Shrimp laminin receptor binds with capsid proteins of two additional shrimp RNA viruses YHV and IMNV

Laminin receptor (Lamr) in shrimp was previously proposed to be a potential receptor protein for Taura syndrome virus (TSV) based on yeast two-hybrid assays. Since shrimp Lamr bound to the VP1 capsid protein of TSV, we were interested to know whether capsid/envelope proteins from other shrimp viruses would also bind to Lamr. Thus, capsid/envelope encoding genes from 5 additional shrimp viruses were examined. These were Penaeus stylirostris densovirus (PstDNV), white spot syndrome virus (WSSV), infectious myonecrosis virus (IMNV), Macrobrachium rosenbergii nodavirus (MrNV), and yellow head virus (YHV). Protein interaction analysis using yeast two-hybrid assay revealed that Lamr specifically interacted with capsid/envelope proteins of RNA viruses IMNV and YHV but not MrNV and not with the capsid/envelope proteins of DNA viruses PstDNV and WSSV. In vitro pull-down assay also confirmed the interaction between Lamr and YHV gp116 envelope protein, and injection of recombinant Lamr (rLamr) protein produced in yeast cells protected shrimp against YHV in laboratory challenge tests.     

Specific monoclonal antibodies raised against Taura syndrome virus (TSV) capsid protein VP3 detect TSV in single and dual infections with white spot syndrome virus (WSSV)

The gene sequence encoding VP3 capsid protein of Taura syndrome virus (TSV) was cloned into pGEX-6P-1 expression vector and transformed into Escherichia coli BL21. After induction, recombinant GST-VP3 (rVP3) fusion protein was obtained and further purified by electro-elution before use in immunizing Swiss mice for production of monoclonal antibodies (MAb). One MAb specific to glutathione-S-transferase (GST) and 6 MAb specific to VP3 were selected using dot blotting and Western blotting. MAb specific to VP3 could be used to detect natural TSV infections in farmed whiteleg shrimp Penaeus vannamei by dot blotting and Western blotting, without cross reaction to shrimp tissues or other shrimp viruses, such as white spot syndrome virus (WSSV), yellow head virus (YHV), monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV). These MAb were also used together with those specific for WSSV to successfully detect TSV and WSSV in dual infections in farmed P. vannamei.     

Historic emergence, impact and current status of shrimp pathogens in Asia

It is estimated that approximately 60% of disease losses in shrimp aquaculture have been caused by viral pathogens and 20% by bacterial pathogens. By comparison, losses to fungi and parasites have been relatively small. For bacterial pathogens, Vibrio species are the most important while for viral pathogens importance has changed since 2003 when domesticated and genetically selected stocks of the American whiteleg shrimp Penaeus (Litopenaeus) vannamei (Boone 1931) replaced the formerly dominant giant tiger or black tiger shrimp Penaeus (Penaeus) monodon (Fabricius 1798) as the dominant cultivated species. For both species, white spot syndrome virus (WSSV) and yellow head virus (YHV) are the most lethal. Next most important for P. vannamei is infectious myonecrosis virus (IMNV), originally reported from Brazil, but since 2006 from Indonesia where it was probably introduced by careless importation of shrimp aquaculture stocks. So far, IMNV has not been reported from other countries in Asia. Former impacts of Taura syndrome virus (TSV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV) on this species have dramatically declined due to the introduction of tolerant stocks and to implementation of good biosecurity practices. Another problem recently reported for P. vannamei in Asia is abdominal segment deformity disease (ASDD), possibly caused by a previously unknown retrovirus-like agent. Next most important after WSSV and YHV for P. monodon is monodon slow growth syndrome (MSGS) for which component causes appear to be Laem Singh virus (LSNV) and a cryptic integrase containing element (ICE). Hepatopancreatic parvovirus (HPV) and monodon baculovirus (MBV) may be problematic when captured P. monodon are used to produce larvae, but only in the absence of proper preventative measures. Since 2009 increasing losses with P. vannamei in China, Vietnam and now Thailand are associated with acute hepatopancreatic necrosis syndrome (AHPNS) of presently unknown cause. Despite these problems, total production of cultivated penaeid shrimp from Asia will probably continue to rise as transient disease problems are solved and use of post larvae originating from domesticated SPF shrimp stocks in more biosecure settings expands.     

