Probes for LONG

Human endogenous retroviruses (HERVs) play pivotal roles in the development of breast cancer. However, the detailed mechanisms of noncoding HERVs remain elusive. Here, our genome-wide transcriptome analysis of HERVs revealed that a primate long noncoding RNA, which we dubbed TROJAN, was highly expressed in human triple-negative breast cancer (TNBC). TROJAN promoted TNBC proliferation and invasion and indicated poor patient outcomes. We further confirmed that TROJAN could bind to ZMYND8, a metastasis-repressing factor, and increase its degradation through the ubiquitin-proteasome pathway by repelling ZNF592. TROJAN also epigenetically up-regulated metastasis-related genes in multiple cell lines. Correlations between TROJAN and ZMYND8 were subsequently confirmed in clinical samples. Furthermore, our study verified that antisense oligonucleotide therapy targeting TROJAN substantially suppressed TNBC progression in vivo. In conclusion, the long noncoding RNA TROJAN promotes TNBC progression and serves as a potential therapeutic target.

Our appreciation for the extent of Epstein Barr virus (EBV) transcriptome complexity continues to grow through findings of EBV encoded microRNAs, new long non-coding RNAs as well as the more recent discovery of over a hundred new polyadenylated lytic transcripts. Here we report an additional layer to the EBV transcriptome through the identification of a repertoire of latent and lytic viral circular RNAs. Utilizing RNase R-sequencing with cell models representing latency types I, II, and III, we identified EBV encoded circular RNAs expressed from the latency Cp promoter involving backsplicing from the W1 and W2 exons to the C1 exon, from the EBNA BamHI U fragment exon, and from the latency long non-coding RPMS1 locus. In addition, we identified circular RNAs expressed during reactivation including backsplicing from exon 8 to exon 2 of the LMP2 gene and a highly expressed circular RNA derived from intra-exonic backsplicing within the BHLF1 gene. While expression of most of these circular RNAs was found to depend on the EBV transcriptional program utilized and the transcription levels of the associated loci, expression of LMP2 exon 8 to exon 2 circular RNA was found to be cell model specific. Altogether we identified over 30 unique EBV circRNAs candidates and we validated and determined the structural features, expression profiles and nuclear/cytoplasmic distributions of several predominant and notable viral circRNAs. Further, we show that two of the EBV circular RNAs derived from the RPMS1 locus are detected in EBV positive clinical stomach cancer specimens. This study increases the known EBV latency and lytic transcriptome repertoires to include viral circular RNAs and it provides an essential foundation and resource for investigations into the functions and roles of this new class of EBV transcripts in EBV biology and diseases.

The productive infection of influenza A virus (IAV) depends on host factors. However, the involvement of long non-coding RNAs (lncRNAs) in IAV infection remains largely uninvestigated. In this work, we have discovered a human lncRNA, named lncRNA-PAAN (PA-associated noncoding RNA) that enhances IAV replication. The level of lncRNA-PAAN increases upon infection of IAV, but not other viruses, nor interferon treatment, suggesting specific up-regulation of lncRNA-PAAN expression by IAV. Silencing lncRNA-PAAN significantly decreases IAV replication through impairing the activity of viral RNA-dependent RNA polymerase (RdRp). This function of lncRNA-PAAN is a result of its association with viral PA protein, a key component of IAV RNA polymerase complex. Consequently, depletion of lncRNA-PAAN prevents the formation of functional RdRp. Together, these results suggest that lncRNA-PAAN promotes the assembly of viral RNA polymerase, thus warranting efficient viral RNA synthesis. Elucidating the functions of lncRNAs in IAV infection is expected to advance our understanding of IAV pathogenesis and open new avenues to the development of novel anti-IAV therapeutics.

Macrophages have long been considered to contribute to HIV infection of the CNS; however, a recent study has contradicted this early work and suggests that myeloid cells are not an in vivo source of virus production. Here, we addressed the role of macrophages in HIV infection by first analyzing monocytes isolated from viremic patients and patients undergoing antiretroviral treatment. We were unable to find viral DNA or viral outgrowth in monocytes isolated from peripheral blood. To determine whether tissue macrophages are productively infected, we used 3 different but complementary humanized mouse models. Two of these models (bone marrow/liver/thymus [BLT] mice and T cell-only mice [ToM]) have been previously described, and the third model was generated by reconstituting immunodeficient mice with human CD34+ hematopoietic stem cells that were devoid of human T cells (myeloid-only mice [MoM]) to specifically evaluate HIV replication in this population. Using MoM, we demonstrated that macrophages can sustain HIV replication in the absence of T cells; HIV-infected macrophages are distributed in various tissues including the brain; replication-competent virus can be rescued ex vivo from infected macrophages; and infected macrophages can establish de novo infection. Together, these results demonstrate that macrophages represent a genuine target for HIV infection in vivo that can sustain and transmit infection.

