Phenotypic Selection of Dairy Cattle Infected with Bovine Leukemia Virus Demonstrates Immunogenetic Resilience through NGS-Based Genotyping of BoLA MHC Class II Genes

Characterization of the bovine leukocyte antigen (BoLA) DRB3 gene has shown that specific alleles associate with susceptibility or resilience to the progression of bovine leukemia virus (BLV), measured by proviral load (PVL). Through surveillance of multi-farm BLV eradication field trials, we observed differential phenotypes within seropositive cows that persist from months to years. We sought to develop a multiplex next-generation sequencing workflow (NGS-SBT) capable of genotyping 384 samples per run to assess the relationship between BLV phenotype and two BoLA genes. We utilized longitudinal results from milk ELISA screening and subsequent blood collections on seropositive cows for PVL determination using a novel BLV proviral load multiplex qPCR assay to phenotype the cows. Repeated diagnostic observations defined two distinct phenotypes in our study population, ELISA-positive cows that do not harbor detectable levels of provirus and those who do have persistent proviral loads. In total, 565 cows from nine Midwest dairy farms were selected for NGS-SBT, with 558 cows: 168 BLV susceptible (ELISA-positive/PVL-positive) and 390 BLV resilient (ELISA-positive/PVL-negative) successfully genotyped. Three BoLA-DRB3 alleles, including one novel allele, were shown to associate with disease resilience, *009:02, *044:01, and *048:02 were found at rates of 97.5%, 86.5%, and 90.3%, respectively, within the phenotypically resilient population. Alternatively, DRB3*015:01 and *027:03, both known to associate with disease progression, were found at rates of 81.1% and 92.3%, respectively, within the susceptible population. This study helps solidify the immunogenetic relationship between BoLA-DRB3 alleles and BLV infection status of these two phenotypic groupings of US dairy cattle.

Human T-Cell Leukemia Virus Type 1 Envelope Protein: Post-Entry Roles in Viral Pathogenesis

Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus that is the causative infectious agent of adult T-cell leukemia/lymphoma (ATL), an aggressive and fatal CD4+ T-cell malignancy, and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a chronic neurological disease. Disease progression in infected individuals is the result of HTLV-1-driven clonal expansion of CD4+ T-cells and is generally associated with the activities of the viral oncoproteins Tax and Hbz. A closely related virus, HTLV-2, exhibits similar genomic features and the capacity to transform T-cells, but is non-pathogenic. In vitro, HTLV-1 primarily immortalizes or transforms CD4+ T-cells, while HTLV-2 displays a transformation tropism for CD8+ T-cells. This distinct tropism is recapitulated in infected people. Through comparative studies, the genetic determinant for this divergent tropism of HTLV-1/2 has been mapped to the viral envelope (Env). In this review, we explore the emerging roles for Env beyond initial viral entry and examine current perspectives on its contributions to HTLV-1-mediated disease development.

Clinical Performance of Friend Leukemia Virus Integration 1 Methylation as a Biomarker for Colorectal Carcinoma

