The recruitment of TRiC chaperonin in rotavirus viroplasms correlates with virus replication.

Rotavirus (RV) replication takes place in the viroplasms, cytosolic inclusions that allow the synthesis of virus genome segments and their encapsidation in the core shell, followed by the addition of the second layer of the virion. The viroplasms are composed of several viral proteins, including NSP5, which serves as the main building block. Microtubules, lipid droplets, and miRNA-7 are among the host components recruited in viroplasms. We investigated the interaction between RV proteins and host components of the viroplasms by performing a pull-down assay of lysates from RV-infected cells expressing NSP5-BiolD2. Subsequent tandem mass spectrometry identified all eight subunits of the tailless complex polypeptide I ring complex (TRiC), a cellular chaperonin responsible for folding at least 10% of the cytosolic proteins. Our confirmed findings reveal that TRiC is brought into viroplasms and wraps around newly formed double-layered particles. Chemical inhibition of TRiC and silencing of its subunits drastically reduced virus progeny production. Through direct RNA sequencing, we show that TRiC is critical for RV replication by controlling dsRNA genome segment synthesis, particularly negative-sense single-stranded RNA. Importantly, cryo-electron microscopy analysis shows that TRiC inhibition results in defective virus particles lacking genome segments and polymerase complex (VP1/VP3). Moreover, TRiC associates with VP2 and NSP5 but not with VP1. Also, VP2 is shown to be essential for recruiting TRiC in viroplasms and preserving their globular morphology. This study highlights the essential role of TRiC in viroplasm formation and in facilitating virion assembly during the RV life cycle. IMPORTANCE: The replication of rotavirus takes place in cytosolic inclusions termed viroplasms. In these inclusions, the distinct 11 double-stranded RNA genome segments are co-packaged to complete a genome in newly generated virus particles. In this study, we show for the first time that the tailless complex polypeptide I ring complex (TRiC), a cellular chaperonin responsible for the folding of at least 10% of the cytosolic proteins, is a component of viroplasms and is required for the synthesis of the viral negative-sense single-stranded RNA. Specifically, TRiC associates with NSP5 and VP2, the cofactor involved in RNA replication. Our study adds a new component to the current model of rotavirus replication, where TRiC is recruited to viroplasms to assist replication.

Hazard potential of Swiss Ixodes ricinus ticks: Virome composition and presence of selected bacterial and protozoan pathogens.

Ticks play an important role in transmitting many different emerging zoonotic pathogens that pose a significant threat to human and animal health. In Switzerland and abroad, the number of tick-borne diseases, in particular tick-borne encephalitis (TBE), has been increasing over the last few years. Thus, it remains essential to investigate the pathogen spectrum of ticks to rapidly detect emerging pathogens and initiate the necessary measures. To assess the risk of tick-borne diseases in different regions of Switzerland, we collected a total of 10'286 ticks from rural and urban areas in ten cantons in 2021 and 2022. Ticks were pooled according to species, developmental stage, gender, and collection site, and analyzed using next generation sequencing (NGS) and quantitative polymerase chain reaction (qPCR). The metagenomic analysis revealed for the first time the presence of Alongshan virus (ALSV) in Swiss ticks. Interestingly, the pool-prevalence of ALSV was higher than that of tick-borne encephalitis virus (TBEV). Furthermore, several TBEV foci have been identified and pool prevalence of selected non-viral pathogens determined.

Cellular state landscape and herpes simplex virus type 1 infection progression are connected.

Prediction, prevention and treatment of virus infections require understanding of cell-to-cell variability that leads to heterogenous disease outcomes, but the source of this heterogeneity has yet to be clarified. To study the multimodal response of single human cells to herpes simplex virus type 1 (HSV-1) infection, we mapped high-dimensional viral and cellular state spaces throughout the infection using multiplexed imaging and quantitative single-cell measurements of viral and cellular mRNAs and proteins. Here we show that the high-dimensional cellular state scape can predict heterogenous infections, and cells move through the cellular state landscape according to infection progression. Spatial information reveals that infection changes the cellular state of both infected cells and of their neighbors. The multiplexed imaging of HSV-1-induced cellular modifications links infection progression to changes in signaling responses, transcriptional activity, and processing bodies. Our data show that multiplexed quantification of responses at the single-cell level, across thousands of cells helps predict infections and identify new targets for antivirals.

