Sequestration of membrane cholesterol by cholesterol-binding proteins inhibits SARS-CoV-2 entry into Vero E6 cells.

Membrane lipids and proteins form dynamic domains crucial for physiological and pathophysiological processes, including viral infection. Many plasma membrane proteins, residing within membrane domains enriched with cholesterol (CHOL) and sphingomyelin (SM), serve as receptors for attachment and entry of viruses into the host cell. Among these, human coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), use proteins associated with membrane domains for initial binding and internalization. We hypothesized that the interaction of lipid-binding proteins with CHOL in plasma membrane could sequestrate lipids and thus affect the efficiency of virus entry into host cells, preventing the initial steps of viral infection. We have prepared CHOL-binding proteins with high affinities for lipids in the plasma membrane of mammalian cells. Binding of the perfringolysin O domain four (D4) and its variant D4E458L to membrane CHOL impaired the internalization of the receptor-binding domain of the SARS-CoV-2 spike protein and the pseudovirus complemented with the SARS-CoV-2 spike protein. SARS-CoV-2 replication in Vero E6 cells was also decreased. Overall, our results demonstrate that the integrity of CHOL-rich membrane domains and the accessibility of CHOL in the membrane play an essential role in SARS-CoV-2 cell entry.

TXM peptides inhibit SARS-CoV-2 infection, syncytia formation, and lower inflammatory consequences.

After three years of the SARS-CoV-2 pandemic, the search and availability of relatively low-cost benchtop therapeutics for people not at high risk for a severe disease are still ongoing. Although vaccines and new SARS-CoV-2 variants reduce the death toll, the long COVID-19 along with neurologic symptoms can develop and persist even after a mild initial infection. Reinfections, which further increase the risk of sequelae in multiple organ systems as well as the risk of death, continue to require caution. The spike protein of SARS-CoV-2 is an important target for both vaccines and therapeutics. The presence of disulfide bonds in the receptor binding domain (RBD) of the spike protein is essential for its binding to the human ACE2 receptor and cell entry. Here, we demonstrate that thiol-reducing peptides based on the active site of oxidoreductase thioredoxin 1, called thioredoxin mimetic (TXM) peptides, can prevent syncytia formation, SARS-CoV-2 entry into cells, and infection in a mouse model. We also show that TXM peptides inhibit the redox-sensitive HIV pseudotyped viral cell entry. These results support disulfide targeting as a common therapeutic strategy for treating infections caused by viruses using redox-sensitive fusion. Furthermore, TXM peptides exert anti-inflammatory properties by lowering the activation of NF-κB and IRF signaling pathways, mitogen-activated protein kinases (MAPKs) and lipopolysaccharide (LPS)-induced cytokines in mice. The antioxidant and anti-inflammatory effects of the TXM peptides, which also cross the blood-brain barrier, in combination with prevention of viral infections, may provide a beneficial clinical strategy to lower viral infections and mitigate severe consequences of COVID-19.

Prevalence of red panda amdoparvovirus infection in European zoos.

Red panda amdoparvovirus (RPAV) was first described in captive red pandas (Ailurus fulgens) at a zoo in the United States in 2018. Subsequently, the prevalence of infection in zoos in the United States was reported to be 50%; however, RPAV prevalence outside the United States remains unstudied. This study was conducted to investigate the prevalence of RPAV in 134 red pandas from zoos in Europe. Overall, RPAV was detected with PCR in 21 of 62 zoos (33.9%), and the virus prevalence among individuals was estimated to be 24.2% (95% confidence interval, 17.4%-32.0%). Remarkably, adult females tested positive for RPAV more frequently than adult males. Zoos where RPAV was detected reported a significantly higher occurrence of alopecia (and clinical signs in general), whereas other commonly reported problems (fecal disorders and dental disease) showed no difference. A repeated pooled sampling of two positive individuals further showed that RPAV excretion in feces is intermittent, with the viral DNA being only detected on 8 out of 14 sampling days. The intermittent nature of excretion implies that RPAV prevalence may be higher than the estimated value.

Rehoming and Other Refinements and Replacement in Procedures Using Golden Hamsters in SARS-CoV-2 Vaccine Research.

