Molecular analysis of the env, LTR, and pX regions of bovine leukemia virus in dairy cattle of Türkiye.

Bovine leukemia virus is a retrovirus that causes enzootic bovine leukosis and is associated with global economic losses in the livestock industry. The aim of this study was to investigate the genotype determination of BLVs from cattle housed in 6 different farms in Türkiye and the characterization of their LTR and pX (tax, rex, R3, and G4 gene) regions. For this purpose, blood samples from 48 cattle infected with BLV were used. The phylogenetic analysis based on the env gene sequences revealed that all BLVs were clustered in genotype 1 (G1), and the sequences of the LTR (n = 48) and the pX region (n = 33) of BLVs were obtained. Also, analysis of these nucleic acid and amino acid sequences allowed assessments similar to those reported in earlier studies to be relevant to transactivation and pathogenesis. This study reports the molecular analysis of the LTR and pX region of BLVs in Türkiye for the first time.

Decoding the mystery of MMTV-like virus and its relationship with breast cancer metastasis.

BACKGROUND: MMTV causes mammary tumors in mice, and it is associated with invasive and aggressive forms of breast cancer in humans. However, the underlying mechanisms are yet unknown. We aimed to determine the MMTV-like virus (MMTV-LV) association with histological types of breast cancer, nodal involvement, and metastasis. METHODS: First, 105 breast cancer biopsies and 15 disease-free biopsies were collected. Details of clinicopathological characteristics were retrieved from patients' records. The status of MMTV-LV was already known for these biopsy samples. Associations of MMTV-LV prevalence with LNM status and metastatic history were determined. Next, quantitative PCR (qPCR) was used to quantify env gene mRNA in biopsies positive for MMTV-LV. Expression of the env gene was compared against different histopathological types of mammary tumor, LNM status, and metastasis by performing Ordinary One Way ANOVA followed by Tukey's multiple comparisons test. RESULTS: MMTV-LV prevalence was found to have no significant association with LNM or metastatic history. As compared to normal control, expression of the env gene was significantly higher (>2.8 folds) in invasive samples (P-value: < 0.01). Expression was also higher (3.28 and 2.89 folds) in patient samples with LNM (P-value: 0.0006) or metastatic history (P-value: < 0.0001), respectively. CONCLUSION: We conclude that MMTV-LV prevalence is not associated with LNM status or breast cancer metastasis; samples with invasive phenotypes, nodal involvement, and metastasis exhibit significantly higher expression of the MMTV-like env gene.

Truncation of the Cytoplasmic Tail of Equine Infectious Anemia Virus Increases Virion Production by Improving Env Cleavage and Plasma Membrane Localization.

Envelope glycoproteins (Envs) of lentiviruses harbor unusually long cytoplasmic tails (CTs). Natural CT truncations always occur in vitro and are accompanied by attenuated virulence, but their effects on viral replication have not been fully elucidated. The Env in equine infectious anemia virus (EIAV) harbors the longest CT in the lentiviral family, and a truncated CT was observed in a live attenuated vaccine. This study demonstrates that CT truncation significantly increased EIAV production, as determined by comparing the virion yields from EIAV infectious clones in the presence and absence of the CT. A significant increase in a cleaved product from the CT-truncated Env precursor, but not the full-length Env, was observed. We further confirmed that the presence of the CT inhibited the cleavage of the Env precursor and found that a functional domain located at the C terminus was responsible for this function. Moreover, CT-truncated Env was mainly localized at the plasma membrane (PM), while full-length Env was mainly localized in the cytoplasm. The CT truncation caused a dramatic reduction in the endocytosis of Env. These results suggest that the CT can modulate the processing and trafficking of EIAV Env and thus regulate EIAV replication. IMPORTANCE The mature lentivirus envelope glycoprotein (Env) is composed of a surface unit (SU) and a transmembrane unit (TM), which are cleaved products of the Env precursor. After mature Env is heterodimerically formed from the cleavage of the Env precursor, it is trafficked to the plasma membrane (PM) for incorporation and virion assembly. Env harbors a long cytoplasmic tail (CT), which has been increasingly found to play multiple roles in the Env biological cycle. Here, we revealed for the first time that the CT of equine infectious anemia virus (EIAV) Env inhibits cleavage of the Env precursor. Simultaneously, the CT promoted Env endocytosis, resulting in weakened Env localization at the PM. We also validated that the CT could significantly decrease EIAV production. These findings suggest that the CT regulates the processing and trafficking of EIAV Env to balance virion production.

