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The furin cleavage site (FCS) in SARS-CoV-2 is unique within the Severe acute respiratory syndrome–related coronavirus (SrC) species. We re-assessed diverse SrC from European horseshoe bats and analyzed the spike-encoding genomic region harboring the FCS in SARS-CoV-2. We reveal molecular features in SrC such as purine richness and RNA secondary structures that resemble those required for FCS acquisition in avian influenza viruses. We discuss the potential acquisition of FCS through molecular mechanisms such as nucleotide substitution, insertion, or recombination, and show that a single nucleotide exchange in two European bat-associated SrC may suffice to enable furin cleavage. Furthermore, we show that FCS occurrence is variable in bat- and rodent-borne counterparts of human coronaviruses. Our results suggest that furin cleavage sites can be acquired in SrC via conserved molecular mechanisms known in other reservoir-bound RNA viruses and thus support a natural origin of SARS-CoV-2.

Coronaviruses (CoV) are widely distributed pathogens of human and animals and can cause mild or severe respiratory and gastrointestinal disease. Antigenic and genetic similarity of some CoVs within the Betacoronavirus genus is evident. Therefore, for the first time in Slovenia, we investigated the genetic diversity of partial 390-nucleotides of RNA-dependent-RNA polymerase gene (RdRp) for 66 human (HCoV) and 24 bovine CoV (BCoV) positive samples, collected between 2010 and 2016 from human patients and cattle with respiratory disease. The characterized CoV strains belong to four different clusters, in three separate human clusters HCoV-HKU1 (n = 34), HCoV-OC43 (n = 31) and HCoV 229E (n = 1) and bovine grouping only as BCoVs (n = 24). BCoVs from cattle and HCoV-OC43 were genetically the most closely related and share 96.4–97.1% nucleotide and 96.9–98.5% amino acid identity.

In Slovenia, the control of bovine viral diarrhoea virus (BVDV) infections started in 1994. Since 2014, a voluntary programme has been running according to the national rules that prescribe the conditions for recognising, acquiring, and maintaining a BVDV-free status for an individual herd. The principle is based on periodical laboratory testing and preventive measures that need to be strictly implemented in a herd. Between 2014 and 2020, a total of 348 herds were included in BVDV antibody testing, and 25.0% of tested herds were detected to be BVDV antibody positive. To recognise the BVDV-free status of the herd, the breeder should provide two consecutive tests with intervals of at least 6 months in all animals in the age from 7 to 13 months, with negative results for BVDV antibodies in ELISA. The BVDV-free status of the herd can be maintained by implementing preventive measures and can be renewed each year with one laboratory test in the age group of animals from 7 to 13 months for antibodies in ELISA. During the 7 years of the voluntary programme, 236 herds were included in the detection of BVDV in individual herds by real-time RT-PCR method and the elimination of positive animals from herds. In 71 (31.3%) herds, at least one BVDV-positive animal was detected, with the identification of a total of 267 persistently infected (PI) animals, representing an average of 2.9% of tested animals. The cost of testing for an average herd, recognised as BVDV-negative, and maintaining its BVDV-free status within the implemented voluntary programme, was €97.64/year, while for the average positive herd, the laboratory costs for elimination of BVDV were €189.59/year. Only limited progress towards eradication at the national level has been achieved in Slovenia since 2014.

To determine the presence and the prevalence of four different honeybee viruses (acute bee paralysis virus—ABPV, black queen cell virus—BQCV, chronic bee paralysis virus—CBPV, deformed wing virus—DWV) in wild bumblebees, pooled randomly selected bumblebee samples were collected from twenty-seven different locations in the territory of Croatia. All samples were prepared and examined using the RT-PCR methods for quantification of mentioned honeybee viruses. Determined prevalence (%) of identified positive viruses were in the following decreasing order: BQCV &gt, DWV &gt, ABPV, CBPV. Additionally, direct sequencing of samples positive for BQCV (n = 24) and DWV (n = 2) was performed, as well as a test of molecular phylogeny comparison with those available in GenBank. Selected positive field viruses’ strains showed 95.7 to 100% (BQCV) and 98.09% (DWV) nucleotide identity with previously detected and deposited honeybee virus strains in the geographic areas in Croatia and neighboring Slovenia. In this article, the first detection of four honeybee viruses with genetic characterization of high diversity strains circulating in wild bumblebees in Croatia is presented.

