Molecular Epidemiological Investigation of Infectious Hypodermal and Hematopoietic Necrosis Virus and Taura Syndrome Virus in Penaeus Vannamei Cultured in China

The Infectious hypodermal and hematopoietic necrosis virus(IHHNV) and Taura syndrome virus(TSV) are two important shrimp viruses in cultured shrimp in America.These two viruses were transmitted to China at the beginning of the 21st century.In this study,214 shrimp samples of Penaeus vannamei were collected from seven different areas of China and tested by PCR for IHHNV and TSV infection.The results showed that there were a high prevalence of IHHNV(65.42%) and low prevalence of TSV(3.27%) in the tested samples.Several samples were found to be co-infected with these two viruses.A 3 kb fragment of 7 positive IHHNV samples and a structure protein region(ORF2) of three TSV positive samples were amplified and sequenced.The sequence comparison indicated that both IHHNV and TSV sequenced in China have a low genetic variations compared with the prototype IHHNV and TSV from Hawaii.Phylogenetic analysis showed that TSV isolates were clustered into two groups,Asia and America group,which was genetically correlated to geographic distribution.     

Experimental infections reveal that common Thai crustaceans are potential carriers for spread of exotic Taura syndrome virus

Taura syndrome virus (TSV) was first reported as a serious cause of shrimp mortality limited to reared Penaeus (Litopenaeus) vannamei in the Americas, where it spread principally through regional and international transfer of live post larvae (PL) and broodstock. Subsequently, through importation of infected broodstock, TSV outbreaks spread to Asia, first to Taiwan and China and then to Thailand, Indonesia and Korea. Since its introduction to Thailand, outbreaks have occasionally been reported from rearing ponds stocked with batches of specific pathogen free (SPF) P. vannamei PL that tested negative for TSV by nested RT-PCR assay. Since it was possible that the outbreaks may have occurred via horizontal transfer of TSV from wild carrier species, we tested 5 common native crustaceans that live in and around shrimp ponds (2 palaemonid shrimp species, Palaemon styliferus and Macrobrachium lanchesteri, and 3 species of crabs, Sesarma mederi, Scylla serrata and Uca vocans) for susceptibility to TSV in experimental challenges. We found that U. vocans, S. serrata and S. mederi did not die but, respectively, gave strong RT-PCR reactions indicating heavy viral load at 5, 10 and 15 d post-injection of TSV and 10, 15 and up to 50 d after feeding with TSV-infected P. vannamei carcasses. Also after feeding, P. styliferus did not die, but a high proportion gave strong RT-PCR reactions at 5 d post-challenge and no reactions at 15 d. Similarly after feeding, M. lanchesteri showed no mortality and gave only light RT-PCR reactions at 2 d, moderate reactions at 5 d and no reaction at 15 d. By contrast, transmission experiments from the TSV-infected crabs and palaemonid shrimp via water or feeding resulted in death of all the exposed P. vannamei from 8 to 12 d post-challenge and all were positive for heavy viral load by RT-PCR assay. Despite the results of these laboratory challenge tests, natural TSV infections were not detected by nested RT-PCR in samples of these species taken from the wild. These results indicated that transmission of TSV from infected crabs and palaemonid shrimp via water or feeding might pose a potential risk to shrimp aquaculture.     

Taura syndrome virus (TSV) in Thailand and its relationship to TSV in China and the Americas

The cultivation of exotic Penaeus vannamei in Thailand began on a very limited scale in the late 1990s, but a Thai government ban on the cultivation of P. monodon in freshwater areas in 2000 led many Thai shrimp farmers to shift to cultivation of P. vannamei. Alarmed by the possibility of Taura syndrome virus (TSV) introduction, the Thai Department of Fisheries required that imported stocks of P. vannamei be certified free of TSV by RT-PCR (Reverse Trasciption Polymerase Chain Reaction) testing. During the interval of allowed importation, over 150,000 broodstock shrimp were imported, 67% of these from China and Taiwan. Despite the safeguards, TSV outbreaks occurred and we confirmed the first outbreak by RT-PCR in early 2003. This resulted in a governmental ban on all shrimp broodstock imports from February 2003, but TSV outbreaks have continued, possibly due to original introductions or to the continued illegal importation of stocks. To determine the origin of the TSV in Thailand, the viral coat protein gene VP1 was amplified by RT-PCR from several shrimp specimens found positive for TSV by RT-PCR from January to November 2003. These included 7 samples from P. vannamei disease outbreaks in Thailand, 3 other non-diseased shrimp samples from Thailand and Burma and 6 samples including P. vannamei and P. japonicus from China. Comparison revealed that the Thai, Burmese and Chinese TSV types formed a clade distinct from a clade of TSV types from the Americas.     

Molecular epidemiological investigation of infectious hypodermal and hematopoietic necrosis virus and Taura syndrome virus in <Emphasis Type="Italic">Penaeus Vannamei</Emphasis> cultured in China

The Infectious hypodermal and hematopoietic necrosis virus (IHHNV) and Taura syndrome virus (TSV) are two important shrimp viruses in cultured shrimp in America. These two viruses were transmitted to China at the beginning of the 21^st century. In this study, 214 shrimp samples of Penaeus vannamei were collected from seven different areas of China and tested by PCR for IHHNV and TSV infection. The results showed that there were a high prevalence of IHHNV (65.42%) and low prevalence of TSV (3.27%) in the tested samples. Several samples were found to be co-infected with these two viruses. A 3 kb fragment of 7 positive IHHNV samples and a structure protein region (ORF2) of three TSV positive samples were amplified and sequenced. The sequence comparison indicated that both IHHNV and TSV sequenced in China have a low genetic variations compared with the prototype IHHNV and TSV from Hawaii. Phylogenetic analysis showed that TSV isolates were clustered into two groups, Asia and America group, which was genetically correlated to geographic distribution. Foundation item: This work was supported by 973 project (2006CB101801)     

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.     

