CAJ | 학술논문

【目的】对首次暴发的大熊猫源性犬瘟热病毒（panda derived-canine distemper virus,P-CDV）全基因组进行克隆测序，以了解大熊猫源 CDV 的全基因组遗传变异情况，进一步追踪感染源，为大熊猫犬瘟热防控提供理论依据。【方法】根据 GenBank 公布的 CDV 全基因组序列设计17对特异性引物，利用 RT-PCR 技术，从感染犬瘟热病毒的大熊猫肺渗出液中分片段扩增 CDV 全基因序列，并克隆到 pMD19-T 载体中；经测序、拼接，获得第一个 P-CDV 全长 cDNA 序列；利用 DNAman 生物学分析软件分别对全基因组序列、H 蛋白基因序列、F 蛋白基因序列、P 蛋白基因序列、M 蛋白基因序列等进行遗传变异分析，构建系统进化树。【结果】经序列测序和拼接，大熊猫源犬瘟热病毒全基因组长15690 nt，GenBank 登录号为 KP677502，主要编码6种蛋白，分别是 N 蛋白、P 蛋白、M 蛋白、F 蛋白、H 蛋白和 L 蛋白，在各个基因及间隔区中未发现碱基插入和缺失。遗传进化分析显示，P-CDV全基因组与20株代表性 CDV 全基因组序列同源性为91.5%—98.7%，P-CDV 与强毒株 MKY-KM08（HM852904）、PS、HLJ1-06（JX681125）、Hebei（KC427278）以及 AC96I-H358（AB753776）在一个大的分支上，与 PS 株（JN896331）亲缘关系最近，同源性为98.7%，与标准野毒株 Strain A75-17（AF164967）同源性为95.7%，与疫苗株 CDV3（EU726268）亲缘关系较远，同源性为91.5%。H 蛋白氨基酸序列分析和系统进化树表明，大熊猫源犬瘟热病毒属于强毒株，基因型为 Asia-I 型。P-CDV 各蛋白基因与20株代表性的毒株相比有9处氨基酸发生了变异，与GenBank 上现有的 CDV 序列相比，其中3处是独有的，分别是：F 蛋白基因的208位由 N（Asn，天冬酰胺，强毒株多为 N）或 K（Lys，赖氨酸，弱毒株多为 K）变成了 S（Ser，丝氨酸），第215位由 S 变成了 A（Ala，丙氨酸），P 蛋白基因的58位由 Q（Gln，谷氨酰胺）变成 K（Lys，赖氨酸）。【结论】成功克隆了大熊猫源犬瘟热病毒的全基因，并完成了序列分析，发现了在 F、P 基因上碱基的重要变异。这些数据将为研究大熊猫犬瘟热的遗传变异和流行特征提供分子生物学依据。【目的】對首次暴髮的大熊貓源性犬瘟熱病毒（panda derived-canine distemper virus,P-CDV）全基因組進行剋隆測序，以瞭解大熊貓源 CDV 的全基因組遺傳變異情況，進一步追蹤感染源，為大熊貓犬瘟熱防控提供理論依據。【方法】根據 GenBank 公佈的 CDV 全基因組序列設計17對特異性引物，利用 RT-PCR 技術，從感染犬瘟熱病毒的大熊貓肺滲齣液中分片段擴增 CDV 全基因序列，併剋隆到 pMD19-T 載體中；經測序、拼接，穫得第一箇 P-CDV 全長 cDNA 序列；利用 DNAman 生物學分析軟件分彆對全基因組序列、H 蛋白基因序列、F 蛋白基因序列、P 蛋白基因序列、M 蛋白基因序列等進行遺傳變異分析，構建繫統進化樹。【結果】經序列測序和拼接，大熊貓源犬瘟熱病毒全基因組長15690 nt，GenBank 登錄號為 KP677502，主要編碼6種蛋白，分彆是 N 蛋白、P 蛋白、M 蛋白、F 蛋白、H 蛋白和 L 蛋白，在各箇基因及間隔區中未髮現堿基插入和缺失。遺傳進化分析顯示，P-CDV全基因組與20株代錶性 CDV 全基因組序列同源性為91.5%—98.7%，P-CDV 與彊毒株 MKY-KM08（HM852904）、PS、HLJ1-06（JX681125）、Hebei（KC427278）以及 AC96I-H358（AB753776）在一箇大的分支上，與 PS 株（JN896331）親緣關繫最近，同源性為98.7%，與標準野毒株 Strain A75-17（AF164967）同源性為95.7%，與疫苗株 CDV3（EU726268）親緣關繫較遠，同源性為91.5%。H 蛋白氨基痠序列分析和繫統進化樹錶明，大熊貓源犬瘟熱病毒屬于彊毒株，基因型為 Asia-I 型。P-CDV 各蛋白基因與20株代錶性的毒株相比有9處氨基痠髮生瞭變異，與GenBank 上現有的 CDV 序列相比，其中3處是獨有的，分彆是：F 蛋白基因的208位由 N（Asn，天鼕酰胺，彊毒株多為 N）或 K（Lys，賴氨痠，弱毒株多為 K）變成瞭 S（Ser，絲氨痠），第215位由 S 變成瞭 A（Ala，丙氨痠），P 蛋白基因的58位由 Q（Gln，穀氨酰胺）變成 K（Lys，賴氨痠）。【結論】成功剋隆瞭大熊貓源犬瘟熱病毒的全基因，併完成瞭序列分析，髮現瞭在 F、P 基因上堿基的重要變異。這些數據將為研究大熊貓犬瘟熱的遺傳變異和流行特徵提供分子生物學依據。
【목적】대수차폭발적대웅묘원성견온열병독（panda derived-canine distemper virus,P-CDV）전기인조진행극륭측서，이료해대웅묘원 CDV 적전기인조유전변이정황，진일보추종감염원，위대웅묘견온열방공제공이론의거。【방법】근거 GenBank 공포적 CDV 전기인조서렬설계17대특이성인물，이용 RT-PCR 기술，종감염견온열병독적대웅묘폐삼출액중분편단확증 CDV 전기인서렬，병극륭도 pMD19-T 재체중；경측서、병접，획득제일개 P-CDV 전장 cDNA 서렬；이용 DNAman 생물학분석연건분별대전기인조서렬、H 단백기인서렬、F 단백기인서렬、P 단백기인서렬、M 단백기인서렬등진행유전변이분석，구건계통진화수。【결과】경서렬측서화병접，대웅묘원견온열병독전기인조장15690 nt，GenBank 등록호위 KP677502，주요편마6충단백，분별시 N 단백、P 단백、M 단백、F 단백、H 단백화 L 단백，재각개기인급간격구중미발현감기삽입화결실。유전진화분석현시，P-CDV전기인조여20주대표성 CDV 전기인조서렬동원성위91.5%—98.7%，P-CDV 여강독주 MKY-KM08（HM852904）、PS、HLJ1-06（JX681125）、Hebei（KC427278）이급 AC96I-H358（AB753776）재일개대적분지상，여 PS 주（JN896331）친연관계최근，동원성위98.7%，여표준야독주 Strain A75-17（AF164967）동원성위95.7%，여역묘주 CDV3（EU726268）친연관계교원，동원성위91.5%。H 단백안기산서렬분석화계통진화수표명，대웅묘원견온열병독속우강독주，기인형위 Asia-I 형。P-CDV 각단백기인여20주대표성적독주상비유9처안기산발생료변이，여GenBank 상현유적 CDV 서렬상비，기중3처시독유적，분별시：F 단백기인적208위유 N（Asn，천동선알，강독주다위 N）혹 K（Lys，뢰안산，약독주다위 K）변성료 S（Ser，사안산），제215위유 S 변성료 A（Ala，병안산），P 단백기인적58위유 Q（Gln，곡안선알）변성 K（Lys，뢰안산）。【결론】성공극륭료대웅묘원견온열병독적전기인，병완성료서렬분석，발현료재 F、P 기인상감기적중요변이。저사수거장위연구대웅묘견온열적유전변이화류행특정제공분자생물학의거。
