Hypoxia increases susceptibility of Pacific white shrimp to whitespot syndrome virus (WSSV) / Condição de hipóxia aumenta a susceptibilidade do camarão branco do Pacíficoao vírus da mancha branca (WSSV)

The present study aimed to evaluate the mortality, reactive oxygen species production (ROS) and total hemocyte counts (THC) of the marine shrimp Litopenaeus vannamei infected with the white spot syndrome virus (WSSV) at three levels of oxygen saturation. For this, 360 shrimp (20±2g) were distributed in 24 tanks (60L), divided in two groups (infected and non-infected), which were subjected to 30, 60 and 100% of dissolved oxygen saturation (in quadruplicate). During 96 hours after infection, daily hemolymph samples were collected for hemato-immunological parameter evaluation (THC and ROS) and dead animals were removed and computed to assess cumulative mortality rates. In the infected group, animals subjected to 100% saturation showed higher ROS production (P<0.05) after 48 hours, while THC was significantly reduced (P<0.05), regardless of oxygen saturation. The hypoxia resulted in high mortality when compared to 100% saturation condition. In the uninfected group, no significant differences were observed in all evaluated parameters. Thus, the hypoxia condition increased the susceptibility of shrimp to the infection of WSSV, which may be partly related to the low ROS production showed by the animals subjected to 30% oxygen saturation.

O presente estudo teve por finalidade avaliar a mortalidade e a contagem total de hemócitos (CTH) e espécies reativas de oxigênio (EROs) de camarão Litopenaeus vannamei infectados com o vírus da mancha branca (WSSV) e submetidos a três níveis de saturação de oxigênio. Para tanto, 360 camarões (20±2g) foram distribuídos em 24 tanques (60L), divididos em dois grupos, infectados e não infectados e submetidos a 30, 60 e 100% de saturação de oxigênio (em quadruplicata). Após a infecção, diariamente foram coletadas amostras de hemolinfa dos animais para avaliação dos parâmetros hematoimunológicos (CTH e EROs) e foi estimada a mortalidade, por 96 horas. No grupo com infecção, os animais submetidos à saturação de 100% apresentaram um aumento na produção de EROs (P<0,05) após 48 horas, ao mesmo tempo em que a CTH demonstrou uma redução (P<0,05) independentemente da saturação do oxigênio, e a condição de hipóxia acarretou maiores mortalidades quando comparada à do grupo com 100% de saturação. No grupo sem infecção, não foram observadas diferenças significativas nos parâmetros avaliados nem mortalidade. Dessa forma, pode-se concluir que a hipóxia aumentou a susceptibilidade do camarão à infecção com o vírus da mancha branca, que pode estar, em parte, relacionada com a baixa contagem de hemócitos e produção de EROs observadas nos animais submetidos a essa condição.

Comparative analysis of variable region of white spot syndrome virus genome in Penaeus vannamei in Guangxi, China / 病毒学报

Comparative analysis of variable region ORF14/15 genes of white spot syndrome virus (WSSV) genome in Guangxi Penaeus vannamei (P. vannamei) could provide useful information for the evaluation of genetic diversity and genetic evolutionary relationship among WSSV isolates from Guangxi, China and other places. Based on geographical and temporal considerations, 40 WSSV-positive P. vannamei samples were collected during the period between May 2010 and July 2013 from Beihai, Qinzhou, and Fangchenggang, which were the main P. vannamei production areas in Guangxi, and the variable region ORF14/15 genes of the WSSV genome from all infected samples were amplified by PCR and then subjected to cloning and sequence analysis. Pairwise and multiple alignment analysis was then conducted to evaluate the degree of genetic divergence between different strains. The variable region ORF14/15 genes from 25 of 40 WSSV positive samples were successfully cloned and sequenced; among the ORF14/15 genes of 25 WSSV-positive strains, 22 was 619 bp in length and 3 was 620 bp. All the 25 Guangxi strains carried a 5949-bp deletion in the ORF14/15 region relative to TH-96-II, which has the longest nucleotide sequence in this region; the deletion of Guangxi strains occurred in the middle region of ORF14/15 gene, with only 190 bp and 429 bp/ 430 bp at 5' and 3' ends, respectively, which were coincident with WSSV-IN-05-I in deletion length and position. Sixteen of 25 Guangxi strains had completely identical nucleotide sequences in the variable re gion, and the homology between other strains also exceeded 97.9%. There were single nucleotide substi tution, deletion, and insertion in the ORF14/15 region of Guangxi strains compared with other strains in GenBank. In the phylogenetic tree based on WSSV variable region ORF14/15, the Guangxi strains were closely related and formed a separate branch with Indian strain IN-05-I, but far from other strains in GenBank. The ORF14/15 gene of WSSV isolates in cultured P. vannamei in Guangxi has a large deletion in the middle of the variable region, and the Guangxi WSSV strains show no significant spatio-temporal differences; the Guangxi strains are closer in genetics to Indian strain IN-05-I than other strains in GenBank.

