{"created":"2023-06-19T07:18:07.942442+00:00","id":3254,"links":{},"metadata":{"_buckets":{"deposit":"b6c25c89-e5c2-416d-bf3b-12f8c6026b9a"},"_deposit":{"created_by":4,"id":"3254","owners":[4],"pid":{"revision_id":0,"type":"depid","value":"3254"},"status":"published"},"_oai":{"id":"oai:az.repo.nii.ac.jp:00003254","sets":["370:15:392"]},"author_link":["16335","16336"],"item_10006_date_granted_11":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"2009-10-26"}]},"item_10006_degree_grantor_9":{"attribute_name":"学位授与機関","attribute_value_mlt":[{"subitem_degreegrantor":[{"subitem_degreegrantor_name":"麻布大学"}]}]},"item_10006_degree_name_8":{"attribute_name":"学位名","attribute_value_mlt":[{"subitem_degreename":"博士（獣医学）"}]},"item_10006_description_22":{"attribute_name":"Abstract","attribute_value_mlt":[{"subitem_description":"The first report on bovine viral diarrhea (BVD) was outbreak in cattle observed in the U.S.A. in 1946. After the outbreak in the U.S.A., a similar but more severe BVD in cattle with mucosal disease (MD) was reported in Canada. Thus, several years later, the disease became officially known as bovine viral diarrhea-mucosal disease. At present, BVD is an economically important disease of cattle having been reported throughout the world and is caused by infection with bovine viral diarrhea virus (BVDV).\n BVDV is a small positive-sense single-stranded RNA virus classified in the genus Pestivirus within the family Flaviviridae. There are currently four recognized species within the pestivirus genus: BVDV 1; BVDV 2; border disease virus and classical swine fever virus, previously known as hog cholera virus. The Pestivirus genome is approximately 12.3 Kb in length. The highest nucleic acid sequence identity among pestiviruses is found in the5' UTR.\n There are two biotypes of BVDV: one is non-cytopathogenic (ncp) BVDV which did not induce cytopathogenic effects (CPE) in any cell culture, sand the other is cytopathogenic (cp) virus, which shows clear CPE in bovine cell cultures. It became generally accepted that only ncp BVDV strain could produce persistently infected (PI) cattle. PI cattle with ncp BVDV suffer from MD if they are superinfected with a cp BVDV.\n Hemorrhages associated with BVDV infection in young veal calves were also observed with increasing frequency in the late 1980s in the northeastern United States. Phylogenetic analysis of the BVDV strains grouped them separately from the BVDV strains commonly used, at that time, in vaccine production, diagnostic tests, and research. The newly recognized group of BVDV 2 and the group containing the early strains was termed BVDV 1. Prevalence of BVDV 2 in North America, Europe and South Africa have been reported. Ideally, genotypes would be associated with practical observations such as geographic distribution, antigenic variation, or variations in virulence. The differentiation between the BVDV 1 and BVDV 2 genotypes meets these practical considerations. Therefore, it is worthwhile to segregate BVDV strains isolated in Japan into BVDV 1 or BVDV 2.\n With the emergence of BVDV 2, the concern for incorporation of BVDV 2 in vaccines escalated with a report that vaccines containing BVDV 1 appeared not to be protective against infection with BVDV 2. The efficacy of Japanese vaccine containing BVDV 1 against BVDV 2 should also be evaluated.