Our aim was to examine the clinicopathological features of squamous cell carcinoma (SCC) of the oral cavity and oropharynx in a group of young patients who were dignosed during a 15-year period (2000-2014). Patients' clinical details, risk factors, and survival were obtained from medical records. Formalin-fixed, paraffin-embedded, tissue was tested for high-risk human papillomavirus (HPV). The results were compared with those of a matching group of older patients. We identified 91 patients who were younger than 45 years old, and the 50 youngest patients were studied in detail. The male:female ratio was 2:1, with more tumours located in the oral cavity than in the oropharynx (35 compared with 15). HPV-related SCC was restricted to the oropharynx. When matched for site, stage and HPV status, five-year overall survival was similar in young and matched older patients (log-rank test, p=0.515). Our findings suggest that young patients with oral SCC have a disease profile similar to that of older patients with the condition. It is plausible that prognostic information generally available for oral cancers is applicable to young patients with the disease.

Abstract
Our laboratory has serially reported on the virologic and immunopathologic features of a cohort of experimental feline immunodeficiency virus (FIV)-infected cats for more than eight years. At 8.09 years post infection (PI), one of these animals entered the terminal stage of infection, characterized by undulating hyperthermia, progressive anorexia, weight loss, and pancytopenia; the animal was not responsive to therapeutic interventions, necessitating euthanasia six weeks later (8.20 years PI). Subsequent analyses indicated that neoplastic lymphocytes infiltrated multiple cervical lymph nodes and a band-like region of the mucosal lamina propria within a segment of the intestine. Immunohistochemistry and T cell clonality testing determined that the nodal and intestinal lesions were independently arising from CD3 T cell lymphomas. In-situ RNA hybridization studies indicated that diffuse neoplastic lymphocytes from the cervical lymph node contained abundant viral nucleic acid, while viral nucleic acid was not detectable in lymphocytes from the intestinal lymphoma lesion. The proviral long terminal repeat (LTR) was amplified and sequenced from multiple anatomic sites, and a common clone containing a single nucleotide polymorphism was determined to be defective in response to phorbol myristate acetate (PMA)-mediated promoter activation in a reporter gene assay. This assay revealed a previously unidentified PMA response element within the FIV U3 region 3’ to the TATA box. The possible implications of these results on FIV-lymphoma pathogenesis are discussed.

Filoviruses are among the most pathogenic infectious agents known to human, with high destructive potential, as evidenced by the recent Ebola virus epidemic in West Africa. As members of the filovirus family, marburgviruses have caused similar devastating outbreaks, albeit with lower case numbers. In this study we compare the pathogenesis of Ravn virus (RAVV) and Marburg virus (MARV) strains Angola, Musoke, and Ozolin in rhesus and cynomolgus macaques, the 2 nonhuman primate species most commonly used in filovirus research. Our results reveal the most pathogenic MARV strain to be Angola, followed by Musoke, whereas Ozolin is the least pathogenic. We also demonstrate that RAVV is highly pathogenic in cynomolgus macaques but less pathogenic in rhesus macaques. Our results demonstrate a preferential infection of endothelial cells by MARVs; in addition, analysis of tissue samples suggests that lymphocyte and hepatocyte apoptosis might play a role in MARV pathogenicity. This information expands our knowledge about pathogenicity and virulence of marburgviruses.

Following the largest Ebola virus disease outbreak from 2013 to 2016, viral RNA has been detected in survivors from semen and breast milk long after disease recovery. However, as there have been few cases of sexual transmission, it is unclear whether every RNA positive fluid sample contains infectious virus. Virus isolation, typically using cell culture or animal models, can serve as a tool to determine the infectivity of patient samples. However, the sensitivity of these methods has not been assessed for the Ebola virus isolate, Makona. Described here is an efficiency comparison of Ebola virus Makona isolation using Vero E6, Huh-7, monocyte-derived macrophage cells, and suckling laboratory mice. Isolation sensitivity was similar in all methods tested. Laboratory mice and Huh-7 cells were less affected by toxicity from breast milk than Vero E6 and MDM cells. However, the advantages associated with isolation in Huh-7 cells over laboratory mice, including cost effectiveness, sample volume preservation, and a reduction in animal use, make Huh-7 cells the preferred substrate tested for Ebola virus Makona isolation.

Porcine circovirus-associated disease encompasses multiple disease syndromes including porcine circovirus 2 systemic diseases, reproductive failure, and porcine dermatitis and nephropathy syndrome. Until recently, porcine circovirus 2 was the only species associated with the porcine circovirus-associated disease. In this report, diagnostic investigations of thirty-six field cases submitted from multiple production systems, numerous sites and varied geographic locations demonstrated porcine circovirus 3 within lesions by in situ hybridization including fetuses with myocarditis, weak-born neonatal piglets with encephalitis and myocarditis, from cases of porcine dermatitis and nephropathy syndrome, and in weaned pigs with systemic periarteritis. Porcine circovirus 3 was detected by PCR in numerous fetuses and perinatal piglets at high viral loads (trillions of genome copies per mL of tissue homogenate). Samples from all cases in this study were assayed and found negative for porcine circovirus 2 by PCR. Metagenomic sequencing was performed on a subset of reproductive cases, consisting of sixteen fetuses/fetal sample pools. PCV3 was identified in all pools and the only virus identified in fourteen pools. Based on these data, porcine circovirus 3 is considered a putative cause of reproductive failure, encephalitis and myocarditis in perinatal piglets, porcine dermatitis and nephropathy syndrome, and periarteritis in swine in the United States.