Due to SARS CoV-2 recombination rates, number of infected people and recent reports of environmental contamination, the possibility of SARS CoV-2 transmission to animals can be expected. We tested samples of dominant free-living and captive wildlife species in Croatia for the presence of anti-SARS CoV-2 antibodies and viral RNA. In total, from June 2020 until February 2021, we tested blood, muscle extract and fecal samples of 422 free-living wild boars (Sus scrofa), red foxes (Vulpes vulpes) and jackals (Canis aureus); blood and cloacal swabs of 111 yellow-legged gulls (Larus michahellis) and fecal samples of 32 zoo animals. A commercially available ELISA (ID.Vet, France) and as a confirmatory test, a surrogate virus neutralization test (sVNT; GenScript, Netherlands) were used. Fecal samples were tested for the presence of viral RNA by a real-time RT–PCR protocol. Fifteen out of 533 (2.8%) positive ELISA results were detected; in wild boars (3.9%), red foxes (2.9%) and jackals (4.6%). However, the positive findings were not confirmed by sVNT. No viral RNA was found. In conclusion, no spillover occurred within the investigated period (second COVID-19 wave). However, further investigation is needed, especially regarding wildlife sample features for serological tests.

To fulfill epidemiological data and investigate possible interspecies transmission, this study shall attempt to sequence representative HEV strains of human, swine and wild boar origin collected from 2010 to 2017 in Croatia. In total, 174 anti-HEV antibody positive human sera samples; 1419 blood or faeces samples of swine, as well as 720 tissue and/or blood samples of wild boar originating from different counties (18 in total) in Croatia were tested for the presence of HEV RNA. HEV RNA was detected in 26 human sera samples (14.9%; 95% CI 10.4–21.0%). HEV RNA was detected in 216 tested swine (15.2%; 95% CI 13.5–17.1%), regardless of age, farm breeding system or geographical origin. Viral RNA was also detectable in faeces samples which prove that swine actively participate in shedding HEV into the environment. Of the total of 720 tested wild boar samples, 83 were HEV RNA positive (11.5, 95% CI 9.4–14.1%) originating from six counties. According to the sequence analysis all strains have shown to be members of Orthohepevirus A genotype HEV-3, regardless of host. The genotyping results confirm grouping of sequences into four subtypes of HEV strains of which subtypes 3a and 3c belong to the general cluster 3abchij, and were predominately detected during the study, while subtypes 3e and 3f fall within cluster 3efg. Strains within subtypes 3a and 3e were found in humans, swine and wild boars; subtype 3c strains were derived from humans and swine, whereas subtype 3f strains were found only in humans. Strains belonging to subtypes 3a and 3c were derived during the entire investigated period and may be considered endemic in Croatia, whereas strains within subtypes 3e and 3f were detected sporadically indicating the possibility of newly imported infections. All detected strains show to be genetically highly related to strains found in humans and/or animals from other European Countries, indicating that trade of live animals or wild boar movement increases the risk of HEV infection spread. Furthermore, homologous strains found in different investigated species within this study indicate interspecies transmission of HEV and/or an existence of an accessible mutual source of infection.

Several arboviruses have emerged in Croatia in recent years. Tick-borne encephalitis is endemic in continental counties; however, new natural micro-foci have been detected. Two autochthonous dengue cases were reported in 2010. West Nile virus emerged in 2012, followed by emergence of Usutu virus in 2013. Although high seroprevalence rates of Toscana virus have been detected among residents of Croatian littoral, the virus remains neglected, with only a few clinical cases of neuroinvasive infections reported. Lymphocytic choriomeningitis virus is a neglected neuroinvasive rodent-borne virus. So far, there are no reports on human clinical cases; however, the seroprevalence studies indicate the virus presence in the Croatian mainland. Puumala and Dobrava hantaviruses are widely distributing rodent-borne viruses with sporadic and epidemic occurrence. Hepatitis E virus is an emerging food-borne virus in Croatia. After the emergence in 2012, cases were regularly recorded. Seropositivity varies greatly by region and population group. Rotaviruses represent a significant healthcare burden since rotavirus vaccination is not included in the Croatian national immunization program. Additionally, rotaviruses are widely distributed in the Croatian ecosystem. A novel coronavirus, SARS-CoV-2, emerged in February 2020 and spread rapidly throughout the country. This review focuses on emerging and neglected viruses of zoonotic importance detected in Croatia.

The influence of cultivation on the expression pattern of canine adipose-derived mesenchymal stem cells (cAD-MSCs) surface markers, contributing to, among others, the promotion of growth, proliferation, differentiation and immunomodulatory mechanisms of an excellent therapeutic, is still unknown. To fill the gap, we investigated CD90, CD44, CD73, CD29, CD271, CD105, CD45 and CD14 patterns of expression at the protein level with flow cytometry and mRNA level using a real-time polymerase chain reaction array. Gentle variations of expression occurred during cultivation, along with increased CD90, CD44 and CD29 expression, low and decreasing CD271 and CD73 expression and a decrease of initially high CD105. As expected, CD45 and CD14 were not expressed by cAD-MSCs. Interestingly, we discovered a significant decrease of CD73 expression, compared to early (P1–P3) to late (P4–P6) passages, although the CD73 gene expression was found to be stable. The percentage of positive cells was found to be higher for all positive markers up to P4. As CD73′s one important feature is a modulation from a pro-inflammatory environment to an anti-inflammatory milieu, the expression of CD73 in our conditions indicate the need to consider the time cells spend in vitro before being transplanted into patients, since it could impact their favourable therapeutical properties.