Cytokine secretion in stem cells of cattle infected with bovine leukaemia virus.

Introduction: Bovine leukaemia virus (BLV) is a Deltaretrovirus responsible for enzootic bovine leukosis, the most common neoplastic disease of cattle. It deregulates the immune system, favouring secondary infections and changes in the blood and lymphatic tissues. Blood homeostasis depends on functional haematopoietic stem cells (HSCs). Bone marrow is populated by these cells, which express CD34+ and CD35+ surface antigens and produce and release cytokines involved in the maintenance of haematopoiesis. The aim of the study was determination of the profile of cytokine production by CD34+ stem cells of cattle naturally infected with BLV. Material and Methods: The HSCs were generated from the blood and lymphoid organs of cows infected with BLV and healthy control cows with immunomagnetic separation and anti-CD34+ monoclonal antibodies. Isolated CD34+ cells were cultivated for two weeks with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor. The levels of IL-6, IL-10, IL-12p40, IL-12p70, interferon gamma (IFN-γ) and tumour necrosis factor alpha (TNF-α) were determined in culture fluid by flow cytometry. Results: The expression of IL-6, IL-12p70 and TNF-α in blood HSCs was higher in BLV+ cows than in the control animals. In bone marrow HSCs of infected cows, IL-12, TNF-α and IFN-γ were more concentrated, but in these cows' spleen HSCs only expression of IL-10 was elevated. In HSCs isolated from the lymph nodes of leukaemic cows, only TNF-α secretion was lower than in control cows, the other cytokines being more potently secreted. Conclusion: Infection with BLV caused statistically significant differences in cytokine expression by HSC CD34+ cells.

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.

Development of a recombinant protein-based ELISA for detection of antibodies against bovine herpesvirus 6 (BoHV6).

Introduction: Bovine herpesvirus 6 (BoHV6) belongs to the Herpesviridae family, Gammaherpesvirinae subfamily and Macavirus genus. It is common in cattle, but was also detected in American bison (Bison bison) and water buffalo (Bubalus bubalis). The aim of the experiment was to develop an ELISA for serological examination of cattle sera for the presence of anti-BoHV6 specific antibodies. Material and Methods: Viral DNA from a BoHV6-positive cow was amplified by qPCR and the resulting fragments of the gB and gH genes encoding glycoproteins B and H (gB and gH) were cloned into the pLATE52 vector to express recombinant gB (rgB) and gH (rgH) in Rosetta (DE3) E. coli. The expressed recombinant proteins were used as antigens in the developed ELISA. Results: The proteins expressed had the expected molecular weight. A total of 143 sera were examined, and 141 of them were positive, according to the chosen cut-off values of 9% and 10% for the sample-to-positive ratios of the rgB and rgH antigens, respectively. Conclusion: The rgB and rgH recombinant antigens of BoHV6 were successfully expressed in E. coli and successfully used in a newly developed ELISA.

An Outbred Calf Model for Determining Innate Immune Sensing and Evolutionary Trajectories of a Cell Culture-Adapted Bovine Foamy Virus Variant.

Bovine foamy virus (BFVbta) displays a very high degree of cell-associated replication which is unprecedented even among the other known foamy viruses. Interestingly, recent studies have shown that it can in fact adapt in vitro to high-titer (HT) cell-free transmission due to genetic changes acquired during repeated rounds of cell-free BFVbta passages in immortalized bovine MDBK cells. Molecular clones obtained from the HT BFVbta Riems cell-free variant (HT BFVbta Riems) have been thoroughly characterized in MDBK cell cultures However, during recent years, it has become increasingly clear that the source of the host cells used for virus growth and functional studies of virus replication and virus-cell interactions plays a paramount role. Established cell lines, mostly derived from tumors, but occasionally experimentally immortalized and transformed, frequently display aberrant features relating, for example. to growth, metabolism, and genetics. Even state-of-the-art organoid cultures of primary cells cannot replicate the conditions in an authentic host, especially those concerning cell diversity and the role of innate and adaptive immunity. Therefore, to determine the overall replication characteristics of the cloned wt and HT BFVbta Riems variant, we conducted a small-scale animal pilot study. The replication of the original wt BFVbta Riems isolate, as well as that of its HT variant, were analyzed. Both BFVbta variants established infection in calves, with proviruses in peripheral blood mononuclear cells and induced Gag-specific antibodies. In addition, a related pattern in the host innate immune reaction was detected in the peripheral blood leukocytes of the BFV-infected calves. Surprisingly, an analysis of the Gag sequence two weeks post-inoculation revealed that the HT BFVbta variant showed a very high level of genetic reversion to the wild type (parental BFVbta genotype).

