Probes for INS

HSV1 encodes an endoribonuclease termed virion host shutoff (vhs) that is produced late in infection and packaged into virions. Paradoxically, vhs is active against not only host but also virus transcripts, and is involved in host shutoff and the temporal expression of the virus transcriptome. Two other virus proteins-VP22 and VP16 -are proposed to regulate vhs to prevent uncontrolled and lethal mRNA degradation but their mechanism of action is unknown. We have performed dual transcriptomic analysis and single-cell mRNA FISH of human fibroblasts, a cell type where in the absence of VP22, HSV1 infection results in extreme translational shutoff. In Wt infection, host mRNAs exhibited a wide range of susceptibility to vhs ranging from resistance to 1000-fold reduction, a variation that was independent of their relative abundance or transcription rate. However, vhs endoribonuclease activity was not found to be overactive against any of the cell transcriptome in Δ22-infected cells but rather was delayed, while its activity against the virus transcriptome and in particular late mRNA was minimally enhanced. Intriguingly, immediate-early and early transcripts exhibited vhs-dependent nuclear retention later in Wt infection but late transcripts were cytoplasmic. However, in the absence of VP22, not only early but also late transcripts were retained in the nucleus by a vhs-dependent mechanism, a characteristic that extended to cellular transcripts that were not efficiently degraded by vhs. Moreover, the ability of VP22 to bind VP16 enhanced but was not fundamental to the rescue of vhs-induced nuclear retention of late transcripts. Hence, translational shutoff in HSV1 infection is primarily a result of vhs-induced nuclear retention and not degradation of infected cell mRNA. We have therefore revealed a new mechanism whereby vhs and its co-factors including VP22 elicit a temporal and spatial regulation of the infected cell transcriptome, thus co-ordinating efficient late protein production.

We present the first documented case of Trypanosoma cruzi-induced orchitis in a rhesus macaque. Additionally, we describe an in situ hybridization-based assay to confirm T. cruzi infection in formalin-fixed tissues.

Equine arteritis virus (EAV) can establish long-term persistent infection in the reproductive tract of stallions and is shed in the semen. Previous studies showed that long-term persistence is associated with a specific allele of the CXCL16 gene (CXCL16S) and that persistent infection is maintained despite the presence of a local inflammatory and humoral and mucosal antibody responses. Here, we demonstrated that equine seminal exosomes (SEs) are enriched in a small subset of miRNAs. Most importantly, we demonstrated that long-term EAV persistence is associated with the downregulation of an SE-associated miRNA (eca-mir-128) and with an enhanced expression of CXCL16 in the reproductive tract, a putative target of eca-mir-128. The findings presented herein suggest that SE eca-mir-128 is implicated in the regulation of the CXCL16/CXCR6 axis in the reproductive tract of persistently infected stallions, a chemokine axis strongly implicated in EAV persistence. This is a novel finding and warrants further investigation to identify its specific mechanism in modulating the CXCL16/CXCR6 axis in the reproductive tract of the EAV long-term carrier stallion.IMPORTANCEEquine arteritis virus (EAV) has the ability to establish long-term persistent infection in the stallion reproductive tract and to be shed in semen, which jeopardizes its worldwide control. Currently, the molecular mechanisms of viral persistence are being unraveled, and these are essential for the development of effective therapeutics to eliminate persistent infection. Recently, it has been determined that long-term persistence is associated with a specific allele of the CXCL16 gene (CXCL16S) and is maintained despite induction of local inflammatory, humoral and mucosal antibody responses. This study demonstrated that long-term persistence is associated with the downregulation of seminal exosome miRNA eca-mir-128 and enhanced expression of its putative target, CXCL16, in the reproductive tract. For the first time, this study suggests complex interactions between eca-mir-128 and cellular elements at the site of EAV persistence and implicates this miRNA in the regulation of the CXCL16/CXCR6 axis in the reproductive tract during long-term persistence.

Outcomes of Zika virus (ZIKV) infection in pregnant women vary from the birth of asymptomatic offspring to abnormal development and severe brain lesions in fetuses and infants. There are concerns that offspring affected in utero and born without apparent symptoms may develop mental illnesses. Therefore, animal models are important to test interventions against in utero infection and health sequelae in symptomatic and likely more widespread asymptomatic offspring. To partially reproduce in utero infection in humans, we directly inoculated selected porcine conceptuses with ZIKV. Inoculation resulted in rapid trans-fetal infections, persistent infection in conceptuses, molecular pathology in fetal brains, fetal antibody and type I interferon responses. Offspring infected in utero showed ZIKV in their fetal membranes collected after birth. Some in utero affected piglets were small, depressed, had undersized brains, showed seizures and potentially increased activity. Our data suggest that porcine model of persistent in utero ZIKV infection has a strong potential for translational research and can be used to test therapeutic interventions in vivo.