\n The observation that fetal bovine sera frequently contained BVDV was an increasing concern in the 1970s. In order to ensure that BVDV-free vaccines are produced, it is therefore essential to use valid tests and to develop effective quality assurance programs, stage by stage, throughout the manufacturing process.\n As mentioned above, BVDV especially new BVDV 2, is a very important agent of livestock hygiene and quality control of vaccines. Regrettably, it is not cleared the presence of BVDV 2 in Japan, efficacy of commercial vaccine used in Japan against BVDV 2 and potentiality of detection of extraneous active BVDV in bovine vaccine by RT-PCR. The purposes of this study were to clarify these uncertainties.\n The present thesis consists of three chapters. In the first chapter, BVDV strains isolated in Japan are segregated into genotypes. In the second chapter, the efficacy of BVD vaccine used in Japan against BVDV 2 strain 890 is evaluated. In the last chapter, it is shown that the active BVDV contaminating bovine live viral vaccines could be detected by means of the detection of negative-sense RNA of BVDV with RT-PCR. Segregation of bovine viral diarrhea virus isolated in Japan into genotypes\n It was demonstrated that BVDV 2 did not have PstI site, which is present in all known BVDV 1, on the 5'-UTR of genome. It made it easy that BVDV was distinguished BVDV 2 from BVDV 1. BVDV infection associated with thrombocytopenia in Japan has not been reported in any literature until now. In the chapter, BVDV strains isolated in Japan are segregated into BVDV 1 or BVDV 2 using genomic criteria, and demonstrated those pathogenecity and antigenicity.\n It was suggested that 3 strains of BVDV isolated from PI calves in Tochigi Prefecture in Japan belonged to BVDV 2. It was recognized lack of PstI site on the 5'-UTR of genome of them. Inoculated with the 3 strains, the calves showed the mild decrease of platelet counts which was specific clinical sign of BVDV 2. It should be reported that the 3 strains were the first BVDV 2 isolated in Japan. Neutralizing antibody titers of the antisera against the 3 strains using laboratory strains as neutralizing virus were lower than those of them using homologous strains. Therefore, it was indicated that the difference between BVDV 1 and BVDV 2 in the antigenicity.\nEfficacy of bovine viral diarrhea vaccine used in Japan against Bovine viral diarrhea virus 2 strain 890\n Antigenic heterogeneity in BVDV has been demonstrated, but their serotypes have not been established yet. Especially, BVDV 2 is antigenically distinct from BVDV 1. In previous chapter, it was reported that three strains of BVDV 2 isolated in Japan antigenically differed from BVDV 1 strain No.12-43, which is the vaccine strain, used in Japan. Therefore, it was feared that BVDV 2, escaping neutralization by vaccine-induced antibody, might contribute to vaccine failure. The purpose of the study reported in this chapter was to determine the efficacy of a Japanese modified-live BVDV vaccine containing ncp biotype of BVDV 1. in protecting calves from diseases caused by a virulent strain of BVDV 2 strain 890 which is a representative strain of BVDV 2\n The vaccinated calves did not develop any clinical sign and hematological change such as observed in unvaccinated calves after the challenge. Furthermore, the challenge virus was not recovered from the vaccinated calves throughout the duration of the experiment, whereas it was recovered from all unvaccinated calves. The BVDV 1 vaccine used in Japan is efficacious against infection with BVDV 2 strain 890.