The bovine leukemia virus-derived long non-coding RNA AS1-S binds to bovine hnRNPM and alters the interaction between hnRNPM and host mRNAs.

Viruses utilize several strategies to cause latent infection and evade host immune responses. Long non-coding RNA (lncRNA), a class of non-protein-encoding RNA that regulates various cellular functions by interacting with RNA-binding proteins, plays important roles for viral latency in several viruses, such as herpesviruses and retroviruses, due to its lack of antigenicity. Bovine leukemia virus (BLV), which belongs to the family Retroviridae, encodes the BLV-derived lncRNA AS1-S, which is a major transcript expressed in latently infected cells. We herein identified bovine heterogeneous nuclear ribonucleoprotein M (hnRNPM), an RNA-binding protein located in the nucleus, as the binding partner of AS1-S using an RNA-protein pull-down assay. The pull-down assay using recombinant hnRNPM mutants showed that RNA recognition motifs (RRMs) 1 and 2, located in the N-terminal region of bovine hnRNPM, were responsible for the binding to AS1-S. Furthermore, RNA immunoprecipitation (RIP) assay results showed that the expression of AS1-S increased the number of mRNAs that co-immunoprecipitated with bovine hnRNPM in MDBK cells. These results suggested that AS1-S could alter the interaction between hnRNPM and host mRNAs, potentially interfering with cellular functions during the initial phase of mRNA maturation in the nucleus. Since most of the identified mRNAs that exhibited increased binding to hnRNPM were correlated with the KEGG term "Pathways in cancer," AS1-S might affect the proliferation and expansion of BLV-infected cells and contribute to tumor progression. IMPORTANCE BLV infects bovine B cells and causes malignant lymphoma, a disease that greatly affects the livestock industry. Due to its low incidence and long latent period, the molecular mechanisms underlying the progression of lymphoma remain enigmatic. Several non-coding RNAs (ncRNAs), such as miRNA and lncRNA, have recently been discovered in the BLV genome, and the relationship between BLV pathogenesis and these ncRNAs is attracting attention. However, most of the molecular functions of these transcripts remain unidentified. To the best of our knowledge, this is the first report describing a molecular function for the BLV-derived lncRNA AS1-S. The findings reported herein reveal a novel mechanism underlying BLV pathogenesis that could provide important insights for not only BLV research but also comparative studies of retroviruses.

A point mutation in GPI-attachment signal peptide accelerates the development of prion disease.

A missense variant from methionine to arginine at codon 232 (M232R) of the prion protein gene accounts for ~ 15% of Japanese patients with genetic prion diseases. However, pathogenic roles of the M232R substitution for the induction of prion disease have remained elusive because family history is usually absent in patients with M232R. In addition, the clinicopathologic phenotypes of patients with M232R are indistinguishable from those of sporadic Creutzfeldt-Jakob disease patients. Furthermore, the M232R substitution is located in the glycosylphosphatidylinositol (GPI)-attachment signal peptide that is cleaved off during the maturation of prion proteins. Therefore, there has been an argument that the M232R substitution might be an uncommon polymorphism rather than a pathogenic mutation. To unveil the role of the M232R substitution in the GPI-attachment signal peptide of prion protein in the pathogenesis of prion disease, here we generated a mouse model expressing human prion proteins with M232R and investigated the susceptibility to prion disease. The M232R substitution accelerates the development of prion disease in a prion strain-dependent manner, without affecting prion strain-specific histopathologic and biochemical features. The M232R substitution did not alter the attachment of GPI nor GPI-attachment site. Instead, the substitution altered endoplasmic reticulum translocation pathway of prion proteins by reducing the hydrophobicity of the GPI-attachment signal peptide, resulting in the reduction of N-linked glycosylation and GPI glycosylation of prion proteins. To the best of our knowledge, this is the first time to show a direct relationship between a point mutation in the GPI-attachment signal peptide and the development of disease.

Upregulation of host genes during disease progression in bovine leukemia virus infection is independent of overexpression of viral transcriptional regulators in vitro.

