Competition of infectious hypodermal and haematopoietic necrosis virus (IHHNV) with white spot syndrome virus (WSSV) for binding to shrimp cellular membrane

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) and white spot syndrome virus (WSSV) are two widespread shrimp viruses. The interference of IHHNV on WSSV was the first reported case of viral interference that involved crustacean viruses and has been subsequently confirmed. However, the mechanisms underlying the induction of WSSV resistance through IHHNV infection are practically unknown. In this study, the interference mechanisms between IHHNV and WSSV were studied using a competitive ELISA. The binding of WSSV and IHHNV to cellular membrane of Litopenaeus vannamei was examined. The results suggested that there existed a mutual competition between IHHNV and WSSV for binding to receptors present on cellular membrane of L. vannamei and that the inhibitory effects of WSSV towards IHHNV were more distinct than those of IHHNV towards WSSV.     

浙江省主要养殖区凡纳滨对虾（Litopenaeus vannamei）红体病病原研究

对2012—2013年浙江省凡纳滨对虾( L itopenaeus vannamei)主要养殖区的红体病虾进行病原鉴定及其分子特征与耐药性研究,并与2011年的结果进行比较分析.结果表明：通过 Vitek 、16S rRNA 序列分析与病毒特异性聚合酶链反应,2012—2013年在红体病虾中同时检测出副溶血弧菌( V ibrio parahaemolyticus)与传染性皮下和造血组织坏死病毒( infectious hypodermal and haematopoietic necrosis virus , IHHNV ),而2011年在红体病虾中仅检测出副溶血弧菌,未检测出 IHHNV ;3年均未检测出桃拉综合征病毒( Taura syndrome virus , TSV)和白斑综合征病毒( white spot syndrome virus , WSSV ).基于 dnaE‐gyrB‐recA‐dtdS‐pntA‐pyrC‐tnaA 的多位点序列分型结果表明,副溶血弧菌分离株具有较高水平的分子多样性：2011—2013年各年的分离株均分属多个序列型( sequence type ,ST ),来自位于进化树不同亚枝、亲缘关系较远的不同克隆;2012—2013年的分离株经历了较高水平的分子变异,其中 ST918与 ST919为首次报道的副溶血弧菌新 ST ;2011年分离株与2012—2013年分离株的等位基因和序列型特征具有较大差异,ST414与 ST114分别代表2011年与2012—2013年红体病的优势 ST .2011—2013年的副溶血弧菌分离株均属于大流行群,并具有相同的毒力基因谱( tlh＋ tdh－ trh－ T3SS1＋ T3SS2－),tdh与 trh的缺失并未影响细菌的致病力.分离株对抗菌药物尤其是氨基糖苷类的耐药性呈现加重趋势.说明本次凡纳滨对虾红体病可能是多种致病因子共同作用的结果,亟须建立红体病病原监测体系,并探明细菌性病原与病毒性病原混合感染对致病性的影响及其互作机制,以提出综合防控方案.     

Evaluation of bone marrow aspirates from multiple sites for staging of canine lymphoma and mast cell tumours

This prospective study evaluated the utility of bone marrow aspirates (BMAs) obtained from multiple sites for staging of canine lymphoma (LSA) and mast cell tumours (MCTs). Forty dogs (LSA, n = 24; MCTs, n = 16) were enrolled, but only 33 (82.5%) had diagnostic bone marrow (BM) aspirates obtained from two sites for inclusion in the study. Nineteen dogs with LSA were included, and 6 (31.6%) had BM involvement. Neoplastic lymphocytes were present in BM from both sites in all of these dogs. Fourteen dogs with MCTs were included, and 3 (21.4%) had BM involvement. Neoplastic mast cells were present at both sites in two dogs and at only one site in the third. These results indicate that BMAs from multiple sites may not be needed for accurate staging of canine LSA patients, but more studies evaluating the pattern of BM infiltration in dogs with high‐grade MCTs are warranted.     

