Experimental transmission and tissue tropism of white spot syndrome virus (WSSV) in two species of lobsters, Panulirus homarus and Panulirus ornatus

The susceptibility of two species of lobsters, Panulirus homarus and Panulirus ornatus to white spot syndrome virus (WSSV) was tested by oral route and intramuscular injection. The results revealed that these lobsters were as highly susceptible as marine shrimp when the WSSV was administered intramuscularly. The WSSV caused 100% mortality in both Panulirus homarus and Panulirus ornatus, at 168 and 120 h, respectively, after intramuscular injection and failed to cause mortality when given orally. The presence of WSSV in moribund lobsters was confirmed by single-step and nested PCR, Western blot, histology, and bioassay test. It was found in eyestalk, gill, head muscle, tail muscle, hemolymph, appendages, and stomach. In lobsters with oral route infection, all tested organs except stomach and head muscle was negative for WSSV by nested PCR at 120 h post-inoculation. The stomach and head muscle was positive by nested PCR at 120 h p.i., but negative at 168 h p.i. Western blot analysis was negative in all the tested organs of both species of lobster at 120 h post-inoculation by oral route.     

A cDNA microarray approach for analyzing transcriptional changes in Penaeus monodon after infection by pathogens

A cDNA microarray comprised of 9990 different ESTs obtained from the Penaeus monodon EST project (http://pmonodon.biotec.or.th) was employed to identify viral (white spot and yellow head viruses) and bacterial (Vibrio harveyi) responsive genes in the hemocytes of P. monodon at 6, 24 and 48 h post-injection (hpi). The number of differentially expressed genes found was highest in shrimps infected with white spot virus (1954 genes) followed by yellow head virus (1136 genes) and V. harveyi (420 genes). Changes in shrimp gene expression were highest at the late infection stage for both viruses, whilst that for V. harveyi induced gene expression was mainly found at the early infection stage, but the repression of genes was mainly found in the mid stage of infection. Shrimp genes specifically upregulated by each particular pathogen are identified and are summarized.     

Primary culture of hemocytes from the Caribbean spiny lobster, Panulirus argus, and their susceptibility to Panulirus argus Virus 1 (PaV1)

Primary cultures of hemocytes from the Caribbean spiny lobster Panulirus argus were developed for studies on the in vitro propagation of Panulirus argus Virus 1 (PaV1). A modified Leibovitz L-15 medium supported the best survival of hemocytes in in vitro primary cultures. However, degradation of the cultures occurred rapidly in the presence of granulocytes. A Percoll step gradient was used to separate hemocytes into three subpopulations enriched in hyalinocytes, semigranulocytes, and granulocytes, respectively. When cultured separately, hyalinocytes and semigranulocytes maintained higher viability ( approximately 80%) after 18 days incubation compared with granulocytes, which degraded over 2-3 days. Susceptibility of the cell types was investigated in challenge studies with PaV1. Hyalinocytes and semigranulocytes were susceptible to PaV1. Cytopathic effects (CPE) were observed as early as 12h post-inoculation, and as the infection progressed, CPE became more apparent, with cell debris and cellular exudates present in inoculated cultures. Cell lysis was noticeable within 24h of infection. The presence of virus within cells was further confirmed by in situ hybridization using a specific DNA probe. The probe gave a unique staining pattern to cells infected with PaV1 24-h post-inoculation. Cells in the control treatment were intact and negative to hybridization. This assay was further applied to the quantification of infectious virus in hemolymph using a 50% tissue culture infectious dose assay (TCID(50)) based on CPE. These tools will now allow the quantification of PaV1 using established culture-based methods.     

Molecular characterization and bioactivity of a CXCL13 chemokine in large yellow croaker Pseudosciaena crocea