Lymphoma is one of the most common malignancies in domestic cats. The lymphomagenic potential of Felis catus gammaherpesvirus 1 (FcaGHV1), a common infection in domestic cats, is unknown. In other species, including humans, cellular transformation by gammaherpesviruses is typically mediated by viral genes expressed during latency. We analysed tumour RNA, from diffuse large B-cell lymphomas (DLBCL) appearing in cats coinfected with FcaGHV1 and feline immunodeficiency virus (FIV) (n = 10). Analysis was done by high throughput transcriptome sequencing and reverse transcription PCR. A limited repertoire of FcaGHV transcripts was identified in five tumors, including homologs of oncogenic latency-associated transcripts, latency-associated nuclear antigen (LANA, ORF73) and vFLIP (F7), lytic genes (ORF50, ORF6, ORF59, F10), and an ORF unique to FcaGHV1, F20. In situ hybridization of FIV-associated DLBCLs (n = 9), post-transplant lymphomas (n = 6) and high-grade B and T-cell intestinal lymphomas (n = 8) identified a single case in which FcaGHV1 nucleic acid was detectable. These results demonstrate that FcaGHV1 transcripts can be detected in some FIV-associated lymphomas, but with a low copy number, precluding assessment of a potential role for FcaGHV1 in lymphomagenesis. Future investigation of the FcaGHV1 transcriptome in clinical samples might employ viral enrichment and greater sequencing depth to enhance the retrieval of viral reads. Our results suggest prioritization of a subset of intestinal T-cell tumors and large granular lymphocyte lymphoma, for study.

Complex Citrus tristeza virus (CTV) populations composed of mixtures of different strains of the virus are commonly found in citrus trees in the field. At present, little is known about how these populations are formed, maintained, and how they are structured within a host. Here we used a novel in situ hybridization approach allowing simultaneous visualization of two different RNA targets with high sensitivity and specificity to examine the distribution of two isolates, T36 and T68-1, representing phylogenetically distinct strains of CTV, in a citrus host in single and mixed infections. Remarkably, in doubly inoculated plants the two virus variants appeared to be well mixed within the infected tissue and showed no spatial segregation. In addition, both CTV variants were often found occupying the same cells. Possible mechanisms involved in shaping CTV populations and the biological significance of the observed lack of structural separation of the individual components are discussed.

Abstract

Objectives

In the present study, we comprehensively analyzed the prevalence of transcriptionally active HPV in tissue samples of Indian patients with leukoplakia - predominantly hyperplastic lesions and HNSCC. In addition, saliva samples from patients with HNSCC were screened for HPV detection.

Study Design

p16 overexpression was analyzed by immunohistochemistry. Leukoplakia (n = 121) and HNSCC (n = 427) tissue samples and the saliva of patients with HNSCC (n = 215) were tested for HPV using nested PCR. Positive samples were sequenced for subtyping. The presence of HPV E6/E7 mRNA was confirmed by RNA in-situ hybridization.

Results

p16 expression and HPV DNA were not detected in any of the leukoplakia specimens. Of the 427 HNSCC tumors, 9 showed p16 overexpression and 7/427 cases were positive for HPV16 DNA, either in saliva and/or tissue. E6/E7 mRNA positivity was observed in eight HNSCC samples, primarily from patients with no habit of tobacco consumption. The prevalence of high-risk HPV was restricted to oropharynx and larynx with very little concordance between p16 overexpression and HPV positivity. All patients with HPV positive saliva samples had transcriptionally active HPV present in their tumors.

Conclusion

Presence of HPV-DNA does not necessarily reflect transcriptionally active virus in tumors; hence, it is important to consider this fact while categorizing HPV associated tumors.

We genetically characterized seven nearly complete genomes in the protoparvovirus genus from the feces of children with diarrhea. The viruses, provisionally named cutaviruses (CutaV), varied by 1-6% nucleotides and shared ~76% and ~82% amino acid identity with the NS1 and VP1 of human bufaviruses, their closest relatives. Using PCR, cutavirus DNA was found in 1.6% (4/245) and 1% (1/100) of diarrhea samples from Brazil and Botswana respectively. In silico analysis of pre-existing metagenomics datasets then revealed closely related parvovirus genomes in skin biopsies from patients with epidermotropic cutaneous T-cell lymphoma (CTCL or mycosis fungoides). PCR of skin biopsies yielded cutavirus DNA in 4/17 CTCL, 0/10 skin carcinoma, and 0/21 normal or noncancerous skin biopsies. In situ hybridization of CTCL skin biopsies detected viral genome within rare individual cells in regions of neoplastic infiltrations. The influence of cutavirus infection on human enteric functions and possible oncolytic role in CTCL progression remain to be determined.

Undifferentiated nasopharyngeal carcinoma (NPC) is a cancer with high metastatic potential that is consistently associated with Epstein-Barr virus (EBV) infection. In this study, we have investigated the functional contribution of sphingosine-1-phosphate (S1P) signalling to the pathogenesis of NPC. We show that EBV infection or ectopic expression of the EBV-encoded latent genes (EBNA1, LMP1 and LMP2A) can up-regulate sphingosine kinase 1 (SPHK1), the key enzyme that produces S1P, in NPC cell lines. Exogenous addition of S1P promotes the migration of NPC cells through the activation of AKT; shRNA knockdown of SPHK1 resulted in a reduction in the levels of activated AKT and inhibition of cell migration. We also show that S1P receptor 3 (S1PR3) mRNA is over-expressed in EBV-positive NPC patient-derived xenografts and a subset of primary NPC tissues, and that knockdown of S1PR3 suppressed the activation of AKT and the S1P-induced migration of NPC cells. Taken together, our data point to a central role for EBV in mediating the oncogenic effects of S1P in NPC and identify S1P signalling as a potential therapeutic target in this disease.