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Matías Cárdenas Claudia Galleguillos Karina Acevedo Catarina Ananias Javiera Alarcón Sofía Michelson Jorge Toledo Margarita Montoya Claudio Meneses Eduardo Castro-Nallar Yesseny Vásquez-Martínez Marcelo Cortez-San Martin 《Journal of fish diseases》2020,43(12):1483-1496
The ISAV has a genome composed of eight segments of (–)ssRNA, segment 6 codes for the hemagglutinin–esterase protein, and has the most variable region of the genome, the highly polymorphic region (HPR), which is unique among orthomyxoviruses. The HPR has been associated with virulence, infectivity and pathogenicity. The full length of the HPR is called HPR0 and the strain with this HPR is avirulent, in contrast to strains with deleted HPR that are virulent to varying degrees. The molecular mechanism that gives rise to the different HPRs remains unclear. Here, we studied in vitro the evolution of reassortant recombinant ISAV (rISAV) in Atlantic salmon head kidney (ASK) cells. To this end, we rescued and cultivated a set of rISAV with different segment 6-HPR genotypes using a reverse genetics system and then sequencing HPR regions of the viruses. Our results show rapid multiple recombination events in ISAV, with sequence insertions and deletions in the HPR, indicating a dynamic process. Inserted sequences can be found in four segments of the ISAV genome (segments 1, 5, 6, and 8). The results suggest intra-segmental heterologous recombination, probably by class I and class II template switching, similar to the proposed segment 5 recombination mechanism. 相似文献
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根据ISAV的基因保守序列,利用LAMP Designer软件设计了6条引物,采用新型的环介导等温扩增设备进行扩增和检测,优化了反应条件,分析了所建立方法的特异性和灵敏度,并与RT-PCR和实时荧光RT-PCR进行比较。研究表明,该方法最适反应温度为64℃,反应10 min就可以观察到明显的扩增。该方法灵敏度高,检测限为78.4 fg RNA,比常规RT-PCR灵敏度高100倍,与实时荧光定量RT-PCR灵敏度相当;特异性好,与传染性胰腺坏死病毒(IPNV)、鲤春病毒血症病毒(SVCV)、出血性败血症病毒(VHSV)、鱼类病毒性神经坏死病病毒(VNNV)、鱼腹水病毒(YAV)等14种主要鱼类病毒没有交叉反应。结果表明,本研究建立了ISAV的实时荧光环介导等温扩增检测方法,实验能对整个扩增过程进行实时监测,提高检测灵敏度的同时,防止由于开盖跑电泳或加染料而导致的污染。 相似文献
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Real-time PCR assays are being increasingly applied to the detection of fish pathogens due to their sensitivity, specificity and potential for high throughput sample processing. Such assays allow for the ready and efficient inclusion of appropriate quality controls which are fundamental to scientific integrity and to satisfying the demands of diagnostic test accreditation. In this article, we report development of a universal positive control strategy for real-time PCR assays, which has been used to support and improve a previously published method for detection of infectious salmon anaemia virus (ISAV). The strategy employed uses an RNA mimic template, which is based on the ISAV segment 8 target sequence but includes an artificial universal positive control sequence. Inclusion of this sequence, which is targeted by a second specific probe carrying a different fluorophore to the primary assay, allows for convenient screening of all real-time PCR reactions for the presence of contaminating positive control material. The development of readily distinguishable artificial positive control material offers distinct advantages to real-time PCR assays over using control material derived from clinical material. 相似文献
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F.O. Mardones B. Martinez-Lopez P. Valdes-Donoso T.E. Carpenter A.M. Perez 《Preventive veterinary medicine》2014
Infectious salmon anemia virus (ISAV) infection is a constant major threat to farmed and wild Atlantic salmon worldwide. Many epidemics have recently been reported in the most important salmon farming regions of the world, including Chile (2007–2009), where ISAV generated the most important disease and economic crisis in history of the salmon industry of the country. The spread of ISAV within a region is most likely by local or neighborhood spread from an infected farm; however, there is evidence that anthropogenic activities, such as movement of live or harvested fish or their byproduct, may have played a more important role than environmental or passive transmission in the 2007–2009 outbreak. Atlantic salmon farms (n = 421) were retrospectively followed from stocking to harvesting in southern Chile at the time of the ISAV epidemic (2007–2009). The effect of husbandry and spatial risk factors, in addition to contact-network risk factors, which were obtained from the social network analyses, on time to first ISAV infection was estimated using a multivariable Cox proportional hazards model. Five variables were retained in the final fitted model: co-existing multiple generations on a farm (hazard ratio [HR] = 2.585), mean smolt weight at stocking greater than 120 g (HR = 1.165), farm area (per km2) (HR = 1.005), and increased number of shipments entering a farm, i.e. the farm input degree (HR = 1.876) were associated with reduced time to infection; whereas time-to-infection was longer for farms located farther from an ongoing ISAV outbreak (HR = 0.943). It was demonstrated that movements of latently infected fish resulted in approximately 7 outbreaks, and potentially explain about 6% of the total number of cases during the epidemic. Results from this study provide new information about the mechanisms of spread of ISAV in one the largest documented ISAV epidemics in the world. Findings may be used to support the design and implementation of risk-based surveillance and control programs that may help to prevent, detect and control future ISAV outbreaks. 相似文献