A large-scale study on the seroprevalence of small ruminant lentiviral infection in the Polish goat population.

A large-scale study was carried out in a Polish goat population in 2014-2022 to determine the herd-level (between-herd) and within-herd seroprevalence of small ruminant lentivirus (SRLV) infection. A total of 8354 adult goats (aged >1 year) from 165 herds located in various regions of Poland were serologically tested using a commercial ELISA. One hundred twenty eight herds were randomly selected while 37 were enrolled based on convenience non-random sampling. At least 1 seropositive result was obtained in 103 / 165 herds. For all these herds the probability that they were truly positive (herd-level positive predictive value) was calculated. It was ≥ 90% in 91 seropositive herds and 73% to < 90% in 12 herds in which only 1-4 goats were seropositive (22 goats in total). The seropositive goats in the latter herds were retested using a different commercial ELISA and 14 goats (9 males and 5 females) from 9 herds were confirmed to be seropositive (serial testing). The true herd-level seroprevalence was estimated at 61% (95% confidence interval [CI 95%]: 53%-68%). It differed significantly between herd size classes (p = 0.003): the highest prevalences were found in the medium (51 - 100 adult goats) and large herds (>100 adult goats) - 72% (CI 95%: 56-84%) and 86% (CI 95%: 67%-95%), respectively, while prevalences in very small (≤ 20 adult goats) and small herds (21 - 50 adult goats) were 46% (CI 95%: 34%-59%) and 57% (CI 95%: 43%-70%), respectively. The true herd-level seroprevalence differed significantly also between geographical regions of Poland (p = 0.003), with the highest values in the north-western and the lowest in the southern region of the country. The true within-herd seroprevalence estimated using a Bayesian approach ranged from 0.7% to 100% with the median (IQR) of 42% (17%-84%), and did not vary significantly between herd size classes (p = 0.393) or geographical regions of Poland (p = 0.570). Concluding, SRLV infection is widespread in the Polish goat population, the north-western region of Poland is most extensively infected, and herds counting > 50 adult goats are more often infected.

Genetic Diversity of the LTR Region of Polish SRLVs and Its Impact on the Transcriptional Activity of Viral Promoters.

A long terminal repeat (LTR) plays an indispensable role in small ruminant lentivirus (SRLV) gene expression. In this study, we present the LTR sequence of Polish SRLVs representing different subtypes, and analyzed their impact on SRLV promoter activity, as measured in transient transfection assays. Although certain nucleotide motifs (AML(vis), TATA box and the polyadenylation site (AATAAA)) were conserved across sequences, numerous mutations within the LTR sequences have been identified. Single nucleotide polymorphisms (SNPs) were detected in both regulatory (AP-1, AP-4, Stat and Gas) and non-regulatory sequences, and subtype-specific genetic diversity in the LTR region of Polish SRLVs was observed. In vitro assays demonstrated subtype-specific functional differences between the LTR regions of distinct SRLV subtypes. Our results revealed that the promoter activity of Polish strains was lower (1.64-10.8-fold) than that noted for the K1514 reference strain; however, the differences in most cases were not statistically significant. The lowest promoter activity was observed for strains representing subtype A5 (mean 69.067) while the highest promoter activity was observed for strain K1514 representing subtype A1 (mean 373.48). The mean LTR activities of strains representing subtypes A12, A17, A23, A18 and A24 were 91.22, 137.21, 178.41, 187.05 and 236.836, respectively. The results of the inter-subtype difference analysis showed that the promoter activity of strains belonging to subtype A5 was significantly lower than that for subtype A12 strains (1.32-fold; p < 0.00). The promoter activities of the A5 strain were 1.98-fold and 2.58-fold less active than that of the A17 and A23 strains, and the promoter activities of A12 strains were 1.955 and 1.5 times lower than the promoter activity of A23 and A17 strains, respectively. Furthermore, the promoter activity of A17 strains was 1.3 lower than the promoter activity of A23 strains. Our findings suggest that subtype-specific genetic diversity, mainly in the transcription factor's binding sites, has an impact on their transcriptional activity, producing a distinct activity pattern for the subtypes. This study provides new information that is important for better understanding the function of the SRLV LTR. However, further research including more strains and subtypes as well as other cell lines is needed to confirm these findings.