\nVerification of the active bovine viral diarrhea virus (BVDV) contaminated in bovine live viral vaccines by means of the detection of negative-sense RNA of BVDV with RT-PCR\n Most of viral vaccine is made from vaccine strain cultured with biological material such as cells and fetal bovine serum. However, it means that the vaccines have some risks of contamination with extraneous infectious agent derived from biological materials. BVDV is well known as contaminant within bovine serum and cells. If extraneous active BVDV is present in a live viral vaccine made with biological materials contaminated with active BVDV, it would cause serious problems for animal hygiene.\n Since the RT-PCR methods has been recognized to be a very sensitive for the detection of BVDV in serum, cell cultures, seed virus and vaccines, those RT-PCR methods are recognized to be powerful tool for detection of viral RNA. However, it is unfortunately impossible to determine whether the PCR products are amplified from RNA in active or inactive virus because of the use of a random primer or a specific antisense primer. Therefore, the RT-PCR method was developed for the detection of an active BVDV by amplification of negative strand viral RNA replicated in cells infected with BVDVL The purpose of this study was to verify that extraneous active BVDV in a bovine live viral vaccine could be detected by the RT-PCR method.\n The RT-PCR method was confirmed to be no less sensitive than the interference method. Moreover, it was also shown that all of the non-cytopathogenic BVDV strains examined in this study were detected by this method. Furthermore, ingredients in bovine live viral vaccines did not interfere with the RT-PCR method. The author concludes that the RT-PCR method is very useful for quality control of bovine live viral vaccines except for BVD live vaccine.\nConclusion\n BVDV isolated causing hemorrhagic syndrome and acute severe BVD formed a new genetic group (genotype) of BVDV distinct from early strains.\nVariation between BVDV 1 and BVDV 2 has practical significance.\nUnderstanding for variation among BVDV is essential to developing a successful control of BVDV. Therefore, BVDV strains isolated in Japan were segregated into BVDV 1 and BVDV 2 using genomic criteria, and demonstrated those pathogenecity and antigenicity .\n Three BVDV strains belonged to BVDV 2. While the pathogenicity of these strains was weak, antigenicity of these strains differs from those of early strains. Therefore, the author examined protection by a Japanese BVDV 1 vaccine in calves against BVDV 2 strain 890. The vaccinated calves did not developclinical signs or fever nor have hematological change (decreased WBC) after challenge. The BVDV 1 vaccine is efficacious against infection with BVDV 2 strain 890.