Expression profiles of human endogenous retrovirus (HERV)-K and HERV-R Env proteins in various cancers.

The vertebrate genome contains an endogenous retrovirus that has been inherited from the past millions of years. Although approximately 8% of human chromosomal DNA consists of sequences derived from human endogenous retrovirus (HERV) fragments, most of the HERVs are currently inactive and noninfectious due to recombination, deletions, and mutations after insertion into the host genome. Several studies suggested that Human endogenous retroviruses (HERVs) factors are significantly related to certain cancers. However, only limited studies have been conducted to analyze the expression of HERV derived elements at protein levels in certain cancers. Herein, we analyzed the expression profiles of HERV-K envelope (Env) and HERV-R Env proteins in eleven different kinds of cancer tissues. Furthermore, the expression patterns of both protein and correlation with various clinical data in each tissue were analyzed. The expressions of both HERV-K Env and HERV-R Env protein were identified to be significantly high in most of the tumors compared with normal surrounding tissues. Correlations between HERV Env expressions and clinical investigations varied depending on the HERV types and cancers. Overall expression patterns of HERV-K Env and HERV-R Env proteins were different in every individual but a similar pattern of expressions was observed in the same individual. These results demonstrate the expression profiles of HERV-K and HERV-R Env proteins in various cancer tissues and provide a good reference for the association of endogenous retroviral Env proteins in the progression of various cancers. Furthermore, the results elucidate the relationship between HERV-Env expression and the clinical significance of certain cancers. [BMB Reports 2021; 54(7): 368-373].

Mouse Mammary Tumor Virus (MMTV)-Like env Sequence in Brazilian Breast Cancer Samples: Implications in Clinicopathological Parameters in Molecular Subtypes.

Background: Breast cancer (BC) is a complex disease in which susceptibility and clinical course depend on multiple factors. Evidence suggests that a mouse mammary tumor virus (MMTV)-homolog may be present in human BCs; however, little is known about its clinical implications. Methods: MMTV-like env nucleotide-sequence was searched in tumor and tumor-adjacent tissues from 217 Brazilian BC patients through nested-PCR and confirmed through PCR-sequencing. Blood samples were also tested for patients with MMTV-like env gene-positive tumors. Correlations with clinicopathological parameters were evaluated. Results: MMTV-like env sequence was detected in tumor and tumor-adjacent tissue samples from 41/217 and 30/196 patients, respectively. In blood, MMTV-like was detected in 17/32 patients. In Luminal-B tumors, MMTV-like in tumor tissue was negatively correlated with tumor size and disease stage, whereas in HER2 tumors it anti-correlated with lymph node metastasis (LNM) and disease stage. Considering blood, MMTV-like env gene positivity negatively correlated with age in general BC, while in Luminal-A tumors it positively correlated with Ki67 but negatively correlated with age and LNM. The associations with decreased LNM frequency were independent of other prognostic factors. Conclusion: MMTV-like env positivity is associated with better prognostic parameters in BC subtypes, which might be explainable by its anti-metastatic potential and by putative activation of immune milieu.

Detection and genotyping of bovine leukemia virus (BLV) in Vietnamese cattle.

Bovine leukemia virus (BLV) belongs to the genus, Deltaretrovirus of the family, Retroviridae and it is the causative agent of enzootic bovine leukosis. The prevalence of BLV in three provinces in the Red River Delta Region in the North of Vietnam, Hanoi, Vinhphuc and Bacninh was studied from April 2017 to June 2018. A total of 275 blood samples collected from cattle were used for serum isolation and DNA extraction. Of these samples, 266 sera were subjected to ELISA test for detecting antibody against BLV gp51 protein and 152 DNA samples were used to detect the 444 bp fragment corresponding to a part of the gp51 region of the env by nested PCR. The results showed that 16.5% (n=44) and 21.1% (n=32) of samples were positive for BLV gp51 antibody and BLV proviral DNA, respectively. Phylogenetic analysis of the partial (423 bp) and complete (913 bp) BLV env-gp51 gene indicated that Vietnamese strains were clustered into genotypes 1, 6 and 10 (G1, G6 and G10). Of those genotypes, G1 genotype was dominant; G6 strains were designated as G6e and G6f subgenotypes; the existence of genotype 10 was confirmed for the first time in Vietnam. The present study provides important information regarding the prevalence of BLV infection and genetic characteristics of BLV strains identified in Vietnam, contributing to promote the establishment of disease control and eradication strategies in Vietnam.

