Genetic Variability of Bovine Leukemia Virus: Evidence of Dual Infection, Recombination and Quasi-Species.

We have characterized the intrahost genetic variation in the bovine leukemia virus (BLV) by examining 16 BLV isolates originating from the Western Siberia-Tyumen and South Ural-Chelyabinsk regions of Russia. Our research focused on determining the genetic composition of an 804 bp fragment of the BLV env gene, encoding for the entire gp51 protein. The results provide the first indication of the quasi-species genetic nature of BLV infection and its relevance for genome-level variation. Furthermore, this is the first phylogenetic evidence for the existence of a dual infection with BLV strains belonging to different genotypes within the same host: G4 and G7. We identified eight cases of recombination between these two BLV genotypes. The detection of quasi-species with cases of dual infection and recombination indicated a higher potential of BLV for genetic variability at the intra-host level than was previously considered.

An immunoinformatics study reveals a new BoLA-DR-restricted CD4+ T cell epitopes on the Gag protein of bovine leukemia virus.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leucosis (EBL), which has been reported worldwide. The expression of viral structural proteins: surface glycoprotein (gp51) and three core proteins - p15 (matrix), p24 (capsid), and p12 (nucleocapsid) induce a strong humoral and cellular immune response at first step of infection. CD4+ T-cell activation is generally induced by bovine leukocyte antigen (BoLA) region- positive antigen-presenting cells (APC) after processing of an exogenous viral antigen. Limited data are available on the BLV epitopes from the core proteins recognized by CD4+ T-cells. Thus, immunoinformatic analysis of Gag sequences obtained from 125 BLV isolates from Poland, Canada, Pakistan, Kazakhstan, Moldova and United States was performed to identify the presence of BoLA-DRB3 restricted CD4+ T-cell epitopes. The 379 15-mer overlapping peptides spanning the entire Gag sequence were run in BoLA-DRB3 allele-binding regions using a BoLA-DRB- peptide binding affinity prediction algorithm. The analysis identified 22 CD4+ T-cell peptide epitopes of variable length ranging from 17 to 22 amino acids. The predicted epitopes interacted with 73 different BoLA-DRB3 alleles found in BLV-infected cattle. Importantly, two epitopes were found to be linked with high proviral load in PBMC. A majority of dominant and subdominant epitopes showed high conservation across different viral strains, and therefore could be attractive targets for vaccine development.

Transcriptome Analysis of Bovine Macrophages (BoMac) Cells after Infection with Bovine Immunodeficiency Virus.

Introduction: Bovine immunodeficiency virus (BIV) is found worldwide in cattle under natural conditions. However, the effect of BIV infection on immune functions has not been fully characterised. Material and Methods: Transcriptome analysis of BoMac cells after in vitro infection with BIV was performed using BLOPlus bovine microarrays. Genes identified as differentially expressed were subjected to functional analysis with the Ingenuity Pathway Analysis software (IPA). Results: Out of 1,743 genes with altered expression, 1,315 were mapped as unique molecules. In total, 718 genes were identified as upregulated and 597 genes as downregulated. Differentially expressed genes were involved in 16 pathways related to immune response. The most enriched canonical pathway was leukocyte extravasation signalling. Interleukin-15 (IL-15) production was indicated as the most activated pathway and the 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4) signalling pathway was the most inhibited one. In addition, the study showed that the inflammatory response was decreased during BIV infection. Conclusion: This is the first report to describe the microarray analysis of changes in gene expression upon BIV infection of bovine macrophages. Our data indicated how BIV influences the expression of genes and signalling pathways engaged in the immune response.

Molecular Characterization of the env Gene of Bovine Leukemia Virus in Cattle from Pakistan with NGS-Based Evidence of Virus Heterogeneity.

