Complete genome sequences of two strains of classical swine fever virus of subgenotype 2.3 detected during outbreaks in 2005 and 2006 in Serbia.

Two strains of classical swine fever virus (CSFV) (SRB/CSFV/1264/2005 and SRB/CSFV/6168/2006), producing serious clinical signs of disease during outbreaks in 2005 and 2006 in Serbia, were isolated on porcine kidney cells, and their complete genomes were determined by next-generation sequencing. This first complete genome characterization of Serbian CSFV strains provides new data about the evolution of CSFV in the Balkan region and enables further detailed phylogenetic studies of the various strains.

Variations in the detection of anti-PEDV antibodies in serum samples using three diagnostic tests - short communication.

Over the last few years several porcine epidemic diarrhoea (PED) outbreaks have been discovered in Europe including the first PED case in Slovenia in January 2015. The aim of this study was to determine when PED virus (PEDV) infection started in Slovenia. Serum samples collected between 2012 and 2016 were tested. Three hundred and seventy-five serum samples were collected from 132 Slovenian small, one-site pig farms. Samples were tested for PEDV antibodies utilising three different serological methods: commercially-available indirect ELISA, in-house blocking ELISA test and Immunoperoxidase Monolayer Assay (IPMA) test. One hundred and seventy (45.33%) tested samples were found positive by the commercially-available ELISA test kit, and 10 (5.68%) of these 170 samples found positive were positive by the in-house blocking ELISA. Only these 10 samples were collected from a farm where clinical signs of PED infection had been observed and PEDV was confirmed by RT-PCR methodology; the other 160 samples were collected randomly. Thirty-two samples with the highest S/P value obtained with the commercial ELISA were all negative with IPMA. Reasons for the high variance in the results obtained remain unclear; more research is required to ensure higher sensitivity and specificity in terms of PEDV antibody tests and other PED diagnostic methods.

The silent spread of Porcine Bocavirus in Croatian pigs: should we be concerned?

A survey was conducted to evaluate the presence and prevalence of Porcine Bocavirus (PBoV) in Croatian domestic pigs by means of PCR targeting the NS1 gene fragment of PBoV. This study included testing of faecal samples collected from 10 small commercial farms and 11 small backyard holdings in Croatia. The presence of PBoV was confirmed by PCR in 24 out of 57 composite faecal samples from small commercial farms and in 12 out of 43 composite faecal samples from small backyard holdings. The PCR products of 18 positive samples were sequenced for genotyping. PBoV sequences grouped into the PBoV-a, PBoV-b and PBoV-c groups with 90.81% to 99.25% nucleotide identity. All Croatian PBoV sequences showed a high nucleotide and amino acid identity with PBoV sequences from China and Hong Kong, the United States, Sweden, and Slovenia. These results clearly show that PBoV is circulating among the domestic pig population in Croatia.

Molecular characterisation of noroviruses detected in mussels (Mytilus galloprovincialis) from harvesting areas in Slovenia.

Noroviruses are a leading cause of viral gastroenteritis in humans and are responsible for many outbreaks worldwide. Mussels are one of the most important foodstuffs connected with norovirus outbreaks, also resulting in multinational dimensions. Two hundred and thirty-eight (238) samples of mussels (Mytilus galloprovincialis) were collected in periods between the years 2006-2008 and 2010-2012 to study the prevalence of noroviruses (NoVs) from harvesting areas along the Adriatic coast of Slovenia. Between 2006 and 2008, 9.1% to 24.6% of mussel samples tested by specific GI and/or GII real-time RT-PCR methods were found to be positive for NoVs while between 2010 and 2012 the percentage of NoV positive samples varied from 12.5% to 22.2%. At the nucleotide level within the RdRp gene fragment the genetic diversity of NoVs detected in mussels ranged between 78.8-81.0% nucleotide identity among GII strains (92.1-99.6% within the GII.P4 genotype), 100% nucleotide identity among GI and 58.4-60.2% among GI and GII strains. Nine of the NoV strains detected from mussels were genotyped as GII.4, while two samples were within GI.P2 and one was a positive sample within genotype GII.P21. This study confirmed that mussels are a potential source of the NoV infection. The detected NoVs share the same topology on the phylogenetic tree within the NoV strains detected in water samples and human patients, not only from Slovenia but also from many different countries worldwide. We can assume that mussels in harvesting areas are not only contaminated from the surrounding area but also by contaminated water and sewage from large transport ships, which are regularly present in the area.

Serum inoculation as a possibility for elimination of porcine reproductive and respiratory syndrome (PRRS) from a farrow-to-finish pig farm.