Expression of Bovine Leukaemia Virus (BLV) Gp51 Protein in Blood and Milk Cells of Cows with Leukosis.

Introduction: Bovine leukaemia virus (BLV) is the retroviral causative agent of enzootic bovine leukosis, the most common neoplastic disease of cattle and a serious problem worldwide. Its diagnosis is commonly by tests for antibodies recognising the p24 capsid protein and structural glycoprotein (gp) 51. With flow cytometry recently having come to veterinary immunology, applications for it may now include BLV. The study determined BLV gp51 expression in blood and milk lymphocytes of naturally infected cows by flow cytometry. Material and Methods: Nineteen Polish Black and White Lowland breed cows aged 4-9 years and naturally infected with BLV and ten uninfected counterparts had blood and milk sampled and cultured. The immunological status of the animals was confirmed with ELISA and PCR. Dual-colour flow cytometry analysis was performed with specific monoclonal antibodies for lymphocyte cluster of differentiation (CD) markers and gp51 viral envelope protein and conjugates labelled with fluorescein isothiocyanate or phycoerythrin. Bovine leukaemia virus gp51 was confirmed in lymphocytes by immunofluorescence with anti-gp51 monoclonal antibodies. Results: The gp51 antigen was detected in blood and milk lymphocytes of infected cows, but the percentage of cells expressing it in milk was much lower than in blood. A depleted number of CD4+ lymphocytes, an augmented number of CD8+ lymphocytes, a lower ratio of CD4+ to CD8+ and a proliferation of CD19+ immunoglobulin M+ cells were also found. Conclusion: These proliferated cells were immature, gave no sign of a tendency to differentiation and were characterised by prolonged vitality.

Occurrence of Equine Foamy Virus Infection in Horses from Poland.

Equine foamy virus (EFVeca) is a foamy virus of non-primate origin and among the least-studied members of this retroviral subfamily. By sequence comparison, EFVeca shows the highest similarity to bovine foamy virus. In contrast to simian, bovine or feline foamy viruses, knowledge about the epidemiology of EFVeca is still limited. Since preliminary studies suggested EFVeca infections among horses in Poland, we aimed to expand the diagnostics of EFVeca infections by developing specific diagnostic tools and apply them to investigate its prevalence. An ELISA test based on recombinant EFVeca Gag protein was developed for serological investigation, while semi-nested PCR for the detection of EFVeca DNA was established. 248 DNA and serum samples from purebred horses, livestock and saddle horses, Hucul horses and semi-feral Polish primitive horses were analyzed in this study. ELISA was standardized, and cut off value, sensitivity and specificity of the test were calculated using Receiver Operating Characteristic and Bayesian estimation. Based on the calculated cut off, 135 horses were seropositive to EFVeca Gag protein, while EFVeca proviral DNA was detected in 85 animals. The rate of infected individuals varied among the horse groups studied; this is the first report confirming the existence of EFVeca infections in horses from Poland using virus-specific tools.

