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Introduction Rabies as a zoonosis threatens public health worldwide. Several thousand people die each year of infections by the rabies virus (RABV). Oral rabies vaccination (ORV) of wildlife was successfully implemented in many European countries and led to rabies being brought under control there. In Poland, ORV was introduced in 1993 using vaccines containing an attenuated strain of the rabies virus. However, attenuated rabies viruses may have residual pathogenicity and cause the disease in target and non-target animals. Material and Methods A red fox carcass was tested as part of national rabies surveillance, and its brain was screened for RABV infection using two conjugates and a fluorescent antibody test (FAT). The rabies virus was isolated in mouse neuroblastoma cells by rabies tissue culture infection test (RTCIT), and viral RNA was detected by heminested reverse transcriptase PCR (hnRT-PCR) as well as by quantitative real-time RT-PCR (rtRT-qPCR). An amplicon of 600 bp was subjected to Sanger sequencing. To differentiate between vaccine and field RABV strains, PCR-restriction fragment length polymorphism (PCR-RFLP) using the Dra I, Msp I, Nla IV and Mbo II restriction endonucleases was performed. Results The rabies virus was detected in the fox’s brain using FAT, RTCIT and molecular tests. The PCR-RFLP revealed of vaccine-induced rabies, and full-length genome analysis showed 100% nucleotide sequence identity of the isolate with the reference sequences of Street Alabama Dufferin Bern (SAD Bern) vaccine strains and other vaccine-induced rabies virus isolates detected in animals and deposited in GenBank. Conclusion We detected vaccine-induced rabies for the first time in Poland in a fox during routine rabies surveillance.

Due to the oral vaccination of foxes against rabies most of the territory of Poland was freed from rabies of non-flying mammals. In January 2021, rabies was diagnosed in fox in the central part of Mazowieckie Voivodeship where rabies has not been detected since last 17 years. Subsequently, in the following months the rabies virus infection spread southward reaching the voivodeship of Świętokrzyskie in November 2021. Emergency actions were implemented aiming at rapid rabies elimination.

In late 2022 and early 2023, SARS-CoV-2 infections were detected on three mink farms in Poland situated within a few km from each other. Whole-genome sequencing of the viruses on two of the farms showed that they were related to a virus identified in humans in the same region 2 years before (B.1.1.307 lineage). Many mutations were found, including in the S protein typical of adaptations to the mink host. The origin of the virus remains to be determined.

Introduction Many countries have reported severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infections in mink, and transmission back to humans has raised the concern of novel variants emerging in these animals. The monitoring system on Polish mink farms detected SARS-CoV-2 infection first in January 2021 and has been kept in place since then. Material and Methods Oral swab samples collected between February 2021 and March 2022 from 11,853 mink from 594 farms in different regions of Poland were screened molecularly for SARS-CoV-2. Isolates from those with the highest loads of viral genetic material from positive farms were sequenced and phylogenetically analysed. Serological studies were also carried out for one positive farm in order to follow the antibody response after infection. Results SARS-CoV-2 RNA was detected in mink on 11 farms in 8 out of 16 Polish administrative regions. Whole genome sequences were obtained for 19 SARS-CoV-2 strains from 10 out of 11 positive farms. These genomes belonged to four different variants of concern (VOC) – VOC-Gamma (20B), VOC-Delta (21J), VOC-Alpha (20I) and VOC-Omicron (21L) – and seven different Pango lineages – B.1.1.464, B.1.1.7, AY.43, AY.122, AY.126, B.1.617.2 and BA.2. One of the nucleotide and amino acid mutations specific for persistent strains found in the analysed samples was the Y453F host adaptation mutation. Serological testing of blood samples revealed a high rate of seroprevalence on the single mink farm studied. Conclusion Farmed mink are highly susceptible to infection with SARS-CoV-2 of different lineages, including Omicron BA.2 VOC. As these infections were asymptomatic, mink may become an unnoticeable virus reservoir generating new variants potentially threatening human health. Therefore, real-time monitoring of mink is extremely important in the context of the One Health approach.

