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

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

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.

Kinetic Study of BLV Infectivity in BLV Susceptible and Resistant Cattle in Japan from 2017 to 2019

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis. Polymorphism in bovine lymphocyte antigen (BoLA)-DRB3 alleles is related to susceptibility to BLV proviral load (PVL), which is a useful index for estimating disease progression and transmission risk. However, whether differential BoLA-DRB3 affects BLV infectivity remains unknown. In a three-year follow-up investigation using a luminescence syncytium induction assay for evaluating BLV infectivity, we visualized and evaluated the kinetics of BLV infectivity in cattle with susceptible, resistant and neutral BoLA-DRB3 alleles which were selected from 179 cattle. Susceptible cattle showed stronger BLV infectivity than both resistant and neutral cattle. The order of intensity of BLV infectivity was as follows: susceptible cattle > neutral cattle > resistant cattle. BLV infectivity showed strong positive correlation with PVL at each testing point. BLV-infected susceptible cattle were found to be at higher risk of horizontal transmission, as they had strong infectivity and high PVL, whereas BLV-infected resistant cattle were low risk of BLV transmission owing to weak BLV infection and low PVL. Thus, this is the first study to demonstrate that the BoLA-DRB3 polymorphism is associated with BLV infection.

Polymorphisms in HTLV-1 Tax-responsive elements in HTLV-1-associated myelopathy/tropical spastic paraparesis patients are associated with reduced proviral load but not with disease progression

Human T-lymphotropic virus type 1 (HTLV-1) provirus expression is mainly directed by Tax-responsive elements (TRE) within the long terminal repeats (LTR). Mutations in TRE can reduce provirus expression and since a high proviral load (PVL) is a risk factor for the development of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), we evaluated polymorphisms in the 5′ LTR and the association with PVL and disease progression. HTLV-1 LTR and tax sequences derived from asymptomatic carriers (AC) and HAM/TSP patients followed in a longitudinal study were analysed according to PVL and clinical severity. Individuals infected with HTLV-1 presenting the canonical TRE, considering strain ATK-1 as the consensus, displayed sustained higher PVL. By contrast, an LTR A125G mutation in TRE was associated with slightly reduced PVL only in HAM/TSP patients, although it did not influence the speed of disease progression. Moreover, this polymorphism was frequent in Latin American strains of the HTLV-1 Cosmopolitan Transcontinental subtype. Therefore, polymorphisms in the 5′ TRE of HTLV-1 may represent one of the factors influencing PVL in HAM/TSP patients, especially in the Latin American population. Indeed, higher PVL in the peripheral blood has been associated with an increased inflammatory activity in the spinal cord and to a poorer prognosis in HAM/TSP. However, this event was not associated with TRE polymorphisms.

Anti-HTLV-1/2 IgG Antibodies in the Breastmilk of Seropositive Mothers

Background: HTLV-1/2 mother-to-child transmission (MTCT) is an important route for the maintenance of HTLV-1/2 within populations and disproportionally contributes to the burden of HTLV-1-associated diseases. Avoidance of breastfeeding is the safest recommendation to prevent MTCT. Due to the benefits of breastfeeding, alternative methods that would allow seropositive mothers to breastfeed their babies are needed. There is limited knowledge about HTLV-1/2 infection and breastmilk. Methods: Paired blood and milk samples collected from HTLV-1/2 seropositive mothers were tested for HTLV-1 proviral load (PVL) quantification and for the detection of anti-HTLV-1/2 IgG. Results: All breastmilk samples had detectable anti-HTLV-1/2 IgG. HTLV-1/2 proviral DNA was detected in all samples except for one. HTLV-1 PVL and IgG binding ratio (BR) was similar in milk and plasma. However, antibody titer was significantly higher in blood (Median (95%CI): Milk:128 (32–512); Plasma:131,584 (16,000–131,584), p < 0.05). There was a strong correlation between HTLV-1 PVL, anti-HTLV-1/2 IgG BR, and titer when comparing milk and blood. PVL did not correlate with antibody BR nor titer in blood or milk. Conclusions: Anti-HTLV-1/2 IgG are present in milk in the same proportion as blood but in lower quantity. PVL in milk correlates with blood.

Genetic Analysis of the pX Region in Bovine Leukemia Virus genotype 1 in Holstein Friesian Cattle with Different Stages of Infection

The pX genetic region of the Bovine Leukemia Virus (BLV) includes four genes with overlapping reading frames that code for the Tax, Rex, R3 and G4 proteins. These proteins are involved in the regulation of transcriptional and post-transcriptional viral expression, as well as having oncogenic potential. Our goal was to determine the pathogenic role associated with BLV genotype 1 pX region genetics in terms of lymphocytosis, lymphomas and proviral load. We screened 724 serological samples from mixed-age Holstein Friesian cattle from six states in Mexico. Once peripheral blood leukocytes were isolated from whole blood with anticoagulant, we extracted genomic DNA using a commercial kit. Then, we designed in silico primers that hybridize in conserved regions of the BLV pX region, which allowed for PCR standardization to detect proviral DNA in infected cells. Positive amplicons were sequenced using the Sanger method, obtaining 1156 nucleotide-long final sequences that included the four pX region genes. The 30 heads of cattle that formed the genetic study population included 12 with lymphocytosis, 12 without lymphocytosis and six with lymphoma. Lymphoma presence was determined in six bovine tumor tissues using histopathology, and we identified BLV presence with in situ hybridization.Phylogenetic analysis determined that the 30 sequences were associated with genotype 1, and genetic distance between the sequences ranged from 0.2% - 2.09%. We identified two sequences in the G4 gene, one with a three-nucleotide deletion (AGU_7488L, in a cow with lymphocytosis), and one with a nine nucleotide deletion (AGU_18A, in a cow without lymphocytosis). PX region analysis identified positive selection in the G4, rex and R3 genes, and we found no difference in proviral load between the studied groups. It was not possible to establish an association between pX region variability and the development of lymphocytosis, lymphoma, asymptomatic status and proviral load in BLV-infected cattle.