Effective vaccines are needed to fight the COVID-19 pandemic. Forty golden hamsters were inoculated with two promising vaccine candidates and eighteen animals were used in pilot trials with viral challenge. ELISA assays were performed to determine endpoint serum titres for specific antibodies and virus neutralisation tests were used to evaluate the efficacy of antibodies. All tests with serum from vaccinated hamsters were negative even after booster vaccinations and changes in vaccination protocol. We concluded that antibodies did not have sufficient neutralising properties. Refinements were observed at all steps, and the in vitro method (virus neutralisation test) presented a replacement measure and ultimately lead to a reduction in the total number of animals used in the project. The institutional animal welfare officer and institutional designated veterinarian approved the reuse or rehoming of the surplus animals. Simple socialization procedures were performed and ultimately 19 animals were rehomed, and feedback was collected. Recently, FELASA published recommendations for rehoming of animals used for scientific and educational purposes, with species-specific guidelines, including mice, rats, and rabbits. Based on our positive experience and feedback from adopters, we concluded that the rehoming of rodents, including hamsters, is not only possible, but highly recommended.

Fatal cerebrovascular accident in a captive red panda (Ailurus fulgens fulgens) with concurrent amdoparvovirus infection.

We report the pathological and molecular findings in an adult male Himalayan red panda (Ailurus fulgens fulgens) whose death was attributed to parenchymal brain haemorrhage (PBH) of the thalamus. Post-mortem examination revealed severe, acute PBH and intraventricular haemorrhage with major involvement of the thalamus, as well as scattered chronic microinfarctions. Vascular disease in the brain and other organs was suggestive of systemic hypertension. Histological lesions included arteriolar hyalinosis and varying degrees of arteriosclerosis, arterial tunica media hypertrophy and hyperplasia and infiltration of arterial walls by lipid-laden macrophages. Other relevant findings included marked myocardial fibrosis, lymphoplasmacytic tubulointerstitial nephritis, lymphoplasmacytic meningoencephalitis and chronic mitral valve degeneration. The changes in the cerebral vasculature were consistent with hypertensive encephalopathy and a cerebrovascular accident, specifically PBH, which has not been previously reported in this species. Additionally, polymerase chain reaction analysis for red panda amdoparvovirus (RPAV) was positive in the brain and kidneys. Preceded by hypertensive vascular changes and brain microinfarctions, sudden death in this animal likely resulted from fatal PBH with intraventricular haemorrhage. The clinicopathological role of RPAV infection is unknown in this case, although its contribution to the chronic renal disease is considered possible in the context of our current understanding of RPAV-associated pathology.

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.

SARS-CoV-2 molecular epidemiology in Slovenia, January to September 2021.

BackgroundSequencing of SARS-CoV-2 PCR-positive samples was introduced in Slovenia in January 2021. Our surveillance programme comprised three complementary schemes: (A) non-targeted sequencing of at least 10% of samples, (B) sequencing of samples positive after PCR screening for variants of concern (VOC) and (C) sequencing as per epidemiological indication.AimWe present the analysis of cumulative data of the non-targeted surveillance of SARS-CoV-2 and variant-dependent growth kinetics for the five most common variants in Slovenia for the first 9 months of 2021.MethodsSARS-CoV-2 PCR-positive samples, from January to September 2021, were selected for sequencing according to the national surveillance plan. Growth kinetics studies were done on Vero E6 cells.ResultsAltogether 15,175 genomes were sequenced and 64 variants were detected, of which three successively prevailed. Variant B.1.258.17 was detected in ca 80% of samples in January and was replaced, within 9 weeks, by the Alpha variant. The number of cases decreased substantially during the summer of 2021. However, the introduction of the Delta variant caused a fourth wave and completely outcompeted other variants. Other VOC were only detected in small numbers. Infection of Vero E6 cells showed higher replication rates for the variants Alpha and Delta, compared with B.1.258.17, B.1.258, and B.1.1.70, which dominated in Slovenia before the introduction of the Alpha and Delta variants.ConclusionInformation on SARS-CoV-2 variant diversity provided context to the epidemiological data of PCR-positive cases, contributed to control of the initial spread of known VOC and influenced epidemiological measures.

Passive Disease Surveillance of Alpine Chamois (Rupicapra r. rupicapra) in Slovenia between 2000 and 2020.

In this paper, we provide an overview of the causes of death of Alpine chamois (Rupicapra r. rupicapra) diagnosed in the national passive health surveillance of chamois in Slovenia. From 2000 to 2020, 284 free-ranging chamois provided by hunters were necropsied at the Veterinary Faculty, University of Ljubljana, Slovenia. Depending on the results of complete necropsy, histopathological, bacteriological, parasitological, and virological examinations, a descriptive data analysis was performed. The most common causes of death in chamois were infectious diseases (82.2%), followed by non-infectious diseases (11.8%). Of all the causes of death, parasitic infections accounted for 70.3%, trauma for 9.7%, and bacterial infections for 9.3% of all cases. Less common diseases were viral infections, neoplasms, winter starvation, and metabolic disorders.

