts (1000 mg/kg/day), and blood urea nitrogen (BUN) (100 and 350, but not 1000 mg/kg/day) were noted. A dose-related increase in renal tubular mineralization, without degenerative or inflammatory reaction, was found in females (all treated groups) and two males (1000 mg/kg/day). Renal mineralization in rats (especially females) is influenced by calcium:phosphorus ratios in the diet. A high level of citicoline consumption resulted in increased phosphorus intake in the rats, and likely explains this result.