Pathogenesis of Junonia coenia densovirus in Spodoptera frugiperda: A route of infection that leads to hypoxia

To evaluate densovirus potential against lepidopteran pests and their capacity to invade new hosts, we have characterised in vivo the infection and pathogenesis of the Junonia coenia densovirus (JcDNV) in the noctuid pest Spodoptera frugiperda. Here we show that infection starts with the ingestion of viral particles that cross the midgut epithelium without replicating. By quantitative PCR we established the kinetic and the route of infection, from virus ingestion to replication in visceral tracheae and hemocytes. JcDNV has a high particle-to-infection ratio mostly due to the barrier function of the midgut. Pathology and cytopathology suggested that infection of tracheal cells impairs oxygen delivery to demanding tissues leading to cytopathic effects in all the tissues. Finally, larval death results from several physiological shocks, including molting arrest and anoxia.     

黑胸大蠊浓核症病毒的研究——Ⅲ.最佳感染液回感效果测定

本文作者已从制备的11种黑胸大蠊浓核症病毒(PfDNV)感染液中,测定出最佳感染液为第1次分离出的上清液,其LT_(50)比其它感染液均短,仅44天;死亡率高达97.92%。     

High amplification of a densovirus-derived vector in larval and adult tissues of Drosophila

The Lepidopteran densovirus‐derived vector, pJlacZΔNS3, is a defective virus genome with an insertion of lacZ DNA in the viral structural protein coding sequence, and a deletion of the sequence coding the non‐structural polypeptide NS3. pJlacZΔNS3 was injected into Drosophila eggs and the maintenance of the viral genome was monitored by expression of β‐galactosidase and by Southern blot hybridizations. Intense β‐galactosidase activity was observed in many somatic tissues of third‐instar larvae and adult flies, in more than 60% of the injected animals. DNA analyses showed that staining in adult tissues correlated with the amplification of the vector. Together, these results suggest the occurrence of early events of integration of the vector into the Drosophila host genome.     

云南省新分离蚊浓核病毒的分子特征研究

对云南省新分离的14株浓核病毒进行基因序列测定和分析,以了解浓核病毒基因组结构及特性.用浓核病毒基因组扩增引物(DNV3F,DNV3R),RT-PCR扩增片段,PCR产物直接测序,拼接后获得基因序列.通过Clustal X(1.8)、DNAStar、GeneDoc (3.2) 等生物学软件进行核苷酸序列及氨基酸序列分析...     

Mosquito cells accommodate balanced, persistent co-infections with a densovirus and Dengue virus

To study persistent viral co-infections in arthropods, we first produced stable, persistently infected C6/36 mosquito cell cultures by serial passage of exponentially growing whole cells infected with either a densovirus (AalDNV) or Dengue virus (DEN-2). We then obtained stable, persistent co-infections by reciprocal super-challenge and similar passaging. Persistently infected cultures did not differ from naïve-cell cultures in growth rate and cell morphology. Nor did they differ in high production of both viruses with high infection rates for naïve C6/36 cells. Immunocytochemistry revealed that 99–100% of the cells were coinfected but that super-infection order had some effect on antigen distribution for the two viruses. Our results combined with existing field information and previously published experimental work suggest that the capacity to support stable, viral co-infections may be a general phenomenon for arthropod cells, and that they may be achieved easily and rapidly by serial passaging of whole cultured cells. Such persistent infections would facilitate studies on interactions between co-infecting viruses.     

云南省文山中越边境地区虫媒病毒调查

目的 对云南省文山县、河口县等中越边境地区开展虫媒病毒调查,以期了解当地蚊虫携带虫媒病毒情况.方法 2007年9月在当地5个采集点共采集蚊虫8326只,包括库蚊6091只,中华按蚊1334只,刺扰伊蚊848只,阿蚊53只,并保存于液氮.经消毒、研磨、离心等处理后进行组织培养细胞接种,分离病毒和对病毒分离物进行血清学和分子生物学鉴定,以软件进行病毒的核苷酸序列比对和系统发生分析.结果 从库蚊分离到4株对C6/36细胞致病变的病毒分离物,其他蚊种没有分离到病毒分离物.鉴定结果表明,标本WS0704-2与版纳病毒抗体反应,PCR鉴定为版纳病毒,该病毒基因组第12片段的进化关系同中国其他版纳病毒有明显差异,位于一条独立的进化枝中,在进化上相对独立.标本WS0704-1、WS0708-1、WS0708-2为浓核病毒.结论 云南省文山中越边境地区存在多种蚊虫媒介并携带版纳病毒等虫媒病毒.     