用逆转录多聚酶链式反应从凡纳对虾中检出传染性皮下组织和造血器官坏死病毒

传染性皮下组织和造血器官坏死病毒 (ＩＨＨＮＶ) ,是一种细小病毒 ,它能感染所有起源于中胚层和外胚层的对虾组织细胞[1] 。在许多养殖对虾的国家都有ＩＨＨＮＶ ,特别是中美洲国家和地区[2 ] 的对虾养殖业深受其害。随着各国间对虾贸易的急剧增长 ,ＩＨＨＮＶ地理分布范围日益广泛 ,迄今为止 ,已扩散到中国台湾、新加坡、马来西亚、泰国、印度尼西亚、澳大利亚、菲律宾、厄瓜多尔、秘鲁等国家和地区[2 -4] 。红额角对虾 (Ｐｅｎａｅｕｓｓｔｙｌｉｒｏｓｔｒｉｓ)、斑节对虾 (Ｐ .ｍｏｎｏｄｏｎ)、短沟对虾 (Ｐ .ｓｅｍｉｓｕｌｃａｔｕ…     

Salmonid fish viruses and cell interactions at early steps of the infective cycle

A flow cytometric virus-binding assay that directly visualizes the binding and entry of infectious pancreatic necrosis virus (IPNV), infectious haematopoietic necrosis virus (IHNV) and virus haemorrhagic septicaemia virus (VHSV) to several cell lines was established. The highest efficiency of binding was shown by the BF-2 cell line and this was used to study, at the attachment level, the interactions of these cells with salmonid fish viruses in coinfections, and to further determine if the earliest stage of the viral growth cycle could explain the previously described loss of infectivity of IHNV when IPNV is present. Our results demonstrated that IPNV binds to around 88% of cells either in single or dual infections, whereas IHNV attachment always decreased in the presence of any of the other viruses. VHSV binding was not affected by IPNV, but coinfection with IHNV reduced the percentage of virus-binding cells, which suggests competition for viral receptors or co-receptors. Internalization of the adsorbed IHNV was not decreased by coinfection with IPNV, so the hypothetical competence could be restricted to the binding step. Treatment of the cells with antiviral agents, such as amantadine or chloroquine, did not affect the binding of IPNV and VHSV, but reduced IHNV binding by more than 30%. Tributylamine affected viral binding of the three viruses to different degrees and inhibited IPNV or IHNV entry in a large percentage of cells treated for 30 min. Tributylamine also inhibited IHNV cytopathic effects in a dose-dependent manner, decreasing the virus yield by 4 log of the 50% endpoint titre, at 10 mm concentration. IPNV was also inhibited, but at a lower level. The results of this study support the hypothesis that IHNV, in contrast to VHSV or IPNV, is less efficient at completing its growth cycle in cells with a simultaneous infection with IPNV. It can be affected at several stages of viral infection and is more sensitive to the action of antiviral compounds.     

Canine Haematopoietic Chimerism Analyses by Semiquantitative Fluorescence Detection of Variable Number of Tandem Repeat Polymorphism

Canine models are successfully applied to the study of haematopoietic stem cell transplantation (HSCT). Monitoring of haematopoietic donor/recipient chimerism is of major significance in detecting and quantifying engraftment or graft rejection of the donor-derived haematopoietic cells after transplantation. Radioactive analyses of polymorphic microsatellite markers are commonly used for chimerism analyses. We describe an improved, non-isotopic method that is based on the analysis of microsatellite markers in donor and recipient cells using capillary electrophoresis and fluorescence detection. Artificial mixtures of donor and recipient DNA that were generated from peripheral blood mononuclear cells from dog leukocyte antigen-identical siblings were used to analyse the sensitivity of the assay. DNA from dogs that had received HSCT were also analysed in order to demonstrate the feasibility of the method in vivo. For chimerism analyses, six different microsatellite loci were systematically amplified using fluorescent PCR primer. The fluorescent polymerase chain reaction products were separated by capillary electrophoresis using POP4 on a 310 ABI Prism Genetic Analyzer. After electrophoresis, fluorescence signals were automatically sized and quantified using GeneScan software. The method described provides an accurate assessment of haematopoietic chimerism in the canine model with significantly reduced hands-on time compared to conventional gel electrophoresis.     