A ten-month diseases survey on wild Litopenaeus setiferus (Decapoda: Penaeidae) from Southern Gulf of Mexico / Diez meses de análisis de las enfermedades en especímenes silvestres de Litopenaeus setiferus en el Sur del Golfo de México

The development of shrimp aquaculture in Mexican coasts of the Gulf of Mexico began to be explored using the Pacific white shrimp Litopenaeus vannamei in the mid 90´s. Many concerns over the risk of disease transmission to the economically important native penaeids, have been the main deterrent for the aquaculture of L. vannamei in the region. Concurrently, more than 10 years of research experience on the aquaculture suitability of the native Litopenaeus setiferus from the Terminos Lagoon, in the Yucatán Peninsula, have been accumulated. The aim of this study was then to determine the seasonal variations of the naturally acquired diseases and the possible detection of exotic pathogens. For this, random subsamples (n~60) of juveniles L. setiferus were collected from monthly captures. In order to detect the widest range of pathogens, including infectious hypodermal and hematopoietic necrosis (IHHNv) and white spot syndrome (WSSv) viruses, both histopathological and molecular methods were employed. Monthly prevalence (%) was calculated for every finding. We were able to detect a total of 16 distinct histological anomalies, most of which the presump- tive aetiological agent was readily identified. PCR results for viruses were negative. For some pathogens and symbionts, the prevalence was significantly different between the adult and juvenile populations. Prevalence of diseases tended to be higher in juvenile shrimp than in adults. The results of this study indicated that L. setiferus carry a wide variety of pathogens and symbionts that seem to be endemic to penaeids of the Gulf of Mexico, and those juveniles were more conspicuous to acquire pathogens and symbionts than adults.

Durante la década de los 90´s se introdujo el camarón blanco del Pacífico Litopenaeus vannamei a los Estados costeros mexicanos del Golfo de México con fines acuícolas, por lo que desde entonces existe preocupación por la posible introducción de enfermedades que puedan afectar a las poblaciones de camarones nativos. La investigación sobre la domesticación de especies nativas para una acuacultura sustentable se ha realizado por más de 10 años, sin embargo, aún existe escasa información sobre las enfermedades que se presentan de manera natural en estas poblaciones y posible trasfaunación. El presente estudio aborda el problema de las enfermedades encontradas en subpoblaciones de jóvenes y adultos de Litopenaeus setiferus del Área natural protegida Laguna de Términos, estado de Campeche, México. Técnicas de histología y biología molecular fueron utilizadas como herramientas de diagnóstico. Se encontró que L. setiferus es portador de patógenos y simbiontes endémicos del Golfo de México, y comparativamente, los jóvenes son más susceptibles en adquirir estas infecciones que los adultos, como probable respuesta al ambiente lacustre que ocupan. No se encontró evidencia de los virus IHHNv y WSSv, aunque en trabajos más recientes en algunos Estados del Norte ya se han detectado en poblaciones silvestres.