Bovine leukemia virus (BLV) is a member of the genus Deltaretrovirus within the family Retroviridae that infects bovine B cells, causing persistent lymphocytosis and enzootic bovine leukosis (EBL) in a small fraction of infected cattle. As changes in the transcriptome of infected cells are important for BLV disease progression, comprehensive analysis of gene expression in different disease states is required. In this study, we performed an RNA-seq analysis using samples from non-EBL cattle with and without BLV infection. Subsequently, a transcriptome analysis was conducted in combination with previously obtained RNA-seq data from EBL cattle. We found several differentially expressed genes (DEGs) between the three groups. After screening and confirmation of target DEGs using real-time reverse transcription polymerase chain reaction, we found that 12 target genes were significantly upregulated in EBL cattle compared to BLV-infected cattle without lymphoma. In addition, the expression levels of B4GALT6, ZBTB32, EPB4L1, RUNX1T1, HLTF, MKI67, and TOP2A were significantly and positively correlated with the proviral load in BLV-infected cattle. Overexpression experiments revealed that these changes were independent of BLV tax or BLV AS1-S expression in vitro. Our study provides additional information on host gene expression during BLV infection and EBL development, which may be helpful for understanding the complexity of transcriptome profiles during disease progression.

Development of droplet digital PCR for quantification of bovine leukemia virus proviral load using unpurified genomic DNA.

Bovine leukemia virus (BLV) is the causative agent of a B-cell tumor called enzootic bovine leukosis. Preventing BLV spreading is required to reduce economic loss related to BLV infection of livestock. To quantify proviral load (PVL) more easily and rapidly, we developed a quantification system of PVL using droplet digital PCR (ddPCR). This method uses a multiplex TaqMan assay of the BLV provirus and housekeeping gene RPP30 for the quantification of BLV in BLV-infected cells. Furthermore, we combined ddPCR with DNA purification-free sample preparation (unpurified genomic DNA). The percentage of BLV-infected cells based on unpurified genomic DNA was highly correlated with that based on purified genomic DNA (correlation coefficient: 0.906). Thus, this new technique is a suitable method to quantify PVL of BLV-infected cattle in a large sample number.

Extended application of the rapid post-mortem test kit for bovine spongiform encephalopathy to chronic wasting disease.

Chronic wasting disease (CWD) is a prion disease affecting cervid species primarily in the United States of America and Canada; however, it is now emerging in Scandinavian countries. Although CWD cases have not been reported in Japan, in case of a CWD outbreak occuring, it is critical to prepare for testing a large number of specimens. The present study showed that a rapid post-mortem test kit, which is used for bovine spongiform encephalopathy surveillance in Japan, is valid for the detection of CWD prion.

Novel single nucleotide polymorphisms in the bovine leukemia virus genome are associated with proviral load and affect the expression profile of viral non-coding transcripts.

Bovine leukemia virus (BLV) infects bovine B-cells and causes malignant lymphoma, resulting in severe economic losses in the livestock industry. To control the spread of BLV, several studies have attempted to clarify the molecular mechanisms of BLV pathogenesis, but the details of the mechanism are still enigmatic. Currently, viral non-coding RNAs are attracting attention as a novel player for BLV pathogenesis because these transcripts can evade the host immune response and are persistently expressed in latent infection. One of the viral non-coding RNA, AS1, is encoded in the antisense strand of the BLV genome and consists of two isoforms, AS1-L and AS1-S. Although the function of the AS1 is still unknown, the AS1 RNA might also have some roles because it keeps expressing in tumor tissues. In the present study, we identified novel single nucleotide polymorphisms (SNPs) located in the AS1 coding region and indicated that individuals infected with BLV with minor SNPs showed low proviral load. To evaluate the effect of identified SNPs, we constructed infectious clones with these SNPs and found that their introduction affected the expression profile of AS1 RNA; the amount of AS1-L isoform increased compared with the wild type, although the total amount of AS1 RNA remained unchanged. Prediction analysis also suggested that the introduction of SNPs changed the secondary structure of AS1 RNA. These results explain part of the relationship between BLV expansion in vivo and the expression profile of AS1, although further analysis is required.

Establishment of a simplified inverse polymerase chain reaction method for diagnosis of enzootic bovine leukosis.

Enzootic bovine leukosis (EBL) is a malignant B-cell lymphoma of cattle caused by infection with bovine leukemia virus (BLV). It is defined by clonal and neoplastic expansion of BLV-infected B cells. Currently, multiple examinations are able to comprehensively diagnose this condition. Inverse polymerase chain reaction (PCR) is a useful method to determine retrovirus integration sites. Here, we established a simplified inverse PCR method, involving the evaluation of clonality and similarity of BLV integration sites, to clinically diagnose EBL, and we also assessed its reliability. We found that the novel BLV inverse PCR could detect clonal expansion of infected cells even if they constituted only 5% of the total number of cells, while not amplifying any fragments from BLV-uninfected cells, thus confirming its sufficient sensitivity and specificity for use in EBL diagnosis. Furthermore, 50 clinical cases of bovine leukemia were analyzed using BLV inverse PCR and other PCR-based methods, wherein our method most efficiently determined virus-dependent bovine leukemia, including unidentified clinical cases observed in a previous report. Following further clinical investigations to enhance its reliability, the proposed BLV inverse PCR method has the potential to be applied to EBL diagnosis.