Challenge studies of European stocks of redfin perch, Perca fluviatilis L., and rainbow trout, Oncorhynchus mykiss (Walbaum), with epizootic haematopoietic necrosis virus

A challenge model for comparison of the virulence of epizootic haematopoietic necrosis virus (EHNV) to European stocks of redfin perch, Perca fluviatilis L., and rainbow trout, Oncorhynchus mykiss (Walbaum), was tested. The model investigated intraperitoneal (IP), bath and cohabitation routes at 10, 15 and 20 °C for 5–6 g fish and 15 °C for 20 g perch. In the IP challenges of perch, significant mortality occurred at 15 °C and 20 °C. In challenge trials for rainbow trout, significant mortalities were observed in IP and bath challenges at 20 °C. The mortality observed in IP challenged 20 g perch was not significantly different from that recorded for 6 g fish challenged IP. No significant mortality was observed in any other treatment groups. Re-isolation of ranavirus was confirmed by IFAT and was consistently associated with dead or moribund fish in the trial groups challenged with EHNV. The findings indicate that EHNV does not pose a high risk for wild perch and trout populations in Europe by natural exposure. Mortality appears to be primarily a function of environmental factors, with temperature playing an important role, and not just the presence of the virus in the fish.     

Pre-exposure to infectious hypodermal and haematopoietic necrosis virus or to inactivated white spot syndrome virus (WSSV) confers protection against WSSV in Penaeus vannamei (Boone) post-larvae

Larvae and post-larvae of Penaeus vannamei (Boone) were submitted to primary challenge with infectious hypodermal and haematopoietic necrosis virus (IHHNV) or formalin-inactivated white spot syndrome virus (WSSV). Survival rate and viral load were evaluated after secondary per os challenge with WSSV at post-larval stage 45 (PL45). Only shrimp treated with inactivated WSSV at PL35 or with IHHNV infection at nauplius 5, zoea 1 and PL22 were alive (4.7% and 4%, respectively) at 10 days post-infection (p.i.). Moreover, at 9 days p.i. there was 100% mortality in all remaining treatments, while there was 94% mortality in shrimp treated with inactivated WSSV at PL35 and 95% mortality in shrimp previously treated with IHHNV at N5, Z1 and PL22. Based on viral genome copy quantification by real-time PCR, surviving shrimp previously challenged with IHHNV at PL22 contained the lowest load of WSSV (0-1x10(3) copies microg-1 of DNA). In addition, surviving shrimp previously exposed to inactivated WSSV at PL35 also contained few WSSV (0-2x10(3) copies microg-1 of DNA). Consequently, pre-exposure to either IHHNV or inactivated WSSV resulted in slower WSSV replication and delayed mortality. This evidence suggests a protective role of IHHNV as an interfering virus, while protection obtained by inactivated WSSV might result from non-specific antiviral immune response.     

First detection and isolation of infectious haematopoietic necrosis virus from farmed rainbow trout in Nyeri County,Kenya

Infectious haematopoietic necrosis virus (IHNV) is the causative agent of infectious haematopoietic necrosis, a disease of salmonid responsible for great economic losses. The disease occurs in most parts of the world where rainbow trout is reared but has not been previously reported in Kenya. In this study, rainbow trout fry and growers from two farms in Nyeri County were screened for IHNV. Whole fry (n = 4 from each farm) and kidney samples from growers (n = 15 and n = 6 from the two farms, respectively) were collected and preserved for cell culture examination or PCR analysis. Screening of samples was done by PCR followed by sequencing of the glycoprotein gene of the virus. Demonstration of the virus was done by propagation in EPC cells followed by the indirect fluorescence antibody test (IFAT). The results revealed the presence of IHNV at low prevalence of 0.1 and 0.4 for the two farms. The virus was confirmed both by IFAT and by partial sequencing of the G gene. Phylogenetic analysis revealed that the Kenyan isolates were identical to those of the J genogroup found mostly in Asia. The findings have implications for biosecurity measures and import regulations for the Kenyan rainbow trout industry.