Viral diseases of penaeid shrimp with particular reference to four viruses recently found in shrimp from Queensland

The culture of penaeid shrimp world-wide is primarily dependent on wild-caught broodstock which has an enormous potential to introduce new pathogens, particularly viruses, into culture systems. Of the 13 viruses described for cultured penaeid shrimp, seven have been described within the past 5 years; the most devastating viral epidemics on record for cultured penaeid shrimp have also occurred within the past 5years. During examination of local wild and cultured shrimp, four new viruses were found. Bennettae baculovirus was discovered in the digestive gland of wild Metapenaeus bennettae. It closely resembles monodon baculovirus (MBV) but has a more slender virion, does not cross-react with a DNA probe for MBV and is not infectious to Penaeus monodon. Two morphologically indistinguishable viruses, one pathogenic (gill-associated virus, GAV) and the other benign (lymphoid organ virus, LOV), were found in cultured P. monodon. LOV and GAV closely resemble yellow head virus (YHV) of Thailand. A parvo-like virus was found recently in dying post-larvae of P. japonicus. As the intensity of shrimp culture world-wide increases, researchers can expect to discover more penaeid viruses. The need to close the life cycle of P. monodon and other cultured species and develop rapid diagnostic methods for viral infections has become imperative.     

Prevalence of three shrimp viruses in Zhejiang Province in 2008

White spot syndrome virus (WSSV), Taura syndrome virus (TSV) and Infectious hypodermal and haematopoietic necrosis virus (IHHNV) are three shrimp viruses responsible for major pandemics affecting the shrimp farming industry. Shrimps samples were collected from 12 farms in Zhejiang province, China, in 2008 and analyzed by PCR to determine the prevalence of these viruses. From the 12 sampling locations, 8 farms were positive for WSSV, 8 for IHHNV and 6 for both WSSV and IHHNV. An average percentage of 57.4% of shrimp individuals were infected with WSSV, while 49.2% were infected with IHHNV. A high prevalence of co-infection with WSSV and IHHNV among samples was detected from the following samples: Bingjiang (93.3%), liuao (66.7%), Jianshan (46.7%) and Xianxiang (46.7%). No samples exhibited evidence of infection with TSV in collected samples. This study provides comprehensive information of the prevalence of three shrimp viruses in Zhejiang and may be helpful for disease prevention control in this region.     

Susceptibility to infection and pathogenicity of White Spot Disease (WSD) in non-model crustacean host taxa from temperate regions

Despite almost two decades since its discovery, White Spot Disease (WSD) caused by White Spot Syndrome Virus (WSSV) is still considered the most significant known pathogen impacting the sustainability and growth of the global penaeid shrimp farming industry. Although most commonly associated with penaeid shrimp farmed in tropical regions, the virus is also able to infect, cause disease and kill a wide range of other decapod crustacean hosts from temperate regions, including lobsters, crabs, crayfish and shrimp. For this reason, WSSV has recently been listed in European Community Council Directive 2006/88. Using principles laid down by the European Food Safety Authority (EFSA) we applied an array of diagnostic approaches to provide a definitive statement on the susceptibility to White Spot Syndrome Virus (WSSV) infection in seven ecologically or economically important crustacean species from Europe. We chose four marine species: Cancer pagurus, Homarus gammarus, Nephrops norvegicus and Carcinus maenas; one estuarine species, Eriocheir sinensis and two freshwater species, Austropotamobius pallipes and Pacifastacus leniusculus. Exposure trials based upon natural (feeding) and artificial (intra-muscular injection) routes of exposure to WSSV revealed universal susceptibility to WSSV infection in these hosts. However, the relative degree of susceptibility (measured by progression of infection to disease, and mortality) varied significantly between host species. In some instances (Type 1 hosts), pathogenesis mimicked that observed in penaeid shrimp hosts whereas in other examples (Types 2 and 3 hosts), infection did not readily progress to disease, even though hosts were considered as infected and susceptible according to accepted principles. Results arising from challenge studies are discussed in relation to the potential risk posed to non-target hosts by the inadvertent introduction of WSSV to European waters via trade. Furthermore, we highlight the potential for susceptible but relatively resistant hosts to serve as models to investigate natural mitigation strategies against WSSV in these hosts. We speculate that these non-model hosts may offer a unique insight into viral handling in crustaceans.     

