Yoko Aida

Genetic polymorphism of the swine major histocompatibility complex ( SLA) class I genes, SLA-1, -2 and -3.

In order to identify and characterize genetic polymorphism of the swine major histocompatibility complex (Mhc: SLA) classxa0I genes, RT-PCR products of the second and third exons of the three SLA classical classxa0I genes, SLA-1, SLA-2 and SLA-3 were subjected to nucleotide determination. These analyses allowed the identification of four, eight and seven alleles at the SLA-1, SLA-2 and SLA-3 loci, respectively, from three different breeds of miniature swine and one mixed breed. Among them, 12 alleles were novel. Construction of a phylogenetic tree using the nucleotide sequences of those 19 alleles indicated that the SLA-1 and -2 genes are more closely related to each other than to SLA-3. Selective forces operating at single amino acid sites of the SLA classxa0I molecules were analyzed by the Adaptsite Package program. Ten positive selection sites were found at the putative antigen recognition sites (ARSs). Among the 14 positively selected sites observed in the human MHC (HLA) classical classxa0I molecules, eight corresponding positions in the SLA classxa0I molecules were inferred as positively selected. On the other hand, four amino acids at the putative ARSs were identified as negatively selected in the SLA classxa0I molecules. These results suggest that selective forces operating in the SLA classxa0I molecules are almost similar to those of the HLA classxa0I molecules, although several functional sites for antigen and cytotoxic T-lymphocyte recognition by the SLA classxa0I molecules may be different from those of the HLA classxa0I molecules.

Importin-α Promotes Passage through the Nuclear Pore Complex of Human Immunodeficiency Virus Type 1 Vpr

ABSTRACT Viral protein R (Vpr) of human immunodeficiency virus type 1 has potent karyophilic properties, but details of the mechanism by which it enters the nucleus remain to be clarified. We reported previously that two regions, located between residues 17 and 34 (αH1) and between residues 46 and 74 (αH2), are indispensable for the nuclear localization of Vpr. Here, we reveal that a chimeric protein composed of the nuclear localization signal of Vpr, glutathione S-transferase, and green fluorescent protein was localized at the nuclear envelope and then entered the nucleus upon addition of importin-α. An in vitro transport assay using a series of derivatives of importin-α demonstrated that the carboxyl terminus was required for this nuclear import process. We also showed that Vpr interacts with importin-α through αH1 and αH2; only the interaction via αH1 is indispensable for the nuclear entry of Vpr. These observations indicate that importin-α functions as a mediator for the nuclear entry of Vpr.

A mutant form of the tax protein of bovine leukemia virus (BLV), with enhanced transactivation activity, increases expression and propagation of BLV in vitro but not in vivo.

ABSTRACT In a previous study, we identified an interesting mutant form of the Tax protein of bovine leukemia virus (BLV), designated D247G. This mutant protein strongly transactivated the long terminal repeat of BLV and was also able to transactivate the cellular proto-oncogene c-fos. This finding suggested that BLV that encode the mutant protein might propagate and induce lymphoma more efficiently than wild-type BLV. To characterize the effects of the strong transactivation activity of the mutant Tax protein, we constructed an infectious molecular clone of BLV that encoded D247G and examined the replication and propagation of the virus in vitro and in vivo. Cultured cells were transfected with the wild-type and mutant BLV, and then levels of viral proteins and particles and the propagation of viruses were compared. As expected, in vitro, mutant BLV produced more viral proteins and particles and was transmitted very effectively. We injected the wild-type and mutant BLV into sheep, which are easily infected with BLV, and monitored the proportion of BLV-positive cells in the blood and the expression of BLV RNA for 28 weeks. By contrast to the results of our analyses in vitro, we found no significant difference in the viral load or the expression of viral RNA between sheep inoculated with wild-type or mutant BLV. Our observations indicate that the mutant D247G Tax protein does not enhance the expansion of BLV and that there might be a dominant mechanism for regulation of the expression of BLV in vivo.

Evidence for cattle major histocompatibility complex (BoLA) class II DQA1 gene heterozygote advantage against clinical mastitis caused by Streptococci and Escherichia species