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A Gregory L A Munro M Snow K L Urquhart A G Murray R S Raynard 《Journal of fish diseases》2009,32(6):481-489
This study investigated infection dynamics of infectious salmon anaemia virus (ISAV) by conducting two experiments to examine minimum infective dose and viral shedding of ISAV. In terms of minimum infective dose, the high variability between replicate tanks and the relatively slow spread of infection through the population at 1 × 101 TCID50 mL−1 indicated this dose is approaching the minimum infective dose for ISAV in seawater salmon populations. A novel qPCR assay incorporating an influenza virus control standard with each seawater sample was developed that enabled the quantity of ISAV shed from infected populations to be estimated in values equivalent to viral titres. Viral shedding was first detected at 7 days post-challenge (5.8 × 10−2 TCID50 mL−1 kg−1 ) and rose to levels above the minimum infective dose (4.2 × 101 TCID50 mL−1 kg−1 ) on day 11 post-challenge, 2 days before mortalities in ISAV inoculated fish started. These results clearly demonstrate that a large viral shedding event occurs before death. Viral titres peaked at 7.0 × 101 TCID50 mL−1 kg−1 15 days post-infection. These data provide important information relevant to the management of ISA. 相似文献
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In vivo virulence and genomic comparison of infectious Salmon Anaemia Virus isolates from Atlantic Canada 
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下载免费PDF全文 Francis LeBlanc Steven Leadbeater Mark Laflamme Nellie Gagné 《Journal of fish diseases》2018,41(9):1373-1384
The infectious salmon anaemia virus (ISAV) is capable of causing a significant disease in Atlantic salmon, which has resulted in considerable financial losses for salmon farmers around the world. Since the first detection of ISAV in Canada in 1996, it has been a high priority for aquatic animal health management and surveillance programmes have led to the identification of many genetically distinct ISAV isolates of variable virulence. In this study, we evaluated the virulence of three ISAV isolates detected in Atlantic Canada in 2012 by doing in vivo‐controlled disease challenges with two sources of Atlantic salmon. We measured viral loads in fish tissues during the course of infection. Sequences of the full viral RNA genomes of these three ISAV isolates were obtained and compared to a high‐virulence and previously characterized isolate detected in the Bay of Fundy in 2004, as well as a newly identified ISAV NA‐HPR0 isolate. All three ISAV isolates studied were shown to be of low to mid‐virulence with fish from source A having a lower mortality rate than fish from source B. Viral load estimation using an RT‐qPCR assay targeting viral segment 8 showed a high degree of similarity between tissues. Through genomic comparison, we identified various amino acid substitutions unique to some isolates, including a stop codon in the segment 8 ORF2 not previously reported in ISAV, present in the isolate with the lowest observed virulence. 相似文献
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Valentin Eckart Takuya Yamaguchi Kati Franzke Sven M. Bergmann Pierre Boudinot Edwige Quillet Motokazu Kawanobe Neila Alvarez de Haro Uwe Fischer 《Journal of fish diseases》2019,42(2):181-187
The production of piscine viruses, in particular of koi herpesvirus (KHV, CyHV‐3) and infectious salmon anaemia virus (ISAV), is still challenging due to the limited susceptibility of available cell lines to these viruses. A number of cell lines from different fish species were compared to standard diagnostic cell lines for KHV and ISAV regarding their capability to exhibit a cytopathic effect (CPE) and to accumulate virus. Two cell lines, so far undescribed, appeared to be useful for diagnostic purposes. Fr994, a cell line derived from ovaries of rainbow trout (Oncorhynchus mykiss), produced constantly high ISA virus (ISAV) titres and developed a pronounced CPE even at high cell passage numbers, while standard cell lines are reported to gradually loose these properties upon propagation. Another cell line isolated from the head kidney of common carp (Cyprinus carpio), KoK, showed a KHV induced CPE earlier than the standard cell line used for diagnostics. A third cell line, named Fin‐4, established from the fin epithelium of rainbow trout did not promote efficient replication of tested viruses, but showed antigen sampling properties and might be useful as an in vitro model for virus uptake or phagocytosis. 相似文献
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