实时荧光定量PCR及RT-PCR与常规PCR及RT-PCR检测对虾病毒效果的比较

常规PCR及RT-PCR已用于对虾DNA及RNA病毒检测,但存在费时、灵敏度较低、不能定量等问题。建立了TaqMan实时荧光定量PCR及RT-PCR方法,分别用于检测白斑综合症病毒(WSSV)、传染性皮下及造血组织坏死病毒(IHHNV)及桃拉综合征病毒(TSV)、黄头病毒(YHV)4种对虾病毒。与常规PCR及RT-PCR比较,所建立的TaqMan实时荧光定量PCR及RT-PCR检测上述4种对虾病毒不仅有很高的特异性,检测灵敏度也提高了10～100倍,同时还具有快速、简便、不污染环境、重复性好、实时定量等优点,可明显提高对虾病毒检验检疫工作质量及效率。     

多重RT-PCR同时检测鉴别三种对虾病毒的研究与应用

根据基因库中对虾桃拉综合征病毒（TSV）、白斑综合征病毒（WSSV）、传染性皮下和造血器官坏死病毒（IHHNV）的基因序列,分别设计了三对特异性引物,通过对多重RT—PCR扩增条件的优化,研究建立了可同时检测鉴别TSV、WSSV和IHHNV的多重RT—PCR。该技术对同一样品中的TSV RNA、WSSV DNA和IHHNV DNA模板进行扩增,结果均同时得到3条大小与实验设计相符的231bp（TSV）、593bp（WSSV）和356bp（IHHNV）的特异性多重RT—PCR扩增带,对其它对虾病原核酸的扩增结果为阴性。敏感性试验结果表明,该技术最低能检测到10pgTSV RNA、100pg WSSV DNA和100pg IHHNV DNA。临床检测试验结果表明,该技术对TSV、WSSV和I—HHNV的检出率明显高于传统的临床症状观察和组织病理学检查,提示该技术适用于这三种病毒的临床快速检测和鉴别诊断。     

Suppression of PmRab7 by dsRNA Inhibits WSSV or YHV Infection in Shrimp

Viral entry into host cells requires endocytosis machineries of the host for viral replication. PmRab7, a Penaeus monodon small GTPase protein, was investigated for its function in vesicular transport during viral infection. The double-stranded RNA of Rab7 was injected into a juvenile shrimp before challenging with white spot syndrome virus (WSSV) or yellow head virus (YHV). PmRab7 mRNA was specifically decreased at 48 h after dsRNA-Rab7 injection. Silencing of PmRab7 dramatically inhibited WSSV-VP28 mRNA and protein expression. Unexpectedly, the silencing of PmRab7 also inhibited YHV replication in the YHV-infected shrimp. These results suggested that PmRab7 is a common cellular factor required for WSSV or YHV replication in shrimp. Because PmRab7 should function in the endosomal trafficking pathway, its silencing prevents successful viral trafficking necessary for replication. Silencing of PmRab7 could be a novel approach to prevent both DNA virus (WSSV) and RNA virus (YHV) infection of shrimp.     