Genome Sequence of Alongshan Virus from Ixodes ricinus Ticks Collected in Switzerland.

Here, we report the detection of an Alongshan virus (ALSV) strain in Switzerland. Next-generation sequencing of homogenates from Ixodes ricinus ticks collected in Canton Grisons, Switzerland, in 2022 yielded a coding-complete ALSV genome.

Fully automated dried blood spot sample handling and extraction for BoHV-1 antibody testing by ELISA.

This study is the first proof of concept of the DBS technology for Bovine alphaherpesvirus 1 (BoHV-1) antibody detection by ELISA after fully automated DBS extraction. DBS were prepared from nine BoHV-1 seropositive plasma samples spiked with erythrocytes. Spots were extracted automatically on a DBS-MS 500 HCT autosampler, as well as manually using a 3.2 mm puncher. DBS were equally prepared from 20 bovine seronegative EDTA-blood samples and extracted automatically. Extracts were tested in a commercial BoHV-1 antibody ELISA and results were compared with those from liquid plasma. Eight seropositive DBS samples were additionally tested in the ELISA after storage for four weeks at different conditions. After automated extraction all DBS samples yielded qualitatively correct results and were in full accordance with those obtained from liquid plasma. Automated extraction using a 6 mm extraction head was more sensitive than a 4 mm head. Stability of DBS was highest at - 20 °C and decreased with increasing temperature. Even after four weeks at 37 °C, most seropositive samples yielded a positive result in the ELISA. The minimal invasiveness, biosafety, and simplicity of DBS collection together with automated extraction represents an interesting, high-throughput compatible alternative to liquid blood samples for BoHV-1 monitoring or eradication programs.

Virus Diversity, Abundance, and Evolution in Three Different Bat Colonies in Switzerland.

Bats are increasingly recognized as reservoirs for many different viruses that threaten public health, such as Hendravirus, Ebolavirus, Nipahvirus, and SARS- and MERS-coronavirus. To assess spillover risk, viromes of bats from different parts of the world have been investigated in the past. As opposed to most of these prior studies, which determined the bat virome at a single time point, the current work was performed to monitor changes over time. Specifically, fecal samples of three endemic Swiss bat colonies consisting of three different bat species were collected over three years and analyzed using next-generation sequencing. Furthermore, single nucleotide variants of selected DNA and RNA viruses were analyzed to investigate virus genome evolution. In total, sequences of 22 different virus families were found, of which 13 are known to infect vertebrates. Most interestingly, in a Vespertilio murinus colony, sequences from a MERS-related beta-coronavirus were consistently detected over three consecutive years, which allowed us to investigate viral genome evolution in a natural reservoir host.

Multiplex real-time PCR for the detection and differentiation of equid gammaherpesvirus 2 and 5.

Equid gammaherpesvirus 2 (EHV-2) and 5 (EHV-5) are widely distributed in the equines. Although their pathogenic potential is not yet fully understood, they appear to play a role in disease patterns like equine multinodular pulmonary fibrosis. In this study, a multiplex real-time PCR (rtPCR) was designed to detect DNA of the glycoprotein H (EHV-2) and E11 gene (EHV-5). Analytical specificity was determined by testing DNA of other herpesviruses by SYBR Green rtPCR and melting curve analysis, as well as Sanger sequencing of positive field samples. Analytical sensitivity was assessed by standard curve generation of serial plasmid dilutions containing the respective target gene. Melting curves and BLAST analysis of the sequences indicated specific detection of the viruses. The lower limit of detection of the singleplex rtPCR was 40 and 29 DNA copies per reaction for EHV-2 and EHV-5, respectively. Comparison of the Ct values of a selection of positive field samples showed only minimal differences between the singleplex and the multiplex assay. The here described multiplex rtPCR protocol allows sensitive and specific detection of EHV-2 and EHV-5. It represents a convenient and rapid tool for future studies to investigate the clinical relevance of EHV-2 and EHV-5 in more detail.