The High Content of Fructose in Human Semen Competitively Inhibits Broad and Potent Antivirals That Target High-Mannose Glycans.

Semen is the primary transmission vehicle for various pathogenic viruses. Initial steps of transmission, including cell attachment and entry, likely occur in the presence of semen. However, the unstable nature of human seminal plasma and its toxic effects on cells in culture limit the ability to study in vitro virus infection and inhibition in this medium. We found that whole semen significantly reduces the potency of antibodies and microbicides that target glycans on the envelope glycoproteins (Envs) of HIV-1. The extraordinarily high concentration of the monosaccharide fructose in semen contributes significantly to the effect by competitively inhibiting the binding of ligands to α1,2-linked mannose residues on Env. Infection and inhibition in whole human seminal plasma are accurately mimicked by a stable synthetic simulant of seminal fluid that we formulated. Our findings indicate that, in addition to the protein content of biological secretions, their small-solute composition impacts the potency of antiviral microbicides and mucosal antibodies.IMPORTANCE Biological secretions allow viruses to spread between individuals. Each type of secretion has a unique composition of proteins, salts, and sugars, which can affect the infectivity potential of the virus and inhibition of this process. Here, we describe HIV-1 infection and inhibition in whole human seminal plasma and a synthetic simulant that we formulated. We discovered that the sugar fructose in semen decreases the activity of a broad and potent class of antiviral agents that target mannose sugars on the envelope protein of HIV-1. This effect of semen fructose likely reduces the efficacy of such inhibitors to prevent the sexual transmission of HIV-1. Our findings suggest that the preclinical evaluation of microbicides and vaccine-elicited antibodies will be improved by their in vitro assessment in synthetic formulations that simulate the effects of semen on HIV-1 infection and inhibition.

Tracking the Fate of Endogenous Retrovirus Segregation in Wild and Domestic Cats.

Endogenous retroviruses (ERVs) of domestic cats (ERV-DCs) are one of the youngest feline ERV groups in domestic cats (Felis silvestris catus); some members are replication competent (ERV-DC10, ERV-DC18, and ERV-DC14), produce the antiretroviral soluble factor Refrex-1 (ERV-DC7 and ERV-DC16), or can generate recombinant feline leukemia virus (FeLV). Here, we investigated ERV-DC in European wildcats (Felis silvestris silvestris) and detected four loci: ERV-DC6, ERV-DC7, ERV-DC14, and ERV-DC16. ERV-DC14 was detected at a high frequency in European wildcats; however, it was replication defective due to a single G → A nucleotide substitution, resulting in an E148K substitution in the ERV-DC14 envelope (Env). This mutation results in a cleavage-defective Env that is not incorporated into viral particles. Introduction of the same mutation into feline and murine infectious gammaretroviruses resulted in a similar Env dysfunction. Interestingly, the same mutation was found in an FeLV isolate from naturally occurring thymic lymphoma and a mouse ERV, suggesting a common mechanism of virus inactivation. Refrex-1 was present in European wildcats; however, ERV-DC16, but not ERV-DC7, was unfixed in European wildcats. Thus, Refrex-1 has had an antiviral role throughout the evolution of the genus Felis, predating cat exposure to feline retroviruses. ERV-DC sequence diversity was present across wild and domestic cats but was locus dependent. In conclusion, ERVs have evolved species-specific phenotypes through the interplay between ERVs and their hosts. The mechanism of viral inactivation may be similar irrespective of the evolutionary history of retroviruses. The tracking of ancestral retroviruses can shed light on their roles in pathogenesis and host-virus evolution.IMPORTANCE Domestic cats (Felis silvestris catus) were domesticated from wildcats approximately 9,000 years ago via close interaction between humans and cats. During cat evolution, various exogenous retroviruses infected different cat lineages and generated numerous ERVs in the host genome, some of which remain replication competent. Here, we detected several ERV-DC loci in Felis silvestris silvestris Notably, a species-specific single nucleotide polymorphism in the ERV-DC14 env gene, which results in a replication-defective product, is highly prevalent in European wildcats, unlike the replication-competent ERV-DC14 that is commonly present in domestic cats. The presence of the same lethal mutation in the env genes of both FeLV and murine ERV provides a common mechanism shared by endogenous and exogenous retroviruses by which ERVs can be inactivated after endogenization. The antiviral role of Refrex-1 predates cat exposure to feline retroviruses. The existence of two ERV-DC14 phenotypes provides a unique model for understanding both ERV fate and cat domestication.