Gene expression and protein levels of thioredoxin in the gills from the whiteleg shrimp (Litopenaeus vannamei) infected with two different viruses: the WSSV or IHHNV

The thioredoxin (TRX) system in crustaceans has demonstrated to act as a cell antioxidant being part of the immune response by dealing with the increased production of reactive oxygen species during bacterial or viral infection. Since the number of marine viruses has increased in the last years significantly affecting aquaculture practices of penaeids, and due to the adverse impact on wild and cultured shrimp populations, it is important to elucidate the dynamics of the shrimp response to viral infections. The role of Litopenaeus vannamei thioredoxin (LvTRX) was compared at both, mRNA and protein levels, in response to two viruses, the white spot syndrome virus (WSSV) and the infectious hypodermal and hematopoietic necrosis virus (IHHNV). The results confirmed changes in the TRX gene expression levels of WSSV-infected shrimp, but also demonstrated a more conspicuous response of TRX to WSSV than to IHHNV. While both the dimeric and monomeric forms of LvTRX were detected by Western blot analysis during the WSSV infection, the dimer on its reduced form was only detected through the IHHNV infectious process. These findings indicate that WSSV or IHHNV infected shrimp may induce a differential response of the LvTRX protein.     

The ecology of viruses that infect eukaryotic algae

Because viruses of eukaryotic algae are incredibly diverse, sweeping generalizations about their ecology are rare. These obligate parasites infect a range of algae and their diversity can be illustrated by considering that isolates range from small particles with ssRNA genomes to much larger particles with 560?kb dsDNA genomes. Molecular research has also provided clues about the extent of their diversity especially considering that genetic signatures of algal viruses in the environment rarely match cultivated viruses. One general concept in algal virus ecology that has emerged is that algal viruses are very host specific and most infect only certain strains of their hosts; with the exception of viruses of brown algae, evidence for interspecies infectivity is lacking. Although some host-virus systems behave with boom-bust oscillations, complex patterns of intraspecies infectivity can lead to host-virus coexistence obfuscating the role of viruses in host population dynamics. Within the framework of population dynamics, host density dependence is an important phenomenon that influences virus abundances in nature. Variable burst sizes of different viruses also influence their abundances and permit speculations about different life strategies, but as exceptions are common in algal virus ecology, life strategy generalizations may not be broadly applicable. Gaps in knowledge of virus seasonality and persistence are beginning to close and investigations of environmental reservoirs and virus resilience may answer questions about virus inter-annual recurrences. Studies of algal mortality have shown that viruses are often important agents of mortality reinforcing notions about their ecological relevance, while observations of the surprising ways viruses interact with their hosts highlight the immaturity of our understanding. Considering that just two decades ago algal viruses were hardly acknowledged, recent progress affords the optimistic perspective that future studies will provide keys to unlocking our understanding of algal virus ecology specifically, and aquatic ecosystems generally.     

The host—parasite relationship of two copepod species and two fish species

A total of 2494 Menidia beryllina and 717 M. peninsulae (Atherinidae) were examined from the Pensacola Bay area for parasitic copepods. M. peninsulae was infested with Bomolochus concinnus (Bomolochidae) and Ergasilus marticatus (Ergasilidae) and had incidences (and intensities) of 12.3% (1.6) and 4.2% (1.3), respectively. Only seven M. peninsulae were infested with both species of parasites. M. beryllina was infested only with E. manicatus and showed different incidences (and intensities) in two areas: Mulatto Bayou, 13.2% (1.9); Catfish Basin, 53.0% (2.3). Fishes with parasites were significantly longer than fishes without parasites and the case of increasing number of parasites with increasing fish length was reinforced. B. concinnus is a warm water parasite on M. peninsulae while E. manicatus acts as a cold water parasite on M. peninsulae and a warm water parasite on M. beryllina . The parasites tended to be overdispersed on their hosts but at the same time showed evidence of negative intraspecific associations within a host. These data suggest intraspecific avoidance by the parasite but active acquisition by the host.     

