Larvae and early post-larvae of Penaeus monodon (Fabricius) experimentally infected with white spot syndrome virus (WSSV) show no significant mortality

The pathogenicity of white spot syndrome virus (WSSV) was tested with different developmental stages of Penaeus monodon, i.e. nauplius, protozoeae, mysis, early post-larvae (PL1-10), late post-larvae (PL11-20) and juveniles. WSSV challenge was done by immersion and oral routes. No disease occurred in the larvae and early post-larvae but they were positive for WSSV by nested polymerase chain reaction (PCR) assay. Significant mortality was observed in late post-larvae and juveniles and both single and nested PCR assays gave positive results with these samples. The results demonstrated that WSSV virulence in P. monodon increases with advancing stages of development and that WSSV infection does not result in disease for larvae and post-larvae younger than PL10.     

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.     

白斑综合征病毒感染克氏原螯虾后的PCR检测及组织病理学研究

采用投喂感染白斑综合征病毒(White Spot Syndrome Virus,WSSV)对虾肌肉的方式,对养殖克氏原螯虾(Procambarus clarkii)进行人工感染,以确定WSSV对养殖克氏原螯虾的易感性。结果发现,投喂病虾感染组螯虾的死亡率达到90%,而对照组未出现死亡。采用PCR对试验组螯虾的肌肉进行WSSV检测,发现投喂感染组的阳性检出率为100%,对照组的阳性检出率均为0。PCR检测结果发现,濒死螯虾的肝胰腺、中肠、肌肉、鳃、性腺、心脏六种组织的PCR结果均为WSSV阳性,而对照组的各组织检测结果均为阴性。组织切片的光镜观察也证实,濒死螯虾的肝胰腺、中肠、肌肉、鳃、性腺、心脏及血淋巴等组织均发生了不同程度的病变。     

中国对虾雄性生殖系统感染WSSV在其垂直传播中的作用

通过人工感染实验,对中国对虾(Fenneropenaeus chinensis)雄性亲虾进行投喂感染。在确定其携带WSSV粒子后,将被WSSV感染的精荚人工移植到健康的雌虾纳精囊内。在无其他病源的情况下,促其产卵繁殖,统计各组子代的受精率、孵化率及无节幼体至溞状幼体的变态率贸?式PCR技术对亲虾及子代进行WSSV检测。结果表明,受WSSV感染的精荚能够把病毒传播给健康雌虾,雌虾能产出携带WSSV的卵子,培育出带毒幼体。各组子代的受精率、孵化率及变态率的统计结果表明,感染组和对照组在受精率上没有明显区别,受WSSV感染的精卵细胞可以正常结合。对照组受精卵的孵化率明显高于感染组,差异显著(P=0.045<0.05)。对照组无节幼体的变态率也高于感染组。说明WSSV的入侵对受精卵及幼体的发育有影响,WSSV感染导致部分受精卵及幼体不能正常发育或死亡。     

Developmental stages of kuruma shrimp, Penaeus japonicus Bate, susceptible to baculoviral mid-gut gland necrosis (BMN) virus

Abstract. The stages of kuruma shrimp susceptible to BMNV were determined by water-borne infection at the fertilized egg, nauplius, zoea, mysis, and 2-day (P-2), 4-day (P-4), 6-day (P-6), 8-day (P-8) and 10-day-old (P-10) post-larval stages. Susceptibility to infection tended to decrease with advancing stages of development from zoea to P-10. The stages from zoea to P-4 were very susceptible, with much higher mortality and lower growth rates in virus inoculated animals compared to controls. P-6 shrimp were also highly susceptible with all inoculated animals becoming infected with the virus. However, this group grew only slightly less well than controls and no mortality was observed. P-8 and P-10 post-larvae were refractory to the disease showing no mortality and no loss of growth, even though some were slightly infected with the virus. Fertilized eggs and nauplii did not become infected with the virus using water-borne inoculation.     

Experimental infection of European crustaceans with white spot syndrome virus (WSSV)

Eight European marine and freshwater crustaceans were experimentally infected with diluted shrimp haemolymph infected with white spot syndrome virus (WSSV). Clinical signs of infection and mortalities of the animals were routinely recorded. Diagnosis was by direct transmission electron microscopy (TEM), DNA hybridization (dot-blot and in situ hybridization) using WSSV probes and by PCR using WSSV specific primers. High mortality rates were noted between 7 to 21 days post-infection for Liocarcinus depurator , Liocarcinus puber , Cancer pagurus , Astacus leptodactylus , Orconectes limosus , Palaemon adspersus and Scyllarus arctus . Mortality reached 100%, 1 week post-infection in P. adspersus . When infection was successful, direct TEM observation of haemolymph revealed characteristic viral particles of WSSV, some observed as complete virions (enveloped), others as nucleocapsids associated with envelope debris. WSSV probes showed strong positive reactions in dot-blots and by in situ hybridization in sections and specific virus DNA fragments were amplified successfully with WSSV primers. White spot syndrome virus was pathogenic for the majority of the crustaceans tested. This underlines the epizootic potential of this virus in European crustaceans.     