Influence of Interspecies Transmission of Atypical Bovine Spongiform Encephalopathy Prions to Hamsters on Prion Characteristics.

Bovine spongiform encephalopathy (BSE) is a prion disease in cattle and is classified into the classical type (C-BSE) and two atypical BSEs, designated as high type (H-BSE) and low type (L-BSE). These classifications are based on the electrophoretic migration of the proteinase K-resistant core (PrPres) of the disease-associated form of the prion protein (PrPd). In a previous study, we succeeded in transmitting the H-BSE prion from cattle to TgHaNSE mice overexpressing normal hamster cellular PrP (PrPC). Further, Western blot analysis demonstrated that PrPres banding patterns of the H-BSE prion were indistinguishable from those of the C-BSE prion in TgHaNSE mice. In addition, similar PrPres glycoprofiles were detected among H-, C-, and L-BSE prions in TgHaNSE mice. Therefore, to better understand atypical BSE prions after interspecies transmission, H-BSE prion transmission from TgHaNSE mice to hamsters was investigated, and the characteristics of classical and atypical BSE prions among hamsters, wild-type mice, and mice overexpressing bovine PrPC (TgBoPrP) were compared in this study using biochemical and neuropathological methods. Identical PrPres banding patterns were confirmed between TgHaNSE mice and hamsters in the case of all three BSE prion strains. However, these PrPres banding patterns differed from those of TgBoPrP and wild-type mice infected with the H-BSE prion. In addition, glycoprofiles of TgHaNSE mice and hamsters infected with the L-BSE prion differed from those of TgBoPrP mice infected with the L-BSE prion. These data indicate that the PrPC amino acid sequences of new host species rather than other host environmental factors may affect some molecular aspects of atypical BSE prions. Although three BSE prion strains were distinguishable based on the neuropathological features in hamsters, interspecies transmission modified some molecular properties of atypical BSE prions, and these properties were indistinguishable from those of C-BSE prions in hamsters. Taken together, PrPres banding patterns and glycoprofiles are considered to be key factors for BSE strain typing. However, this study also revealed that interspecies transmission could sometimes influence these characteristics.

PrPSc with Seeding Activity Extensively Overlaps with Proteinase-Resistant PrPSc Rather than Infectious PrPSc.

The disease-associated prion protein (PrPSc) has the ability to seed the conformational conversion of normal prion proteins into the amyloid fibril form. This prion seeding activity can be measured using an in vitro amplification assay termed real-time quaking-induced conversion (RT-QuIC). There is a strong correlation between RT-QuIC positivity and prion infection; however, the relationship between seeding activity and infectivity remains elusive. In this study, we used endpoint dilution RT-QuIC on the brain homogenates from wild-type mice with mouse-adopted bovine spongiform encephalopathy (mBSE) at defined intervals during the incubation period and evaluated the temporal relationship among prion seeding dose, levels of proteinase-resistant PrPSc (PrPres), and infectious titer. We found that the infectious titer reached a plateau by 100 days postinfection, whereas seeding dose and PrPres levels were continuously elevated. Our calculation showed that the doubling time (dt) for seeding dose from 40 to 100 days postinoculation was closer to the dt for PrPres levels than to the dt for prion titer. Although an uncoupling of seeding doses and PrPres levels was observed at end-stage disease in this model, our findings suggest that there is substantial but not complete overlap between PrPSc with seeding activity and PrPres rather than infectious PrPSc.

Eliminating transmissibility of bovine spongiform encephalopathy by dry-heat treatment.

Bovine spongiform encephalopathy (BSE) prion is more resistant to heat inactivation compared to other prions, but the effect of heat inactivation has been reported to differ depending on the BSE-contaminated tissue state or heating type. We aimed to evaluate the secure level of inactivation of original BSE transmissibility by dry-heating. Cattle tissues affected with BSE were subjected to dry-heat treatment for 20 min at various temperatures ranging from 150 to 1000 °C. To assess the inactivation effect, we conducted protein misfolding cyclic amplification (PMCA) and follicular dendritic cell (FDC) assays in transgenic mice expressing bovine prion protein genes. Under dry-heating at 600 °C or higher, BSE cattle tissues lost their transmissibility in transgenic mice. In contrast, transmissibility was detected in the cattle tissues treated at temperatures of 400 °C or lower through the FDC assay combined with PMCA. In this study, we confirmed that transmissibility was eliminated in BSE-affected cattle tissues by dry-heating at 600 °C or higher.

