Cryptic SARS-CoV-2 lineage identified on two mink farms as a possible result of long-term undetected circulation in an unknown animal reservoir, Poland, November 2022 to January 2023.

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.

First detection of bluetongue virus serotype 14 in Poland.

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.

Prevalence of Astroviruses in Different Animal Species in Poland.

Astroviruses (AstVs) are small RNA viruses characterized by a high mutation rate, the ability to recombine, and interspecies transmission, which allows them to infect a multitude of hosts including humans, companion animals, and farmed animals as well as wildlife. AstVs are stable in the environment, and their transmission is usually through the fecal-oral route or via contaminated water and food. Although direct zoonotic transmission was not confirmed, interspecies transmission events have occurred or have been indicated to occur in the past between wild and domestic animals and humans. They cause large economic losses, mainly in the poultry sector, due to gastroenteritis and mortality. In young children, they are the second most common cause of diarrhea. This study involved 166 intestine samples and pools of spleen, lymph node, and kidney samples collected from 352 wild animals, 52 pigs, and 31 companion animals. Astroviruses were detected in the intestine samples and were separately detected in pools of tissue samples prepared for individual animals using a heminested RT-PCR protocol. Amplicons were subjected to Sanger sequencing, and a phylogenetic analysis of 320 nt RNA-dependent RNA polymerase (RdRp) fragments referring to known nt sequences of astroviruses was performed. Astroviral RNA was detected in the intestine samples and/or tissue pools of red foxes (nine positive intestines and six positive tissue pools), rats (two positive intestines and three positive tissue pools), a cat (one AstV detected in an intestine sample), pigs (eight positive tissue pools), and wild boars (two positive pools of spleens, kidneys, and lymph nodes). No astroviral RNA was detected in wild mustelids, dogs, or other small wild animals including rodents. A phylogenetic analysis revealed that the astroviruses detected during this study were mostly host-specific, such as porcine, canine, and rat astroviruses that were highly homologous to the sequences of reference strains. In one of two wild boars, an AstV distinct to porcine species was found with the highest nt identity to Avastroviruses, i.e., turkey astroviruses, which suggests potential cross-species transmission of the virus, as previously described. Here, we present the first detection of astroviruses in the population of wild animals, companion animals, and pigs in Poland, confirming that astroviruses are frequent pathogens circulating in animals in the field. Our study also suggests potential cross-species transmission of Avaastrovirus to wild boars; however, further molecular characterization is needed.

Re-emergence of rabies in Mazowieckie Voivodeship, Poland, 2021.

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 Swietokrzyskie in November 2021. Emergency actions were implemented aiming at rapid rabies elimination.

The Genetic Characterization of the First Detected Bat Coronaviruses in Poland Revealed SARS-Related Types and Alphacoronaviruses.

Bats are a major global reservoir of alphacoronaviruses (alphaCoVs) and betaCoVs. Attempts to discover the causative agents of COVID-19 and SARS have revealed horseshoe bats (Rhinolophidae) to be the most probable source of the virus. We report the first detection of bat coronaviruses (BtCoVs) in insectivorous bats in Poland and highlight SARS-related coronaviruses found in Rhinolophidae bats. The study included 503 (397 oral swabs and 106 fecal) samples collected from 20 bat species. Genetically diverse BtCoVs (n = 20) of the Alpha- and Betacoronavirus genera were found in fecal samples of two bat species. SARS-related CoVs were in 18 out of 58 lesser horseshoe bat (Rhinolophus hipposideros) samples (31%, 95% CI 20.6-43.8), and alphaCoVs were in 2 out of 55 Daubenton's bat (Myotis daubentonii) samples (3.6%, 95% CI 0.6-12.3). The overall BtCoV prevalence was 4.0% (95% CI 2.6-6.1). High identity was determined for BtCoVs isolated from European M. daubentonii and R. hipposideros bats. The detection of SARS-related and alphaCoVs in Polish bats with high phylogenetic relatedness to reference BtCoVs isolated in different European countries but from the same species confirms their high host restriction. Our data elucidate the molecular epidemiology, prevalence, and geographic distribution of coronaviruses and particularly SARS-related types in the bat population.

Vaccine-induced Rabies in a Red Fox in Poland.

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.

SARS-CoV-2 Monitoring on Mink Farms in Poland.

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.

First Detection of Bat Astroviruses (BtAstVs) among Bats in Poland: The Genetic BtAstVs Diversity Reveals Multiple Co-Infection of Bats with Different Strains.

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 7.1-20.2) bat intestines, six bat kidneys (6.1%, 95% CI 2.8-12.7) and two bat livers (2.0%, 95% CI 0.4-7.1). 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.

Mink SARS-CoV-2 Infection in Poland - Short Communication.

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.

Serological Survey of Lyssaviruses in Polish Bats in the Frame of Passive Rabies Surveillance Using an Enzyme-Linked Immunosorbent Assay.

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.

Population- and Variant-Based Genome Analyses of Viruses from Vaccine-Derived Rabies Cases Demonstrate Product Specific Clusters and Unique Patterns.

Rabies in wildlife has been successfully controlled in parts of Europe and North America using oral rabies vaccination, i.e., the distribution of baits containing live-attenuated virus strains. Occasionally, these vaccines caused vaccine virus-induced rabies cases. To elucidate the mechanisms of genetic selection and the effect of viral populations on these rabies cases, a next generation sequencing approach as well as comprehensive data analyses of the genetic diversity of Street Alabama Dufferin (SAD) and ERA vaccine virus strains and vaccine-induced rabies cases from Canada and several European countries were conducted. As a result, twelve newly generated sets of sequencing data from Canada and Poland were added to a pool of previously investigated samples. While the population-based analysis showed a segregation of viruses of ERA vaccine-induced rabies cases from those of SAD Bern original (SAD Bernorig)-derived rabies cases, the in-depth variant analysis revealed three distinct combinations of selected variants for the ERA vaccine-induced cases, suggesting the presence of multiple replication-competent haplotypes in the investigated ERA-BHK21 vaccine. Our findings demonstrate the potential of a deep sequencing approach in combination with comprehensive analyses on the consensus, population, and variant level.

The effect of selected molecules influencing the detrimental host immune response on a course of rabies virus infection in a murine model.

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.

The effect of combined drugs therapy on the course of clinical rabies infection in a murine model.

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/ß, 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/ß 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/ß.

Combination drug treatment prolongs survival of experimentally infected mice with silver-haired bat rabies virus.

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.

Inhibition of caspase-1 prolongs survival of mice infected with rabies virus.

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.

Bokeloh bat lyssavirus isolation in a Natterer's bat, Poland.

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.

Diagnostic Reliability of Different RT-PCR Protocols for the Detection of Bluetongue Virus Serotype 14 (BTV-14).

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.