A CXCL13-like chemokine cDNA was isolated from large yellow croaker (Pseudosciaena crocea) by expressed sequence tag (EST) analysis (LycCXCL13). The full-length cDNA of LycCXCL13 is 796 nucleotides (nt) encoding a protein of 97 amino acids (aa), with a putative molecular weight of 10.7 kDa. The deduced LycCXCL13 contains a 24-aa signal peptide and a 73-aa mature polypeptide, which possesses the typical arrangement of four cysteines as found in other known CXC chemokines (C²⁵, C²⁷, C⁵² and C⁶⁸). It shares 35, 36 and 39% aa sequence identities to green puffer CXCL13-like, Atlantic salmon CXCL13 and Japanese flounder CXCL13 chemokines, and 24–29% identities to CXCL13 chemokines in mammals, respectively. Phylogenetic analysis showed that LycCXCL13 is more closely related to the CXCL13 subgroup than to any other CXC chemokine subgroups. LycCXCL13 gene was constitutively expressed in all tissues examined, except for intestine. Upon induction with poly(I:C) or inactivated trivalent bacterial vaccine, LycCXCL13 gene expression was significantly up-regulated in spleen, head kidney, heart and gills at 24 h post-injection. Real-time PCR results showed that LycCXCL13 gene expression reached peak level in spleen and head kidney at 12 h after induction by poly(I:C), while its expression increased to the highest level in head kidney at 24 h or in spleen at 48 h by bacterial vaccine. Recombinant LycCXCL13 protein produced in E. coli BL21 exhibited obvious chemotaxis to the peripheral blood leucocytes (PBLs) from large yellow croaker. These results suggest that LycCXCL13 may be involved in inflammatory responses as well as homeostatic processes in large yellow croaker.     

Oral susceptibility of Aedes albopictus to dengue type 2 virus: a study of infection kinetics, using the polymerase chain reaction for viral detection

Female Aedes albopictus mosquitoes, aged 1 week, were infected with DEN-2 dengue virus. The kinetics of infection in mosquito brain and mesenteron were monitored using DNA probes with polymerase chain reaction (PCR) amplification of target DNA sequences coding for DEN-2 virus envelope protein, compared with the standard immunofluorescence assay technique (IFA). Rates of virus detection in the mesenteron of orally infected mosquitoes rose to 38% by day 4 post-inoculation, then declined until day 8, followed by irregular peaks around days 11-14 and subsequently. In mosquito head squashes, virus was detected from day 4 onwards, reaching 38% positive by day 18. Salivary glands of all the same females were found to be positive for virus by day 8 onwards. Parenterally infected Ae.albopictus females were all positive for DEN-2 in the brain and salivary glands 8 days post-inoculation. In every case, results obtained with the PCR matched those from the IFA. Our DNA probe with PCR procedure can therefore be utilized as a sensitive and reliable method for studies of DEN-2 vectors.     

The transcriptome of Fusarium graminearum during the infection of wheat

Fusarium graminearum causes head blight disease in wheat and barley. To help understand the infection process on wheat, we studied global gene expression of F. graminearum in a time series from 24 to 196 h after inoculation, compared with a noninoculated control. The infection was rapid and, after 48 h, over 4,000 fungal genes were expressed. The number of genes expressed increased over time up to 96 h (>8,000 genes), and then declined at the 144- and 192-h post-inoculation time points. After subtraction of genes found expressed on complete medium, during carbon or nitrogen starvation, and on barley, only 355 were found exclusively expressed in wheat, mostly genes with unknown function (72.6%). These genes were mainly found in single-nucleotide polymorphism-enriched islands on the chromosomes, suggesting a higher evolutionary selection pressure. The annotated genes were enriched in functional groups predicted to be involved in allantoin and allantoate transport, detoxification, nitrogen, sulfur and selenium metabolism, secondary metabolism, carbohydrate metabolism, and degradation of polysaccharides and ester compounds. Several putative secreted virulence factors were also found expressed in wheat.     

Development of a novel bioassay for estimation of median lethal concentrations (LC50) and doses (LD50) of the entomopathogenic fungus Beauveria bassiana, against western flower thrips, Frankliniella occidentalis

To conduct laboratory experiments aimed at quantifying secondary acquisition of fungal conidia by western flower thrips (Frankliniella occidentalis), an efficient assay technique using Beauveria bassiana as the model fungus was developed. Various application protocols were tested and it was determined that the percent mortality did not vary among protocols. Peak mortality of second-instar nymphs, under constant exposure to conidia, occurred 5 days post-inoculation. Second-instar thrips that were exposed to conidia within 24 h of the molt to second instar were more susceptible to Beauveria bassiana than thrips exposed after times greater than 24 h post-molt. Conidia efficacy, which was monitored at 24 h intervals, did not differ significantly within 72 h. A test of the final bioassay system was conducted in a series of assays aimed at determining the LD50 of B. bassiana technical powder against second-instar western flower thrips. It was determined that B. bassiana (strain GHA) is highly effective at very low doses (LD50 of 33-66 conidia/insect).     

Characterization of varicella-zoster virus enhancement by the pesticide carbaryl.