Objectives Accumulating evidence supports the reliability of molecular biomarkers and cancer, the current situation is lacking. This present study was aimed to investigate clinical performance of friend leukemia virus integration 1 (FLI1) methylation as a biomarker for colorectal cancer (CRC). Methods The datasets of UALCAN, GEPIA, cBioPortal, STRING, TIMER2.0, GEO, and KOBAS were utilized in this present study. In order to testify the methylation function of FLI1 in CRC, we studied 6 CRC cell lines and 20 pairs of tumor tissues. The transcriptional levels of FLI1 were tested by reverse transcription PCR quantitatively and western blot. Cell viability, transwell assays and plate clone assay were utilized to assess the cell function. Results FLI1 was up-regulated in the GBM, KIRC, LAML, etc. Meanwhile, it was down-regulated in BLCA, BRCA, CESC, etc (p<0.05). The CPTAC dataset showed higher total protein in the primary tissues of KIRC, lower protein in the BRCA, OV, LUAD, UCEC than normal tissues(p<0.05). Highly expressed FLI1 was linked to poor prognosis of overall survival (OS) in LGG, UVM (p<0.05). Low expression of FLI1 was associated with short OS in KIRC, LUAD, SKCM (p<0.05). Compared with normal tissues, the methylation level of FLI1 was increased in BRCA, CESC, CHOL, COAD, etc (p<0.05). In the contrary, it was decreased in KIRP, PCPG obviously (p<0.05). Moreover, it showed a reduced phosphorylation for selected probes (BRCA: NP_002008.2:S39, NP_002008.2:S3241; OV: NP_002008.2:S39; LUAD: NP_002008.2:S79, NP_002008.2:S3241; all p<0.05). Meanwhile, it showed an enhanced phosphorylation level of FLI1 in KIRC for selected probes (NP_002008.2:S241, p<0.05). Besides, a statistical negative correlation was found between FLI1 and Treg cells, neutrophils, monocytes, macrophages, NK cells, cancer-associated fibroblasts, and common immune checkpoint gene levels (p<0.05). Besides, in vivo experience showed that 5-Aza-2’-deoxycytidine diminished FLI1 methylation level and restored transcriptional levels (p>0.05) in CRC. In vitro experiments demonstrated that the proliferation, colony formation, invasion and migration of CRC cells were inhibited by FLI1 overexpression through FMNL1 (p<0.05). Conclusions This present study offers a comprehensive understanding of FLI1 in carcinomas with oncogenesis and immunotherapeutic implications. Moreover, FLI1 reduced the proliferation, colony formation, invasion and migration of CRC by FMNL1.

Distribution of Human Immunodeficiency Virus and Human T-Leukemia Virus Co-infection in Bahia, Brazil

Human Immunodeficiency Virus (HIV) and Human T-Leukemia Virus (HTLV) are retroviruses that share similar routes of transmission. In Brazil, the prevalence of HIV and HTLV varies according to geographic region. The state of Bahia, located in the Northeast region, is considered endemic for both retroviruses. The present study aimed to characterize the frequency of HIV/HTLV coinfection and evaluate the geographic distribution of coinfection throughout the state. This cross-sectional study was conducted at the state's Central Laboratory of Public Health (LACEN-BA) and included all samples from 2004 to 2013 submitted to serological testing for anti-HIV and anti-HTLV-1/2, screened by chemiluminescence/ELISA and confirmed by Western blot. Infection rates are expressed as the number of infected individuals per 100,000 inhabitants from each municipality. A total of 129,158 samples originating from 358/417 (85.8%) municipalities in Bahia were evaluated. HTLV was detected in 2.4% of the HIV-positive samples (n = 42) compared to 0.5% of those with negative HIV serology (n = 677) (OR: 4.65; CI: 3.39–6.37). HIV/HTLV coinfection was more frequent in women (69.0%); the median age of coinfected individuals was 47.2 years [interquartile range (IQR): 41.6–55.4 years]. In the 14/417 (3.4%) municipalities where at least one case of HIV/HTLV coinfection was detected, the overall HTLV coinfection rate in HIV-positive samples was 0.25 (range: 0.17–13.84) per 100,000 inhabitants. Most cases of HIV/HTLV-1 coinfection (21/37, 57%) were concentrated in the municipality of Salvador. Isolated instances (one or two cases) of HIV/HTLV-1 coinfection were distributed across municipalities known to be endemic for HTLV infection.