Introduction Since April 2020, when the first SARS-CoV-2 infection was reported in mink and subsequently in mink farm workers in the Netherlands, it has been confirmed that human-to-mink and mink-to-human transmission can occur. Later, SARS-CoV-2 infections in mink were reported in many European and North American countries. Material and methods Samples from 590 mink from a total of 28 farms were tested by real-time RT-PCR. Whole genome sequences from one positive farm were generated and genetic relatedness was established. Results SARS-CoV-2 RNA was detected on a breeder farm with stock of 5,850 mink. Active viraemia was confirmed in individually tested samples with Ct values respectively between 19.4 and 29.6 for E and N gene fragments. Further testing of samples from culled animals revealed 70% positivity in throat swabs and 30% seropositivity in blood samples. Phylogenetic analysis of full-length nucleotide sequences of two SARS-CoV-2 isolates revealed that they belong to the 20B Nextstrain clade. Several nucleotide mutations were found in analysed samples compared to the reference Wuhan HU-1 strain and some of them were nonsynonymous. Conclusion We report the infection of mink with SARS-CoV-2 on one farm in Poland and the results of subsequent analysis of virus sequences from two isolates. These data can be useful for assessment of the epidemiological situation of SARS-CoV-2 in Poland and how it endangers public health.

Mononegavirales is an order of viruses with a genome in the form of a non-segmented negative-strand RNA that encodes several proteins. The functional polymerase complex of these viruses is composed of two proteins: a large protein (L) and a phosphoprotein (P). The replication of viruses from this order depends on Hsp90 chaperone activity. Previous studies have demonstrated that Hsp90 inhibition results in the degradation of mononegaviruses L protein, with exception of the rabies virus, for which the degradation of P protein was observed. Here, we demonstrated that Hsp90 inhibition does not affect the expression of rabies L and P proteins, but it inhibits binding of the P protein and L protein into functional viral polymerase. Rabies and the vesicular stomatitis virus, but not the measles virus, L proteins can be expressed independently of the presence of a P protein and in the presence of an Hsp90 inhibitor. Our results suggest that the interaction of L proteins with P proteins and Hsp90 in the process of polymerase maturation may be a process specific to particular viruses.

Background: Astroviruses (AstVs) are common pathogens of a wide range of animal hosts, including mammals and avians, causing gastrointestinal diseases, mainly gastroenteritis and diarrhea. They prompt a significant health problem in newborns and young children and economic losses in the poultry sector and mink farms. Recent studies revealed a growing number of bat species carrying astroviruses with a noticeable prevalence and diversity. Here, we demonstrate the first detection of bat astroviruses (BtAstVs) circulating in the population of insectivorous bats in the territory of Poland. Results: Genetically diverse BtAstVs (n = 18) were found with a varying degree of bat species specificity in five out of 15 bat species in Poland previously recognized as BtAstV hosts. Astroviral RNA was found in 12 out of 98 (12.2%, 95% CI 7.1&ndash, 20.2) bat intestines, six bat kidneys (6.1%, 95% CI 2.8&ndash, 12.7) and two bat livers (2.0%, 95% CI 0.4&ndash, 7.1). Deep sequencing of the astroviral RNA-dependent RNA polymerase (RdRp) region revealed co-infections in five single bat individuals with highly distinct astrovirus strains. Conclusions: The detection of highly distinct bat astroviruses in Polish bats favors virus recombination and the generation of novel divergent AstVs and creates a potential risk of virus transmission to domestic animals and humans in the country. These findings provide a new insight into molecular epidemiology, prevalence of astroviruses in European bat populations and the risk of interspecies transmission to other animals including humans.