The large heterogeneity among porcine reproductive and respiratory syndrome virus (PRRSV) isolates is probably the main obstacle to its effective control using current commercial vaccines. Intentionally exposing all breeding pigs to PRRSV circulating on the farm could eliminate porcine reproductive and respiratory syndrome (PRRS) from the herd. The objective of this study was to eliminate PRRS from a farrow-to-finish pig farm by serum inoculation. The owner was acquainted with the strict biosecurity measures. Breeding pigs were immunised with serum, which was obtained from PRRSV-positive weaners from the same farm. The percent of antibody high positive breeding pigs decreased six months after serum inoculation, while 34 months after serum inoculation no more antibody high positive pigs were detected and 56.8% of breeding pigs and all other categories were free of antibodies. In the breeding herd no virus was detected during all testing while PRRSV circulated in 2-month-old weaners until 12 months after serum inoculation. Later all tested samples from weaners, growers and fatteners were negative. Herd closure and the adoption of strict biosecurity measures are essential. Serum inoculation of the breeding herd proved to be a successful measure for eliminating PRRS from this farrow-to-finish farm.

Winter Hive Debris Analysis Is Significant for Assessing the Health Status of Honeybee Colonies (Apis mellifera).

Honeybee diseases are one of the most significant and most common causes of honeybee colonies' weakness and death. An early diagnosis of subclinical infections is necessary to implement precautionary and control measures. Sampling debris from hive bottom boards is simple, non-invasive, and cheap. In this study, we collected winter debris samples in apiaries located in the continental part of Croatia. We used molecular methods, PCR and qPCR, for the first time to analyze those samples. Laboratory results were compared with the health condition and strength of honeybee colonies at an apiary in spring. Our study successfully identified the presence and quantity of various pathogens, including the presence of Vairimorpha spp. (Nosema spp.), quintefied Paenibacillus larvae, Acute Bee Paralysis Virus (ABPV), Black Queen Cell Virus (BQCV), Deformed Wing Virus (DWV), and Sacbrood Virus (SBV). However, our analysis did not detect Melissococcus plutonius, Crithidia mellificae, Lotmaria passim, and Aethina tumida. Samples of winter debris were also examined for the presence and quantification of the V. destructor mites, and their natural mite fall was observed in spring. Honeybee colonies were simultaneously infected by an average of four to six pathogens. Some observed honeybee colonies developed characteristic symptoms, while others did not survive the winter.

Eggs sampling as an effective tool for identifying the incidence of viruses in honey bees involved in artificial queen rearing.

The Carniolan honey bee (Apis mellifera carnica) plays an essential role in crop pollination, environment diversity, and the production of honey bee products. However, the health of individual honey bees and their colonies is under pressure due to multiple stressors, including viruses as a significant threat to bees. Monitoring various virus infections could be a crucial selection tool during queen rearing. In the present study, samples from all developmental stages (eggs, larvae, pupae, and queens) were screened for the incidence of seven viruses during queen rearing in Slovenia. The screening of a total of 108 samples from five queen breeders was performed by the RT-qPCR assays. The results showed that the highest incidence was observed for black queen cell virus (BQCV), Lake Sinai virus 3 (LSV3), deformed wing virus B (DWV-B), and sacbrood virus (SBV). The highest viral load was detected in queens (6.07 log10 copies/queen) and larvae (5.50 log10 copies/larva) for BQCV, followed by SBV in larvae (5.47 log10 copies/larva). When comparing all the honey bee developmental stages, the eggs exhibited general screening for virus incidence and load in queen mother colonies. The results suggest that analyzing eggs is a good indicator of resilience to virus infection during queen development.

Genetic diversity of acute bee paralysis virus in Slovenian honeybee samples.

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.

Discovery of a novel bat lyssavirus in a Long-fingered bat (Myotis capaccinii) from Slovenia.

Lyssaviruses are the causative agents of rabies, a zoonotic, fatal disease that is thought to be ancestral to bats. In the last decade, the detection of bat associated lyssaviruses is increasing also in Europe. Within a retrospective bat associated lyssavirus surveillance study a total of 225 dead bats of 21 bat species were collected in Slovenia between 2012 and 2019 and tested by specific real-time RT-PCR method. The first lyssavirus positive sample in bats in Slovenia was detected using the real-time RT-PCR, the fluorescent antibody test, and next generation sequencing, while the rabies tissue culture inoculation test was unsuccessful due to sample degradation and storage conditions. The nearly complete genome of Divaca bat lyssavirus from Slovenia consists of 11,871 nucleotides and reflects the characteristic gene organization known for lyssaviruses, encoding the five viral proteins. Phylogenetic analysis of Divaca bat lyssavirus revealed that it belongs to phylogroup I lyssaviruses and is most closely related to Kotalahti bat lyssavirus (KBLV) with 87.20% nucleotide and 99.22% amino acid identity. Together with KBLV, Khujand virus, European bat lyssavirus 2, Bakeloh bat lyssavirus, and Aravan virus, Divaca bat lyssavirus was detected in the genus Myotis suggesting its key role in the transmission and maintenance of certain lyssaviruses.