Detection of feline immunodeficiency virus subtypes A and B circulating in the city of Buenos Aires.

Feline immunodeficiency virus (FIV), genus Lentivirus, is responsible for feline immunodeficiency syndrome in domestic cats. FIV has been classified into six subtypes: A, B, C, D, E and F, based on regions of the env gene as well as the gag gene. In Argentina, the circulation of subtypes B and E was reported more than two decades ago. The objective of this work was to study the FIV variants circulating presently in the city of Buenos Aires in naturally infected cats utilizing a nested PCR targeting the gag gene. A phylogenetic comparison with representative sequences of five previously published subtypes shows a clustering with subtypes A and B. This is the first report of FIV subtype A in Argentina.

Mouse mammary tumor viral env sequences are not present in the human genome but are present in breast tumors and normal breast tissues.

In 1936, John Joseph Bittner identified mouse mammary tumor virus (MMTV), a milk transmitted beta retrovirus, a form of single-stranded positive-sense RNA virus. A retrovirus inserts a copy of its genome into the DNA of a host cell, thus altering the cell's genome. In the current analysis, we searched for MMTV sequences within the human genome. To compare the MMTV genome to the human genome, we used BLAT, the Blast-Like Alignment Tool of the UCSC Genome Browser. BLAT can align a user sequence of 25 bases or more to the genome. 60 MMTV sequences were in the human genome. Of 56 sequences from the MMTV POL gene, 36 POL sequences were from the same part of the gene, beginning at viral nucleotide 4800 but of different lengths. 8 viral sequences began at nucleotide ∼3430 of the POL gene. Four viral sequences were from GAGdUTPase, encoded by the MMTV PRO gene. Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) is an enzyme present in several major retroviral families. In MMTV dUTPase may be essential for viral replication. Since BLAT identified no MMTV envelope (env) sequence in the human genome, the env sequences from breast tumors and normal breast tissue found in other studies may have come from an MMTV infection. However, no one is certain how MMTV could enter human cells, since the cells do not have a cellular receptor for MMTV, as do mouse cells.

Effects of the SOS (A501C/T605C) and DS (I201C/A433C) Disulfide Bonds on HIV-1 Membrane Envelope Glycoprotein Conformation and Function.

Most broadly neutralizing antibodies and many entry inhibitors target the pretriggered (state 1) conformation of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env). Here we examine two previously reported Env mutants designed to be stabilized in this conformation by the introduction of artificial disulfide bonds: A501C/T605C (called SOS) and I201C/A433C (called DS). SOS Env supported virus entry and cell-cell fusion only after exposure to a reducing agent, dithiothreitol (DTT). Deletion of the Env cytoplasmic tail improved the efficiency with which the SOS Env supported virus infection in a reducing environment. The antigenicity of the SOS Env was similar to that of the unmodified Env, except for greater sensitivity to some state 1-preferring ligands. In contrast, viruses with the DS Env were not infectious, even after DTT treatment. The proteolytic maturation of the DS Env on both cell surfaces and virions was severely compromised compared with that of the unmodified Env. The DS Env exhibited detectable cell-fusing activity when DTT was present. However, the profiles of cell-surface Env recognition and cell-cell fusion inhibition by antibodies differed for the DS Env and the unmodified Env. Thus, the DS Env appears to be stabilized in an off-pathway conformation that is nonfunctional on the virus. The SOS change exerted more subtle, context-dependent effects on Env conformation and function.IMPORTANCE The human immunodeficiency virus type 1 (HIV-1) envelope proteins (Envs) bind receptors on the host cell and change shape to allow the virus to enter the cell. Most virus-inhibiting antibodies and drugs recognize a particular shape of Env called state 1. Disulfide bonds formed by cysteine residues have been introduced into soluble forms of the flexible envelope proteins in an attempt to lock them into state 1 for use in vaccines and as research tools. We evaluated the effect of these cysteine substitutions on the ability of the membrane Env to support virus entry and on susceptibility to inhibition by antibodies and small molecules. We found that the conformation of the envelope proteins with the cysteine substitutions differed from that of the unmodified membrane envelope proteins. Awareness of these effects can assist efforts to create stable HIV-1 Env complexes that more closely resemble the state 1 conformation.