Rabies is a fatal disease of all mammals causing almost 60,000 human deaths every year. To date, there is no effective treatment of clinical rabies once the symptoms appear. Here, we describe the promising effect of combination therapy composed of molecules that target replication of the rabies virus (RV) at different stages of life cycle and molecules that inhibit some pathways of the innate host immune response accompanied by a blood-brain barrier opener on the outcome of RV infection. The study reports statistically significant extension of survival of mice treated with the drug cocktail containing T-705, ribavirin, interferon α/β, caspase-1 inhibitor, TNF-α inhibitor, MAPKs inhibitor and HRIG compared to the survival of mice in the virus control group (p = 0.0312). Furthermore, the study points to the significant impact of interferon α/β on the survival of RV-infected mice. We have shown a significant down regulation of pro-inflammatory molecules (caspase-1 and TNF-a) in the CNS in RV-infected mice treated with a combination of drugs including interferon α/β.

Rabies virus infects almost all mammals resulting in lethal disease. To date there is no treatment available for symptomatic rabies and there is an urgent need to develop treatment strategies that would prolong survival, thereby providing a window of opportunity for the host to mount a protective immune response. We hypothesized that both virus and excessive immune response contribute to disease and that interfering with both is necessary to prevent lethal disease. Here, we have inhibited the pro-inflammatory response associated with pyroptosis and showed that inhibition of CASP-1 had a beneficial effect on survival time. Our results confirm that some inflammatory responses may be involved in the pathogenesis of severe disease and the results suggest that effective intervention includes inhibition of virus and host response.

The 2 predominant circulating subtypes of influenza A virus in the dog population, equine-origin H3N8 and avian-origin H3N2, constitute a potential zoonotic risk. We determined the prevalence of influenza A antibodies in 496 dogs in Poland and found 2.21% of sera positive by commercial ELISA. Hemagglutination inhibition (HI) assays indicated 7.25% of sera positive using equine H3N8, swine H3N2, and pandemic H1N1 antigens, with the most frequently detected immune response being to H3N2. Considering interspecies transfer, reassortment ability, and close contact between dogs and humans, infections of dogs with influenza A virus should be monitored.

Until 2020, the rabies epizootic situation in Poland was favourable. This was interrupted, however, by a rabies outbreak in Mazowieckie voivodeship, which caused a drastic increase in rabies cases among animals, mainly red foxes. Here, we present data on the species and geographical distribution of rabies cases in domestic and wild animals, including bats, in Poland in 2021. In order to limit the transmission of the rabies virus, the Veterinary Inspectorate has introduced appropriate measures, including emergency vaccination and additional oral rabies vaccination (ORV) campaigns for foxes. Nevertheless, strict and comprehensive ORV and rabies surveillance are required to eliminate rabies from the area of rabies outbreak in 2021.

Introduction: The reverse transcription polymerase chain reaction (RT-PCR) is one of the most extensively used methods for identification of animals infected with bluetongue virus (BTV). There are several RT-PCR protocols published and several real-time RT-PCR (rtRT-PCR) commercial kits available on the market. Because Poland faced BTV-14 infection in 2012, different protocols were implemented in the country to confirm the RT-PCR results positive for this virus. The article presents a comparative study of several RT-PCR protocols and discusses their diagnostic reliability and applicability. Material and Methods: Six rtRT-PCR/RT-PCR protocols were compared for the laboratory diagnostic of fourteen BTV-14 isolates circulating in Poland in 2012–2014. Results: All 14 isolates were positive in the protocols of Shaw et al. (18), a commercial LSI NS3 kit, and Eschbaumer et al. (5). Four out of fourteen BTV-14 isolates gave positive results in Hoffmann’s 2 and 6 protocols and none of the 14 isolates yielded positive results in Maan et al. (8) method. Phylogenetic study of a short fragment of 450 nt of BTV segment 2 (258–696 positions) revealed 100% identity within Polish variants and with Russian and Spanish isolates. Conclusion: The paper points to the possible false negative results in the diagnosis of BTV infections depending on the protocol used.