High efficacy of white spot syndrome virus replication in tissues of freshwater rice‐field crab,Paratelphusa hydrodomous (Herbst)

An attempt was made to determine the replication efficiency of white spot syndrome virus (WSSV) of shrimp in different organs of freshwater rice‐field crab, Paratelphusa hydrodomous (Herbst), using bioassay, PCR, RT‐PCR, ELISA, Western blot and real‐time PCR analyses, and also to use this crab instead of penaeid shrimp for the large‐scale production of WSSV. This crab was found to be highly susceptible to WSSV by intramuscular injection. PCR and Western blot analyses confirmed the systemic WSSV infection in freshwater crab. The RT‐PCR analysis revealed the expression of VP28 gene in different organs of infected crab. The indirect ELISA was used to quantify the VP28 protein in different organs of crab. It was found that there was a high concentration of VP28 protein in gill tissue, muscle, haemolymph and heart tissue. The copy number of WSSV in different organs of infected crab was quantified by real‐time PCR, and the results revealed a steady increase in copy number in different organs of infected crab during the course of infection. The viral inoculum prepared from different organs of infected crab caused significant mortality in tiger prawn, Penaeus monodon (Fabricius). The results revealed that this crab can be used as an alternate host for WSSV replication and production.     

Quantitative analysis of an experimental white spot syndrome virus (WSSV) infection in Penaeus monodon Fabricius using competitive polymerase chain reaction

White spot syndrome virus (WSSV) has been a major pathogen of cultured Penaeus monodon Fabricius in Malaysia since 1994. As quantitative study on the replication of WSSV is in its infancy, competitive polymerase chain reaction (PCR) was used for quantitative study of an experimental WSSV infection per os in growout P. monodon . Gills, abdominal integument and abdominal muscle were selected for viral quantification. Infection was detectable as early as 14 h postinfection (h p.i.) in both gills and integument, but the infection in muscle was only detected at 24 h p.i. Gill tissue had the highest viral load, followed by integument and muscle. Typical viral growth curves were obtained for all organs with distinct phases of eclipse (0–24 h p.i.), logarithmic (24–48 h p.i.) and the plateau (48–120 h p.i.). Cumulative mortality rapidly increased from 48 h p.i. and reached 100% at the end of the plateau phase at 120 h p.i. Gross signs of white spots and reddish discoloration were also obvious in moribund individuals from the plateau phase. Based on the three phases of viral growth, WSSV infection was classified into light, moderate and heavy infection stages.     

Experimental host range and histopathology of white spot syndrome virus (WSSV) infection in shrimp, prawns, crabs and lobsters from India

Experimental studies were conducted by injecting or feeding white spot syndrome virus (WSSV) derived from infected shrimp, Penaeus monodon (Fabricius), collected from the south-east coast of India, to five species of shrimp, two species of freshwater prawns, four species of crabs and three species of lobsters. All species examined were susceptible to the virus. Experimental infections in the shrimp had the same clinical symptoms and histopathological characteristics as in naturally infected P. monodon . A cumulative mortality of 100% was observed within 5–7 days in shrimp injected with WSSV and 7–9 days in shrimp fed with infected tissue. Two species of mud crab, Scylla sp., survived the infection for 30 days without any clinical symptoms. All three species of lobsters, Panulirus sp., and the freshwater prawn, Macrobrachium rosenbergii (De Man), survived the infection for 70 days without clinical symptoms. However, bioassay and histology using healthy P. monodon revealed that crabs, prawns and lobsters may act as asymptomatic carriers/reservoir hosts of WSSV. This is the first report to suggest the carrier/reservoir capacity of these hosts through histological and bioassay evidences. Ultrastructural details of the virus in experimentally infected shrimp, P. vannamei , (Boone), were also studied.     

Quantitative real time PCR for the measurement of white spot syndrome virus in shrimp

Quantitative real time PCR, recently developed in molecular biology, is applied in this paper to quantify the white spot syndrome virus (WSSV) in infected shrimp tissue. The WSSV content in moribund shrimp of all species tested ( Penaeus stylirostris, P. monodon, P. vannamei ) ranged from 2.0 × 10⁴ to 9.0 × 10¹⁰ WSSV copies μg^–1 of total DNA ( n =26). In whole moribund post-larvae, 4.3 × 10⁹ WSSV copies μg^–1 of DNA were detected which is equivalent to 5.7 × 10¹⁰ WSSV copies g^–1 of post-larvae. The comparison of WSSV content between different tissues showed that muscle and hepatopancreas tissues contained 10 times less virus than gills, pleopods and haemolymph. With inocula of known virus content, bioassays by immersion challenge showed that a minimum of five logs of WSSV copies was necessary to establish disease in the challenged shrimp. In contrast, five logs of WSSV copies injected into shrimp muscle produced a LT-50 of 52 h. This real time polymerase chain reaction (PCR) technique is sensitive (four copies), specific (negative with DNA from shrimp baculoviruses and parvoviruses), dynamic (seven logs) and easy to perform (96 tests in <4 h).