First case of atypical scrapie in a goat in Japan.

Atypical scrapie in goats has only been reported in European countries. The present study reports the identification of the first case of atypical scrapie in goats in Japan. The genotype of the animal was ALRQ/ALHQ at codons 136, 141, 154, and 171 in prion protein (PrP). Western blot analysis showed a multiplex proteinase K-resistant prion protein (PrP-res) band pattern with a band <15 kDa that was clearly distinguishable from the triplet PrP-res band pattern observed in classical scrapie cases. Histopathological and immunohistological examination showed mild vacuolation and fine granular to globular immunolabelling of disease-associated PrP in the dorsal horn of cervical spinal cord. Collectively, our results confirmed that this goat was affected by atypical scrapie.

Estimation of prion infectivity in tissues of cattle infected with atypical BSE by real time-quaking induced conversion assay.

Atypical bovine spongiform encephalopathy (BSE), first identified in 2004, poses a threat due to the potential to spread the disease to cattle and other animals, including humans. Here, we estimated prion titers in various tissues of cattle infected with atypical BSE using a real-time quaking-induced conversion assay that detects amyloid seeding activity of a disease-specific prion protein, PrPSc, a major component of prions. PrPSc was detected both in and outside of nerve tissues, and some of the peripheral nerve tissues contained relatively high prion titers. Low titers of prions were also observed in masseter, jejunum, and adrenal glands. Quantitative data on prion infectivity in tissues of atypical BSE-affected cattle is useful to assess the risk of atypical BSE.

Sequential detection of pseudocowpox virus and bovine papular stomatitis virus in a same calf in Japan.

We detected parapoxviruses from environmental samples and calves with and without intraoral clinical signs and conducted molecular and serological analyses. Pseudocowpox virus (PCPV) was detected from a calf showing anorexia, frothy salivation, and erosion in the mucosa of the lip and tongue. At the time that PCPV was detected, bovine papular stomatitis viruses (BPSVs) were detected in environmental samples as well as in calves without intraoral clinical signs. BPSV, but not PCPV, was detected in the same calf after 22 days. Phylogenetic analysis revealed that genetically different PCPV strains exist in Japan. This is the first report on the detection of PCPV and BPSV sequentially in the same calf and coexistence of PCPV and BPSV in the same farm in Japan.

A domain responsible for spontaneous conversion of bank vole prion protein.

Bank vole is a small rodent that shows high susceptibility to infection with diverse prion strains. To determine whether the increased susceptibility of bank voles to prion diseases can be attributed to the intrinsic nature of bank vole prion protein (PrP) or to host factors other than PrP, we produced transgenic mice overexpressing bank vole PrP. These transgenic mice spontaneously developed neurological illness with spongiform changes and the accumulation of abnormal PrP in the brain. Then, we produced transgenic mice overexpressing chimeric mouse/bank vole PrP, which differs from mouse PrP only at two residues located at the C-terminus, to determine the minimum essential domain for the induction of spontaneous generation of abnormal PrP. These transgenic mice also developed spontaneous neurological illness with spongiform changes and the accumulation of abnormal PrP in the brain. In addition, knock-in mice expressing bank vole PrP at the same level as that of wild-type mice did not develop spontaneous disease but showed high susceptibility to infection with diverse prion strains, similarly to bank voles. Taken together, these findings show that bank vole PrP has a high propensity for the conformational conversion both in spontaneous disease and in prion infection, probably due to the characteristic structural properties of the C-terminal domain.

Interspecies transmission to bovinized transgenic mice uncovers new features of a CH1641-like scrapie isolate.

In animal prion diseases, including bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease in cervids, and scrapie in sheep and goats, a disease-associated isoform of prion protein (PrPd) accumulates in the brains of affected animals. Although the CH1641 scrapie isolate was experimentally established in the UK, a few natural CH1641-like scrapie cases have been reported in France and the UK. The molecular mass of the unglycosylated protease-resistant core of PrPd (PrPres) is known to be similar between CH1641-like scrapie and experimental BSE in sheep. We previously established an experimental CH1641-like scrapie isolate (Sh294) from a natural classical scrapie case. Here, we demonstrated that the Sh294 isolate was independent of both classical and atypical BSEs by cross-species transmission to bovine PrP overexpressing (TgBoPrP) mice and wild-type mice. Interestingly, we found that the Sh294 isolate altered its host range by the transmission to TgBoPrP mice, and we succeeded in the first stable reproduction of CH1641-like scrapie specific PrPres banding patterns with the ~12-kDa small C-terminal fragment in wild-type mice. This study provides new insight into the relationship between CH1641-like scrapie isolates and BSEs. In addition, interspecies transmission models such as we have demonstrated here could be a great help to investigate the origin and host range of animal prions.