Background: Bats are known to host a number of nonpathogenic viruses, as well as highly pathogenic viruses causing fatal diseases like rabies. Serological surveys as part of active and passive bat rabies surveillance mainly use seroneutralization assays, demonstrating the presence of lyssavirus-specific antibodies in a variety of European bats, particularly against European bat lyssaviruses type 1 (EBLV-1). Here, we present the first serological survey in European bats of this kind during which European bats from Poland collected in the frame of passive rabies surveillance between 2012 and 2018, as well as Serotine bats (Eptesicus serotinus) and North American Big Brown bats (Eptesicus fuscus) from previous experimental studies, were tested using a commercial ELISA kit for the detection of anti-lyssavirus antibodies. Results: Lyssavirus-specific antibodies were detected in 35 (30.4%) out of 115 Polish bats of both sexes, representing nine out of 13 identified bat species endemic mainly to Central Southern Europe and Western Asia, i.e., Eptesicus serotinus, Nyctalus noctula, Myotis daubentonii, Plecotus auritus, Vespertillo murinus, Pipistrellus pipistrellus, Pipistrellus pipilstrellus/Pipistrellus pygmaeus, Myotis brandtii, and Barbastella barbastellus. Seroprevalence was highest in bat species of Nyctalus noctula, Eptesicus serotinus, Plecotus auritus, and Myotis daubentonii. More than 60% of the ELISA seropositive bats originated from the voivodeships of Silesia, Lower-Silesian, Warmian-Mazurian, and Mazowian. Rabies-specific antibodies were also found in Eptesicus fuscus bats from North America. Conclusions: The study demonstrates the principal application of the BioPro Rabies ELISA Ab Kit for the detection of anti-lyssavirus specific antibodies in body fluids and serum samples of bats. However, results may only be reliable for North American bats, whereas interpretation of results for European bats per se is difficult because proper validation of the test is hampered by the protected status of these species.

Introduction High-throughput sequencing (HTS) identifies random viral fragments in environmental samples metagenomically. High reliability gains it broad application in virus evolution, host-virus interaction, and pathogenicity studies. Deep sequencing of field samples with content of host genetic material and bacteria often produces insufficient data for metagenomics and must be preceded by target enrichment. The main goal of the study was the evaluation of HTS for complete genome sequencing of field-case rabies viruses (RABVs). Material and Methods The material was 23 RABVs isolated mainly from red foxes and one European bat lyssavirus-1 isolate propagated in neuroblastoma cells. Three methods of RNA isolation were tested for the direct metagenomics and RABV-enriched approaches. Deep sequencing was performed with a MiSeq sequencer (Illumina) and reagent v3 kit. Bioinformatics data were evaluated by Kraken and Centrifuge software and de novo assembly was done with metaSPAdes. Results Testing RNA extraction procedures revealed the deep sequencing scope superiority of the combined TRIzol/column method. This HTS methodology made it possible to obtain complete genomes of all the RABV isolates collected in the field. Significantly greater rates of RABV genome coverages (over 5,900) were obtained with RABV enrichment. Direct metagenomic studies sequenced the full length of 6 out of 16 RABV isolates with a medium coverage between 1 and 71. Conclusion Direct metagenomics gives the most realistic illustration of the field sample microbiome, but with low coverage. For deep characterisation of viruses, e.g. for spatial and temporal phylogeography during outbreaks, target enrichment is recommended as it covers sequences much more completely.

International audience; The most effective and sustainable method to control and eliminate rabies in wildlife is the oral rabies vaccination (ORV) of target species, namely foxes and raccoon dogs in Europe. According to WHO and OIE, the effectiveness of oral vaccination campaigns should be regularly assessed via disease surveillance and ORV antibody monitoring. Rabies antibodies are generally screened for in field animal cadavers, whose body fluids are often of poor quality. Therefore, the use of alternative methods such as the enzyme-linked immunosorbent assay (ELISA) has been proposed to improve reliability of serological results obtained on wildlife samples. We undertook an international collaborative study to determine if the commercial BioPro ELISA Rabies Ab kit is a reliable and reproducible tool for rabies serological testing. Our results reveal that the overall specificity evaluated on naive samples reached 96.7%, and the coefficients of concordance obtained for fox and raccoon dog samples were 97.2% and 97.5%, respectively. The overall agreement values obtained for the four marketed oral vaccines used in Europe were all equal to or greater than 95%. The coefficients of concordance obtained by laboratories ranged from 87.2% to 100%. The results of this collaborative study show good robustness and reproducibility of the BioPro ELISA Rabies Ab kit.

