First molecular confirmation of cyprinid herpesvirus 1 (CyHV1) in diseased carp in Serbia.

Mass mortality of farmed 1 yr old common carp Cyprinus carpio occurred at a carp farm in April 2022. In addition to high mortality, diseased fish exhibited papillomatous growths on the skin and fins, characteristic of carp pox. To investigate a possible viral cause, tissue samples were collected and nucleic acid was extracted using standard procedures. In a pooled sample from the gills and kidneys, carp edema virus (CEV) was detected by real-time PCR. In a skin tissue sample with papillomatous growths, cyprinid herpesvirus 1 (CyHV1) was detected by a conventional PCR targeting a conserved region of the DNA polymerase of cyprinid herpesviruses. PCR products were visualized through agarose gel electrophoresis, and the presence of CyHV1 DNA was confirmed by Sanger sequencing. This represents the first molecular confirmation of CyHV1 in common carp in Serbia.

Protein sequence features of H1N1 swine influenza A viruses detected on commercial swine farms in Serbia.

Introduction: Swine influenza A viruses (swIAVs) are characterised by high mutation rates and zoonotic and pandemic potential. In order to draw conclusions about virulence in swine and pathogenicity to humans, we examined the existence of molecular markers and accessory proteins, cross-reactivity with vaccine strains, and resistance to antiviral drugs in five strains of H1N1 swIAVs. Material and Methods: Amino acid (AA) sequences of five previously genetically characterised swIAVs were analysed in MEGA 7.0 software and the Influenza Research Database. Results: Amino acid analysis revealed three virus strains with 590S/591R polymorphism and T271A substitution within basic polymerase 2 (PB2) AA chains, which cause enhanced virus replication in mammalian cells. The other two strains possessed D701N and R251K substitutions within PB2 and synthesised PB1-F2 protein, which are the factors of increased polymerase activity and virulence in swine. All strains synthesised PB1-N40, PA-N155, PA-N182, and PA-X proteins responsible for enhanced replication in mammalian cells and downregulation of the immune response of the host. Mutations detected within haemagglutinin antigenic sites imply the antigenic drift of the five analysed viruses in relation to the vaccine strains. All viruses show susceptibility to neuraminidase inhibitors and baloxavir marboxil, which is important in situations of incidental human infections. Conclusion: The detection of virulence markers and accessory proteins in the analysed viruses suggests their higher propensity for replication in mammalian cells, increased virulence, and potential for transmission to humans, and implies compromised efficacy of influenza vaccines.

Phylogenetic analysis of spring viraemia of carp virus isolated in Serbia.

Spring viraemia of carp (SVC) is an infectious disease responsible for severe economic losses for various cyprinid species, particularly common carp (Cyprinus carpio carpio). The causative agent is the Rhabdovirus carpio or SVC virus (SVCV), a member of the Sprivivirus genus, within the Rhabdoviridae family. Phylogenetically, SVCV is divided into four genogroups (SVCV a, SVCV b, SVCV c and SVCV d), which have a reasonable correlation with the geographical distribution of the virus. In the late twentieth century, the disease was widespread in Serbian aquaculture and caused massive deaths in common carp. This study aimed to molecularly characterize the circulating SVCV isolates in Serbia over a 17-year period. The genetic relationships between 21 SVCV isolates from common carp and rainbow trout in Serbia between 1992 and 2009 were determined based on the partial nucleotide sequence of the glycoprotein gene (G gene). The phylogenetic analysis showed that the dominant SVCV isolates in Serbia belong to the SVCV d genogroup, with only one isolate belonging to genogroup SVCV b. The SVCV strains circulating in Serbia exhibited high homogeneity, as several isolates shared 100% similarity within these genogroups. Most Serbian isolates belonged to SVCV d1 and d2 subgroups, with one isolate notably different and included in a new subgroup SVCV d5. Understanding the SVCV genetic variants circulating in Serbia would be helpful in future epizootic investigations.

Genetic analysis of influenza A viruses of swine from commercial farms in Serbia.

Swine influenza presents a very important health and economic issue in pig productions worldwide. Viruses that cause the disease are genetically very diverse but usually belong to the H1N1, H1N2 and H3N2 subtype of influenza A viruses. In this study, we sequenced and analyzed the full genome of viruses detected in swine from seven commercial farms. Through the analysis of the complete sequences of internal gene cassette together with previously characterized HA and NA genes we found three different genotypes amongst five completely sequenced viruses. Two viruses possessed a completely H1avN1 genotype (40%) and belonged to the H1avN1 lineage, which is prevalent in European swine populations. The other three viruses have arisen through the reassortment of the genes of H1avN1 and H1N1pdm09 lineages. In one sample we detected coinfection with viruses of H3N2 subtype with genes of H1avN1, H1N1pdm09 and A/swine/Gent/1/1984-like H3N2 lineages that presents a potential environment for the generation of a triple reassortant virus. The presence of the H1N1pdm09 origin M gene in this sample implies the potential risk of the introduction of these viruses into the human population. Phylogenetic analysis of internal gene cassette revealed slower evolution within genes of H1N1pdm09 lineage than those of H1avN1 lineage.

First confirmation of salmonid alphavirus infection in rainbow trout in Bosnia and Herzegovina and Serbia.