Transcriptomic Analysis of MDBK Cells Infected with Cytopathic and Non-Cytopathic Strains of Bovine Viral Diarrhea Virus (BVDV).

Bovine viral diarrhea virus (BVDV) belongs to the Flaviviridae family and the Pestivirus genus. Infection with BVDV causes a disease with a wide spectrum of clinical symptoms, most often mild, although infections with this virus constitute a serious economic problem all over the world. The virus is characterized by a high genetic variability, while the accumulation of single mutations leads to the formation of its new variants. The aim of this study was to better understand the complicated pathogenesis of this disease at the molecular level via the analysis of the transcriptome of cells infected with this virus. The bovine kidney cell line (MDBK), the cytopathic (cp) reference strain, and two non-cytopathic (ncp) BVD virus field strains were used in transcriptomic studies. The cell transcriptome was tested 24 and 72 h after infection. The results of the microarray analysis revealed changes in the expression levels of numerous genes. Genes with changed expression as a result of infection with the cp strain caused changes in the expression levels of a large number of genes and enriched a number of pathways. Genes with increased expression levels were enriched among other pathways involved in the cell cycle, while genes with reduced expression levels enriched pathways mostly related to metabolism. Genes with increased expression levels as a result of infection with ncp strains enriched a much smaller number of pathways, among them, pathways related to signaling activity 24 h post-infection and serine biosynthetic pathways both 24 and 72 h post-infection. Pathways enriched by genes with reduced expression levels were related to the innate immune response (72 h post-infection) or metabolism (24 and 72 h post-infection). The results of microarray studies can help us to better understand the host's response to BVDV infection.

Molecular Characterization of Small Ruminant Lentiviruses Isolated from Polish Goats with Arthritis.

Previous studies revealed that the small ruminant lentivirus (SRLV) population in Poland is highly heterogeneous. All SRLVs detected from Polish sheep and goats so far have belonged to subtypes B1, B2, A1, A5, A12, A13, A16, A17, A18, A23 and A24. However, all characterized strains originated from asymptomatic animals. This is the first study that characterizes the molecular properties of SRLVs isolated from different organs of six arthritic goats. Segments from three genomic regions (gag, LTR and env) were analyzed. In addition, we quantified the SRLV proviral load in the blood and different organs and examined its association with different degrees of histopathological lesions. All sequences obtained from the goats involved in this study were homogeneous, showing an average degree of variability of 4.8%, 3.7% and 8.8% for gag, LTR and env, respectively. Phylogenetic analysis revealed that the sequences from the analyzed goats were clustered within SRLVs group A and formed a new subtype within this group, tentatively named A27. The histopathological examination of the lung, mammary gland, synovial membranes of joints and brain of the analyzed goats revealed evidence of inflammatory processes associated with SRLV infection, which was confirmed by positive immunohistochemistry assays. No significant correlation was observed between histological features and alterations in the sequences from different tissues. No tissue-specific signature pattern was identified. It was shown that animals with a higher proviral load showed more lesion severity in various SRLV-affected tissues, indicating a positive association between these two parameters. Our results also revealed differences in the SRLV load between animals even though the sequences derived from all of the goats were closely related, suggesting that host factors may restrict and control viral replication. This study provides new information about SRLV variants isolated from arthritic goats; however, more studies, including the isolation and characterization of biological properties of these viruses, should be performed to evaluate their pathogenic potential.

Molecular Characterization of Bovine Leukemia Virus with the Evidence of a New Genotype Circulating in Cattle from Kazakhstan.