Detection of white spot syndrome virus in Brazil using negative staining, immunoelectron microscopy and immunocytochemistry techniques / Detección del virus del síndrome de mancha blanca en el Brasil utilizando inmunomicroscopía e inmunomarcación con partículas de oro coloidal

In this study thirty shrimp samples from commercial marine shrimp (L. vannamei) farms of southern region of Brazil were obtained. Hepatopancreas and shell scrapings fragments collected in these animals were processed by transmission electron microscopy using negative staining (rapid preparation), immunoelectron microscopy and immunocytochemistry (immunolabelling with colloidal gold particles) techniques. On the transmission electron microscopy a great number of white spot virus particles, ovoid or bacilliform-to-ellipsoid, measured 230-290 nm in length and 80-160 nm in diameter with intra-nuclear projections were visualized by the negative staining technique in 27 (90 percent) out of 30 samples examined. Using immunoelectron microscopy technique, the anti-VP 664 serum agllutinated a large number of particles formed by antigen-antibody interaction. In the immunocytochemistry technique, the antigen-antibody reaction was styrongly marked by the particles of colloidal gold over the virus. Notably, this is the first report, to our knowledge, describing use of these microscopy techniques to study Brazilian L. vannamei marine shrimp samples; moreover, this methodology also appears to be a viable complementary tool for diagnosing the presence of the white spot virus within shrimp tissues. Importantly, these are the first photoelectron micrographs of the WSSV in Brazil.

Se obtuvieron para el estudio 30 muestras de camarones marinos comerciales (L. vannamei) de las granjas de la región sur de Brasil. Fueron procesados fragmentos de hepatopáncreas y raspados internos del cefalotórax recogidos en estos animales por microscopía electrónica de transmisión con tinción negativa (preparación rápida), inmunomicroscopía y técnicas de inmunocitoquímica (inmunomarcación con partículas de oro coloidal). En la microscopía electrónica de transmisión de un gran número de partículas de virus de la mancha blanca, ovoide o elipsoidal a baciliformes, medían 230-290 nm de longitud y 80-160 nm de diámetro. En 27 (90 por ciento) de las 30 muestras examinadas intra-nuclear proyecciones se visualizaron mediante la técnica de tinción negativa. Utilizando una técnica de inmunomicroscopía electrónica, el anti-suero VP 664 reunió a un gran número de partículas formadas por la interacción antígeno-anticuerpo. En la técnica de inmunocitoquímica, la reacción antígeno-anticuerpo fue fuertemente reforzada por las partículas de oro coloidal en los virus. En particular, en Brasil este es el primer informe, a nuestro entender, que describe el uso de estas técnicas de microscopía en muestras de camarón marino L. vanamei. Además, esta metodología también parece ser una herramienta complementaria viable para diagnosticar la presencia del virus de la mancha blanca en tejidos de camarón. Es importante destacar que estas son las primeras fotos en microscopia electrónica del WSSV obtenidas en Brasil.

Neutralization of white spot syndrome virus (WSSV) for Penaeus chinensis by antiserum raised against recombinant VP19.

This study was carried out to neutralize the WSSV one of the most virulent pathogen causing large economic damage in shrimp culture industry using the antiserum produced against recombinant WSSV envelope protein VP19 (rVP19) as a tool to evaluate WSSV infection mechanism. A fragment of VP19 was expressed in Sf21 insect cell using baculovirus expression system as fusion protein with 6 His-tag. Then, polyclonal antiserum against rVP19 was raised in white rabbit. A constant amount of WSSV (at 10(4) diluted stock) was incubated with various antiserum concentrations and injected into shrimp, Penaeus chinensis, for the neutralization challenge. At 9 days post injection, the shrimp in the positive control injected with WSSVshowed 100% mortality The shrimps injected with WSSV preincubated with preimmune serum showed 83.3% mortality at 15 days post injection. The shrimps injected with the WSSV preincubated with 1 microl, 5 microl or 10 microl r VP19 antiserum and shrimp mortalities showed 66.6%, 40.0% and 26.6% at 15 days post injection, respectively The high concentration of antiserum group showed lower mortality than those of the low concentration of antiserum group. This indicates that the WSSV can be neutralized by the rVP19 antiserum in a dose-dependent manner. The neutralization challenge result suggested that VP19 might play an important role in WSSV infection to shrimp.