Reduced Folate Carrier: an Entry Receptor for a Novel Feline Leukemia Virus Variant.

Feline leukemia virus (FeLV) is horizontally transmitted among cats and causes a variety of hematopoietic disorders. Five subgroups of FeLV, A to D and T, each with distinct receptor usages, have been described. Recently, we identified a new FeLV Env (TG35-2) gene from a pseudotyped virus that does not belong to any known subgroup. FeLV-A is the primary virus from which other subgroups have emerged via mutation or recombination of the subgroup A env gene. Retrovirus entry into cells is mediated by the interaction of envelope protein (Env) with specific cell surface receptors. Here, phenotypic screening of a human/hamster radiation hybrid panel identified SLC19A1, a feline reduced folate carrier (RFC) and potential receptor for TG35-2-phenotypic virus. RFC is a multipass transmembrane protein. Feline and human RFC cDNAs conferred susceptibility to TG35-2-pseudotyped virus when introduced into nonpermissive cells but did not render these cells permissive to other FeLV subgroups or feline endogenous retrovirus. Moreover, human cells with genomic deletion of RFC were nonpermissive for TG35-2-pseudotyped virus infection, but the introduction of feline and human cDNAs rendered them permissive. Mutation analysis of FeLV Env demonstrated that amino acid substitutions within variable region A altered the specificity of the Env-receptor interaction. We isolated and reconstructed the full-length infectious TG35-2-phenotypic provirus from a naturally FeLV-infected cat, from which the FeLV Env (TG35-2) gene was previously isolated, and compared the replication of the virus in hematopoietic cell lines with that of FeLV-A 61E by measuring the viral RNA copy numbers. These results provide a tool for further investigation of FeLV infectious disease.IMPORTANCE Feline leukemia virus (FeLV) is a member of the genus Gammaretrovirus, which causes malignant diseases in cats. The most prevalent FeLV among cats is FeLV subgroup A (FeLV-A), and specific binding of FeLV-A Env to its viral receptor, thiamine transporter feTHTR1, is the first step of infection. In infected cats, novel variants of FeLV with altered receptor specificity for viral entry have emerged by mutation or recombination of the env gene. A novel FeLV variant arose from a subtle mutation of FeLV-A Env, which altered the specific interaction of the virus with its receptor. RFC, a folate transporter, is a potential receptor for the novel FeLV variant. The perturbation of specific retrovirus-receptor interactions under selective pressure by the host results in the emergence of novel viruses.

Capture of a Hyena-Specific Retroviral Envelope Gene with Placental Expression Associated in Evolution with the Unique Emergence among Carnivorans of Hemochorial Placentation in Hyaenidae.