Bovine leukemia virus (BLV) is a retrovirus that causes enzootic bovine leukosis (EBL) and has worldwide distribution. Infections with BLV have been reported in cattle from Kazakhstan but the virus has not yet been thoroughly characterized. In this study, we detect and estimate the level of BLV proviral DNA by qPCR in DNA samples from 119 cattle naturally infected with BLV, from 18 farms located in four different geographical regions of Kazakhstan. Furthermore, we conducted the phylogenetic and molecular analysis of 41 BLV env-gp51 gene sequences from BLV infected cattle. Phylogenetic analysis showed the affiliation of sequences to two already known genotypes G4 and G7 and also to a new genotype, classified as genotype G12. In addition, a multivariate method was employed for analysis of the association between proviral load and different variables such as the geographical location of the herd, cattle breeds, age of animals, and the presence of particular BLV genotypes. In summary, the results of this study provide the first evidence on molecular characterization of BLV circulating in cattle from Kazakhstan.

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.

Phylogenetic Analysis of Small Ruminant Lentiviruses Originating from Naturally Infected Sheep and Goats from Poland Based on the Long Terminal Repeat Sequences.

Introduction: Previous gag and env sequence studies placed Polish small ruminant lentiviruses (SRLVs) isolated from sheep and goats in subtypes B1, B2, A1, A5, A12, A13, A16-A18, A23, A24 and A27. This study extended the genetic/phylogenetic analysis of previously identified Polish SRLV strains by contributing long terminal repeat (LTR) sequences. Material and Methods: A total of 112 samples were analysed. Phylogenetic analyses were carried out on the LTR fragment using the neighbour-joining, maximum likelihood, and unweighted pair group method with arithmetic mean methods. Results: Polish caprine and ovine LTR sequences clustered within group A and grouped in at least 10 clusters (subtypes A1, A5, A12, A13, A16-A18, A23, A24 and A27). Most of the Polish strains (78%) belonged to the same subtype by the indication of the gag, env and LTR genomic regions. Discrepancies in affiliation depending on the particular sequence were observed in 24 (21%) strains, most of which came from mixed-species flocks where more than one SRLV genotype circulated. Sequences of the LTR reflected subtype-specific patterns. Several subtype-specific markers were identified, e.g. a unique substitution of T to A in the fifth position of the TATA box in A17, A27, A20 and B3. Conclusion: This study provides valuable insights into the genetic diversity of SRLV field strains in Poland, their phylogenetic relationships and their position in the recently established SRLV classification. Our results confirmed the existence of the ten subtypes listed and the readier emergence of new SRLV variants in mixed-species flocks.

Quasispecies Composition of Small Ruminant Lentiviruses Found in Blood Leukocytes and Milk Epithelial Cells.

Small ruminant lentiviruses (SRLVs) exist as populations of closely related genetic variants, known as quasispecies, within an individual host. The privileged way of SRLVs transmission in goats is through the ingestion of colostrum and milk of infected does. Thus, characterization of SRLV variants transmitted through the milk, including milk epithelial cells (MEC), may provide useful information about the transmission and evolution of SRLVs. Therefore, the aim of this study was to detect SRLVs in peripheral blood leukocytes (PBLs) and milk epithelial cells of goats naturally infected with SRLVs and perform single nucleotide variations analysis to characterize the extent of genetic heterogeneity of detected SRLVs through comparison of their gag gene sequences. Blood and milk samples from 24 seropositive goats were tested in this study. The double immunolabeling against p28 and cytokeratin demonstrated that milk epithelial cells originated from naturally infected goats were infected by SRLVs. Moreover, PCR confirmed the presence of the integrated SRLVs proviral genome indicating that MECs may have a role as a reservoir of SRLVs and can transmit the virus through milk. The blood and MEC derived sequences from 7 goats were successfully sequenced using NGS and revealed that these sequences were genetically similar. The MEC and blood-derived sequences contained from 3 to 30 (mean, 10.8) and from 1 to 10 (mean, 5.4) unique SNVs, respectively. In five out of seven goats, SNVs occurred more frequent in MEC derived sequences. Non-synonymous SNVs were found in both, PBLs and MEC-derived sequences of analyzed goats and their total number differed between animals. The results of this study add to our understanding of SRLVs genomic variability. Our data provides evidence for the existence of SRLVs quasispecies and to our knowledge, this is the first study that showed quasispecies composition and minority variants of SRLVs present milk epithelial cells.