Characterization of the global genetic diversity of the bovine leukemia virus (BLV) is an ongoing international research effort. Up to now BLV sequences have been classified into eleven distinct genotypes. Although BLV genotyping and molecular analysis of field isolates were reported in many countries, there is no report describing BLV genotypes present in cattle from Pakistan. In this study we examined 27 env gene sequences from BLV-infected cattle coming from four farms located in Khyber Pakhtunkwa, Gilgit Baltisan and Punjab provinces. Phylogenetic analyses revealed the classification of Pakistani sequences into genotypes G1 and G6. The alignment with the FLK-BLV sequence revealed the presence of 45 mutations, namely, seven in genotype G1 and 33 in genotype G6. Five mutations were found in both, G1 and G6 genotypes. Twelve amino acid substitutions were found in the analyzed sequences, of which only one P264S was specific for sequences from Pakistan. Furthermore, a certain degree of nucleotide heterogeneity was identified by NGS. These results highlight the need for further study on the importance of genetic variability of BLV, especially in the context of its pathogenicity and potential effect on serological detection.

Analysis of Nucleotide Sequence of Tax, miRNA and LTR of Bovine Leukemia Virus in Cattle with Different Levels of Persistent Lymphocytosis in Russia.

Bovine Leukemia Virus (BLV) is the etiological agent of enzootic bovine leucosis (EBL), a lymphoproliferative disease of the bovine species. In BLV-infected cells, the long terminal repeat (LTR), the viral Tax protein and viral miRNAs promote viral and cell proliferation as well as tumorigenesis. Although their respective roles are decisive in BLV biology, little is known about the genetic sequence variation of these parts of the BLV genome and their impact on disease outcome. Therefore, the objective of this study was to assess the relationship between disease progression and sequence variation of the BLV Tax, miRNA and LTR regions in infected animals displaying either low or high levels of persistent lymphocytosis (PL). A statistically significant association was observed between the A(+187)C polymorphism in the downstream activator sequence (DAS) region in LTR (p-value = 0.00737) and high lymphocytosis. Our study also showed that the mutation A(-4)G in the CAP site occurred in 70% of isolates with low PL and was not found in the high PL group. Conversely, the mutations G(-133)A/C in CRE2 (46.7%), C(+160)T in DAS (30%) and A(310)del in BLV-mir-B4-5p, A(357)G in BLV-mir-B4-3p, A(462)G in BLV-mir-B5-5p, and GA(497-498)AG in BLV-mir-B5-3p (26.5%) were often seen in isolates with high PL and did not occur in the low PL group. In conclusion, we found several significant polymorphisms among BLV genomic sequences in Russia that would explain a progression towards higher or lower lymphoproliferation. The data presented in this article enabled the classification between two different genotypes; however, clear association between genotypes and the PL development was not found.

Effects of Naturally Occurring Mutations in Bovine Leukemia Virus 5'-LTR and Tax Gene on Viral Transcriptional Activity.

Bovine leukemia virus (BLV) is a deltaretrovirus infecting bovine B cells and causing enzootic bovine leucosis (EBL). The long terminal repeat (LTR) plays an indispensable role in viral gene expression. The BLV Tax protein acts as the main transactivator of LTR-driven transcription of BLV viral genes. The aim of this study was to analyze mutations in the BLV LTR region and tax gene to determine their association with transcriptional activity. LTRs were obtained from one hundred and six BLV isolates and analyzed for their genetic variability. Fifteen variants were selected and characterized based on mutations in LTR regulatory elements, and further used for in vitro transcription assays. Reporter vectors containing the luciferase gene under the control of each variant BLV promoter sequence, in addition to variant Tax expression vectors, were constructed. Both types of plasmids were used for cotransfection of HeLa cells and the level of luciferase activity was measured as a proxy of transcriptional activity. Marked differences in LTR promoter activity and Tax transactivation activity were observed amongst BLV variants. These results demonstrate that mutations in both the BLV LTR and tax gene can affect the promoter activity, which may have important consequences on proviral load, viral fitness, and transmissibility in BLV-infected cattle.

Regulation of Expression and Latency in BLV and HTLV.

