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 Małopolska 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

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

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

Compartmentalization of Subtype A17 of Small Ruminant Lentiviruses between Blood and Colostrum in Infected Goats Is Not Exclusively Associated to the env Gene

The compartmentalization of small ruminant lentiviruses (SRLVs) subtype A17 was analyzed in colostrum and peripheral blood leukocyte cells of three naturally infected goats. This study aimed to analyze heterogeneity of the SRLV env (V4V5) gene, which encodes neutralizing epitopes of SU glycoprotein, the gag gene encoding capsid protein (CA), and LTR, a noncoding region, responsible for determination of cell tropism. Compartmentalization was assessed using six established tree or distance-based methods, including permutation test to determine statistical significance. We found statistical evidence of compartmentalization between blood and colostrum in all infected goats although phylogenetic evidence of such compartmentalization was not obvious. Our study demonstrated that compartmentalization is not exclusively specific to the env gene, as we revealed that gag and LTR sequences are also compartmentalized between blood and colostrum. The work also confirms the combined use of different methods as essential for reliable determination of intrahost viral compartmentalization. Identifying and characterizing distinct viral subpopulations and the genetic evolution of SRLV in specific anatomical sites enhances our overall understanding of SRLV pathogenesis, immune control, and particularly virus transmission

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

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

EFFECTS OF OCHRATOXIN A, FOR DNA REPAIR IN CULTURES OF SWINE LYMPHOCYTES AND KIDNEY CELLS

Abstract Cytogenetic and cytotoxic effects of ochratoxin A (OTA) on in vitro cultures of swine lymphocytes and kidney cells (SK-6) were investigated. DNA synthesis and DNA repair, as well as cell viability and cell cytotoxicity, following OTA treatment were determined. In cultured swine lymphocytes, the OTA induced unscheduled DNA synthesis (UDS) only in a narrow concentration range. Using a culture medium supplemented with 1% foetal calf serum at 1µM OTA a week, induction of DNA repair was observed. Concentration above 1 µM OTA was cytotoxic and no induction occurred. In cultured swine kidney cells, a target organ of the mycotoxin OTA, induced UDS in a concentration-dependent manner, and at 2 µM OTA the maximum repair of DNA was observed, furthermore it was several times higher than that in lymphocytes. Above 2 µM OTA was cytotoxic in a manner the same as in lymphocytes. This amount was comparable to control cultures incubated with 0.1 µM of aflatoxin B 1 , which is known as one of the strongest genotoxic compounds. These results show that induction of UDS in lymphocytes is relatively weak, whereas this effect was significant in kidney cells. Whether this effect is due to OTA metabolites formed locally in kidney cells, cannot be excluded, since these cells have been shown to be able to metabolise xenobiotics independently from the liver

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 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