Relationship between the transcriptional expression of PIM1 and local control in patients with head and neck squamous cell carcinomas treated with radiotherapy

Purpose Proviral integration site for Moloney murine leukemia virus (PIMs) are proto-oncogenes encoding serine/threonine kinases that phosphorylate a variety of substrates involved in the regulation of cellular processes. Elevated expression of PIM-1 has been associated with poor prognosis in several types of cancer. There are no studies that have analyzed the response to radiotherapy in patients with head and neck squamous cell carcinoma (HNSCC) according to the expression of PIM-1. The aim of our study was to analyze the relationship between the transcriptional expression of PIM-1 and local response to radiotherapy in HNSCC patients. Methods We determined the transcriptional expression of PIM-1 in 135 HNSCC patients treated with radiotherapy, including patients treated with chemoradiotherapy (n = 65) and bioradiotherapy (n = 15). Results During the follow-up, 48 patients (35.6%) had a local recurrence of the tumor. Patients with local recurrence had a higher level of PIM-1 expression than those who achieved local control of the disease (P = 0.017). Five-year local recurrence-free survival for patients with a high expression of PIM-1 (n = 43) was 44.6% (95% CI 29.2–60.0%), and for patients with low expression (n = 92) it was 71.9% (95% CI 62.5–81.3%) (P = 0.007). According to the results of multivariate analysis, patients with a high PIM-1 expression had a 2.2-fold increased risk of local recurrence (95% CI 1.22–4.10, P = 0.009). Conclusion Patients with elevated transcriptional expression levels of PIM-1 had a significantly higher risk of local recurrence after radiotherapy.

Endogenous Retroviruses Provide Protection Against Vaginal HSV-2 Disease

Endogenous retroviruses (ERVs) are genomic sequences that originated from retroviruses and are present in most eukaryotic genomes. Both beneficial and detrimental functions are attributed to ERVs, but whether ERVs contribute to antiviral immunity is not well understood. Here, we used herpes simplex virus type 2 (HSV-2) infection as a model and found that Toll-like receptor 7 (Tlr7-/-) deficient mice that have high systemic levels of infectious ERVs are protected from intravaginal HSV-2 infection and disease, compared to wildtype C57BL/6 mice. We deleted the endogenous ecotropic murine leukemia virus (Emv2) locus on the Tlr7-/- background (Emv2-/-Tlr7-/-) and found that Emv2-/-Tlr7-/- mice lose protection against HSV-2 infection. Intravaginal application of purified ERVs from Tlr7-/- mice prior to HSV-2 infection delays disease in both wildtype and highly susceptible interferon-alpha receptor-deficient (Ifnar1-/-) mice. However, intravaginal ERV treatment did not protect Emv2-/-Tlr7-/- mice from HSV-2 disease, suggesting that the protective mechanism mediated by exogenous ERV treatment may differ from that of constitutively and systemically expressed ERVs in Tlr7-/- mice. We did not observe enhanced type I interferon (IFN-I) signaling in the vaginal tissues from Tlr7-/- mice, and instead found enrichment in genes associated with extracellular matrix organization. Together, our results revealed that constitutive and/or systemic expression of ERVs protect mice against vaginal HSV-2 infection and delay disease.

Epigenetic Regulation of Human T-Cell Leukemia Virus Gene Expression

Viral and cellular gene expression are regulated by epigenetic alterations, including DNA methylation, histone modifications, nucleosome positioning, and chromatin looping. Human T-cell leukemia virus type 1 (HTLV-1) is a pathogenic retrovirus associated with inflammatory disorders and T-cell lymphoproliferative malignancy. The transforming activity of HTLV-1 is driven by the viral oncoprotein Tax, which acts as a transcriptional activator of the cAMP response element-binding protein (CREB) and nuclear factor kappa B (NFκB) pathways. The epigenetic effects of Tax and the induction of lymphoproliferative malignancy include alterations in DNA methylation and histone modifications. In addition, alterations in nucleosome positioning and DNA looping also occur in HTLV-1-induced malignant cells. A mechanistic definition of these effects will pave the way to new therapies for HTLV-1-associated disorders.

