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.

Background: There is growing evidence that equine papillomavirus type 2 (EcPV2) infection is causally associated with the development of equine genital squamous cell carcinomas (SCCs). Early stages of disease present clinically as plaques or wart-like lesions which can gradually progress to tumoural lesions. Histologically these lesions are inconsistently described as benign hyperplasia, papilloma, penile intraepithelial neoplasia (PIN), carcinoma in situ (CIS) or SCC. Guidelines for histological classification of early SCC precursor lesions are not precisely defined, leading to potential misdiagnosis. The aim of this study was to identify histologic criteria and diagnostic markers allowing for a more accurate diagnosis of EcPV2-associated equine penile lesions. Results: A total of 61 archived equine penile lesions were histologically re-assessed and classified as benign hyperplasia, papilloma, CIS or SCC. From these, 19 representative lesions and adjacent normal skin were comparatively analysed for the presence of EcPV2 DNA and transcripts using PCR and RNA in situ hybridisation (RISH). All lesional samples were positive by EcPV2 PCR and RISH, while adjacent normal skin was negative. RISH analysis yielded signal distribution patterns that allowed distinction of early (hyperplasia, papilloma) from late stage lesions (CIS, SCC). Subsequently, the 19 lesions were further assessed for expression of p53, Ki67, MCM7 and MMP1 by immunohistochemistry (IHC). All four proteins were expressed in both normal and lesional tissue. However, p53 expression was up-regulated in basal keratinocyte layers of papillomas, CIS and SCCs, as well as in upper keratinocyte layers of CIS and SCCs. MCM7 expression was only up-regulated in upper proliferating keratinocyte layers of papillomas, CIS and SCCs. Conclusion: This study proposes combining a refined histological protocol for analysis of equine penile lesions with PCR- and/or RISH based EcPV2-screening and p53/MCM7 IHC to more accurately determine the type of lesion. This may help to guide the choice of optimum treatment strategy, especially at early stages of disease.

Papillomaviruses can cause proliferative skin lesions ranging from benign hyperplasia to squamous cell carcinoma (SCC). However, asymptomatic infection is also possible. Several groups have detected Felis domesticus Papillomavirus type 2 (FdPV2) DNA in association with feline Bowenoid in situ carcinoma (BISC). Therefore, a causative connection has been suggested. However, the knowledge about FdPV2 epidemiology is limited. The aim of this study was to describe the genoprevalence and seroprevalence of FdPV2 in healthy cats. For this purpose an FdPV2-specific quantitative (q)PCR assay was developed and used to analyse Cytobrush samples collected from 100 dermatologically healthy cats. Moreover, an ELISA was established to test the sera obtained from the same cats for antibodies against the major capsid protein (L1) of FdPV2. The genoprevalence of FdPV2 was to 98 %. Surprisingly, the quantities of viral DNA detected in some samples from the healthy cats exceeded the amounts detected in control samples from feline BISC lesions. The seroprevalence was much lower, amounting to 22 %. The concentrations of antibodies against FdPV2 were relatively low in healthy cats, whereas they were very high in control cats with BISC. These observations suggest that FdPV2 is highly prevalent, even among healthy cats. However, cats that carry it on their skin mount in most instances no antibody response. It might be hypothesized that FdPV2 is only rarely productively replicating or its replication is only rarely exposed to the immune system.

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.

Rotaviruses (RVs) are important enteric pathogens of newborn humans and animals, causing diarrhea and in rare cases death, especially in very young individuals. Rotavirus vaccines presently used are modified live vaccines that lack complete biological safety. Previous work from our laboratory suggested that vaccines based on in situ produced, non-infectious rotavirus-like particles (RVLPs) are efficient while being entirely safe. However, using either vaccine, active mucosal immunization cannot induce protective immunity in newborns due to their immature immune system. We therefore hypothesized that offspring from vaccinated dams are passively immunized either by transfer of maternal antibodies during pregnancy or by taking up antibodies from milk. Using a codon optimized polycistronic gene expression cassette packaged into herpesvirus particles, the simultaneous expression of the RV capsid genes led to the intracellular formation of RVLPs in various cell lines. Vaccinated dams developed a strong RV specific IgG antibody response determined in sera and milk of both mother and pups. Moreover, sera of naïve pups nursed by vaccinated dams also had RV specific antibodies suggesting a lactogenic transfer of antibodies. Although full protection of pups was not achieved in this mouse model, our observations are important for the development of improved vaccines against RV in humans as well as in various animal species.

Equine papillomavirus type 2 (EcPV2) was discovered only recently, but it is found consistently in the context of genital squamous cell carcinomas (SCCs). Since neither cell cultures nor animal models exist, the characterization of this potential disease agent relies on the analysis of patient materials. To analyse the host and viral transcriptome in EcPV2-affected horses, genital tissue samples were collected from horses with EcPV2-positive lesions as well as from healthy EcPV2-negative horses. It was determined by RNA-seq analysis that there were 1957 differentially expressed (DE) host genes between the SCC and control samples. These genes were most abundantly related to DNA replication, cell cycle, extracellular matrix (ECM)–receptor interaction and focal adhesion. By comparison to other cancer studies, MMP1 and IL8 appeared to be potential marker genes for the development of SCCs. Analysis of the viral reads revealed the transcriptional activity of EcPV2 in all SCC samples. While few reads mapped to the structural viral genes, the majority of reads mapped to the non-structural early (E) genes, in particular to E6, E7 and E2/E4. Within these reads a distinct pattern of splicing events, which are essential for the expression of different genes in PV infections, was observed. Additionally, in one sample the integration of EcPV2 DNA into the host genome was detected by DNA-seq and confirmed by PCR. In conclusion, while host MMP1 and IL8 expression and the presence of EcPV2 may be useful markers in genital SCCs, further research on EcPV2-related pathomechanisms may focus on cell cycle-related genes, the viral genes E6, E7 and E2/E4, and integration events.