Serological Survey of Small Ruminant Lentivirus Infections in Free-Ranging Mouflon and Chamois in Slovenia.

Small ruminant lentiviruses (SRLVs) belong to the genus Lentivirus in the Retroviridae family, which are responsible for the diseases maedi-visna and caprine arthritis-encephalitis in sheep and goats worldwide and are also widespread in Slovenian sheep and goats. SRLVs cause lifelong infections with chronic inflammatory lesions in various organ systems. Cross-species transmission of SRLV strains in sheep and goats is well documented, but there are few data on the ability of these viruses to infect wild ruminants. The objective of this study was to investigate whether SRLVs circulate among wild small ruminants in Slovenia. During the 2017-2018 hunting season, a total of 38 blood samples were collected from free-ranging chamois (Rupicapra rupicapra) and European mouflon (Ovis ammon musimon). The serum samples were tested for antibodies against SRLV by enzyme-linked immunosorbent assay (ELISA). The serological tests revealed that of all tested mouflons, 1 animal (11.1%) was seropositive, while all samples from chamois were negative. Based on the results of this study and considering the results of previous studies in which SRLV infections were detected in mouflons with low seroprevalence, it is very likely that the detected seropositive animal was an incidental spillover host for SRLV. Although no seropositive samples were found in chamois, we cannot speculate on whether chamois may not be a host for SRLV infection because of the small sample size and the disadvantages of the ELISA assay used when applied to samples from chamois.

Detection of Herpesviruses in Wild Bird Casualties in Slovenia.

The complete host range of avian herpesviruses in wild birds is unknown, and information about nucleotide sequences is available only in limited cases. The aim of this study was to detect the presence of herpesviruses in wild birds and to gain more information about their phylogenetic relationship. Oropharyngeal and cloacal swabs from 447 wild birds from 15 different orders presented as wildlife casualties were examined for herpesvirus presence with PCR targeting a fragment of the DNA polymerase gene. Herpesviruses were detected in oropharyngeal and/or cloacal swabs in 34 (7.5%) birds belonging to 11 species from six different avian orders: Accipitriformes, Charadriiformes, Columbiformes, Falconiformes, Passeriformes, and Strigiformes. The results of phylogenetic analysis showed that various herpesviruses sequences are present in the wild bird population. Some herpesviruses are host species-specific, whereas in some cases very similar sequences were detected through different avian orders, which confirms findings that herpesviruses are not always restricted to bird species. It seems that herpesvirus transmission could occur by predation from avian prey, and even by superpredation-for example, large owls, such as the Eurasian eagle owl (Bubo bubo) or Ural owl (Strix uralensis), preying on smaller raptors. This can lead to greater infection exposure and is in line with the fact that raptors were the most infected species group. Nevertheless, the individual or simultaneous detection of herpesviruses in oropharyngeal and cloacal swabs shows that both swab samples should be used for herpesvirus detection in wild birds.

Herpesvirus Infection in a Breeding Population of Two Coexisting Strix Owls.

Birds are a frequent host of a large variety of herpesviruses, and infections in them may go unnoticed or may result in fatal disease. In wild breeding populations of owls, there is very limited information about the presence, impact, and potential transmission of herpesvirus. The herpesvirus partial DNA polymerase gene was detected using polymerase chain reaction in oropharyngeal swabs of 16 out of 170 owls examined that were captured in or near nest boxes. Herpesvirus was detected in Ural owls (Strix uralensis), in both adults and young, but not in tawny owls (Strix aluco). In yellow-necked mice (Apodemus flavicollis), as the main prey of tawny owls and Ural owls in the area, herpesvirus was detected in the organs of 2 out of 40 mice captured at the same locations as the owls. Phylogenetic analysis showed that the herpesvirus sequences detected in the Ural owls differed from the herpesvirus sequences detected in the yellow-necked mice. The results indicate that herpesvirus infection exists in the breeding wild Ural owl population. However, herpesvirus-infected owls did not show any clinical or productivity deviances and, based on a phylogenetic comparison of detected herpesvirus sequences and sequences obtained from Genbank database, it seems that mice and other rodents are not the source of owl infections. The most probable transmission pathway is intraspecific, especially from adults to their chicks, but the origin of herpesvirus in owls remains to be investigated.

Identification of a Novel Papillomavirus Type (MfoiPV1) Associated with Acrochordon in a Stone Marten (Martes foina).