[Objective] The objective of this study is to analyze the genetic variation of the newly emerged giant panda canine distemper virus by sequencing its full-length genome, thereby to track the source of infection and provide a theoretical basis for the prevention and control of giant panda CDV infection. [Method] According to the CDV genomic sequences published in GenBank, 17 pairs of oligonucleotide primers were designed, the overlapping fragments spanning whole genome of CDV were amplified by RT-PCR from lung exudate of the infected giant panda and then cloned into vector pMD19-T and sequenced, respectively, the determined sequences were spliced and assembled by using biological software DNAman. Based on above efforts, the first complete genome sequence of P-CDV isolated from giant panda was accomplished, and was further characterized by alignment with 20 published CDV whole-genome sequences from GenBank. Moreover, to track the source of infection, different phylogenetic trees were constructed according to complete genome as well as genes H, F, P and M, respectively.[Result]The genome of the giant panda CDV is 15 690 nucleotides in length (GenBank accession No. KP677502), and mainly encodes six genes in the order of 3′-N-P-M-F-H-L-5′. No nucleotides insert and deletion were observed in encoding and intergenic regions compared to other CDV complete genome sequences. Phylogenetic analysis of the giant panda CDV genome showed 91.5%-98.7% nucleotide identity with 20 representative CDV isolates. Along with virulent strains MKY-KM08 (accession no. HM852904), PS, HLJ1-06 (accession no. JX681125), Hebei (accession no. KC427278) and AC96I-H358 (accession no. AB753776), P-CDV belongs to one large homogeneous clade, and is phylogenetically most closely related to the isolate PS with 98.7% identity at nucleotide. It shares 95.7% nucleotide identity with wild strain A75-17, and exhibits the lowest nucleotide similarity with standard vaccine strain CDV3 (91.5%). Sequence analysis based on H gene indicates that the giant panda CDV is a virulent strain, it belongs to genotype Asia-I. There are a total of nine amino acid (aa) substitutions, among them three amino acid substitutions were firstly found and are unique to the giant panda CDV. Of these 3 aa substitutions, 2 substitutions (N/K208S, S215A) occur in F open reading frame (ORF), one substitution (Q58K) occurs in P ORF. [Conclusion]The complete genomic sequence of this newly emerged giant panda CDV was successfully characterized, the key site mutations in F and P genes were recognized. The data will provide valuable theoretical reference for the prevention and control of giant panda CDV infection.