Molecular Characterization of Small Ruminant Lentiviruses in Polish Mixed Flocks Supports Evidence of Cross Species Transmission, Dual Infection, a Recombination Event, and Reveals the Existence of New Subtypes within Group A.

Small ruminant lentiviruses (SRLVs) are a group of highly divergent viruses responsible for global infection in sheep and goats. In a previous study we showed that SRLV strains found in mixed flocks in Poland belonged to subtype A13 and A18, but this study was restricted only to the few flocks from Malopolska region. The present work aimed at extending earlier findings with the analysis of SRLVs in mixed flocks including larger numbers of animals and flocks from different part of Poland. On the basis of gag and env sequences, Polish SRLVs were assigned to the subtypes B2, A5, A12, and A17. Furthermore, the existence of a new subtypes, tentatively designed as A23 and A24, were described for the first time. Subtypes A5 and A17 were only found in goats, subtype A24 has been detected only in sheep while subtypes A12, A23, and B2 have been found in both sheep and goats. Co-infection with strains belonging to different subtypes was evidenced in three sheep and two goats originating from two flocks. Furthermore, three putative recombination events were identified within gag and env SRLVs sequences derived from three sheep. Amino acid (aa) sequences of immunodominant epitopes in CA protein were well conserved while Major Homology Region (MHR) had more alteration showing unique mutations in sequences of subtypes A5 and A17. In contrast, aa sequences of surface glycoprotein exhibited higher variability confirming type-specific variation in the SU5 epitope. The number of potential N-linked glycosylation sites (PNGS) ranged from 3 to 6 in respective sequences and were located in different positions. The analysis of LTR sequences revealed that sequences corresponding to the TATA box, AP-4, AML-vis, and polyadenylation signal (poly A) were quite conserved, while considerable alteration was observed in AP-1 sites. Interestingly, our results revealed that all sequences belonging to subtype A17 had unique substitution T to A in the fifth position of TATA box and did not have a 11 nt deletion in the R region which was noted in other sequences from Poland. These data revealed a complex picture of SRLVs population with ovine and caprine strains belonging to group A and B. We present strong and multiple evidence of dually infected sheep and goats in mixed flocks and present evidence that these viruses can recombine in vivo.

Transcriptome Analysis for Genes Associated with Small Ruminant Lentiviruses Infection in Goats of Carpathian Breed.

Small ruminant lentiviruses (SRLV) are economically important viral pathogens of sheep and goats. SRLV infection may interfere in the innate and adaptive immunity of the host, and genes associated with resistance or susceptibility to infection with SRLV have not been fully recognized. The presence of animals with relatively high and low proviral load suggests that some host factors are involved in the control of virus replication. To better understand the role of the genes involved in the host response to SRLV infection, RNA sequencing (RNA-seq) method was used to compare whole gene expression profiles in goats carrying both a high (HPL) and low (LPL) proviral load of SRLV and uninfected animals. Data enabled the identification of 1130 significant differentially expressed genes (DEGs) between control and LPL groups: 411 between control and HPL groups and 1434 DEGs between HPL and LPL groups. DEGs detected between the control group and groups with a proviral load were found to be significantly enriched in several gene ontology (GO) terms, including an integral component of membrane, extracellular region, response to growth factor, inflammatory and innate immune response, transmembrane signaling receptor activity, myeloid differentiation primary response gene 88 (MyD88)-dependent toll-like receptor signaling pathway as well as regulation of cytokine secretion. Our results also demonstrated significant deregulation of selected pathways in response to viral infection. The presence of SRLV proviral load in blood resulted in the modification of gene expression belonging to the toll-like receptor signaling pathway, the tumor necrosis factor (TNF) signaling pathway, the cytokine-cytokine receptor interaction, the phagosome, the Ras signaling pathway, the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) (PI3K-Akt) signaling pathway and rheumatoid arthritis. It is worth mentioning that the most predominant in all pathways were genes represented by toll-like receptors, tubulins, growth factors as well as interferon gamma receptors. DEGs detected between LPL and HPL groups were found to have significantly enriched regulation of signaling receptor activity, the response to toxic substances, nicotinamide adenine dinucleotide (NADH) dehydrogenase complex assembly, cytokine production, vesicle, and vacuole organization. In turn, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway tool classified DEGs that enrich molecular processes such as B and T-cell receptor signaling pathways, natural killer cell-mediated cytotoxicity, Fc gamma R-mediated phagocytosis, toll-like receptor signaling pathways, TNF, mammalian target of rapamycin (mTOR) signaling and forkhead box O (Foxo) signaling pathways, etc. Our data indicate that changes in SRLV proviral load induced altered expression of genes related to different biological processes such as immune response, inflammation, cell locomotion, and cytokine production. These findings provide significant insights into defense mechanisms against SRLV infection. Furthermore, these data can be useful to develop strategies against SRLV infection by selection of animals with reduced SRLV proviral concentration that may lead to a reduction in the spread of the virus.