Capture of retroviral envelope genes from endogenous retroviruses has played a role in the evolution of mammals, with evidence for the involvement of these genes in the formation of the maternofetal interface of the placenta. It has been shown that the diversity of captured genes is likely to be responsible for the diversity of placental structures, ranging from poorly invasive (epitheliochorial) to highly invasive (hemochorial), with an intermediate state (endotheliochorial) as found in carnivorans. The latter recapitulate part of this evolution, with the hyena being the sole carnivoran with a hemochorial placenta. In this study, we performed RNA sequencing on hyena placental transcripts and searched for endogenous retroviral envelope genes that have been captured specifically in the Hyaenidae clade and are not found in any other carnivoran. We identified an envelope gene that is expressed in the placenta at the level of the maternofetal interface, as evidenced by in situ hybridization/immunohistochemistry. The gene entry is coincidental with the emergence of the Hyaenidae clade 30 million years ago (Mya), being found at the same genomic locus in all 4 extant hyena species. Its coding sequence has further been maintained during all of Hyaenidae evolution. It is not found in any of the 30 other carnivorans-both Felidae and Canidae-that we screened. This envelope protein does not disclose any fusogenic activity in ex vivo assays, at variance with the syncytin-Car1 gene, which is found in all carnivorans, including the hyena, in which it is still present, transcriptionally active in the placenta, and fusogenic. Together, the present results illustrate the permanent renewal of placenta-specific genes by retroviral capture and de facto provide a candidate gene for the endotheliochorial to hemochorial transition of Hyaenidae among carnivorans.IMPORTANCE The placenta is the most diverse organ among mammals, due in part to stochastic capture of retroviral envelope genes. In carnivorans, capture of syncytin-Car1 took place 80 Mya. It is fusogenic, expressed at the syncytialized placental maternofetal interface, and conserved among all carnivorans, consistent with their shared endotheliochorial placenta. Hyenas are a remarkable exception, with a highly invasive hemochorial placenta, as found in humans, where disruption of maternal blood vessels results in maternal blood bathing the syncytial maternofetal interface. In this study, we identified a retroviral envelope gene capture and exaptation that took place about 30 Mya and is coincident with the emergence of the Hyaenidae, being conserved in all extant hyena species. It is expressed at the maternofetal interface in addition to the shared syncytin-Car1 gene. This new env gene, not present in any other carnivoran, is a likely candidate to be responsible for the specific structure of the hyena placenta.

Computational analysis of envelope glycoproteins from diverse geographical isolates of bovine leukemia virus identifies highly conserved peptide motifs.

BACKGROUND: Bovine leukemia virus (BLV) is a deltaretrovirus infecting bovine B cells and causing enzootic bovine leucosis. The SU or surface subunit, gp51, of its envelope glycoprotein is involved in receptor recognition and virion attachment. It contains the major neutralizing and CD4+ and CD8+ T cell epitopes found in naturally infected animals. In this study, we aimed to determine global variation and conservation within gp51 in the context of developing an effective global BLV vaccine. RESULTS: A total of 256 sequences extracted from the NCBI database and collected in different parts of the world, were studied to identify conserved segments along the env gene sequences that encode the gp51 protein. Using the MEME server and the conserved DNA Region module for analysis within DnaSP, we identified six conserved segments, referred to as A-F, and five semi-conserved segments, referred to as G-K. The amino acid conservation ranged from 98.8 to 99.8% in conserved segments A to F, while segments G to K had 89.6-95.2% conserved amino acid sequence. Selection analysis of individual segments revealed that residues of conserved segments had undergone purifying selection, whereas, particular residues in the semi-conserved segments are currently undergoing positive selection, specifically at amino acid positions 48 in segment K, 74 in segment G, 82 in segment I, 133 and 142 in segment J, and residue 291 in segment H. Each of the codons for these six residues contain the most highly variable nucleotides within their respective semi-conserved segments. CONCLUSIONS: The data described here show that the consensus amino acid sequence constitutes a strong candidate from which a global vaccine can be derived for use in countries where eradication by culling is not economically feasible. The most conserved segments overlap with amino acids in known immunodeterminants, specifically in epitopes D-D', E-E', CD8+ T-cell epitopes, neutralizing domain 1 and CD4+ T-cell epitopes. Two of the segments reported here represent unique segments that do not overlap with previously identified antigenic determinants. We propose that evidence of positive selection in some residues of the semi-conserved segments suggests that their variation is involved in viral strategy to escape immune surveillance of the host.

A G-quadruplex motif in an envelope gene promoter regulates transcription and virion secretion in HBV genotype B.