Mastitis is an inflammatory response of the mammary gland to irritation, injury, or infectious agents and is a major problem in the dairy industry. We genotyped bovine major histocompatibility complex (BoLA)-DRB3 and BoLA-DQA1 genes in 120 Holstein cattle with clinical mastitis and 85 randomly selected Holstein cattle in Japan by polymerase chain reaction-sequence-based typing. The mastitis cattle were divided into four groups according to the bacterial species that caused the mastitis (Staphylococcus aureus, Streptococci, Escherichia, and coagulase-negative staphylococci). The BoLA-DRB3 and BoLA-DQA1 heterozygosity of each group was compared with that of the control cattle, while the expected heterozygosities based on Hardy-Weinberg proportions and the observed heterozygosities for each locus were compared for each group. The Escherichia-induced and Streptococci-induced mastitis groups showed significant differences between their expected and observed heterozygosities with regard to their BoLA-DQA1 genes. No differences were observed for any group with regard to the BoLA-DRB3 genes. We then found that two BoLA-DQA1 alleles promoted susceptibility to Streptococci-induced mastitis, namely BoLA-DQA1*0101 and BoLA-DQA1*10012 and that the homozygous BoLA-DQA1*0101/0101 and BoLA-DQA1*10011/10011 genotypes promoted susceptibility to mastitis caused by Streptococci and Escherichia, respectively. This is the first report showing that heterozygosity of the BoLA-DQA1 gene is associated with resistance to mastitis progression.

The influence of ovine mhc class ii drb1 alleles on immune response in bovine leukemia virus infection

We have reported previously that the alleles of the ovine leukocyte antigen (OLA)‐DRB1 gene that encode the Arg‐Lys (RK) motif and the Ser‐Arg (SR) motif at positions β70/71 of the OLA‐DRβ1 domain are associated with resistance and susceptibility, respectively, to development of bovine leukemia virus (BLV)‐induced ovine lymphoma. Here, to investigate the different immune response in sheep that carried alleles associated with resistance and susceptible for 30 weeks after infection with BLV, we selected sheep that had the RK/RK or SR/SR genotype among the 52 sheep analyzed by polymerase chain reaction‐restriction fragment length polymorphism and DNA sequencing of PCR product for the OLA‐DRB1 exon 2 and infected them with BLV. Although the number of BLV‐infected cells and virus titer had been maintaining low levels throughout the experimental period, the sheep with the RK/RK genotype could induce expansion of CD5− B‐cells and rapid production of neutralizing antibody in the early phase of infection. The level of incorporation of [3H]thymidine by peripheral blood mononuclear cells from the sheep with RK/RK genotype gave a strong response to BLV virion antigen and synthetic antigenic peptides that corresponded to T‐helper epitope of the BLV envelope glycoprotein gp51. In contrast, the sheep with SR/SR genotype showed a strong response to BLV virion antigen and synthetic antigenic peptides that corresponded to T‐cytotoxic and B‐cell epitopes. In such cases, the animals with the RK/RK strongly expressed IFN‐7, the animals with SR/SR genotype strongly expressed IL‐2. To determine the proliferating cells, we tried a blocking assay with monoclonal antibodies such as anti‐CD4, ‐CD8 and ‐DR molecule. We found that these proliferating cells were MHC‐restricted CD4+ T‐cells.

Latency of Viral Expression In Vivo Is Not Related to CpG Methylation in the U3 Region and Part of the R Region of the Long Terminal Repeat of Bovine Leukemia Virus

ABSTRACT Bovine leukemia virus (BLV) is silent in most cells detectable in vivo, and the repression of its expression allows BLV to evade the hosts immune response. In this study, we examined whether CpG methylation of DNA might be involved in the regulation of the expression of BLV in vivo. To investigate the effects of CpG methylation on the activity of the long terminal repeat (LTR) of BLV, we measured the transactivation activity of this region after treatment with the CpG methyltransferase SssI by using a luciferase reporter system. The activity of methylated LTR was significantly lower than that of nonmethylated LTR. Therefore, we examined the extent of CpG methylation of the U3 region and part of the R region of the LTR in BLV-infected cattle and in experimentally BLV-infected sheep at various clinical stages by the bisulfite genomic sequencing method. We detected no or minimal CpG methylation at all stages examined in cattle and sheep, and our results indicate that CpG methylation probably does not participate in the silencing of BLV in vivo.

Distribution and origin of bovine major histocompatibility complex class II DQA1 genes in Japan

We sequenced the major histocompatibility complex (MHC) class II DQA1 gene in 352 Japanese cattle (95 Japanese Black, 91 Holstein, 102 Japanese Shorthorn and 64 Jersey cattle) using a new sequence-based typing method. In total, 19 bovine MHC (BoLA)-DQA1 alleles, of which two were novel alleles, were detected. The Holstein, Jersey, Japanese Shorthorn and Japanese Black breeds had 13, 12, 10 and 15 alleles, respectively. The dendrogram that was constructed by the neighbor-joining method on the basis of the DQA1 gene allele frequencies of the four Japanese cattle breeds showed that the Holstein and Japanese Black breeds were closest to each other, with Jersey being farther from these two breeds than Japanese Shorthorn. In addition, Wu-Kabat analysis showed that the DQA1 alleles of the Holstein and Japanese Black were the most and least polymorphic, respectively. Phylogenetic analyses indicated that the DQA1 gene of Bovidae such as cattle, sheep, bison and goat were more similar to pig SLA-DQA genes than to human HLA-DQA1 and dog DLA-DQA genes. The cattle, goat, bison, sheep, human and pig DQA1 molecules had similar rates of amino acid sequence polymorphism, but the distribution of their polymorphic residues differed from that in the dog DQA1 protein. However, the Bovidae DQA1 molecule had more polymorphic residues than the human, pig and dog DQA molecules at two regions, namely positions 52-53 and 65-66. This indicates that the Bovidae DQA1 locus is more polymorphic than the DQA loci of other species.