Here, we present the first detected cases of bluetongue virus (BTV) in native cattle from Poland. The virus was found in animals located near the Polish-Belarusian and Polish-Lithuanian borders. The positive animals were detected through an official epidemiological surveillance program. A combination of type-specific real-time RT-PCR and phylogenetic tests revealed the presence of BTV serotype 14 (BTV-14). This serotype is highly homologous to the vaccine strain and BTV-14 present in Russia, Lithuania, and Spain (from an animal imported from Lithuania). The most probable route of virus introduction to Poland was transmission through midges. All of the cases were subclinical.

Rabies is a lethal disease in humans and animals, killing approximately 60,000 people every year. Currently, there is no treatment available, except post-exposure prophylaxis (PEP) that can be administered whenever exposure to a rabid animal took place. Here we describe the beneficial effects of a combination treatment initiated at day 4 post infection, containing anti-viral drugs and immune modulators in infected mice. Combination therapy resulted in significant increase in survival time (P < 0.05) and significantly lowers viral RNA in the brain and spinal cord (P < 0.05). Furthermore, treatment influenced markers of pyroptosis and apoptosis and early inflammatory response as measured by the levels of TNF-α. Morphological lesions were absent in rabies virus infected mice with few signs of inflammation. However, these were not significant between the different groups.

Rabies is invariably fatal, when post-exposure prophylaxis is administered after the onset of clinical symptoms. In many countries, rabies awareness is very low and the availability of post-exposure prophylaxis, as recommended by WHO guidelines, is very limited or non-existent, probably as a consequence of high cost. Therefore, new concepts for rabies therapy are needed. Innate immune mechanisms involving the production of pro-inflammatory cytokines and chemokines, activated after rabies infection, are thought to be involved in the neuropathogenesis of rabies. These mechanisms can contribute to a detrimental host response to the rabies virus (RABV) infection. The use of inhibitors of cytokines/chemokines are supposed to extend the survival of a sick individual. Inhibitors of TNF-α, IL-6 and MAPKs were used in RABV inoculated mice to define their influence on the survival time of rabid mice. The study demonstrated that all inhibitors extended mice survival, but at different rates. A log-rank test confirmed the statistically significant survival of mice treated with TNF-α (p = .0087) and MAPKs inhibitors (p = .0024). A delay in the time of onset of rabies was also recorded, in mice given TNF-α and MAPKs inhibitors. The highest virus load was found in the spinal cord and the lowest in the cortex, regardless of the experimental group. Significant TNF-α (p ≤ .0001) and IL-6 (p ≤ .0001) gene upregulation was observed in mice, as a consequence of RABV infection. Regarding MAPKs pathways, there was significant upregulation of the caspase 3 (p = .012, p = .0026) and Mcl-1 (p = .0348, p = .0153) genes, whereas significant downregulation of the cytochrome C (p ≤ .0001), Bcl2 (p = .0002, p = .0007) and JNK3 (p = .042) genes. Rabies pathogenesis is multifactorial, involving both virus and host influences on the course of the infection.

In recent years, Bokeloh bat lyssavirus (BBLV), a member of the novel lyssavirus genus Bokeloh bat lyssavirus in the family Rhabdoviridae, has been detected in Germany (five cases) and France (two cases). Here, we report the isolation of BBLV in a Natterer's bat (Myotis nattereri) in Poland. The bat brain tested positive for rabies using classical diagnostics tests (FAT and RTCIT) and then subsequently confirmed by molecular techniques. Viral RNA was found in all peripheral organs tested, and the highest viral loads were detected in brain, the salivary gland and bladder. Phylogenetic analysis performed on complete viral genome sequences revealed the closest homology to representatives of BBLV lineage B, isolated previously in southern Germany. This case provides further evidence that BBLV is widespread in Europe.