蚊浓核病毒非结构蛋白1的生物信息学分析

目的 通过生物信息学分析软件对蚊浓核病毒(mosquito densovirus,MDV)菲结构蛋白1(Nostructual proteinl,NS1)的结构与功能进行分析预测.方法 应用EXPASY pmtparam tool、ClagtalX1.83、Bioedit、MEGA3.1、ScanProsite、Motifscan等分析软件和在线生物信息学分析工具对MDVNS1的理化特性、同源性、进化关系、二级结构与主要功能结构域进行分析预测.结果 MDV NSI属不稳定亲水性蛋白,氨基酸序列高度保守,与虾传染性皮下及造血组织坏死病毒(Infectious hypodermal and hematopoietic necrosis virus,IHHNV)进化距离较近;MDV NSI具有小分子DNA病毒编码的解螺旋酶超家族3(Saperfamily 3 helicage of DNA viruses)的结构域,该结构域包含有NTP-binding区域,使其具有金属离子依赖的ATPase的活性,蛋白靠近N端包含有病毒滚筒复制rolling-circle replication(RCR)起始蛋白的结构域并具有单链切口酶的生物学功能.结论 生物信息学预测结果提示MDV NS1蛋白在病毒的复制、包装等各个阶段起关键性作用.     

Markedly reduced severity of Dengue virus infection in mosquito cell cultures persistently infected with Aedes albopictus densovirus (AalDNV)

AalDNV-infected C6/36 cells serially passaged for over 10 weeks showed a decline in percentage of anti-AalDNV-positive cells (APC) from an initial 92% to approximately 20%. Cultures of persistent APC were indistinguishable from uninfected cultures by direct microscopy but most stained cells from early APC passages had enlarged nuclei with eosinophilic inclusions, while late APC passages had few and naive cells none. Super challenge of persistent APC cultures did not increase percentage APC and supernatants from persistent APC cultures gave low APC (40%) in naive C6/36 cell cultures. When challenged with dengue virus serotype 2 (DEN-2), naive C6/36 cells showed severe cytopathic effects (CPE) and high mortality within 4 days, as did early passage APC cultures. Remarkably, DEN-2 infections in persistent APC cultures were much less severe, being characterized by reduced DEN-2 infection percentage, retarded DEN-2 virion production, no CPE and no significant mortality. Reasons for rapid reduction in APC and resistance to superinfection upon serial passage remain unproven but may relate to production of AalDNV-defective interfering particles (DIP) by molecular mechanisms still open to speculation. More difficult to explain is cross-protection against DEN-2-induced mortality seen in persistent APC cultures. However, by comparison to work on shrimp viruses, we speculate that this may involve blockage of viral-triggered apoptosis. The phenomena described raise questions regarding the potential for persistent infections by unknown viruses to confound experimental results with insect cell lines.     

黑胸大蠊浓核病毒病(PfDNV)的研究——Ⅱ.回感试验

本文报道我们于1990年发现黑胸大蠊浓核病毒病后,按照Koch's定理,将该病毒病进行了回感试验。结果表明:从自然死亡的黑胸大蠊中分离的DNV,回感健康的黑胸大蠊,可引起同样的病症,并从中重新分离到完全一样的DNV,说明DNV是黑胸大蠊若虫的致死病原体。该病毒的回感力极强,LT_(50)为39天,最终死亡率可达99%以上,是防治黑胸大蠊的一种高毒力的病原体,为黑胸大蠊生物防治提供了一种有效的新途径和极好的病毒资源,值得加以研究和开发利用。