Further characterization of the viral enhancement system of varicella-zoster virus by the pesticide carbaryl (1-naphthyl-N-methylcarbamate) is presented. It was necessary to expose cells to the enhancing chemical during the period of virus replication to detect enhancement. The optimum time for the pretreatment is 20 to 24 h. Maximum enhancement of virus expression occurs 48 to 72 h post-inoculation. Treated cells cannot pass on to daughter cells the ability to produce increased amounts of virus. alpha-Naphthol, a metabolite of carbaryl, is also capable of enhancing virus replication; Guthion, another pesticide tested, did not enhance varicella-zoster virus. The stable cell line HEP-2 can be used in place of human embryonic lung cells to detect enhancement. Differences in enhancement levels were not due to cell lot, cell passage, or a change in the stability of the cell membrane to sonic disruption.     

Surface Ultrastructural Studies on the Germination,Penetration and Conidial Development of Aspergillus Flavus Link : Fries Infecting Silkworm,Bombyx Mori Linn

Aspergillosis is a common disease of the silkworm Bombyx mori Linn., caused by an insect mycopathogen Aspergillus flavus Link:Fries. The present study reveals the germination, penetration and conidial development of A. flavus on the larval integument of B. mori under SEM. Four different strains (NB18, KA, NB4D2 and NB7) of B. mori was surface inoculated with ca. of 1 x 10(6) conidia/ml. Each conidium germinated on the cuticle approximately 6 h after inoculation, forming a humpy or suctorial appressoria within 24 h. The hyphae which entered into haemocoel 2 day post-inoculation, grew and multiplied extensively, forming a mycelial complex, causing death of the host larva in about 4-5 days. This occurred with minimal breakdown of the internal tissues. Death of the host was followed by ramification of the fungus through the mesodermal and epidermal tissues, leading to larval mummification about 5-6 days after inoculation. Extensive fungal growths on the entire larval body followed, consisting of aerial hyphae, which developed branched conidiophores. The aerial hyphae with abundant conidiophores formed a confluent yellowish green fungal mat over the entire larval body in 6-7 days of post-inoculation. The tip of each emerging conidiophores gradually dilated and developed to become a bulbous head known as the vesicle. A large number of conidiogenous cells were produced over the entire surface of vesicle, which later developed into finger-like projections termed as sterigmata or phialides. The phialides matured within 2 days after the aerial hyphae emerged as evidenced by chains of conidia at their tips. The conidia were globose with externally roughened walls. The life cycle of the fungus on B. mori was completed in six to seven days.     

The Swiss/ICR (Ha) albino mouse as an experimental host for infectious pancreatic necrosis virus of trout

Intracranial inoculation of infectious pancreatic necrosis virus (IPNV), a pathogen of several species of trout, produced pancreatic necrosis in suckling Swiss albino mice. Peri-nuclear halos, cytoplasmic vacuoles, and necrosis were found in histologic sections of pancreas taken from mice killed 21 days post-inoculation. Virus was recovered from the pancreas of mice killed as early as 10 days post-inoculation. Rivers' postulates were fulfilled. Virus recovered from the infected mouse pancreas was neutralized by IPNV specific antiserum. The significance of the mouse as an experimental host is discussed.     

Characterization of varicella-zoster virus enhancement by the pesticide carbaryl

Further characterization of the viral enhancement system of varicella-zoster virus by the pesticide carbaryl (1-naphthyl-N-methylcarbamate) is presented. It was necessary to expose cells to the enhancing chemical during the period of virus replication to detect enhancement. The optimum time for the pretreatment is 20 to 24 h. Maximum enhancement of virus expression occurs 48 to 72 h post-inoculation. Treated cells cannot pass on to daughter cells the ability to produce increased amounts of virus. alpha-Naphthol, a metabolite of carbaryl, is also capable of enhancing virus replication; Guthion, another pesticide tested, did not enhance varicella-zoster virus. The stable cell line HEP-2 can be used in place of human embryonic lung cells to detect enhancement. Differences in enhancement levels were not due to cell lot, cell passage, or a change in the stability of the cell membrane to sonic disruption.     