Rabies is a zoonosis caused by RNA viruses belonging to the genus Lyssavirus, which includes 16 species. In Europe, the majority of rabies cases in bats are caused by European bat 1 lyssavirus (EBLV-1) associated mainly with serotine bats (Eptesicus serotinus and Eptesicus isabellinus). In 2010, Bokeloh bat lyssavirus (BBLV) was detected in Natterer’s bat (Myotis nattereri) in Germany and it was isolated several times in Germany and France in the following years. As required by the programme of passive rabies surveillance, the dead bat was sent to the laboratory and identified as Natterer’s bat on the basis of morphological criteria, which was confirmed by genetic identification using the sequencing of the cytochrome b gene. Brain tissue and salivary glands were collected for examination. In the brain, the direct immunofluorescence test revealed lyssavirus antigen. The presence of the infectious virus in brain tissue was confirmed by a tissue culture inoculation test. High levels of viral RNA were found in the brain and salivary glands. In order to determine the virus species, a complete genome sequence was obtained by next-generation sequencing of RNAs. The analysis of the full virus genome sequence confirmed the occurrence of BBLV in Poland and revealed that it showed the highest similarity (99.3%) to German isolates from Bavaria.

Twelve National Reference Laboratories (NRLs) for rabies have undertaken a comparative assay to assess the comparison of fluorescent antibody test (FAT) results using five coded commercial anti-rabies conjugates (Biorad, Bioveta, Fujirebio, Millipore, and SIFIN conjugates). Homogenized positive brain tissues infected with various lyssavirus species as well as negative samples were analyzed blindly using a standardized FAT procedure. Conjugates B, C, D, and E were found to be significantly more effective than conjugate A for GS7 (French RABV) diluted samples (1/8 and 1/100) while the frequency of concordant results of conjugates C and D differ significantly from conjugates A, B and E for CVS 27. For detection of EBLV-1 strains, conjugates C and D also presented a significantly lower frequency of discordant results compared to conjugates A, B and E. Conjugates B, C and D were found to be significantly more effective than conjugates E and A for EBLV-2 and ABLV samples. In view of these results, conjugates C and D set themselves apart from the others and appeared as the most effective of this 5-panel conjugates. This study clearly demonstrates that the variability of conjugates used by National Reference Laboratories can potentially lead to discordant results and influence assay sensitivity. In case of false negative results this could have a dramatic impact if the animal under investigation is responsible for human exposure. To avoid such situations, confirmatory tests should be implemented.

To investigate the circulation of rabies virus in Ukraine, 78 rabies virus isolates were acquired from 14 states in 2002 and 2008-2010 for characterization. Partial sequences of nucleoprotein (359 nt) and glycoprotein (344 nt) genes were compared with those from neighbouring countries. The analysis identified 39 unique nucleoprotein genes and two geographically distinct RV variants belonging to the cosmopolitan lineage. The Ukrainian samples were similar to the North-East European lineage (NEE) (n = 19) and Russian group C (n = 20). The group C viruses were mainly isolated in Eastern Ukraine, from 9 regions, and from two other regions in Western Ukraine, suggesting the presence of group C throughout the country. These group C viruses are intermixed in bordering regions along the Dnieper River with viruses of group NEE, which were mainly isolated in six regions in Western Ukraine. Both nucleoprotein and glycoprotein gene analyses suggested evidence for cross-border movements of rabies virus.

The evolution of rabies viruses of predominantly European origin was studied by comparing nucleotide sequences of the nucleoprotein and glycoprotein genes, and by typing isolates using RFLP. Phylogenetic analysis of the gene sequence data revealed a number of distinct groups, each associated with a particular geographical area. Such a pattern suggests that rabies virus has spread westwards and southwards across Europe during this century, but that physical barriers such as the Vistula river in Poland have enabled localized evolution. During this dispersal process, two species jumps took place – one into red foxes and another into raccoon dogs, although it is unclear whether virus strains are preferentially adapted to particular animal species or whether ecological forces explain the occurrence of the phylogenetic groups.