Rotavirus Spike Protein VP4 Mediates Viroplasm Assembly by Association to Actin Filaments.

Rotavirus (RV) viroplasms are cytosolic inclusions where both virus genome replication and primary steps of virus progeny assembly take place. A stabilized microtubule cytoskeleton and lipid droplets are required for the viroplasm formation, which involves several virus proteins. The viral spike protein VP4 has not previously been shown to have a direct role in viroplasm formation. However, it is involved with virus-cell attachment, endocytic internalization, and virion morphogenesis. Moreover, VP4 interacts with actin cytoskeleton components, mainly in processes involving virus entrance and egress, and thereby may have an indirect role in viroplasm formation. In this study, we used reverse genetics to construct a recombinant RV, rRV/VP4-BAP, that contains a biotin acceptor peptide (BAP) in the K145-G150 loop of the VP4 lectin domain, permitting live monitoring. The recombinant virus was replication competent but showed a reduced fitness. We demonstrate that rRV/VP4-BAP infection, as opposed to rRV/wt infection, did not lead to a reorganized actin cytoskeleton as viroplasms formed were insensitive to drugs that depolymerize actin and inhibit myosin. Moreover, wild-type (wt) VP4, but not VP4-BAP, appeared to associate with actin filaments. Similarly, VP4 in coexpression with NSP5 and NSP2 induced a significant increase in the number of viroplasm-like structures. Interestingly, a small peptide mimicking loop K145-G150 rescued the phenotype of rRV/VP4-BAP by increasing its ability to form viroplasms and hence improve virus progeny formation. Collectively, these results provide a direct link between VP4 and the actin cytoskeleton to catalyze viroplasm assembly. IMPORTANCE The spike protein VP4 participates in diverse steps of the rotavirus (RV) life cycle, including virus-cell attachment, internalization, modulation of endocytosis, virion morphogenesis, and virus egress. Using reverse genetics, we constructed for the first time a recombinant RV, rRV/VP4-BAP, harboring a heterologous peptide in the lectin domain (loop K145-G150) of VP4. The rRV/VP4-BAP was replication competent but with reduced fitness due to a defect in the ability to reorganize the actin cytoskeleton, which affected the efficiency of viroplasm assembly. This defect was rescued by adding a permeable small-peptide mimicking the wild-type VP4 loop K145-G150. In addition to revealing a new role of VP4, our findings suggest that rRV harboring an engineered VP4 could be used as a new dual vaccination platform providing immunity against RV and additional heterologous antigens.

Adeno-associated virus type 2 (AAV2) uncoating is a stepwise process and is linked to structural reorganization of the nucleolus.

Nucleoli are membrane-less structures located within the nucleus and are known to be involved in many cellular functions, including stress response and cell cycle regulation. Besides, many viruses can employ the nucleolus or nucleolar proteins to promote different steps of their life cycle such as replication, transcription and assembly. While adeno-associated virus type 2 (AAV2) capsids have previously been reported to enter the host cell nucleus and accumulate in the nucleolus, both the role of the nucleolus in AAV2 infection, and the viral uncoating mechanism remain elusive. In all prior studies on AAV uncoating, viral capsids and viral genomes were not directly correlated on the single cell level, at least not in absence of a helper virus. To elucidate the properties of the nucleolus during AAV2 infection and to assess viral uncoating on a single cell level, we combined immunofluorescence analysis for detection of intact AAV2 capsids and capsid proteins with fluorescence in situ hybridization for detection of AAV2 genomes. The results of our experiments provide evidence that uncoating of AAV2 particles occurs in a stepwise process that is completed in the nucleolus and supported by alteration of the nucleolar structure.

Differential Viral Genome Diversity of Healthy and RSS-Affected Broiler Flocks.