Human T-lymphotrophic virus type 1 (HTLV-1) and Bovine leukemia virus (BLV) belong to the Deltaretrovirus genus. HTLV-1 is the etiologic agent of the highly aggressive and currently incurable cancer adult T-cell leukemia (ATL) and a neurological disease HTLV-1-associated myelopathy (HAM)/tropical spastic paraparesis (TSP). BLV causes neoplastic proliferation of B cells in cattle: enzootic bovine leucosis (EBL). Despite the severity of these conditions, infection by HTLV-1 and BLV appear in most cases clinically asymptomatic. These viruses can undergo latency in their hosts. The silencing of proviral gene expression and maintenance of latency are central for the establishment of persistent infection, as well as for pathogenesis in vivo. In this review, we will present the mechanisms that control proviral activation and retroviral latency in deltaretroviruses, in comparison with other exogenous retroviruses. The 5' long terminal repeats (5'-LTRs) play a main role in controlling viral gene expression. While the regulation of transcription initiation is a major mechanism of silencing, we discuss topics that include (i) the epigenetic control of the provirus, (ii) the cis-elements present in the LTR, (iii) enhancers with cell-type specific regulatory functions, (iv) the role of virally-encoded transactivator proteins, (v) the role of repressors in transcription and silencing, (vi) the effect of hormonal signaling, (vii) implications of LTR variability on transcription and latency, and (viii) the regulatory role of non-coding RNAs. Finally, we discuss how a better understanding of these mechanisms may allow for the development of more effective treatments against Deltaretroviruses.

Balance between Retroviral Latency and Transcription: Based on HIV Model.

The representative of the Lentivirus genus is the human immunodeficiency virus type 1 (HIV-1), the causative agent of acquired immunodeficiency syndrome (AIDS). To date, there is no cure for AIDS because of the existence of the HIV-1 reservoir. HIV-1 infection can persist for decades despite effective antiretroviral therapy (ART), due to the persistence of infectious latent viruses in long-lived resting memory CD4+ T cells, macrophages, monocytes, microglial cells, and other cell types. However, the biology of HIV-1 latency remains incompletely understood. Retroviral long terminal repeat region (LTR) plays an indispensable role in controlling viral gene expression. Regulation of the transcription initiation plays a crucial role in establishing and maintaining a retrovirus latency. Whether and how retroviruses establish latency and reactivate remains unclear. In this article, we describe what is known about the regulation of LTR-driven transcription in HIV-1, that is, the cis-elements present in the LTR, the role of LTR transcription factor binding sites in LTR-driven transcription, the role of HIV-1-encoded transactivator protein, hormonal effects on virus transcription, impact of LTR variability on transcription, and epigenetic control of retrovirus LTR. Finally, we focus on a novel clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/dCas9)-based strategy for HIV-1 reservoir purging.

Proinflammatory Cytokine Changes in Bronchoalveolar Lavage Fluid Cells Isolated from Pigs Infected Solely with Porcine Reproductive and Respiratory Syndrome Virus or Co-infected with Swine Influenza Virus.

INTRODUCTION: The study evaluated the patterns of local innate immune response in bronchoalveolar lavage fluid (BALF) cells of pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV) alone or co-infected with swine influenza virus (SIV). MATERIAL AND METHODS: The study was performed on 26 seven-week-old pigs in three groups: PRRSV-infected (n = 11), PRRSV and SIV-infected (n = 11), and control (n = 4). BALF was collected post euthanasia at 2 and 4 dpi (three piglets per inoculated group) and at 21 dpi (all remaining pigs). Expression of IFN-α, IFN-γ, IL-1ß, IL-6, IL-8, and IL-10 mRNA was quantified in BALF cells. PRRSV RNA was quantified in BALF samples using a commercial real-time RT-PCR kit. RESULTS: The three cytokines IFN-α, IFN-γ, and IL-1ß presented significant expression changes in all experimental pigs. In PRRSV-infected animals IL-8 also did, but in co-infected subjects IL-6 and IL-10 were the additional upregulated cytokines. The highest number of differentially expressed genes was observed at 4 dpi, and significant differences in cytokine gene expression did not occur between the experimental groups at any other time point. The mean PRRSV load in the BALF of PRRSV-infected pigs was higher than that of co-infected pigs at each time point, having statistical significance only at 4 dpi. CONCLUSION: The results of the study indicate that infection with PRRSV alone as well as with SIV interferes with innate and adaptive immune response in the infected host. They also showed that co-infection demonstrates additive effects on IL-6 and IL-10 mRNA expression levels.

Bovine leukemia virus long terminal repeat variability: identification of single nucleotide polymorphisms in regulatory sequences.