Promoter activity of different promoters in recombinant baculovirus-infected Sf9 cells / 生物工程学报

To compare the activity of different promoter in baculovirus-insect system, a series of recombinant baculoviruses were generated harboring the E-GFP reporter gene under the control of one of 5 promoters, including the ie1 promoter of shrimp white spot syndrome virus (WSSV), the truncated ie1 (mie1) promoter, the ETL promoter of the baculovirus, the elongated ETL (mETL) promoter, and the polyhedron promoter (P(PH)) of the baculovirus. The expression efficiency of the E-GFP reporter gene in the recombinant baculovirus-infected Sf9 cells was determined by flow cytometry. The results showed that both ie1 and mETL promoters had a strong promoter activity at early phase, while P(PH) showed a strong promoter activity at late phase. The ie1 promoter suggested the strongest promoter activity. The homologous region 1 (hr1) was also found to enhance the ETL promoter activity.

Selection of a novel single-chain variable fragment antibody specifically against a linear epitope of white spot syndrome virus / 生物工程学报

White spot syndrome virus (WSSV) is one of the most important pathogens in shrimp farm throughout the world. Many researches on WSSV have been done, but no efficient approach has been gained to protect and cure the disease. In this study, we constructed a single-chain fragment variable (scFv) antibody library displayed on phage using spleen cells from mice immunized with denatured WSSV. After several rounds of panning respectively against purified intact WSSV virions and purified VP28 expressed in Escherichia coli, five novel scFv antibodies specifically against WSSV were selected, one of which, clone P75E8, recognized a linear epitope. The location in virions of the epitopes recognized by the five scFv clones was determined by immunoelectron microscopy. This study provides a new way to obtain more different antibodies specifically binding to WSSV, and especially provides a new strategy to obtain scFvs against linear epitopes.

Comparison of variable region genes of shrimp White Spot Syndrome Virus (WSSV) in different areas in China / 病毒学报

According to the conservative sequence in the epitaxial variable region of Thailand strain of WSSV published in GenBank,a pair of primers were designed to amplify the variable region genes of 5 local WSSV strains (ZHSH, ZHJ, HN, QD1, QD2) by PCR and then cloned. In accordance with the CN, the results indicated that the number of nucleotide of 5 strains were deleted more than 591bp of the TW and TH strains. The ZHSH and HN strains that deleted 591bp at the 3' end, and 454 bp at the 5' end of variable gene was highly homologous with CN strain about 99.3%. 229bp of ZHJ strain at the 5' end was homologous with CN about 99.3%, and deleted of 816bp at the 3' end. 97bp at the 5' end and 171bp at the 3' end of QD1 and QD2 strains were homologous with CN strain about 99.3%, and about 777bp were absent in the middle. The above data showed that the variable region genes of WSSV had mutated more in China. The variable region gene of QD2 strain was coincidence with that of QD1 after propagating 10 generations in crayfish. The results indicated that the crayfish inoculation did not result in mutation of variable region genes of WSSV.

Shrimps survive white spot syndrome virus challenge following treatment with Vibrio bacterin.

Taking an innovative approach, a vaccination study using five bacterial strains viz. Vibrio campbelli (B60), V. alginolyticus (B73), V. parahaemolyticus-like (B79), V. parahaemolyticus (R8) and V. harveyi (RG203) was conducted in Penaeus monodon against white spot syndrome virus (WSSV) infection, considered as one of the serious pathogens of shrimps. Oral challenge with shrimps infected with WSSV showed a relative percentage survival of 5 and 47% in the P. monodon juveniles vaccinated with V. parahaemolyticus and V. harveyi, respectively. Results showed that there is a possibility of specifically immunising the shrimps against WSSV using bacterin prepared out of Vibrio harveyi isolates taken from shrimps infected with WSSV. Also, there was a level of protection attained by the shrimps due to immunisation with Vibrio strains.