The intestinal virus community contributes to health and disease. Runting and stunting syndrome (RSS) is associated with enteric viruses and leads to economic losses in the poultry industry. However, many viruses that potentially cause this syndrome have also been identified in healthy animals. To determine the difference in the virome of healthy and diseased broilers, samples from 11 healthy and 17 affected broiler flocks were collected at two time points and analyzed by Next-Generation Sequencing. Virus genomes of Parvoviridae, Astroviridae, Picornaviridae, Caliciviridae, Reoviridae, Adenoviridae, Coronaviridae, and Smacoviridae were identified at various days of poultry production. De novo sequence analysis revealed 288 full or partial avian virus genomes, of which 97 belonged to the novel genus Chaphamaparvovirus. This study expands the knowledge of the diversity of enteric viruses in healthy and RSS-affected broiler flocks and questions the association of some viruses with the diseases.

Display of Heterologous Proteins in Bacillus Subtilis Biofilms for Enteric Immunization.

One of the foremost goals in vaccine development is the design of effective, heat-stable vaccines that simplify the distribution and delivery while conferring high levels of protective immunity. Here, we describe a method for developing a live, oral vaccine that relies on the biofilm-forming properties of the spore-former bacterium Bacillus subtilis. The amyloid protein TasA is an abundant component of the extracellular matrix of the biofilms formed by B. subtilis that can be genetically fused to an antigen of interest. Spores of the recombinant strain are then prepared and applied via the oral route in an animal model. Due to the intrinsic resistance of the spores, they can bypass the stomach barrier, germinate, and subsequently colonize the gut, where they develop into biofilms, expressing the antigen of interest. We describe here the steps necessary to produce spores, immunization, and downstream analysis of the vaccine efficacy.

Genetic Diversity of Hepatitis E Virus Type 3 in Switzerland-From Stable to Table.

Hepatitis E caused by hepatitis E viruses of the genotype 3 (HEV-3) is a major health concern in industrialized countries and due to its zoonotic character requires a "One Health" approach to unravel routes and sources of transmission. Knowing the viral diversity present in reservoir hosts, i.e., pigs but also wild boars, is an important prerequisite for molecular epidemiology. The aim of this study was to gain primary information on the diversity of HEV-3 subtypes present along the food chain in Switzerland, as well as the diversity within these subtypes. To this end, samples of domestic pigs from slaughterhouses and carcass collection points, as well as from hunted wild boars, were tested for HEV RNA and antibodies. HEV positive meat products were provided by food testing labs. The HEV subtypes were determined using Sanger and next generation sequencing. The genetic analyses confirmed the predominance of a Swiss-specific cluster within subtype HEV-3h in pigs, meat products, and wild boars. This cluster, which may result from local virus evolution due to the isolated Swiss pig industry, supports fast differentiation of domestic and imported infections with HEV.

Genome Sequence of Equid Alphaherpesvirus 1 (EHV-1) from a Nasal Swab of a Swiss Horse Associated with a Major EHV-1 Outbreak following a Show Jumping Event in Valencia, Spain.

We present the genome sequence of equid alphaherpesvirus 1 (EHV-1) sequenced directly from the nasal swab of a Swiss horse that attended an international equestrian event in Valencia, Spain, the origin of an outbreak of neurological disorders in horses in several European countries in February 2021.

In Vitro Comparison of the Internal Ribosomal Entry Site Activity from Rodent Hepacivirus and Pegivirus and Construction of Pseudoparticles.

The 5' untranslated region (5' UTR) of rodent hepacivirus (RHV) and pegivirus (RPgV) contains sequence homology to the HCV type III internal ribosome entry sites (IRES). Utilizing a monocistronic expression vector with an RNA polymerase I promoter to drive transcription, we show cell-specific IRES translation and regions within the IRES required for full functionality. Focusing on RHV, we further pseudotyped lentivirus with RHV and showed cell surface expression of the envelope proteins and transduction of murine hepatocytes and we then constructed full-length RHV and RPgV replicons with reporter genes. Using the replicon system, we show that the RHV NS3-4A protease cleaves a mitochondrial antiviral signaling protein reporter. However, liver-derived cells did not readily support the complete viral life cycle.

Establishment of a Three-Dimensional In Vitro Model of Equine Papillomavirus Type 2 Infection.