Sleeping disease is a highly infectious viral disease caused by salmonid alphavirus subtype 2 (SAV2 FW), affecting mainly rainbow trout Oncorhynchus mykiss farmed in freshwater. During March to May 2014, disease episodes with clinical signs of sleeping disease in rainbow trout fingerlings occurred almost simultaneously in 2 trout farms located in Bosnia and Herzegovina (BiH) and Serbia. The infection of rainbow trout with SAV2 FW in 2 farms was confirmed by virus isolation and molecular methods. This is the first isolation and molecular characterization of SAV2 FW in BiH and Serbia.

First detection of infectious haematopoietic necrosis virus in farmed rainbow trout in North Macedonia.

Infectious haematopoietic necrosis virus (IHNV) is the causative agent of infectious haematopoietic necrosis (IHN), a disease of salmonids responsible for great economic losses. The disease occurs in most parts of the world where rainbow trout is reared but has not been previously reported in North Macedonia. In this study, 150 pooled samples in total, each consisting of organ mix of 10 freshly killed rainbow trout Oncorhynchus mykiss, were collected from 50 trout farms by the Food and Veterinary Agency of North Macedonia as part of the annual surveillance plan for IHN and viral haemorrhagic septicaemia (VHS) control. Screening of samples was done by cell culture and real-time RT-PCR (qRT-PCR). All 150 tested samples were VHS virus (VHSV) qRT-PCR negative. Two samples from different trout farms were IHNV qRT-PCR positive. On cell culture, 1 IHNV qRT-PCR positive sample caused cytopathic effect after 2 passages on EPC cells. The virus, isolated from an asymptomatic rainbow trout fry, was identified by qRT-PCR and designated as MAKIHNV1. The phylogenetic reconstruction indicates that the isolated virus belongs to the European E genogroup, more specifically within the E-1 clade, and is similar to the German, Italian and Iranian isolates. This study has revealed for the first time the presence of IHNV in rainbow trout in North Macedonia. However, it is not possible to make interpretations about the source of infection from the phylogenetic analysis, and the origin of MAKIHNV1 remains unclear.

Molecular detection of black queen cell virus and Kashmir bee virus in honey.

Considering the intensive trading nowadays, the honey from the local market was tested for the presence of the six most common bee viruses. To prove the suitability of honey as a sample for the bee viruses detection, the set of different sample types taken directly from the hives we comparatively tested. The study included 30 samples of domestic and 5 samples of imported honey. Additionally, we tested 40 sets of samples including live bees, dead bees, and the honey taken from four apiaries for the evaluation of honey suitability for the virus detection, Two out of the six most common bee viruses were detected in the samples of honey from the market. Black queen cell virus (BQCV) genome was found in 24 domestic honey samples and Kashmir bee virus (KBV) genome was detected in one sample of imported honey. The nucleotide sequences of 24 BQCV isolates showed the highest identity (86.4%) with strains from Europe at the polyprotein gene, whilst the Serbian isolates between each other showed 98.5% similarity. By comparative testing of the different type of samples, in three out of four apiaries BQCV genome was detected in both bees and honey. Evaluating the suitability of honey for the detection of the viral disease by simultaneous testing of live, dead bees, and honey from the same hive, it was shown that the honey can be successfully used for the detection of BQCV. Since, as of yet, there has been no evidence of KBV circulation in Serbia, after its detection in imported honey, there is a substantial risk of its introduction and consequently the need for its surveillance. Therefore, the programs of bee diseases screening should be included in the regular control procedures for the international trade. In addition to this benefit, honey gives an opportunity to beekeepers for continuous monitoring of bees' health status.

Bovine viral diarrhea virus infection in wild boar.

Bovine viral diarrhea (BVD) is one of the most economically important diseases of cattle. With its very high prevalence, cattle kept on pastures become a source of the virus for the wildlife which, due to their susceptibility, then easily can serve as a source for re-infections of cattle. Therefore, we investigated the BVDV infection in Serbian wild boar and assessed the role of wild boar in BVDV epidemiology including possible spreading to domestic species. This study was based on examination of 50 spleen samples which were collected from wild boars located in Eastern Serbia during the hunting season 2016/2017. BVDV genome was detected in 4 of 50 samples (8%). Phylogenetic analysis based on 5'UTR revealed that BVDV strains from wild boars shared 100% identity. Belonging to the BVDV 1f subgenotype, the most common in cattle, we showed that BVDV infections of wild boar occurred as a result of either direct or indirect contact with domestic animals. Therefore, the occurrence of infectious disease in wildlife emphasizes the need to study the pathogens shared by wildlife and domestic animals by investigating the incidence of pathogens and disease patterns of those populations.

First Serbian isolates of bovine herpesvirus 4 (BoHV-4) from a herd with a history of postpartum metritis.

Two farms in the Belgrade area have experienced serious problems with postpartal metritis. Serological examination of BoHV-4 infection was done using ELISA test and vaginal swabs were used for virus isolation. Average seroprevalence of BoHV-4 in these farms was 84.37%. BoHV-4 isolation was successful from three vaginal swabs on the MDBK cell line. Rising values of BoHV-4 antibodies were recorded in nine of ten cows with clinical signs of postpartal metritis. PCR and restriction analysis were used for better characterisation and isolate classification. Two isolates showed similarity with MOVAR 33/63 virus type, but one differed in polyrepetitive and other parts of DNA. This was the first isolation and characterisation of BoHV4 from Serbian herds.