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 shr...     

The role of selective breeding and biosecurity in the prevention of disease in penaeid shrimp aquaculture

About 3.5 million metric tons of farmed shrimp were produced globally in 2009 with an estimated value greater than USD$14.6 billion. Despite the economic importance of farmed shrimp, the global shrimp farming industry continues to be plagued by disease. There are a number of strategies a shrimp farmer can employ to mitigate crop loss from disease, including the use of Specific Pathogen Free (SPF), selectively bred shrimp and the adoption of on-farm biosecurity practices. Selective breeding for disease resistance began in the mid 1990s in response to outbreaks of Taura syndrome, caused by Taura syndrome virus (TSV), which devastated populations of farmed shrimp (Litopenaeus vannamei) throughout the Americas. Breeding programs designed to enhance TSV survival have generated valuable information about the quantitative genetics of disease resistance in shrimp and have produced shrimp families which exhibit high survival after TSV exposure. The commercial availability of these selected shrimp has benefitted the shrimp farming industry and TSV is no longer considered a major threat in many shrimp farming regions. Although selective breeding has been valuable in combating TSV, this approach has not been effective for other viral pathogens and selective breeding may not be the most effective strategy for the long-term viability of the industry. Cost-effective, on-farm biosecurity protocols can be more practical and less expensive than breeding programs designed to enhance disease resistance. Of particular importance is the use of SPF shrimp stocked in biosecure environments where physical barriers are in place to mitigate the introduction and spread of virulent pathogens.     

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.     

Effect of chronic Taura syndrome virus infection on salinity tolerance of Litopenaeus vannamei

Taura syndrome virus (TSV) is one of the most important shrimp viruses affecting farmed shrimp worldwide. After an acute phase during which the likelihood of mortality is elevated, infected shrimp enter a chronic phase during which shrimp appear to resume normal behavior and display no gross signs of infection. This study was designed to determine if chronically TSV-infected shrimp Litopenaeus vannamei are compromised by the infection. Specifically we investigated whether chronically infected shrimp could tolerate a drop in salinity as strongly as uninfected shrimp. The study consisted of 3 trials that compared survival of uninfected and chronically TSV-infected L. vannamei after drops in salinity from 24 ppt to salinities varying from 18 to 0 ppt. Logistic regression detected a significant effect of TSV infection on survival of chronically infected shrimp (p < 0.05). Salinity drops from 24 ppt to 3 and 6 ppt resulted in statistically different survivals (p < 0.05). Survival rates were similar among groups for salinity drops to greater than 6 ppt or less than 3 ppt. Salinities at which 50% of the shrimp died (LC50) were 3.06 ppt for the uninfected and 6.65 ppt for the chronically infected groups. Moreover, histopathological analysis of chronically infected shrimp that were moribund or recently dead showed no signs of having reverted to the acute stage of the disease. These results suggest that chronically infected shrimp are not able to tolerate a salinity drop as strongly as uninfected shrimp.     

Taura syndrome virus loads in Litopenaeus vannamei hemolymph following infection are related to differential mortality

Taura syndrome is an economically important disease that can cause catastrophic losses of farmed shrimp. Without effective treatments for Taura syndrome virus (TSV), one approach to managing the problem is to selectively breed shrimp populations with increased disease resistance. To better understand why some shrimp can survive exposure to TSV, information is needed on how viral loads progress and persist following infection. Data reported here show that mortalities occurring mostly within 1 wk of infection are associated with high viral titers, and titers as high as 10(8.7) genome copies per microl hemolymph can persist for up to 3 wk in survivors. Thereafter, and up to approximately 9 wk post-exposure, most surviving shrimp remain chronically infected with TSV loads ranging from 10(4) to 10(8) genome copies per microl hemolymph. Challenging shrimp from families with varying TSV resistance showed that in shrimp from less resistant families, the TSV load in hemolymph increased earlier and reached higher peaks than in shrimp from more resistant families. Although TSV loads in moribund shrimp from families differing in resistance did not differ significantly, infection loads among survivors were lower in shrimp from more resistant families. Taken together, the data suggest that lethal infection loads can occur in both more and less susceptible shrimp and that survivors represent shrimp in which viral expansion is better contained.     

Ultrastructure of the replication site in Taura syndrome virus (TSV)-infected cells

Taura syndrome virus (TSV) is a member of the family Dicistroviridae that infects Pacific white shrimp Litopenaeus vannamei (also called Penaeus vannamei), and its replication strategy is largely unknown. To identify the viral replication site within infected shrimp cells, the viral RNA was located in correlation with virus-induced membrane rearrangement. Ultrastructural changes in the infected cells, analyzed by transmission electron microscopy (TEM), included the induction and proliferation of intracellular vesicle-like membranes, while the intracytoplasmic inclusion bodies and pyknotic nuclei indicative of TSV infection were frequently seen. TSV plus-strand RNA, localized by electron microscopic in situ hybridization (EM-ISH) using TSV-specific cDNA probes, was found to be associated with the membranous structures. Moreover, TSV particles were observed in infected cells by TEM, and following EM-ISH, they were also seen in close association with the proliferating membranes. Taken together, our results suggest that the membranous vesicle-like structures carry the TSV RNA replication complex and that they are the site of nascent viral RNA synthesis. Further investigations on cellular origins and biochemical compositions of these membranous structures will elucidate the morphogenesis and propagation strategy of TSV.     

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.     

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。