Slovenia has a long tradition of beekeeping and a high density of honeybee colonies, but less is known about bumblebees and their pathogens. Therefore, a study was conducted to define the incidence and prevalence of pathogens in bumblebees and to determine whether there are links between infections in bumblebees and honeybees. In 2017 and 2018, clinically healthy workers of bumblebees (Bombus spp.) and honeybees (Apis mellifera) were collected on flowers at four different locations in Slovenia. In addition, bumblebee queens were also collected in 2018. Several pathogens were detected in the bumblebee workers using PCR and RT-PCR methods: 8.8% on acute bee paralysis virus (ABPV), 58.5% on black queen cell virus (BQCV), 6.8% on deformed wing virus (DWV), 24.5% on sacbrood bee virus (SBV), 15.6% on Lake Sinai virus (LSV), 16.3% on Nosema bombi, 8.2% on Nosema ceranae, 15.0% on Apicystis bombi and 17.0% on Crithidia bombi. In bumblebee queens, only the presence of BQCV, A. bombi and C. bombi was detected with 73.3, 26.3 and 33.3% positive samples, respectively. This study confirmed that several pathogens are regularly detected in both bumblebees and honeybees. Further studies on the pathogen transmission routes are required.

In the period from 2015 to 2020, an entomological survey for the presence of West Nile virus (WNV) and Usutu virus (USUV) in mosquitoes was performed in northwestern Croatia. A total of 20,363 mosquitoes were sampled in the City of Zagreb and Međimurje county, grouped in 899 pools and tested by real-time RT-PCR for WNV and USUV RNA. All pools were negative for WNV while one pool each from 2016 (Aedes albopictus), 2017 (Culex pipiens complex), 2018 (Cx. pipiens complex), and 2019 (Cx. pipiens complex), respectively, was positive for USUV. The 2018 and 2019 positive pools shared 99.31% nucleotide homology within the USUV NS5 gene and both clustered within USUV Europe 2 lineage. The next-generation sequencing of one mosquito pool (Cx. pipiens complex) collected in 2018 in Zagreb confirmed the presence of USUV and revealed several dsDNA and ssRNA viruses of insect, bacterial and mammalian origin.

Hepatitis E is a zoonotic viral disease of pigs with increasing public health concern in industrialized countries. Presented broad study of hepatitis E virus (HEV) presence in pigs in Slovenia is the first attempt to overview the HEV situation in pigs entering a slaughterhouse and, further, to analyse the possibility of HEV entering into the food supply chain. 2433 samples from 811 clinically healthy pigs were collected at four slaughterhouses in Slovenia. Sampling covered three different age groups of pigs and three different types of samples (faeces, bile and liver) important for tracing HEV in a pig population. In addition, 63 swab samples were collected systematically from three different sites on the slaughter line, as well as 22 samples of minced meat and 30 bratwurst samples. All the samples were screened for the presence of HEV nucleic acids by specific real-time RT-PCR assay. In the group of three month old pigs 13.7% of faeces, 13.0% of bile and 2.1% of liver samples were HEV positive. In the group of six months old pigs only 0.25% of liver and 0.25% of bile samples were positive. In the category of sows, no positive samples were found. Two out of 63 swab samples collected on the slaughter line were HEV positive. All tested samples of minced meat and bratwurst were negative. The phylogenetic analysis of 50 HEV positive samples, with comparison of 366 nucleotides in ORF1 region, revealed high diversity of identified strains of HEV in pigs, belonging into subtypes 3a, 3b, 3c and 3e.

Bovine viral diarrhea virus (BVDV) subtypes 1f and 1d were isolated for the first time in Slovenia in 1999 and detected later in a majority of BVDV-infected cattle herds. Here, we report the first nearly complete genome sequences of noncytopathogenic BVDV-1f strain SLO/1170/2000 and cytopathogenic BVDV-1d strain SLO/2416/2002, isolated in Slovenia.

The epidemiology of West Nile (WNV) and Usutu virus (USUV) has changed dramatically over the past two decades. Since 1999, there have been regular reports of WNV outbreaks and the virus has expanded its area of circulation in many Southern European countries. After emerging in Italy in 1996, USUV has spread to other countries causing mortality in several bird species. In 2009, USUV seroconversion in horses was reported in Italy. Co-circulation of both viruses was detected in humans, horses and birds. The main vector of WNV and USUV in Europe is Culex pipiens, however, both viruses were found in native Culex mosquito species (Cx. modestus, Cx. perexiguus). Experimental competence to transmit the WNV was also proven for native and invasive mosquitoes of Aedes and Culex genera (Ae. albopictus, Ae. detritus, Cx. torrentium). Recently, Ae. albopictus and Ae. japonicus naturally-infected with USUV were reported. While neuroinvasive human WNV infections are well-documented, USUV infections are sporadically detected. However, there is increasing evidence of a role of USUV in human disease. Seroepidemiological studies showed that USUV circulation is more common than WNV in some endemic regions. Recent data showed that WNV strains detected in humans, horses, birds, and mosquitoes mainly belong to lineage 2. In addition to European USUV lineages, some reports indicate the presence of African USUV lineages as well. The trends in WNV/USUV range and vector expansion are likely to continue in future years. This mini- review provides an update on the epidemiology of WNV and USUV infections in Southern Europe within a multidisciplinary "One Health" context.