Papillomaviruses (PVs) are an extremely large group of viruses that cause skin and mucosal infections in humans and various domestic and wild animals. Nevertheless, there is limited knowledge about PVs in wildlife hosts, including mustelid species. This study describes a case in stone marten (Martes foina) with a clinical manifestation of skin tumor, which is rather atypical for infections with PVs. The result of the papillomavirus PCR performed on the skin tumor sample was positive, and the complete PV genome was determined in the studied sample using next-generation sequencing technology. The analysis of the PV genome revealed infection of the stone marten with a putative new PV type belonging to the Dyonupapillomavirus genus. The proposed new stone marten PV type was named MfoiPV1.

Screening of Mosquitoes for West Nile Virus and Usutu Virus in Croatia, 2015-2020.

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 Medimurje 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.

Twenty Years of Passive Disease Surveillance of Roe Deer (Capreolus capreolus) in Slovenia.

In this paper, we provide an overview of the causes of death of roe deer (Capreolus capreolus) diagnosed within the national passive health surveillance of roe deer in Slovenia. From 2000 to 2019, postmortem examinations of 510 free-ranging roe deer provided by hunters were conducted at the Veterinary Faculty, Slovenia. A comprehensive necropsy was performed. According to the results of the necropsy, the samples were subjected to microscopic, histopathological, bacteriological, parasitological, or virological examination. The most frequent causes of death in roe deer were infectious diseases (67%), followed by noninfectious diseases (28%). Of all deaths, parasitic infections represented 48%, bacterial infections 14.8%, trauma 12.5%, and metabolic disorders 9.8%. Less frequent causes were diseases like neoplasia and mycotic infections, winter starvation, hernias, and lightning strike. This study covered an estimated 1% of the total disease-related mortality of roe deer in Slovenia. Comparisons of sex/age structure indicated that hunters did not provide random samples (e.g., young males were disproportionately represented). Therefore, such monitoring does not ensure an unbiased assessment of the significance of the individual disease for the mortality of the population; however, it can provide credible evidence of whether or not a particular disease is present in a population. We show that no identified disease in roe deer in Slovenia can be considered a significant health threat to roe deer, other wildlife species, or humans.

A Comprehensive Study of Cutaneous Fibropapillomatosis in Free-Ranging Roe Deer (Capreolus capreolus) and Red Deer (Cervus elaphus): from Clinical Manifestations to Whole-Genome Sequencing of Papillomaviruses.

Papillomaviruses (PVs) are an extremely large group of viruses that cause skin and mucosa infections in humans and various animals. In roe deer and red deer, most PVs belong to the Deltapapillomavirus genus and cause neoplastic changes that are generally described as fibropapillomas. Despite the wide distribution of roe and red deer throughout Europe and beyond, the data in the scientific literature regarding the widespread distribution of PVs and the genetic variability of PV genomes in these species are rather scarce. This study describes cutaneous fibropapillomatosis cases in roe and red deer with clinical manifestations that are typical of infections with PVs. In all cases, the presence of PV DNA was confirmed using PCR, followed by Sanger sequencing of the partial L1 gene. The complete PV genomes were determined in all the investigated samples using next-generation sequencing technology, revealing infections of roe deer with the CcaPV1-type and red deer with the CePV1v-type variant. A comparison of the complete CcaPV1-type and CePV1v-type variant genome sequences reported here with already available complete genome sequences in GenBank revealed their great genetic stability across time and space.

First Complete Genome of Lake Sinai Virus Lineage 3 and Genetic Diversity of Lake Sinai Virus Strains From Honey Bees and Bumble Bees.

The complete genome of Lake Sinai virus 3 (LSV3) was sequenced by the Ion Torrent next-generation sequencing (NGS) technology from an archive sample of honey bees collected in 2010. This strain M92/2010 is the first complete genome sequence of LSV lineage 3. From October 2016 to December 2017, 56 honey bee samples from 32 different locations and 41 bumble bee samples from five different locations were collected. These samples were tested using a specific reverse transcriptase-polymerase chain reaction (RT-PCR) method; 75.92% of honey bee samples and 17.07% of bumble bee samples were LSV-positive with the RT-PCR method. Phylogenetic comparison of 557-base pair-long RNA-dependent RNA polymerase (RdRp) genome region of selected 23 positive samples of honey bees and three positive bumble bee samples identified three different LSV lineages: LSV1, LSV2, and LSV3. The LSV3 lineage was confirmed for the first time in Slovenia in 2010, and the same strain was later detected in several locations within the country. The LSV strains detected in bumble bees are from 98.6 to 99.4% identical to LSV strains detected among honey bees in the same territory.