\n The removal of PI animals is considered to be integral to an effective control strategy. Unfortunately, PI animal may be born from cow injected with a vaccine contaminated ncp BVDV. Accordingly, it is very important to examine for the presence of extraneous BVDV in a bovine viral vaccine. So, it is shown that extraneous active BVDV in a bovine live viral vaccine could be detected by the RT-PCR method. As expected, the RT-PCR method is very useful for quality control of bovine live viral vaccine except for BVD live vaccine.\n The author concludes that the present works are possible contribute to develop a successful control of BVDV 2 in Japan and quality control of a BVD live vaccine, which is a very important tool prevention of bovine infectious diseases.\n","subitem_description_type":"Other"}]},"item_10006_description_7":{"attribute_name":"抄録","attribute_value_mlt":[{"subitem_description":"牛ウイルス性下痢症(BVD)は1946年にアメリカで初めて報告され、その後カナダで粘膜病を伴う重篤なBVDが報告された。BVDはその数年後に牛ウイルス性下痢-粘膜病として広く知られるようになった。現在、BVDは牛ウイルス性下痢ウイルス(BVDV)の感染による重要な疾病として世界中で報告され畜産経営に甚大な被害を及ぼしている。\n　BVDVは、フラビウイルス科ペスチウイルス属に属するプラス1本鎖RNAウイルスである。ペスチウイルス属は、BVDV1、BVDV2、ボーダー病ウイルスおよび豚コレラウイルスの4種からなり、その遺伝子の長さは約12.3Kbである。5'末端の非翻訳領域の塩基配列は属内で最も保存され、特異的であるため遺伝子検査に適している。\n　BVDVには2つのバイオタイプがあり、1つは培養細胞に細胞変性効果(CPE)を引き起こす細胞病原性株で、もう1つはCPEを引き起こさない非細胞病原性株である。一般的に非細胞病原性株のみが牛に持続感染し、その持続感染牛に細胞病原性株が重感染すると粘膜病を発症するとされている。\n　1980年代後半、北米で食用子牛に出血を主徴とする高病原性を呈するBVDV感染が頻繁に発生した。その分離株について遺伝子の系統樹解析を行ったところ、従来の野外流行株、ワクチン株、診断用抗原、若しくは研究材料として一般的に用いられていた株とは異なるグループであることが判明した。このグループは新しい種とされBVDV2と命名され、既知の株が属するグループはBVDV1と命名された。その後、BVDV2の流行は北米だけにとどまらず欧州や南米にも拡がった。したがって、我が国においても早急にBVDVを型別し、BVDV2の抗原性状、病原性について検討する必要が生じた。また、BVDV1を製造用株とする現行ワクチンはBVDV2感染を予防できないとする報告があったことから、ワクチンの製造用株にBVDV2を加えることが言及されるようになった。そこで、著者は我が国で使われているBVDV1を製造用株としているワクチンがBVDV2感染症の予防に対して有効かどうかを明らかにする必要があると考えた。\n　一方、1970年代から細胞培養に使用していた牛胎子血清にBVDVが混入するケースが増加傾向にあり、培地および培養細胞への汚染が目立つようになった。このため、BVDVが迷入していないワクチンの製造を確保するためには、ワクチンの各製造工程における適切な検査の実施が効果的な品質の確保に必要不可欠であると思われた。\n　上述のように、BVDV、とりわけ新たなBVDV2は家畜衛生およびワクチンの晶質管理において重要な病原因子である。そこで、我が国におけるBVDV2の確認およびBVDV2に対する現行ワクチンの効果について究明し、並びにRT-PCRによる牛用ワクチン中に迷入する活性BVDV検出法の確立を目的として以下の研究を行った。\n　本論文は3章から構成される。第1章において我が国で分離されたBVDVを遺伝子型別した。第2章では、我が国で使用されているBVDワクチンのBVDV2に対する効果を評価した。第3章では、BVDVの複製過程で生成されるマイナス鎖RNAをRT-PCRにより検出する方法を確立し、牛用生ウイルスワクチン中に迷入する活性BVDVの検出に有用か検証した。\n\n第1章　我が国で分離された牛ウイルス性下痢ウイルスの遺伝子型別\n　BVDV2の5'末端非翻訳領域にはBVDV1に存在するPstIサイトがない。このことを指標にBVDVは容易にBVDV1とBVDV2に型別できる。そこで、著者は我が国で分離されたBVDVをBVDV1若しくはBVDV2に型別し、検出されたBVDV2の病原性および抗原性を検討した。\n　その結果、野外の持続感染牛から分離された3株の5'末端非翻訳領域にPstIサイトがなかったことから、これら3株はBVDV2と型別された。これら3株を牛に実験感染させたところ、発熱およびロイコペニーと共にBVDV2に特徴的な血小板減少が認められた。以上の結果から、これらの3株は、我が国において初めて確認されたBVDV2であった。また、実験感染牛の血清中和抗体価を測定したところ、ホモの株に対する抗体価とBVDV1である既知株に対する抗体価に差が認められ、抗原性の相違が明らかとなった。\n\n第2章　牛ウイルス性下痢ウイルス2-890株に対する我が国で使用されている牛ウイルス性下痢症ワクチンの有効性\n　前章においてBVDV2であることが明らかとなった3株の抗原性はワクチン株であるNo.12-43株のそれと異なることを示した。このことから、BVDV2がワクチン抗体をすり抜けることで既存のワクチンが無効となることが危惧された。そこで、BVDV1を製造用株とし我が国で汎用されているBVDワクチンのBVDV2の代表株かつ強毒株である890株に対する有効性を調べた。ワクチン接種牛および未接種牛に890株を攻撃したところ、ワクチン接種牛には未接種牛に診られた臨床症状や血液学的変化が観察されず、攻撃株も分離されなかった。我が国のBVDワクチンはBVDV2感染に対しても有効であることが示唆された。\n\n第3章　牛用ウイルスワクチンに迷入する活性牛ウイルス性下痢ウイルスの迅速検出法の検証\n　多くのウイルスワクチンは細胞や牛胎子血清などの生物由来原料を使って培養したウイルスをワクチン株として製造されている。しかし、それは生物由来原料を介して感染因子が迷入するリスクを常に持っているということを意味するものである。BVDVは牛血清や細胞に混入することがよく知られている。活性BVDVに汚染された生物由来原料を使ってワクチンを製造した場合、それは家畜衛生上、非常に重篤な問題が生じることになるかもしれない。