The viral loads of acute bee paralysis virus (ABPV), black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), deformed wing virus (DWV), Lake Sinai virus 3 (LSV3), and sacbrood bee virus (SBV) were determined in samples with the use of quantitative TaqMan real-time reverse transcription and polymerase chain reaction (RT-qPCR). A total of 108 samples of healthy adult honeybees from four differently located apiaries and samples of honeybees showing different clinical signs of viral infections from 89 apiaries were collected throughout Slovenia. The aim of this study was to discover correlations between viral loads and clinical signs in adult honeybees and confirm previously set threshold viral load levels between healthy and clinically affected honeybees. Within this study, two new RT-qPCR assays for quantification of LSV3 and SBV were developed. Statistically significant differences in viral loads of positive samples were identified between healthy and clinically affected honeybees for ABPV, CBPV, DWV, and SBV, while for BQCV and LSV3, no statistical differences were observed between both groups. Despite high detected LSV3 prevalence and viral loads around 6.00 log10 viral copies/bee, this lineage probably has a limited impact on the health status of honeybee colonies. The determined viral loads between 3.94 log10 and 13.17 log10 in positive samples for six viruses, collected over 10 consecutive months, including winter, present additional information of high viral load variations in healthy honeybee colonies.

The lumpy skin disease virus (LSDV) isolate SERBIA/Bujanovac/2016 consists of 150,661 nucleotides and has a 99.95% nucleotide identity with the Neethling Warmbaths LW strain isolated in South Africa in 1999. This is the first complete LSDV genome determined in Serbia and also in the Balkan area.

Chronic bee paralysis virus (CBPV) causes an infectious and contagious disease of adult honeybees. Here, we report the complete genome sequence of CBPV strain SLO/M92/2010. This is the first published complete genome of CBPV in Apis mellifera carnica , which provides important additional knowledge about the divergence of the CBPV genome.

Summary Reasons for performing this study In 2009, a surprisingly high number of animals seropositive for equine infectious anaemia virus (EIAV; 26 horses from 13 farms) were detected in Slovenia. Objectives To develop a polymerase chain reaction (PCR) assay for the detection of the proviral nucleic acid, to phylogenetically characterise the Slovenian EIAV strains and to investigate whether transmission in utero occurred. Study design Cross-sectional clinical study. Methods In total, 26 horses (including 2 foals and 4 pregnant mares) and 4 fetuses were examined in this study. A PCR assay using the EIAV F1 and EIAV R1 primers was designed and tested using genomic DNA extracted from 28 spleen samples, 18 whole blood samples and 17 peripheral blood leucocyte samples. Amplicons of 22 PCRs obtained from the spleen samples were subjected to direct DNA sequencing and phylogenetic analysis. Results All spleen samples from 22 adult animals were positive for EIAV by PCR, whereas whole blood and the peripheral blood leucocyte samples were positive from only 4 animals. Spleen samples from foals and fetuses were negative by PCR. The Slovenian EIAV sequences could be mapped to 9 different branches of the phylogenetic tree. Conclusions The PCR was able to detect different EIAV strains from spleen samples of seropositive animals detected in Slovenia. Phylogenetic analysis revealed high genetic diversity of the EIAV strains detected in Slovenia, with their closest relatives being European strains. In utero transmission in pregnant mares did not occur.

Bovine viral diarrhea virus (BVDV) subgenotype 1e was isolated for the first time in Slovenia in 2006. Here, we report the complete genome sequence of BVDV-1e, strain SLO/2407/2006. The published genome will increase our understanding of the molecular characteristics of the BVDV-1e strains circulating in Europe.

Porcine epidemic diarrhea virus (PEDV) was detected for the first time in Slovenia in January 2015. The complete genome sequence of PEDV strain SLO/JH-11/2015, obtained from a fecal sample of a fattening pig with diarrhea in September 2015, is closely related to recently detected European strains.

Bats have been identified as a natural reservoir for an increasing number of emerging zoonotic viruses, such as Hendra virus, Nipah virus, Ebola virus, Marburg virus, rabies and other lyssaviruses. Recently, a large number of viruses closely related to members of the genus Coronavirus have been associated with severe acute respiratory syndrome (SARS) and detected in bat species. In this study, samples were collected from 106 live bats of seven different bat species from 27 different locations in Slovenia. Coronaviruses were detected by RT-PCR in 14 out of 36 horseshoe bat (Rhinolophus hipposideros) fecal samples, with 38.8% virus prevalence. Sequence analysis of a 405-nucleotide region of the highly conserved RNA polymerase gene (pol) showed that all coronaviruses detected in this study are genetically closely related, with 99.5-100% nucleotide identity, and belong to group 2 of the coronaviruses. The most closely related virus sequence in GenBank was SARS bat isolate Rp3/2004 (DQ071615) within the SARS-like CoV cluster, sharing 85% nucleotide identity and 95.6% amino acid identity. The potential risk of a new group of bat coronaviruses as a reservoir for human infections is highly suspected, and further molecular epidemiologic studies of these bat coronaviruses are needed.