大黄鱼TRIM25基因克隆和表达分析

三重基序蛋白25 (Tripartite motif-containing protein 25, TRIM25)属于E3泛素连接酶家族, 在先天免疫反应中发挥重要作用。为研究TRIM25基因在大黄鱼(Larimichthys crocea)先天抗病毒免疫反应中的作用, 研究鉴定并克隆大黄鱼TRIM25基因(命名为LcTRIM25)。LcTRIM25基因编码序列2097 bp (GenBank登录号: MK327541), 编码698个氨基酸。蛋白结构域预测发现LcTRIM25包括保守的RING结构域、B-box2结构域、Coiled-coil结构域和可变的C末端PRY/SPRY结构域。多序列比对以及系统进化树分析表明LcTRIM25基因与斜带石斑鱼同源性高, 与哺乳动物、爬行动物、两栖动物和鸟类同源性相对低, 这说明不同物种受到来自环境不同的选择压力, 导致进化程度不同。应用实时荧光定量PCR方法分析大黄鱼TRIM25基因的表达水平。结果分析发现LcTRIM25基因在健康大黄鱼的9个组织中均有广泛表达, 且在肝脏中表达量最高, 在心脏中表达量最低。在poly(I:C)刺激后, 在外周血、头肾、脾脏和肝脏中LcTRIM25基因表达量迅速且明显上调, 均出现上升达到峰值后下降的趋势。LcTRIM25基因表达量在头肾和脾脏中6h达到最高表达量, 在肝脏中12h达到峰值, 外周血中在24h达到最高表达量。上述结果表明, 不同组织中LcTRIM25基因表达模式具有差异性。研究结果推测大黄鱼TRIM25基因参与抗病毒免疫反应且发挥十分关键的作用, 为进一步了解大黄鱼抗病毒免疫机制提供理论基础。     

Intradermally administered yellow fever vaccine at reduced dose induces a protective immune response: a randomized controlled non-inferiority trial

Background

Implementation of yellow fever vaccination is currently hampered by limited supply of vaccine. An alternative route of administration with reduced amounts of vaccine but without loss of vaccine efficacy would boost vaccination programmes.

Methods and Findings

A randomized, controlled, non-inferiority trial was conducted in a Dutch university center between August 2005 and February 2007. A total of 155 primary vaccinated and 20 previously vaccinated volunteers participated. Participants were randomly assigned in a 1∶1 ratio to receive intradermal (i.d.) vaccination with live attenuated yellow fever 17D vaccine at a reduced dose (1/5^th; 0·1 mL) or the conventional subcutaneous (s.c.) vaccination (0·5 mL). Antibody neutralization titers were determined at 2, 4 and 8 weeks and 1 year after vaccination by counting the reduction in virus-induced plaques in the presence of serial serum dilutions. Adverse events were documented in a 3-week dairy. Viraemia was measured 5 days after vaccination. From 2 weeks up to one year after vaccination, the maximum serum-dilution at which 80% of the virus plaques were neutralized, which indicates protection against yellow fever, did not differ between those given a reduced i.d. dose or standard s.c. dose of vaccine. In all cases the WHO standard of seroprotection (i.e. 80% virus neutralization) was reached (in 77/77 and 78/78, respectively). Similar results were found in the previously vaccinated individuals. Viraemia was detected in half of the primary vaccinated participants, which was not predictive of serological response. In revaccinees no viraemia was detected.

Conclusions

Intradermal administration of one fifth of the amount of yellow fever vaccine administered subcutaneously results in protective seroimmunity in all volunteers. Albeit this vaccination route should enable vaccination of five-times as many individuals at risk for disease, these results should now be confirmed in field studies in areas with potential yellow fever virus transmission to change vaccination policy.

Trial Registration

Nederlands Trial Register ISRCTN46326316     

Chitosan and its quaternized derivative as effective long dsRNA carriers targeting shrimp virus in Spodoptera frugiperda 9 cells

RNA interference (RNAi) is a promising strategy to combat shrimp viral pathogens at lab-scale experiments. Development of effective orally delivered agents for double-stranded (ds)RNA is necessary for RNAi application at farm level. Since continuous shrimp cell lines have not been established, we are developing a dsRNA-delivery system in Spodoptera frugiperda (Sf9) cells for studying in vitro RNAi-mediated gene silencing of shrimp virus. Sf9 cells challenged with yellow head virus (YHV) were used for validating nanoparticles as effective dsRNA carriers. Inexpensive and biodegradable polymers, chitosan and its quarternized derivative (QCH4), were formulated with long dsRNA (>100 bp) targeting YHV. Their morphology and physicochemical properties were examined. When treated with chitosan- and QCH4-dsRNA complexes, at least 50% reduction in YHV infection in Sf9 cells relative to the untreated control was evident at 24h post infection with low cytoxicity. Inhibitory effects of chitosan- and QCH4-dsRNA complexes were comparable to that of dsRNA formulated with Cellfectin(?), a commercial lipid-based transfection reagent. The natural and quaternized chitosan prepared in this study can be used for shrimp virus-specific dsRNA delivery in insect cultures, and have potential for future development of dsRNA carriers in shrimp feed.     