MHC class II DR classification based on antigen-binding groove natural selection.

Major histocompatibility complex (MHC) genes are highly polymorphic and play key roles in immune susceptibility and resistance to pathogens. While the immunological and structural functions of several human and murine alleles have been analyzed, little is known about the MHC molecules of other animals. Here, we could classify five mammalian species into three groups (human, cow and dog, and cat and pig) on the basis of DRB nucleotide sequences, synonymous and nonsynonymous mutation rates, and natural selection of individual residues. These observations, along with the locations of the positively and negatively selected residues in three-dimensional DR structures, suggest that the antigen-recognition sites of swine and feline DR molecules have been negatively selected while those of bovine and canine DR molecules have been positively selected. Human DR molecules show evidence of high negative and positive selection. Our observations suggest that MHC-DR molecules are under different selective force depending on each species.

Association of BoLA-DRB3 alleles identified by a sequence-based typing method with mastitis pathogens in Japanese Holstein cows.

The association of the polymorphism of bovine leukocyte antigen (BoLA-DRB3) genes identified by the polymerase chain reaction sequence-based typing (PCR-SBT) method with resistance and susceptibility to mastitis caused by pathogenic bacteria was investigated. Blood samples for DNA extraction were collected from 194 Holstein cows (41 healthy cows and 153 mastitis cows including 24 mixed-infection cows infected with 2 or 3 species of pathogens) from 5 districts of Chiba prefecture, Japan. Sixteen BoLA-DRB3 alleles were detected. The 4 main alleles of DRB3*0101, *1501, *1201, and *1101 constituted 56.8% of the total number of alleles detected. Mastitis cows were divided into 2 groups: group 1 with single-infection cows and group 2 with all mastitis cows including 24 mixed-infection cows. The differences in the frequencies of BoLA-DRB3 alleles and the number of cows homozygous or heterozygous for each BoLA-DRB3 allele between healthy cows and the 2 groups of mastitis cows were evaluated. Furthermore, similar comparisons were performed between healthy cows and the 2 groups of mastitis cows for each mastitis pathogen. It was considered that the 4 alleles, namely, DRB3*0101, *1501, *1201, and *1101 had specific resistance and susceptibility to 4 different mastitis pathogens. Thus, DRB3*0101 might be associated with susceptibility to coagulase-negative Staphylococci and Escherichia coli, and DRB3*1501 might be associated with susceptibility to Escherichia coli. However, DRB3*1101 might be associated with resistance to Streptococci and coagulase-negative Staphylococci, and DRB3*1201, with resistance to Streptococci, Escherichia coli, and Staphylococcus aureus.

Association of the amino acid motifs of BoLA-DRB3 alleles with mastitis pathogens in Japanese Holstein cows.

The association of the polymorphism of bovine leukocyte antigen (BoLA-DRB3) genes, identified by the polymerase chain reaction sequence-based typing (PCR-SBT) method, with resistance and susceptibility to mastitis caused by Streptococci, coagulase-negative Staphylococci, Escherichia coli and Staphylococcus aureus was investigated. Blood samples for DNA extraction were collected from 170 Holstein cows (129 mastitis and 41 healthy cows) from 5 districts in Chiba prefecture, Japan. Susceptibility or resistance to the mastitis-causing pathogens was thought to vary by the presence of amino acid substitutions at the 9, 11, 13, and 30 positions. DRB3*0101 and DRB3*1501 had amino acid motifs of Glu(9), Ser(11), Ser(13), and Tyr(30), and they were considered to have susceptibility to all 4 mastitis pathogens. In contrast, DRB3*1101 and DRB3*1401 had amino acid motifs of Gln(9), His(11), Gly(13), and His(30) in these positions, and they also had Val(86), so these alleles were considered to have resistance to Streptococcal and coagulase-negative Staphylococcal mastitis. However, in the case of Escherichia coli mastitis, amino acid substitutions at the 9, 11, 13, and 30 positions had little effect, but rather substitutions at the 47, 67 positions of pocket 7, and at the 71, 74 positions of pocket 4, Tyr(47), Ile(67), Ala(71), and Ala(74), were associated with resistance. This motif was present in DRB3*1201.