A single step RT-PCR was tested for detection of foot and mouth disease virus (FMDV) and immunoenzymatic determination of amplified products in a microplate hybridization assay. Inactivated reference strains (ELISA antigen) of all seven serotypes were used to optimize the test. Oligonucleotide primers were selected from two different genomic regions coding for RNA polymerase and VP1 protein, respectively. The RT-PCR used to amplify the polymerase gene specific RNA detected FMDV strains A, C, O, Asial and SAT1, and the identity of the fragments obtained was confirmed with a specific internal biotin-labelled capture probe. For the amplification of the VP1 genome region, two sets of oligonucleotide primers were used. One primer pair was successfully applied for the detection of serotypes A, C, O and Asial and a second one for serotypes SAT1, SAT2, SAT3. The specific probe enabled the detection of all the amplified products in a PCR ELISA test. By comparison with antigen ELISA, the PCR ELISA method allowed the detection of smaller amounts of FMDV in the inactivated material examined. The application of molecular diagnostic methods to inactivated antigens offers a good alternative procedure for developing and optimizing a sensitive method for detection of FMDV in laboratories that are not allowed to work with viable FMDV.

The results of an investigation on bovine viral diarrhoea virus (BVDV) in fetal calf serum (FCS), whole blood and pathological material obtained from sick or dead cattle in Serbia are presented. Whole blood and FCS from sick animals were screened for BVDV antigen (Erns) by ELISA. ELISA positive samples and pathological material from dead animals were inoculated into primary cell cultures of fetal calf testis (FTTe). After threefold passage in FTTe cells, BVDV was detected by direct immunofluorescence and indirect immunoperoxidase tests and by reverse transcriptase-polymerase chain reaction (RT-PCR). Among 64 individual samples of FCS, two were positive for noncytopathogenic BVDV. One cytopathogenic BVDV was isolated from a whole blood sample from a heifer with clinical signs of mucosal disease. The 5'-untranslated region (5'-UTR) of three Serbian BVDV isolates was amplified by RT-PCR, sequenced and, together with 15 recent Slovenian BVDV isolates characterized by phylogenetic analysis. All isolates were classified as BVDV genotype 1 viruses. The majority of the BVDV isolates were of the 1f (Serbia - 2 isolates, Slovenia - 7 isolates) and 1d subtypes (Slovenia - 7 isolates) whilst one Serbian and one Slovenian isolate were genotyped as BVDV 1b.

The genetic diversity of acute bee paralysis virus (ABPV) in honeybees was studied in Slovenia. A total of 248 honeybee samples obtained from 134 different apiaries in Slovenia were tested for the presence of ABPV by RT-PCR. Specific 398-base pair (bp) products were generated with primers amplifying the ORF2 region and 452-base pair (bp) products with primers amplifying the ORF1 region of the viral genome. To characterise the overall nucleotide diversity among the ABPV sequences, phylogenetic trees with 54 and 29 samples were constructed from 357 nucleotides from ORF2 and 408 nucleotides from ORF1, respectively. The nucleotide comparison of Slovenian ABPV strains revealed two distinct clusters in ORF2 and ORF1, showing 91.2–92.5% and 96.7–97.2% nucleotide identity, respectively. Comparison of data regarding the geographical location of the ABPV-positive samples with the constructed phylogenetic trees revealed the random distribution of the two clusters throughout Slovenia.

Two polymerase chain reaction (PCR) assays specific for glycoprotein B (gB) and glycoprotein E (gE) gene detection, respectively, were adopted for the detection of bovine herpesvirus-1 (BHV-1) in naturally infected bulls. The methods were tested on bovine semen artificially inoculated with BHV-1 and were compared with an optimised virus isolation method. Raw and extended semen samples were diluted in minimal essential medium (MEM) and spiked with equal dose of BHV-1. The extended semen was found to be more toxic for the cells than the raw semen, while the viral DNA could be detected by the PCR method in all tested dilutions of raw and extended semen samples. The sensitivity of both methods was compared also for BHV-1 detection in semen, nasal swabs and leucocytes of a seropositive bull in a different time period after virus reactivation with dexamethasone treatment. The sensitivity of virus detection by the PCR method was equivalent to that of virus isolation in cell culture. However, PCR was shown to be faster and easier to perform and may be a good alternative to virus isolation especially when bovine semen has to be screened for BHV-1 prior to artificial insemination.