Genetic analysis reveals multiple intergenic region and central variable region in the African swine fever virus variants circulating in Serbia.

This study provides the first comprehensive report on the molecular characteristics of African swine fever virus (ASFV) variants in Serbia between 2019 and 2022. Since its first observation in July 2019, the disease has been found in wild boar and domestic swine. The study involved the analysis of 95 ASFV-positive samples collected from 12 infected administrative districts in Serbia. Partial four genomic regions were genetically characterized, including B646L, E183L, B602L, and the intergenic region (IGR) between the I73R-I329L genes. The results of the study suggest that multiple ASFV strains belonging to genotype II are circulating in Serbia, as evidenced by the analysis of the IGR between I73R-I329L genes that showed the most differences. Furthermore, the phylogenetic analysis of the B602L gene showed three different clades within the CVR I group of ASFV strains. Regarding the IGR, 98.4% were grouped into IGR II, with only one positive sample grouped into the IGR III group. These findings provide essential insights into the molecular characteristics of ASFV variants in Serbia and contribute to the knowledge of circulating strains of ASFV in Europe. However, further research is necessary to gain a better understanding of ASFV spread and evolution.

Study of the genetic variability of porcine circovirus type 2 detected in Serbia and Slovenia.

Recent variants of porcine circovirus type 2 (PCV2) were obtained from tissues of domestic pigs with porcine circovirus associated disease and from randomly selected wild boar samples from Serbia and Slovenia. A 450-base-pair nucleotide sequence was obtained by PCR from the ORF2. The derived nucleotide and amino acid sequences were aligned and compared to the corresponding region of closely related PCV2 sequences determined in previous years and retrieved from the GenBank. The 30 Serbian and 17 Slovenian PCV2 sequences clustered into three previously determined genotypes (PCV2a: 7), (PCV2b: 38) and (PCV2d: 2). Three major variable regions, concerning 29 amino acid position substitutions within the ORF2, were observed, which further supports the segregation of the detected strains into three separate genotypes. This study indicates that PCV2b is the predominant genotype in Serbia and Slovenia and the detected PCV2 strains are closely related to those previously described in Europe and in other parts of the world.

First Detection of Honeybee Pathogenic Viruses in Butterflies.

Several pathogens are important causes of the observed pollinator decline, some of which could be transmitted between different pollinator species. To determine whether honeybee viruses can be transmitted to butterflies, a total of 120 butterflies were sampled at four locations in Slovenia. At each location, butterflies from three families (Pieridae, Nymphalidae, Hesperiidae/Lycenidae) and Carniolan honeybees (Apis mellifera carnica) were collected. The RNA of six honeybee viruses, i.e., acute bee paralysis virus (ABPV), black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), deformed wing virus A (DWV-A), Sacbrood bee virus (SBV), and Lake Sinai virus 3 (LSV3), was detected by a specific quantitative method (RT-PCR). The presence of ABPV, BQCV, LSV3, and SBV was detected in both butterflies and honeybees. All butterfly and bee samples were negative for CBPV, while DWV-A was detected only in honeybees. The viral load in the positive butterfly samples was much lower than in the positive bee samples, which could indicate that butterflies are passive carriers of bee viruses. The percentage of positive butterfly samples was higher when the butterflies were collected at sampling sites with a higher density of apiaries. Therefore, we believe that infected bees are a necessary condition for the presence of viruses in cohabiting butterflies. This is the first study on the presence of pathogenic bee viruses in butterflies.

Genomic determinants of Furin cleavage in diverse European SARS-related bat coronaviruses.

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.

Detection of Vibrio parahaemolyticus in Mediterranean mussels (Mytilus galloprovincialis) in Slovenia.

