MDPV和GPV感染宿主致病性差异的分子基础

Muscovy duck parvovirus (MDPV) and Goose parvovirus (GPV) are classified in the Dependovirus genus of the Parvoviridae family. Both viruses cause serious loss in geese and ducks industry. The goose parvoviral disease is often called “Derzsy’s disease” or “Gosling plaque” in China, and the muscovy parvoviral disease is also named “three-week disease”, which imply the infection often occur in 3-week-old muscovy ducklings. In case of B strain of GPV and FM strain of MDPV, they share 82% homologies at the genome level. Comparisons of the coding proteins between MDPV and GPV show that the Rep protein and the VP1 protein display 90.6% and 87.7% identities at the amino acid level, respectively. Irrespective of the high similarity between GPV and MDPV, two viruses have different host ranges. MDPV infects muscovy ducklings only, while GPV is infectious for both goslings and muscovy ducklings, what’s more, infection of GPV in muscovy ducklings generally produce the higher morbidity and mortality than infection of MDPV. Undoubtedly, the differential pathogenicity exhibited in muscovy ducklings and goslings caused by MDPV and GPV results from the genome differences between two viruses, but which genome regions actually contribute the differential pathogenicity for the two intimate waterfowl parvoviruses are not understood clearly..In a preliminary study, the recombinant plasmid containing the entire genome of the virulent strain LH of GPV was constructed, and the infectious virus carrying the genetic marker was rescued successfully by transfection of the pLH plasmid in embryonated goose eggs via the chorioallantic membrane route. In this study, we plan to further construct the infectious plasmid pNY that contain the complete genome of the virulent strain NY of MDPV, and perform the rescue in embryonated muscovy duck eggs. The whole genome of strain NY will be sequenced and compared with that of strain LH. The highly variable regions distributed in the genomes that may play the key roles in differential pathogenicity between NY and LH will be identified for further analysis. The variable DNA regions will be swapped between the plasmid pNY and pLH, resulting in a series of chimeric plasmids. After transfection of the chimeric plasmids in embryonated muscovy ducks or goose eggs, the recombinant chimeric viruses will be rescued. Animal challenge experiments will be performed on 1-day-old muscovy ducklings or goslings to evaluate the pathogenicity of the chimeric viruses, and the experimental results will allow us to map the DNA regions correlated with the differential pathogenicity of MDPV and GPV. Based on the obtained data and analysis, we will continue to implement mutations on key amino acids or nucleotides sites, and testify their roles in the pathogenicity. Elucidation of the molecular basis of the differential pathogenicity of MDPV and GPV will lay a solid foundation for us to further explore the pathogenesis of waterfowl parvoviruses.

MDPV和GPV在基因组水平上具有很高同源性，但在感染谱上却差异明显。MDPV仅感染雏番鸭而不感染雏鹅，GPV不仅感染雏鹅也能感染雏番鸭，而且对后者致病力还要强于MDPV，这一致病性差异的机理一直未予阐明。先前研究中，我们构建了GPV 强毒株LH的感染性质粒pLH，本研究中，拟进一步构建MDPV 强毒株NY的感染性质粒pNY。在对MDPV和GPV基因组序列比对基础上，拟在pNY和pLH质粒之间互换差异DNA片段，从而获得一系列嵌合对方DNA片段的重组质粒，通过绒毛尿囊膜途径转染番鸭胚或鹅胚来拯救嵌合病毒，并在雏番鸭或雏鹅上进行毒力试验来评估嵌合病毒的致病性，据此可以精确定位哪些DNA区域与MDPV和GPV的致病性差异相关联。在此基础上，拟对关键氨基酸或碱基予以定点突变并验证这些位点在致病性差异中的作用。阐明MDPV和GPV致病性差异的分子基础对深入揭示水禽细小病毒的致病机理将发挥指引作用。

番鸭细小病毒（MDPV）与鹅细小病毒（GPV）之间同源性较高，但在对不同宿主的致病性上差异明显，GPV对雏鹅和雏番鸭均可致病，而MDPV仅使雏番鸭致病而不感染雏鹅。在本项目研究中，拟在构建的MDPV感染性质粒pYY和先前构建的GPV强毒株LH株的感染性质粒pLH基础上，通过片段交换重组，再通过转染拯救生成嵌合病毒，通过研究嵌合病毒对雏番鸭和雏鹅的致病性，进而推断出哪些基因片段在MDPV和GPV对雏番鸭和雏鹅的致病性差异上发挥关键作用。为了实现上述目标，首先构建了MDPV强毒株YY的感染性质粒pYY。pYY质粒转染11日龄番鸭胚成功拯救出感染性病毒，拯救病毒具有与亲本病毒相似的致病性，从而建立了感染性质粒构建-转染拯救病毒-动物感染实验的技术路线。在pYY质粒构建基础上，本研究通过overlap PCR交换pYY和pLH之间的基因片段，构建了系列嵌合质粒，分别为pYY-gRep1、pYY-gVP1、pYY-gVP3、pYY-gRC、pLH-mVP1和pLH-mRep1，嵌合质粒分别转染番鸭胚拯救出嵌合病毒，对嵌合病毒分别感染雏番鸭和雏鹅，统计15天观察期内的致死率和生长体重。.雏鹅感染试验表明，携带MDPV VP1基因的pLH-mVP1病毒不仅对雏鹅表现出明显致病性，甚至对雏鹅的致死率还要高于携带GPV VP1基因的pLH-mRep1病毒，但仍然不及pLH病毒对雏鹅的致病性。而pYY-gVP1病毒对雏鹅的致死率可达75%，死亡产生在接毒后9-13天，但无法观察到雏鹅感染GPV后肠道纤维素性渗出和肠栓的特征性病变。感染试验表明MDPV和GPV在雏鹅上表现出的致病性差异，不仅与VP1蛋白相关，Rep蛋白和Rep-ITR的互作在其中也发挥重要作用。.雏番鸭感染试验表明，5个重组病毒对番鸭均具有致病性，但都不及GPV pLH病毒对雏番鸭的致病性强，表现为致死率不高，大多表现为发育迟缓受阻。其中，携带GPV VP1基因的pYY-gVP1的致病性高于pYY-gRep1和pYY-gRC病毒；而携带MDPV编码基因的pLH-mRep1致病性略高于pLH-mVP1。感染试验的结果可推断：GPV对番鸭的致病性既与VP1相关，也与Rep蛋白以及其他顺式作用元件（ITR、启动子）之间的互作有关。