A molecular survey, whole genome sequencing and phylogenetic analysis of astroviruses from roe deer.

BACKGROUND: Although astroviruses (AstV) have been detected in a variety of host species, there are only limited records of their occurrence in deer. One of the most important game species in Europe, due to its meat and antlers, is roe deer. Infected game animals can pose a threat to the health of other animals and of humans, so more attention needs to be focused on understanding the diversity of viruses in wildlife. The complete genome and organization of the roe deer AstV genome have not so far been described. RESULTS: In our study, 111 game animals were screened for the presence of AstV. While no AstVs were detected in red deer, wild boar, chamois and mouflon, AstV RNA was present in three samples of roe deer. They were further subjected to whole genome sequencing with next generation sequencing. In this study, two AstV genomes were assembled; one in sample D5-14 and one in sample D12-14, while, in sample D45-14, no AstV sequences were identified. The complete coding sequences of the AstV SLO/D5-14 strain genome and of the almost complete genome of the AstV SLO/D12-14 strain were determined. They showed a typical Mamastrovirus organization. Phylogenetic analyses and amino acid pairwise distance analysis revealed that Slovenian roe deer AstV strains are closely related to each other and, also, related to other deer, bovine, water buffalo, yak, Sichuan takin, dromedary, porcine and porcupine AstV strains - thus forming a highly supported group of currently unassigned sequences. CONCLUSIONS: Our findings suggest the existence of a new Mamastrovirus genogroup might be constituted while this aforementioned group is distantly related to Mamastrovirus genogroups I and II. In this study, additional data supporting a novel taxonomic classification are presented.

Genome sequence of an aichivirus detected in a common pipistrelle bat (Pipistrellus pipistrellus).

The family Picornaviridae includes important human and animal pathogens that are associated with a wide range of diseases and, in some cases, have zoonotic potential. During epidemiological surveillance of bats, we identified, by next-generation sequencing (NGS) techniques, the presence of picornavirus RNA in a common pipistrelle bat (Pipistrellus pipistrellus). By coupling NGS, primer-walking strategies, and sequence-independent protocols to obtain the sequences of the 5' and 3' termini, we reconstructed the genome sequence of picornavirus strain ITA/2017/189/18-155. The genome of the bat picornavirus is 8.2 kb in length and encodes a polyprotein of 2462 amino acids. A comparison of polyprotein sequences revealed that this virus is distantly related (65.1% and 70.9% sequence identity at the nucleotide and amino acid level, respectively) to a bat aichivirus identified in 2010. Phylogenetic analysis showed that this picornavirus clustered closely with members of the genus Kobuvirus, which also includes human and animal aichiviruses. The identification of aichiviruses in several animal hosts is providing hints that will lead to an understanding of their origin and evolutionary patterns.

Post-epidemic investigation of Schmallenberg virus in wild ruminants in Slovenia.

Schmallenberg virus (SBV) is a vector-borne virus belonging to the genus Orthobunyavirus within the Bunyaviridae family. SBV emerged in Europe in 2011 and was characterized by epidemics of abortions, stillbirths and congenital malformations in domestic ruminants. The first evidence of SBV infection in Slovenia was from an ELISA-positive sample from a cow collected in August 2012; clinical manifestations of SBV disease in sheep and cattle were observed in 2013, with SBV RNA detected in samples collected from a total of 28 herds. A potential re-emergence of SBV in Europe is predicted to occur when population-level immunity declines. SBV is also capable of infecting several wild ruminant species, although clinical disease has not yet been described in these species. Data on SBV-positive wild ruminants suggest that these species might be possible sources for the re-emergence of SBV. The aim of this study was to investigate whether SBV was circulating among wild ruminants in Slovenia and whether these species can act as a virus reservoir. A total of 281 blood and spleen samples from wild ruminants, including roe deer, red deer, chamois and European mouflon, were collected during the 2017-2018 hunting season. Serum samples were tested for antibodies against SBV by ELISA; the overall seroprevalence was 18.1%. Seropositive samples were reported from all over the country in examined animal species from 1 to 15 years of age. Spleen samples from the seropositive animals and serum samples from the seronegative animals were tested for the presence of SBV RNA using real-time RT-PCR; all the samples tested negative. Based on the results of the seropositive animals, it was demonstrated that SBV was circulating in wild ruminant populations in Slovenia even after the epidemic, as almost half (23/51) of the seropositive animals were 1 or 2 years old.

Nearly Complete Genome Sequences of Two Bovine Viral Diarrhea Virus Isolates, Subtype 1f Strain SLO/1170/2000 and Subtype 1d Strain SLO/2416/2002.

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.