In vitro inhibition of Salmonella enterica serovars choleraesuis and typhimurium,Escherichia coli F-18, and Escherichia coli O157:H7 by a porcine continuous-flow competitive exclusion culture

A competitive exclusion (CE) culture of porcine cecal bacteria was developed as a continuous-flow culture in chemostats, was designated RPCF, and was used as a model to determine its usefulness against in vitro colonization by Salmonella enterica serovars Typhimurium and Choleraesuis, Escherichia coli strain F-18, and E. coli serotype O157:H7 (933). Chemostats with or without RPCF were inoculated with 10⁶ colony-forming units (CFU)/ml of Typhimurium, Choleraesuis, F-18, or O157:H7. Chemostats were sampled for salmonellae and E. coli at 15 min, 7 h, and every 24 h thereafter. In control chemostats without RPCF, Typhimurium, Choleraesuis, F-18, and O157:H7 rapidly established colonization and had concentrations of 10⁶ CFU/ml for 96–120 h post-inoculation. In the chemostats that contained RPCF, reductions (P < 0.05) of Choleraesuis, F-18, and O157:H7 were observed at 24 h post-inoculation. Typhimurium was decreased (P < 0.05) at 48 h post-inoculation, and by 120 h post-inoculation, all chemostats were negative for the four challenge microorganisms. These results demonstrate that RPCF cultures were able to inhibit the growth of Typhimurium, Choleraesuis, and E. coli strains F-18 and O157:H7 in vitro and suggest the potential for the use of CE in swine to prevent disease induced by these microorganisms. Received: 2 October 2001 / Accepted: 31 December 2001     

Relations of carrot red leaf and carrot mottle viruses with their aphid vector, Cavariella aegopodii

Studies with Scottish isolates of carrot red leaf (CRLV) and carrot mottle (CMotV) viruses confirmed the dependency of CMotV on CRLV for transmission by the aphid Cavariella aegopodii. CMotV was transmitted by aphids only when the two viruses were present in the same source plant, and its transmission was not assisted by anthriscus yellows virus, which acts as a helper for parsnip yellow fleck virus. Some test plants became infected with CRLV alone, and a few with CMotV alone. In winter, aphid transmission of CRLV and CMotV was greatly increased when the source plants received supplementary lighting whereas the CMotV infectivity of sap was not increased. C. aegopodii acquired CRLV and CMotV after minimum acquisition access times of 30 min and inoculated them after minimum inoculation access times of 2 min. There was a minimum latent period of 7–18 h. The viruses were retained by the aphid after moulting and are therefore circulative in the vector, but were not transmitted to progeny insects. Aphids allowed 24 h to acquire the viruses continued to transmit them for at least 12 days, but some aphids allowed 6 h or less for virus acquisition ceased to transmit after 3 or 4 days. CRLV is considered a tentative member of the luteovirus group.     

Comparative analysis of expressed sequence tags in resistant and susceptible ecotypes of Arabidopsis thaliana infected with cucumber mosaic virus

Arabidopsis thaliana ecotype Columbia (Col-0) is susceptible to the yellow strain of cucumber mosaic virus [CMV(Y)], whereas ecotype C24 is resistant to CMV(Y). Comprehensive analyses of approximately 9,000 expressed sequence tags in ecotypes Col-0 and C24 infected with CMV(Y) suggested that the gene expression patterns in the two ecotypes differed. At 6, 12, 24 and 48 h after CMV(Y) inoculation, the expression of 6, 30, 85 and 788 genes, respectively, had changed in C24, as opposed to 20, 80, 53 and 150 genes in CMV(Y)-infected Col-0. At 12, 24 and 48 h after CMV(Y) inoculation, the abundance of 3, 10 and 55 mRNAs was altered in both ecotypes. However, at 6 h after CMV(Y) inoculation, no genes were co-induced or co-suppressed in both ecotypes. This differential pattern of gene expression between the two ecotypes at an early stage of CMV(Y) infection indicated that the cellular response for resistance may differ from that resulting in susceptibility at the level detectable by the macroarray. According to the expression pattern at various stages of infection, the expression of many genes could be grouped into clusters using cluster analysis. About 100 genes that encode proteins involved in chloroplast function were categorized into clusters 1 and 4, which had a differentially lower expression in CMV(Y)-inoculated C24. The expression of various genes encoding proteins in the endomembrane system belonged to clusters 2 and 4, which were induced in CMV(Y)-inoculated C24 and Col-0 leaves. Characterization of CMV(Y)-altered gene expression in the two ecotypes will contribute to a better understanding of the molecular basis of compatible and incompatible interactions between virus and host plants.