Experimental Infection of Cattle With a Novel Prion Derived From Atypical H-Type Bovine Spongiform Encephalopathy.

H-type bovine spongiform encephalopathy (H-BSE) is an atypical form of BSE in cattle. During passaging of H-BSE in transgenic bovinized (TgBoPrP) mice, a novel phenotype of BSE, termed BSE-SW emerged and was characterized by a short incubation time and host weight loss. To investigate the biological and biochemical properties of the BSE-SW prion, a transmission study was conducted in cattle, which were inoculated intracerebrally with brain homogenate from BSE-SW-infected TgBoPrP mice. The disease incubation period was approximately 15 months. The animals showed characteristic neurological signs of dullness, and severe spongiform changes and a widespread, uniform distribution of disease-associated prion protein (PrPSc) were observed throughout the brain of infected cattle. Immunohistochemical PrPSc staining of the brain revealed the presence of intraglial accumulations and plaque-like deposits. No remarkable differences were identified in vacuolar lesion scores, topographical distribution patterns, and staining types of PrPSc in the brains of BSE-SW- vs H-BSE-infected cattle. PrPSc deposition was detected in the ganglia, vagus nerve, spinal nerve, cauda equina, adrenal medulla, and ocular muscle. Western blot analysis revealed that the specific biochemical properties of the BSE-SW prion, with an additional 10- to 12-kDa fragment, were well maintained after transmission. These findings indicated that the BSE-SW prion has biochemical properties distinct from those of H-BSE in cattle, although clinical and pathologic features of BSW-SW in cattle are indistinguishable from those of H-BSE. The results suggest that the 2 infectious agents, BSE-SW and H-BSE, are closely related strains.

Selective propagation of mouse-passaged scrapie prions with long incubation period from a mixed prion population using GT1-7 cells.

In our previous study, we demonstrated the propagation of mouse-passaged scrapie isolates with long incubation periods (L-type) derived from natural Japanese sheep scrapie cases in murine hypothalamic GT1-7 cells, along with disease-associated prion protein (PrPSc) accumulation. We here analyzed the susceptibility of GT1-7 cells to scrapie prions by exposure to infected mouse brains at different passages, following interspecies transmission. Wild-type mice challenged with a natural sheep scrapie case (Kanagawa) exhibited heterogeneity of transmitted scrapie prions in early passages, and this mixed population converged upon one with a short incubation period (S-type) following subsequent passages. However, when GT1-7 cells were challenged with these heterologous samples, L-type prions became dominant. This study demonstrated that the susceptibility of GT1-7 cells to L-type prions was at least 105 times higher than that to S-type prions and that L-type prion-specific biological characteristics remained unchanged after serial passages in GT1-7 cells. This suggests that a GT1-7 cell culture model would be more useful for the economical and stable amplification of L-type prions at the laboratory level. Furthermore, this cell culture model might be used to selectively propagate L-type scrapie prions from a mixed prion population.

Oral Transmission of L-Type Bovine Spongiform Encephalopathy Agent among Cattle.

To determine oral transmissibility of the L-type bovine spongiform encephalopathy (BSE) prion, we orally inoculated 16 calves with brain homogenates of the agent. Only 1 animal, given a high dose, showed signs and died at 88 months. These results suggest low risk for oral transmission of the L-BSE agent among cattle.

Identification of the first case of atypical scrapie in Japan.

A Corriedale ewe was confirmed as the first atypical scrapie case during an active surveillance program for transmissible spongiform encephalopathies in small ruminants in Japan. The animal was homozygous for the AF141RQ haplotype of PRNP. The animal showed clinical neurological signs possibly due to listeriosis before culling. Western blot analysis showed an unusual multiple banded pattern with a low-molecular fragment at ~7 kDa. Histopathology revealed suppurative meningoencephalitis caused by listeriosis in the brainstem. Fine granular to globular immunostaining of disease-associated prion proteins was mainly detected in the neuropil of the spinal tract of the trigeminal nerve and in the white matter of the spinocerebellar tract. Based on these results, this case was conclusively diagnosed as atypical scrapie with encephalitic listeriosis.