BACKGROUND: Limited data are available on the incidence of variations in nucleotide sequences of long terminal repeat (LTR) regions of Bovine Leukemia Virus (BLV). Consequently, the possible impact of SNPs on BLV LTR function are poorly elucidated. Thus, a detailed and representative study of full-length LTR sequences obtained from sixty-four BLV isolates from different geographical regions of Poland, Moldova, Croatia, Ukraine and Russia were analyzed for their genetic variability. METHODS: Overlap extension PCR, sequencing and Bayesian phylogenetic reconstruction of LTR sequences were performed. These analyses were followed by detailed sequence comparison, estimation of genetic heterogeneity and identification of transcription factor binding site (TFBS) modifications. RESULTS: Phylogenetic analysis of curated LTR sequences and those available in the GenBank database reflected the acknowledged env gene classification of BLV into 10 genotypes, and further clustered analysed sequences into three genotypes - G4, G7 and G8. Additional molecular studies revealed the presence of 97 point mutations distributed at 89 positions throughout all 64 LTR sequences. The highest rate of variability was noted in U3 and U5 subregions. However, the variability in regulatory sequences (VR) was assessed as lower than the variability within non-regulatory sequences (VNR) for both, U3 and U5 subregions. In contrast, VR value for R subregion, as well as for the total LTR, was higher than the VNR suggesting the existence of positive selection. Twelve unique SNPs for these LTR sequences localized in regulatory and non-regulatory elements were identified. The presence of different types of substitutions lead to the abrogation of present or to the creation of additional TFBS. CONCLUSION: This study represents the largest study of LTR genetic variability of BLV field isolates from Eastern part of Europe. Phylogenetic analysis of LTRs supports the clustering BLV variants based on their geographic origin. The SNP screening showed variations modifying LTR regulatory sequences, as well as altering TFBS. These features warrant further exploration as they could be related to proviral load and distinctive regulation of BLV transcription and replication.

Transcriptional profiles of PBMCs from pigs infected with three genetically diverse porcine reproductive and respiratory syndrome virus strains.

Porcine reproductive and respiratory syndrome virus is the cause of reproductive failure in sows and respiratory disease in young pigs, which has been considered as one of the most costly diseases to the worldwide pig industry for almost 30 years. This study used microarray-based transcriptomic analysis of PBMCs from experimentally infected pigs to explore the patterns of immune dysregulation after infection with two East European PRRSV strains from subtype 2 (BOR and ILI) in comparison to a Danish subtype 1 strain (DAN). Transcriptional profiles were determined at day 7 post infection in three tested groups of pigs and analysed in comparison with the expression profile of control group. Microarray analysis revealed differential regulation (> 1.5-fold change) of 4253 and 7335 genes in groups infected with BOR and ILI strains, respectively, and of 12518 genes in pigs infected with Danish strain. Subtype 2 PRRSV strains showed greater induction of many genes, especially those involved in innate immunity, such as interferon stimulated antiviral genes and inflammatory markers. Functional analysis of the microarray data revealed a significant up-regulation of genes involved in processes such as acute phase response, granulocyte and agranulocyte adhesion and diapedesis, as well as down-regulation of genes enrolled in pathways engaged in protein synthesis, cell division, as well as B and T cell signaling. This study provided an insight into the host response to three different PRRSV strains at a molecular level and demonstrated variability between strains of different pathogenicity level.

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.

Dissemination and genetic diversity of chlamydial agents in Polish wildfowl: Isolation and molecular characterisation of avian Chlamydia abortus strains.

Wild birds are considered as a reservoir for avian chlamydiosis posing a potential infectious threat to domestic poultry and humans. Analysis of 894 cloacal or fecal swabs from free-living birds in Poland revealed an overall Chlamydiaceae prevalence of 14.8% (n = 132) with the highest prevalence noted in Anatidae (19.7%) and Corvidae (13.4%). Further testing conducted with species-specific real-time PCR showed that 65 samples (49.2%) were positive for C. psittaci whereas only one was positive for C. avium. To classify the non-identified chlamydial agents and to genotype the C. psittaci and C. avium-positive samples, specimens were subjected to ompA-PCR and sequencing (n = 83). The ompA-based NJ dendrogram revealed that only 23 out of 83 sequences were assigned to C. psittaci, in particular to four clades representing the previously described C. psittaci genotypes B, C, Mat116 and 1V. Whereas the 59 remaining sequences were assigned to two new clades named G1 and G2, each one including sequences recently obtained from chlamydiae detected in Swedish wetland birds. G1 (18 samples from Anatidae and Rallidae) grouped closely together with genotype 1V and in relative proximity to several C. abortus isolates, and G2 (41 samples from Anatidae and Corvidae) grouped closely to C. psittaci strains of the classical ABE cluster, Matt116 and M56. Finally, deep molecular analysis of four representative isolates of genotypes 1V, G1 and G2 based on 16S rRNA, IGS and partial 23S rRNA sequences as well as MLST clearly classify these isolates within the C. abortus species. Consequently, we propose an expansion of the C. abortus species to include not only the classical isolates of mammalian origin, but also avian isolates so far referred to as atypical C. psittaci or C. psittaci/C. abortus intermediates.