Hydrogen Sulfide Inhibits Human T-cell Leukemia Virus Type-1 (HTLV-1) Protein Expression via Regulation of ATG4B

Background: Hydrogen sulfide(H 2 S)is a redox gasotransmitter. It has been shown that H 2 S has a key role in host antiviral defense by inhibiting interleukin (IL)-6 production and S-sulfhydrating Keap1 lead to Nrf2/ARE pathway activation. However, it is yet unclear whether H 2 S can play an antiviral role by regulating autophagy. Results: In this research, we found that exogenous H 2 S decreased the expression of HTLV-1 protein and HTLV-1 induced autophagosomes accumulation. Transmission electron microscope assays indicated that autophagosomes accumulation decreased after H 2 S administration. HTLV-1-transformed T-cell lines had a high level of CSE (H 2 S endogenous enzyme) which could be induced in Hela by HTLV-1 infection. Immunoblot demonstrated that overexpression of CSE inhibited HTLV-1 protein expression and autophagy. And we got the opposite after CSE knockdown. Meanwhile, H 2 S could not restrain the autophagy when ATG4B had a mutant at its site of 89. Conclusion: In a word, these results suggested that H 2 S modulated HTLV-1 protein expression via ATG4B. Meanwhile, our findings suggested a new mechanism by which H 2 S defended against virus infection.

Vaccination against the Koala Retrovirus (KoRV): Problems and Strategies

The koala retrovirus (KoRV) is spreading in the koala population from the north to the south of Australia and is also in the process of endogenization into the koala genome. Virus infection is associated with tumorigenesis and immunodeficiency and is contributing to the decline of the animal population. Antibody production is an excellent marker of retrovirus infection; however, animals carrying endogenous KoRV are tolerant. Therefore, the therapeutic immunization of animals carrying endogenous KoRV seems to be ineffective. Using the recombinant transmembrane (TM) envelope protein of the KoRV, we immunized goats, rats and mice, obtaining in all cases neutralizing antibodies which recognize epitopes in the fusion peptide proximal region (FPPR), and in the membrane-proximal external region (MPER). Immunizing several animal species with the corresponding TM envelope protein of the closely related porcine endogenous retrovirus (PERV), as well as the feline leukemia virus (FeLV), we also induced neutralizing antibodies with similar epitopes. Immunizing with the TM envelope protein in addition to the surface envelope proteins of all three viruses resulted in higher titers of neutralizing antibodies. Immunizing KoRV-negative koalas with our vaccine (which is composed of both envelope proteins) may protect these animals from infection, and these may be the starting points of a virus-free population.

A Pragmatic Pharmacophore Informatics Strategy to Discover New Potent Inhibitors Against Pim-3

Pim-3 (proviral integration site moloney murine leukemia virus-3) is an oncogene which encodes proteins belonging to serine/threonine kinase family, and PIM subfamily. It is generally over expressed in epithelial and hematological tumors. It is known to involve in numerous cellular functions such as cell growth, differentiation, survival, tumorigenesis and apoptosis. It also plays a crucial role in regulation of signal transduction cascades. Therefore it emerged as a hopefultherapeutic target for cancer treatment. In current study, indole derivatives having potent inhibitory activity against Pim 3 were taken and pharmacophore based virtual screening was carried out. A five point pharmacophore hypothesis with one hydrogen bond acceptor, one hydrogen bond donor and three aromatic rings i.e., ADRRR was developed with acceptable R2and Q2 values of 0.913 and 0.748 respectively. It was employed as a query and screening was conducted against Asinex and Otava lead library databases to screen out potent drug like candidates. The obtained compounds were subjected to SP, XP docking using 3D model of pim-3 which was constructed through comparative homology modelling and finally binding free energies were calculated for top hits. The docking and binding free energy studies revealed that six hit molecules showed higher binding energy in comparison to the best active molecule. Finally, MD simulations of the top hit with highest binding energy was carried out which indicated that the obtained hit N1 formed a stable complex with pim-3. We believe that these combined protocols will be helpful and cooperative to discover and design more potent pim-3 inhibitors in near future.