There is growing evidence that equine papillomavirus type 2 (EcPV2) infection is etiologically associated with the development of genital squamous cell carcinoma (SCC) and precursor lesions in equids. However, the precise mechanisms underlying neoplastic progression remain unknown. To allow the study of EcPV2-induced carcinogenesis, we aimed to establish a primary equine cell culture model of EcPV2 infection. Three-dimensional (3D) raft cultures were generated from equine penile perilesional skin, plaques and SCCs. Using histological, molecular biological and immunohistochemical methods, rafts versus corresponding natural tissue sections were compared with regard to morphology, presence of EcPV2 DNA, presence and location of EcPV2 gene transcripts and expression of epithelial, mesenchymal and tumor/proliferation markers. Raft cultures from perilesional skin harboring only a few EcPV2-positive (EcPV2+) cells accurately recapitulated the differentiation process of normal skin, whilst rafts from EcPV2+ penile plaques were structurally organized but showed early hyperplasia. Rafts from EcPV2+ SCCs exhibited pronounced hyperplasia and marked dysplasia. Raft levels of EcPV2 oncogene transcription (E6/E7) and expression of tumor/proliferation markers p53, Ki67 and MCM7 expression positively correlated with neoplastic progression, again reflecting the natural situation. Three-dimensional raft cultures accurately reflected major features of corresponding ex vivo material, thus constituting a valuable new research model to study EcPV2-induced carcinogenesis.

Porcine teschovirus, sapelovirus, and enterovirus in Swiss pigs: multiplex RT-PCR investigation of viral frequencies and disease association.

Porcine teschovirus (PTV), sapelovirus (PSV-A), and enterovirus (EV-G) are enteric viruses that can infect pigs and wild boars worldwide. The viruses have been associated with several diseases, primarily gastrointestinal, neurologic, reproductive, and respiratory disorders, but also with subclinical infections. However, for most serotypes, proof of a causal relationship between viral infection and clinical signs is still lacking. In Switzerland, there has been limited investigation of the occurrence of the 3 viruses. We used a modified multiplex reverse-transcription PCR protocol to study the distribution of the viruses in Swiss pigs by testing 363 fecal, brain, and placental or abortion samples from 282 healthy and diseased animals. We did not detect the 3 viruses in 94 placental or abortion samples or in 31 brain samples from healthy pigs. In brain tissue of 81 diseased pigs, we detected 5 PSV-A and 4 EV-G positive samples. In contrast, all 3 viruses were detected at high frequencies in fecal samples of both healthy and diseased pigs. In healthy animals, PTV was detected in 47%, PSV-A in 51%, and EV-G in 70% of the 76 samples; in diseased animals, frequencies in the 81 samples were 54%, 64%, and 68%, respectively. The viruses were detected more frequently in fecal samples from weaned and fattening pigs compared to suckling piglets and sows. Co-detections of all 3 viruses were the most common finding. Based on clinical and pathology data, statistical analysis yielded no evidence for an association of virus detection and disease. Further research is required to determine if pathogenicity is linked to specific serotypes of these viruses.

Herpes simplex virus co-infection facilitates rolling circle replication of the adeno-associated virus genome.

Adeno-associated virus (AAV) genome replication only occurs in the presence of a co-infecting helper virus such as adenovirus type 5 (AdV5) or herpes simplex virus type 1 (HSV-1). AdV5-supported replication of the AAV genome has been described to occur in a strand-displacement rolling hairpin replication (RHR) mechanism initiated at the AAV 3' inverted terminal repeat (ITR) end. It has been assumed that the same mechanism applies to HSV-1-supported AAV genome replication. Using Southern analysis and nanopore sequencing as a novel, high-throughput approach to study viral genome replication we demonstrate the formation of double-stranded head-to-tail concatemers of AAV genomes in the presence of HSV-1, thus providing evidence for an unequivocal rolling circle replication (RCR) mechanism. This stands in contrast to the textbook model of AAV genome replication when HSV-1 is the helper virus.

Metagenomic analysis of fecal and tissue samples from 18 endemic bat species in Switzerland revealed a diverse virus composition including potentially zoonotic viruses.