HBV genotypes differ in pathogenicity. In addition, genotype-specific differences in the regulation of transcription and virus replication exist in HBV, but the underlying mechanisms are unknown. Here, we show the presence of a G-quadruplex motif in the promoter of the preS2/S gene; this G-quadruplex is highly conserved only in HBV genotype B but not in other HBV genotypes. We demonstrate that this G-quadruplex motif forms a hybrid intramolecular G-quadruplex structure. Interestingly, mutations disrupting the G-quadruplex in HBV genotype B reduced the preS2/S promoter activity, leading to reduced hepatitis B surface antigen (HBsAg) levels. G-quadruplex ligands stabilized the G-quadruplex in genotype B and enhanced the preS2/S promoter activity. Furthermore, mutations disrupting the G-quadruplex in the full-length HBV genotype B constructs were associated with impaired virion secretion. In contrast to typical G-quadruplexes within promoters which are negative regulators of transcription the G-quadruplex in the preS2/S promoter of HBV represents an unconventional positive regulatory element. Our findings highlight (a) G-quadruplex mediated enhancement of transcription and virion secretion in HBV and (b) a yet unknown role for DNA secondary structures in complex genotype-specific regulatory mechanisms in virus genomes.

Sequencing the Biology of Entry: The Retroviral env Gene.

The surface envelope protein of any virus is major determinant of the host cell that is infected and as a result a major determinant of viral pathogenesis. Retroviruses have a single surface protein named Env. It is a trimer of heterodimers and is responsible for binding to the host cell receptor and mediating fusion between the viral and host membranes. In this review we will discuss the history of the discovery of the avian leukosis virus (ALV) and human immunodeficiency virus type 1 (HIV-1) Env proteins and their receptor specificity, comparing the many differences but having some similarities. Much of the progress in these fields has relied on viral genetics and genetic polymorphisms in the host population. A special feature of HIV-1 is that its persistent infection in its human host, to the point of depleting its favorite target cells, allows the virus to evolve new entry phenotypes to expand its host range into several new cell types. This variety of entry phenotypes has led to confusion in the field leading to the major form of entry phenotype of HIV-1 being overlooked until recently. Thus an important part of this story is the description and naming of the most abundant entry form of the virus: R5 T cell-tropic HIV-1.

Molecular characterization of bovine leukemia virus from Moldovan dairy cattle.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL), a disease that has worldwide distribution. Whilst it has been eradicated in most of Western Europe and Scandinavia, it remains a problem in other regions, particularly Eastern Europe and South America. For this study, in 2013, 24 cattle from three farms in three regions of Moldova were screened by ELISA and nested PCR. Of these cattle, 14 which were PCR positive, and these were molecularly characterized based on the nucleotide sequence of the env gene and the deduced amino acid sequence of the encoded gp51 protein. Our results demonstrated a low level of genetic variability (0-2.9%) among BLV field strains from Moldova, in contrast to that observed for other retroviruses, including human immunodeficiency virus (HIV) (20-38%) Mason IL (Trudy vologod moloch Inst 146-164, 1970) and equine infectious anemia virus (EIAV) (~40%) Willems L et al (AIDS Res Hum Retroviruses 16(16):1787-1795, 2000), where the envelope gene exhibits high levels of variation Polat M et al (Retrovirology 13(1):4, 2016). Sequence comparisons and phylogenetic analysis revealed that BLV genotype 7 (G7) is predominant in Moldova and that the BLV population in Moldovan cattle is a mixture of at least three new sub-genotypes: G7D, G7E and G4C. Neutrality tests revealed that negative selection was the major force operating upon the 51-kDa BLV envelope surface glycoprotein subunit gp51, although one positively selected site within conformational epitope G was detected in the N-terminal part of gp51. Furthermore, two functional domains, linear epitope B and the zinc-binding domain, were found to have an elevated ratio of nonsynonymous to synonymous codon differences. Together, these data suggest that the evolutionary constraints on epitopes G and B and the zinc-binding domains of gp51 differ from those on the other domains, with a tendency towards formation of homogenous genetic groups, which is a common concept of global BLV diversification during virus transmission that may be associated with genetic drift.

Serologically silent, occult equine infectious anemia virus (EIAV) infections in horses.