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.

Transcriptomic microarray analysis of BoMac cells after infection with bovine foamy virus.

Bovine foamy virus (BFV) infections are highly prevalent among cattle worldwide. However, relatively little is known about the impact of this virus on the host immune system. In our study, we focused on a bovine macrophage cell line (BoMac) and examined changes in the BoMac transcriptome after in vitro infection with BFV using bovine BLOPlus oligo microarrays. One hundred twenty-four genes showed significant changes in expression level. The biological process categories found to be enriched include metabolic processes, cell communication, transport, immune system processes, and response to extracellular stimuli. RT-qPCR was applied to confirm the results obtained for representative genes.

The molecular characterization of bovine leukaemia virus isolates from Eastern Europe and Siberia and its impact on phylogeny.

Recent studies have shown that bovine leukemia virus (BLV) sequences can be classified into seven distinct genotypes based on full gp51 sequence. This classification was based on available sequence data that mainly represented the BLV population that is circulating in cattle from the US and South America. In order to aid with a global perspective inclusion of data from Eastern Europe is required. In this study we examined 44 BLV isolates from different geographical regions of Poland, Belarus, Ukraine, and Russia. Phylogenetic analysis based on a 444bp fragment of env gene revealed that most of isolates belonged to genotypes 4 and 7. Furthermore, we confirmed the existence of a new genotype, genotype 8, which was highly supported by phylogenetic computations. A significant number of amino acid substitutions were found in the sequences of the studied Eastern European isolates, of which 71% have not been described previously. The substitutions encompassed mainly the C-part of the CD4+ epitope, zinc binding peptide region, CD8+ T cell epitope, and overlapping linear epitope E. These observations highlight the use of sequence data to both elucidate phylogenetic relationships and the potential effect on serological detection of geographically diverse isolates.

[Review of the effectiveness of an empirical antibiotic therapy in suspected ventilator-associated pneumonia]. / Ocena skutecznosci rekomendowanych scheatów antybiotykoterapii emprirycznej VAP w oparciu o analize in vitro lekowraziliwosci glównych patogenów.

BACKGROUND: Ventilator-associated pneumonia (VAP) occurs in approximately 10-20% of mechanically ventilated patients, and is associated with an extremely high mortality rate (up to 70%). The purpose of the study was to determine the susceptibility spectrum of Klebsiella, Pseudomonas and Acinetobacter strains isolated from VAP patients. METHODS: We analysed 81 strains of microorganisms isolated from bronchoalveolar lavages (BAL) of VAP patients. The minimal inhibitory concentrations (MIC) of antibiotics recommended for empirical therapy were determined using an automated VITEK 2 system, and for the MIC of doripenem - the Etest assay. Results were analysed following the guidelines of the Clinical and Laboratory Standards Institute. RESULTS: For infections caused by the group of bacteria under investigation, the most successful regimen was monotherapy with carbapenems (doripenem, meropenem and imipenem). Cephalosporins (cefepim and ceftazidim) were less effective in vitro. The worst results were obtained with the combination of piperacillin/tazobactam with aminoglycosides (amikacin or gentamicin) or fluoroquinolones (ciprofloxacin). CONCLUSIONS: Antibiotic monotherapy proved to be more effective in VAP patients than combined therapy; the best results were achieved with carbapenems. Doripenem showed strong activity in vitro against P. aeruginosa and Klebsiella sp. and should be considered for empirical VAP therapy; however, carbapenems may be less effective against Acinetobacter baumannii. The wide range of bacteria, and their broad range of susceptibility to antibiotics, suggests the need for modification of current recommendations.