Many recent disease outbreaks in humans had a zoonotic virus etiology. Bats in particular have been recognized as reservoirs to a large variety of viruses with the potential to cross-species transmission. In order to assess the risk of bats in Switzerland for such transmissions, we determined the virome of tissue and fecal samples of 14 native and 4 migrating bat species. In total, sequences belonging to 39 different virus families, 16 of which are known to infect vertebrates, were detected. Contigs of coronaviruses, adenoviruses, hepeviruses, rotaviruses A and H, and parvoviruses with potential zoonotic risk were characterized in more detail. Most interestingly, in a ground stool sample of a Vespertilio murinus colony an almost complete genome of a Middle East respiratory syndrome-related coronavirus (MERS-CoV) was detected by Next generation sequencing and confirmed by PCR. In conclusion, bats in Switzerland naturally harbour many different viruses. Metagenomic analyses of non-invasive samples like ground stool may support effective surveillance and early detection of viral zoonoses.

Herpes Simplex Virus 1 Coinfection Modifies Adeno-associated Virus Genome End Recombination.

Wild-type adeno-associated virus (AAV) can only replicate in the presence of helper factors, which can be provided by coinfecting helper viruses such as adenoviruses and herpesviruses. The AAV genome consists of a linear, single-stranded DNA (ssDNA), which is converted into different molecular structures within the host cell. Using high-throughput sequencing, we found that herpes simplex virus 1 (HSV-1) coinfection leads to a shift in the type of AAV genome end recombination. In particular, open-end inverted terminal repeat (ITR) recombination was enhanced, whereas open-closed ITR recombination was reduced in the presence of HSV-1. We demonstrate that the HSV-1 protein ICP8 plays an essential role in HSV-1-mediated interference with AAV genome end recombination, indicating that the previously described ICP8-driven mechanism of HSV-1 genome recombination may be underlying the observed changes. We also provide evidence that additional factors, such as products of true late genes, are involved. Although HSV-1 coinfection significantly changed the type of AAV genome end recombination, no significant change in the amount of circular AAV genomes was identified. IMPORTANCE Adeno-associated virus (AAV)-mediated gene therapy represents one of the most promising approaches for the treatment of genetic diseases. Currently, various GMP-compatible production methods can be applied to manufacture clinical-grade vector, including methods that employ helper factors derived from herpes simplex virus 1 (HSV-1). Yet, to date, we do not fully understand how HSV-1 interacts with AAV. We observed that HSV-1 modulates AAV genome ends similarly to the genome recombination events observed during HSV-1 replication and postulate that further improvements of the HSV-1 production platform may enhance packaging of the recombinant AAV particles.

The HSV-1 Transcription Factor ICP4 Confers Liquid-Like Properties to Viral Replication Compartments.

Herpes Simplex Virus Type-1 (HSV-1) forms progeny in the nucleus within distinct membrane-less inclusions, the viral replication compartments (VRCs), where viral gene expression, DNA replication, and packaging occur. The way in which the VRCs maintain spatial integrity remains unresolved. Here, we demonstrate that the essential viral transcription factor ICP4 is an intrinsically disordered protein (IDP) capable of driving protein condensation and liquid-liquid phase separation (LLPS) in transfected cells. Particularly, ICP4 forms nuclear liquid-like condensates in a dose- and time-dependent manner. Fluorescence recovery after photobleaching (FRAP) assays revealed rapid exchange rates of EYFP-ICP4 between phase-separated condensates and the surroundings, akin to other viral IDPs that drive LLPS. Likewise, HSV-1 VRCs revealed by EYFP-tagged ICP4 retained their liquid-like nature, suggesting that they are phase-separated condensates. Individual VRCs homotypically fused when reaching close proximity and grew over the course of infection. Together, the results of this study demonstrate that the HSV-1 transcription factor ICP4 has characteristics of a viral IDP, forms condensates in the cell nucleus by LLPS, and can be used as a proxy for HSV-1 VRCs with characteristics of liquid-liquid phase-separated condensates.