Expression of single chain fragment variable P1D3 antibody against shrimp white spot syndrome virus in Pichia pastoris / 生物工程学报

White spot syndrome virus (WSSV) is a major pathogen in aquaculture penaeid shrimp, which caused catastrophic economic losses in the worldwide. No adequate treatments against WSSV are available. In order to study infection mechanism of WSSV, a phage display scFv cDNA library against WSSV was constructed and a neutralizing antibody of scFv P1D3 was selected in our lab previously. In this study, scFv P1D3 was expressed successfully in yeast. Firstly, the original expression vector of P1D3, M13 phagmid, was used as a template to design primers with restriction sites of SnaB I and EcoR I . Then the gene of P1D3 was amplified by PCR. After digested by SnaB I and EcoR I , the fragment of scFv P1D3 with E-tag was inserted into yeast and E. coli shuttle plasmid pPIC9k. The recombinant plasmid pPIC9k-scFv P1D3-Etag was linearized with Bgl II and then transformed into Pichia pastoris GS115 by electroporation. Positive clones were selected and verified by PCR and DNA sequencing. The scFv PID3 was induced to express in yeast by methanol. The results of ELISA demonstrate that scFv P1D3 expressed in yeast still has high specificity to bind on WSSV and the binding activity is higher than that expressed in E. coli TG1. After several optimizing experiments, the results show that the expression amount of scFv P1D3 can reach to 302 mg/L in yeast culture supernatant. This experiment has offered a new source of antibody for the researches on passive immunology for shrimp.

White spot syndrome virus isolates of tiger shrimp Penaeus monodon (Fabricious) in India are similar to exotic isolates as revealed by polymerase chain reaction and electron microscopy.

Microbiological analysis of samples collected from cases of white spot disease outbreaks in cultured shrimp in different farms located in three regions along East Coast of India viz. Chidambram (Tamil Nadu), Nellore (Andhra Pradesh) and Balasore (Orissa), revealed presence of Vibrio alginolyticus, Vibrio parahaemolyticus, and Aeromonas spp. but experimental infection trials in Penaeus monodon with these isolates did not induce any acute mortality or formation of white spots on carapace. Infection trials using filtered tissue extracts by oral and injection method induced mortality in healthy P. monodon with all samples and 100% mortality was noted by the end of 7 day post-inoculation. Histopathological analysis demonstrated degenerated cells characterized by hypertrophied nuclei in gills, hepatopancreas and lymphoid organ with presence of intranuclear basophilic or eosino-basophilic bodies in tubular cells and intercellular spaces. Analysis of samples using 3 different primer sets as used by other for detection of white spot syndrome virus (WSSV) generated 643, 1447 and 520bp amplified DNA products in all samples except in one instance. Variable size virions with mean size in the range of 110 x 320 +/- 20 nm were observed under electron microscope. It could be concluded that the viral isolates in India involved with white spot syndrome in cultured shrimp are similar to RV-PJ and SEMBV in Japan, WSBV in Taiwan and WSSV in Malaysia, Indonesia, Thailand, China and Japan.

The expression of VP19 gene from prawn white spot syndrome virus in silkworm, Bombyx mori using host range-expanded HyNPV / 生物工程学报

Prawn white spot syndrome is caused by the pathogen prawn white spot syndrome virus (WSSV). VP19 is a vesicle membrane protein of WSSV. HyNPV (Hybrid of AcNPV and BmNPV) constructed by the recombination of BmNPV and AcNPV is a new hybrid virus having both of their advantages. The recombinant transfer vector pBlueBicHisC-vp19 and recombinant baculovirus HyNPV-VP19 were constructed on the basis of the successful cloning of VP19. Newly-molted silkworms Bombyx mori of fifth instar were inoculated by the recombinant virus. SDS-PAGE and Western blotting analysis showed a specific band, about 21kD, which was consistent with the expectation suggesting that the WSSV-VP19 gene was successfully expressed in silkworm bodies.