The aim of this study was to determine the prevalence of Vibrio parahaemolyticus in shellfish samples harvested along the Slovenian coast. Shellfish samples of Mediterranean mussels (Mytilus galloprovincialis) were collected along the Slovenian coast at four locations (Seca, Piran, Strunjan and Debeli Rtic) between 2006 and 2008. Samples were examined and analysed for the presence of V. parahaemolyticus by conventional and molecular methods. The presence of Vibrio in the samples was examined by conventional methods on plate grown bacterial cells before and after enrichment in alkaline saline peptone water (ASPW). PCR methods were used for the detection of V. parahaemolyticus-specific toxR and tlh genes and of the virulence-associated tdh and trh genes. Out of 168 samples examined, 24 were positive for toxR and tlh genes by PCR from enrichment broth. Five out of 62 (8.1%), 4 out of 32 (12.5%) and 15 out of 74 (20.2%) samples were positive in 2006, 2007 and 2008, respectively. Colonies of V. parahaemolyticus were isolated from only one sample positive for V. parahaemolyticus by PCR.

The Successful Elimination of Sylvatic Rabies Using Oral Vaccination of Foxes in Slovenia.

Sylvatic rabies was present in Slovenia between 1973 and 2013, with the red fox as the main reservoir of the rabies virus. The first oral rabies vaccination (ORV) control program in foxes started in 1988, using the manual distribution of baits. Significant improvement of fox vaccination was achieved with the aerial distribution of baits, starting in 1995 and successfully finished with the final, fifty-ninth vaccination campaign in 2019. Between 1979 and 2019, a total of 86,471 samples were tested, and 10,975 (12.69%) rabies-positive animals were identified. Within the ORV, two different vaccines were used, containing modified live virus strain Street Alabama Dufferin (SAD) B19 and SAD Bern, while the last ORV campaigns were completed in 2019, with a vaccine containing a genetically modified strain of SPBN GASGAS. Molecular epidemiological studies of 95 rabies-positive samples, originating from red foxes, badgers, cattle, dogs, martens, cats, and horses, revealed a low genetic diversity of circulating strains and high similarity to strains from neighboring countries. During the elimination program, few vaccine-induced rabies cases were detected: three in red foxes and one case in a marten, with no epidemiological relevance. Slovenia has been officially declared a country free of rabies since 2016.

The Comparison of Honeybee Viral Loads for Six Honeybee Viruses (ABPV, BQCV, CBPV, DWV, LSV3 and SBV) in Healthy and Clinically Affected Honeybees with TaqMan Quantitative Real-Time RT-PCR Assays.

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 First Detection and Genetic Characterization of Four Different Honeybee Viruses in Wild Bumblebees from Croatia.

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 > DWV > 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.

Review of Infections With Bovine Herpesvirus 1 in Slovenia.

In the 1950s, infectious bovine rhinotracheitis/infectious pustular vulvovaginitis (IBR/IPV) disease was clinically detected and documented in cattle for the first time in Slovenia. The bovine herpes virus 1 (BoHV-1) was confirmed several times from infected herds by virus isolation on cell cultures. To keep the IC virus-free, high biosecurity measures were introduced. Before entering the IC, all calves are serologically tested and quarantined. Bulls in Slovenian insemination centres (IC) have been negative for IBR /IPV infection since 1979. From 1985 to 1991, few large-scale studies of the prevalence of IBR/IPV were carried out. In 1985, a high percentage (56.9%) of serologically positive animals were found in large state farms with Holstein Friesian cattle. Epidemiological studies in farm with bulls' mother herds were also carried out in the farms with Simmental and Brown cows. Antibodies against BoHV-1 were detected in the serum of 2.3% of Brown cattle and 3.5% of Simmental cattle. In the year 2000, 3.4% of bulk tank milk samples from 13,349 dairy farms were detected BoHV-1 antibodies positive. The highest percentage of positive animals was found in regions with an intensive grazing system (6.2% positive) and the lowest percentage in the east part of Slovenia (0.9% positive) on farms with mostly Simmental cattle. In 2006, a total 204,662 sera of cattle older than 24 months were tested for the presence of BoHV-1 antibodies and positive cattle were detected in 3.6% of tested farms. These farms kept 34,537 animals that were potential carriers of the BoHV-1. Most of the positive farms kept Holstein Friesian cattle, descendants from the state-owned farms, which were privatised or closed after 1990. In 2015, the Administration of the Republic of Slovenia for Food Safety, Veterinary and Plant Protection issued a rule that describes the conditions for granting and maintaining the status of BoHV-1 free holdings. The rule provides a voluntary control programme for breeders who want to obtain BoHV-1 free status and are willing to cover all the cost of acquiring and maintaining that status. There has been very little response from breeders.

The Principles of the Voluntary Programme for the Control and Elimination of Bovine Viral Diarrhoea Virus (BVDV) From Infected Herds in Slovenia.

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.

The Pathogens Spillover and Incidence Correlation in Bumblebees and Honeybees in Slovenia.

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.