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.

Single Nucleotide Polymorphisms in Genes Encoding Toll-Like Receptors 7 and 8 and Their Association with Proviral Load of SRLVs in Goats of Polish Carpathian Breed.

Toll-like receptors (TLRs) 7 and 8 are important in single-stranded viral RNA recognition, so genetic variation of these genes may play a role in SRLVs infection and disease progression. Present study aimed to identify SNPs in genes encoding TLR7 and TLR8 in goats of Carpathian breed and analyze their association with the SRLVs provirus concentration as index of disease progression. A total of 14 SNPs were detected, 6 SNPs in the TLR7 gene locus and 8 SNPs in the TLR8 gene. Nine of the 14 identified polymorphisms, 4 in the TLR7 gene and 5 in TLR8 gene, were significantly associated with the SRLVs proviral concentration. These SNPs were located in 3'UTR, 5'UTR and intron sequences as well as in the coding sequences, but they led to silent changes. Homozygous genotypes of three TLR7 SNPs (synonymous variant 1:50703293, 3'UTR variant 1:50701297 and 5'UTR variant 1:50718645) were observed in goats with lower provirus copy number as well as in seronegative animals. The results obtained in this study suggest that SNPs of TLR7/TLR8 genes may induce differential innate immune response towards SRLVs affecting proviral concentration and thereby disease pathogenesis and progression. These findings support a role for genetic variations of TLR7 and TLR8 in SRLVs infection and warrants further studies on the effect of TLR7/TLR8 polymorphisms on SRLVs infection in different populations.

Bayesian Estimation of the True Seroprevalence and Risk Factor Analysis of Bovine Leukemia Virus Infection in Pakistan.

The objective of this study was to determine the true seroprevalence of bovine leukemia virus (BLV) infection in dairy cattle from Pakistan at the animal and herd-level. We tested 1380 dairy cattle from 451 herds and 92 water buffalo. The sera were tested by ELISA and the results were analyzed using Bayesian inference. The median posterior estimate of the herd level true BLV prevalence was 1.4%, with a 95% credible interval (CI) 0.7-3.1, whereas the median posterior estimate of the within-farm true seroprevalence was 3.8% with a 95% CI 2.8-4.8. All 92 sera collected from water buffalo were negative. Several risk factors potentially associated with seropositivity to BLV infections in Pakistan were analyzed using logistic regression model based on calculation of an odds ratio (OR). The study showed an association between seropositivity and medium herd (≥50) size (OR = 23.57, 95% CI: 3.01-103.48). Common housing of indigenous cattle with exotic-breed cattle (OR = 0.67, 95% CI: 06-2.35) or housing indigenous or their crossbred cattle with exotic-breed cattle (OR = 0.95, 95% CI: 0.14-3.01) had no effect on the BLV seroprevalence. Similarly, common housing of cattle and water buffalo was not risk factor for increased BLV seropositivity (OR = 27.10, 95% CI: 0.63-119.34).