Molecular and serological techniques for Equine Infectious Anemia Virus (EIAV) diagnosis were compared using samples from 59 clinically normal horses stabled on five farms in the Santa Fe Province of Argentina. Of these 26 (44.1%) were positive in official AGID tests and/or gp45/gp90-based ELISA. Surprisingly 18 of the 33 seronegative horses were positive in a PCR against viral sequences encoding gp45 (PCR-positive/AGID-negative) with all but one remaining EIAV-antibody negative throughout a two year observation period. The gp45 PCR results are supported by fact that 7/18 of these horses were positive in the Office International des Epizooties (OIE) recommended EIAV gag gene specific PCR plus 2 of this 7 also reacted in a PCR directed predominantly against the 5' untranslated region of the viral genome. Furthermore sufficient quantities of serum were available from 8 of these horses to verify their seronegative status in sensitive Western Blot tests and demonstrate by ELISA the absence of EIAV-specific antibodies was not attributable to abnormalities in total IgG concentration. Studies involving 7 of the PCR-positive/AGID-negative horses to measure lymphocyte proliferation in the presence of PHA showed no significant differences between this group and control animals. In addition, lymphocytes from 2 of these 7 horses responded to peptides derived from gp90 and gp45. Together these results demonstrate that apparently clinically normal horses with no gross signs of immunodeficiency in terms of total IgG concentration or T helper-cell function can remain seronegative for at least 24 months while harboring EIAV specific nucleic acid sequences.

HIV-1 epidemiology and circulating subtypes in the countryside of South Brazil

INTRODUCTION: Human immunodeficiency virus type 1 (HIV-1) has spread worldwide, with several subtypes and circulating recombinant forms. Brazil has an incidence of 20.5 HIV-1/acquired immunodeficiency syndrome (AIDS) patients per 100,000 inhabitants; however, the Southernmost State of Rio Grande do Sul (RS) has more than twice the number of HIV-1-infected people (41.3/100,000 inhabitants) and a different pattern of subtype frequencies, as previously reported in studies conducted in the capital (Porto Alegre) and its metropolitan region. This study examined HIV-1/AIDS epidemiological and molecular aspects in the countryside of Rio Grande do Sul. METHODS: Socio-demographic, clinical and risk behavioral characteristics were obtained from HIV-1-positive adult patients using a structured questionnaire. HIV-1 subtypes were determined by nested-polymerase chain reaction (PCR) and sequencing of the pol and env genes. RESULTS: The study sample included 149 (55% women) patients with a mean age of 41.8 ± 11.9 years. Most (73.8%) patients had a low education level and reported heterosexual practices as the most (91.9%) probable transmission route. HIV-1 subtypes were detected in 26 patients: 18 (69.2%) infected with subtype C, six (23.1%) infected with subtype B and two (7.7%) infected with BC recombinant forms. CONCLUSIONS: These data highlight the increasing number of HIV-1 subtype C infections in the countryside of South Brazil. .

Genetic diversity in the feline leukemia virus gag gene.

Feline leukemia virus (FeLV) belongs to the Gammaretrovirus genus and is horizontally transmitted among cats. FeLV is known to undergo recombination with endogenous retroviruses already present in the host during FeLV-subgroup A infection. Such recombinant FeLVs, designated FeLV-subgroup B or FeLV-subgroup D, can be generated by transduced endogenous retroviral env sequences encoding the viral envelope. These recombinant viruses have biologically distinct properties and may mediate different disease outcomes. The generation of such recombinant viruses resulted in structural diversity of the FeLV particle and genetic diversity of the virus itself. FeLV env diversity through mutation and recombination has been studied, while gag diversity and its possible effects are less well understood. In this study, we investigated recombination events in the gag genes of FeLVs isolated from naturally infected cats and reference isolates. Recombination and phylogenetic analyses indicated that the gag genes often contain endogenous FeLV sequences and were occasionally replaced by entire endogenous FeLV gag genes. Phylogenetic reconstructions of FeLV gag sequences allowed for classification into three distinct clusters, similar to those previously established for the env gene. Analysis of the recombination junctions in FeLV gag indicated that these variants have similar recombination patterns within the same genotypes, indicating that the recombinant viruses were horizontally transmitted among cats. It remains to be investigated whether the recombinant sequences affect the molecular mechanism of FeLV transmission. These findings extend our understanding of gammaretrovirus evolutionary patterns in the field.