\n　従来用いられたRT-PCRは、血清や培養細胞、シードウイルス、ワクチンからBVDVを検出できる非常に感度の高い方法であるが、これまでのRT-PCRはランダムプライマー若しくは特異的なアンチセンスプライマーを用いるため、その混入しているBVDVが活性を有しても有していなくてもPCR産物が増幅される。そこで、BVDVが感染細胞で複製する際に生じるマイナス鎖RNAに対するセンスプライマーを用いて増幅することで活性を有するBVDVのみを検出するRT-PCR法を開発し、この新しいRP-PCR法を用いて牛用生ウイルスワクチン中に迷入する活性BVDVの検出について試験した。\n　その結果、新たに開発したRT-PCR法は、動物用生物学的製剤基準において採用されている干渉法に比べ簡便かつ迅速で、同程度の感度であることが確認された。さらに、供試した全ての非細胞病原性BVDVを検出することができ、牛用ウイルス生ワクチン中に含まれる成分の干渉を受けなかった。\nこのRTP-CR法は牛用ウイルス生ワクチンの品質管理法として有用であると考えられた。\n\n　我が国で初めて著者がBVDV2と型別した3株の病原性は北米で分離されたウイルスに比べて弱いものであったが、抗原性は既知のBVDV1とは異なるものであった。そこで、著者は我が国のBVDワクチンがBVDV2の発症を防御できるか明らかにするために、ワクチン接種牛および未接種牛にBVDV2-890株を攻撃した。その結果、ワクチン接種牛には未接種牛に診られた臨床症状や血液学的変化が観察されず、攻撃株も分離されなかった。このことから我が国のBVDワクチンはBVDV2に対しても有効であることが示唆された。\n　BVDをコントロールする上で最も効果的なことは持続感染牛の摘発淘汰である。非細胞病原性のBVDVが迷入したワクチンを妊娠牛に接種すると、不幸にも持続感染牛が生まれてくる恐れがある。したがって、牛用ウイルスワクチンにBVDVが迷入していないか調べることは重要なことである。そこで、著者は新しいRT-PCR法を開発し、牛用生ウイルスワクチン中に迷入した活性BVDVを検出できるか検証したところ、このRT-PCR法は牛用生ウイルスワクチンの品質管理に有用であることが示唆された。\n　この研究成果は我が国におけるBVDV2感染症の防疫対策を可能にし、牛の感染症対策において重要なツールである牛用ワクチンの品質管理に応用可能で、家畜衛生の向上に大きく貢献できるものである。\n","subitem_description_type":"Abstract"}]},"item_10006_dissertation_number_12":{"attribute_name":"学位授与番号","attribute_value_mlt":[{"subitem_dissertationnumber":"乙第423号"}]},"item_10006_version_type_18":{"attribute_name":"著者版フラグ","attribute_value_mlt":[{"subitem_version_resource":"http://purl.org/coar/version/c_ab4af688f83e57aa","subitem_version_type":"AM"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"嶋﨑, 智章"}],"nameIdentifiers":[{"nameIdentifier":"16335","nameIdentifierScheme":"WEKO"}]},{"creatorNames":[{"creatorName":"Shimazaki, Tomoaki","creatorNameLang":"en"}],"nameIdentifiers":[{"nameIdentifier":"16336","nameIdentifierScheme":"WEKO"}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2013-02-20"}],"displaytype":"detail","filename":"diss_dv_otsu0423.pdf","filesize":[{"value":"27.3 MB"}],"format":"application/pdf","licensetype":"license_note","mimetype":"application/pdf","url":{"label":"diss_dv_otsu0423","url":"https://az.repo.nii.ac.jp/record/3254/files/diss_dv_otsu0423.pdf"},"version_id":"a62172dd-04d1-4561-9c81-e60d1147b9af"},{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2014-08-19"}],"displaytype":"detail","filename":"diss_dv_otsu0423_jab&rev.pdf","filesize":[{"value":"357.5 kB"}],"format":"application/pdf","licensetype":"license_note","mimetype":"application/pdf","url":{"label":"diss_dv_otsu0423_jab&rev","url":"https://az.repo.nii.ac.jp/record/3254/files/diss_dv_otsu0423_jab&rev.pdf"},"version_id":"d833e676-3e31-4996-bf9f-c2136de3e0ca"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"eng"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"thesis","resourceuri":"http://purl.org/coar/resource_type/c_46ec"}]},"item_title":"牛ウイルス性下痢ウイルス2の抗原性および病原性並びに検出法に関する研究","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"牛ウイルス性下痢ウイルス2の抗原性および病原性並びに検出法に関する研究"},{"subitem_title":"Studies on antigenic and pathogenic characterizations and detection of bovine viral diarrhea virus 2","subitem_title_language":"en"}]},"item_type_id":"10006","owner":"4","path":["392"],"pubdate":{"attribute_name":"公開日","attribute_value":"2013-02-13"},"publish_date":"2013-02-13","publish_status":"0","recid":"3254","relation_version_is_last":true,"title":["牛ウイルス性下痢ウイルス2の抗原性および病原性並びに検出法に関する研究"],"weko_creator_id":"4","weko_shared_id":4},"updated":"2023-06-19T08:13:50.655088+00:00"}