Viruses,biosecurity and specific pathogen-free stocks in shrimp aquaculture

The greatest threat to the future of world shrimp aquaculture is disease, in particular the virulent untreatable viruses, infectious hypodermal and haematopoietic necrosis virus (IHHNV), taura syndrome virus (TSV), yellow head virus (YHV), and white spot syndrome virus (WSSV). To overcome these hazards, the industry of the future must be based on: (i) specific pathogen-free and genetically improved shrimp stocks; (ii) biosecure systems including enclosed, reduced water-exchange/increased water-reuse culture systems; (iii) biosecure management practices; and (iv) co-operative industry-wide disease control strategies. Specific pathogen-free shrimp are those that are known to be free of specified pathogens and such stocks will ensure that seed shrimp are not the conduit for introduction of pathogens and that if pathogens are encountered the stocks will not be severely affected. Commercially acceptable biosecure culture systems that are under cover and use recirculated sea water will need to be developed for shrimp production. Adherence to operating protocols that incorporate strict biosecurity practices, including restricted access and disinfection strategies, will need to become standard. Co-operative efforts will include: early warning surveillance; co-ordination of harvest and water exchange schedules of contaminated ponds; processor co-operation to ensure that processing wastes are not threats; quick response to outbreaks.     

A model for apoptotic interaction between white spot syndrome virus and shrimp

White spot syndrome virus (WSSV) is an enveloped, large dsDNA virus that mainly infects penaeid shrimp, causing serious damage to the shrimp aquaculture industry. Like other animal viruses, WSSV infection induces apoptosis. Although this occurs even in by-stander cells that are free of WSSV virions, apoptosis is generally regarded as a kind of antiviral immune response. To counter this response, WSSV has evolved several different strategies. From the presently available literature, we construct a model of how the host and virus both attempt to regulate apoptosis to their respective advantage. The basic sequence of events is as follows: first, when a WSSV infection occurs, cellular sensors detect the invading virus, and activate signaling pathways that lead to (1) the expression of pro-apoptosis proteins, including PmCasp (an effecter caspase), MjCaspase (an initiator caspase) and voltage-dependent anion channel (VDAC); and (2) mitochondrial changes, including the induction of mitochondrial membrane permeabilization and increased oxidative stress. These events initiate the apoptosis program. Meanwhile, WSSV begins to express its genes, including two anti-apoptosis proteins: AAP-1, which is a direct caspase inhibitor, and WSV222, which is an E3 ubiquitin ligase that blocks apoptosis through the ubiquitin-mediated degradation of shrimp TSL protein (an apoptosis inducer). WSSV also induces the expression of a shrimp anti-apoptosis protein, Pm-fortilin, which can act on Bax to inhibit mitochondria-triggered apoptosis. This is a life and death struggle because the virus needs to prevent apoptosis in order to replicate. If WSSV succeeds in replicating in sufficient numbers, this will result in the death of the infected penaeid shrimp host.     

A review of the parasitic dinoflagellates Hematodinium species and Hematodinium-like infections in marine crustaceans

Parasitic dinoflagellates in the genus Hematodinium are important parasites of marine Crustacea. Outbreaks of these parasites have damaged commercial stocks of Norway lobster Nephrops norvegicus, snow crab Chionoecetes opilio, Tanner crab C. bairdi, American blue crab Callinectes sapidus, and velvet swimming crab Necora puber. Species of Hematodinium can reach high enough levels to regulate their host populations, but mortalities are also centred on the unfished juveniles and females, hosts not normally sampled by fisheries; hence impacts are often underreported. Seasonal prevalences of up to 85 % occur annually in many host populations; in effect, these parasites form cryptic blooms in the water column with crabs and other crustaceans at risk of disease. We review the biology and ecology of Hematodinium spp. infections in crustaceans. Included is a comparison of the different infections, a synthesis of what is known, and an attempt to highlight fruitful areas for continued research.