G. H. Kou

Detection of white spot baculovirus (WSBV) in giant freshwater prawn, Macrobrachium rosenbergii, using polymerase chain reaction

Abstract White spot baculovirus (WSBV) is the causative agent of a disease which decimated some cultured penaeid shrimp populations and inflicted severe economic damage in Taiwan. Until very recently, the giant freshwater prawn Macrobrachium rosenbergii was thought to be unaffected by this virus, but now signs closely resembling white spot syndrome (WSS) have been observed on its exoskeleton. In this paper, WSBV was established as the causative agent by using the diagnostic polymerase chain reaction (PCR) with WSBV-specific primers. WSBV was found in M. rosenbergii larvae, postlarvae, juveniles, and adults. The amplified product from the DNA of the naturally-infected WSS M. rosenbergii was similar to that of WSBV-infected Penaeus monodon. Furthermore, comparison of the restriction profiles of these two PCR products by HaeIII, HpaII, RsaI, and Sau3AI revealed no differences, suggesting that WSBVs from the infected P. monodon and M. rosenbergii are closely related, if not identical. A homogenate positive in one-step WSBV diagnostic PCR was prepared from frozen P. monodon for the challenge experiment. Dilutions were added to tanks of healthy M. rosenbergii larvae and postlarvae. After 2 days, some of the dead specimens were positive for WSBV by diagnostic PCR.

Analysis of a genomic segment of white spot syndrome virus of shrimp containing ribonucleotide reductase genes and repeat regions

White spot syndrome is a worldwide disease of penaeid shrimp. The disease agent is a bacilliform, enveloped virus, white spot syndrome virus (WSSV), with a double-stranded DNA genome that probably contains well over 200 kb. Analysis of a 12.3 kb segment of WSSV DNA revealed eight open reading frames (ORFs), including the genes for the large (RR1) and small (RR2) subunits of ribonucleotide reductase. The rr1 and rr2 genes were separated by 5760 bp, containing several putative ORFs and two domains with multiple sequence repeats. The first domain contained six direct repeats of 54 bp and is part of a coding region. The second domain had one partial and two complete direct repeats of 253 bp at an intergenic location. This repeat, located immediately upstream of rr1, has homologues at several other locations on the WSSV genome. Phylogenetic analysis of RR1 and RR2 indicated that WSSV belongs to the eukaryotic branch of an unrooted parsimonious tree and, further, seems to suggest that WSSV and baculoviruses probably do not share an immediate common ancestor. The present analysis of WSSV favours the view that this virus is either a member of a new genus (Whispovirus) within the Baculoviridae or a member of an entirely new virus family.

Studies on transmission of white spot syndrome associated baculovirus (WSBV) in Penaeus monodon and P. japonicus via waterborne contact and oral ingestion

WSBV (white spot syndrome associated baculovirus) is considered to be the main causative agent of a recently reported disease which has resulted in serious mortality among cultured penaeid shrimp in Taiwan and is characterized by obvious white spots on the body. Shrimp infectivity tests of WSBV were carried out by means of waterborne contact and oral ingestion. Healthy juvenile P. monodon and P. japonicus and healthy P. penicillatus postlarvae were immersed in filtrates prepared from either diseased P. japonicus or diseased P. monodon, both of which exhibited marked white spot signs. Cumulative mortalities of the three tested shrimp species reached 100% within 4–6 days. Using PCR with a specific primer set, WSBV was first detected in the previously healthy P. monodon immersed in filtrate from diseased P. monodon 6 h postinoculation (h p.i.). At 24 h p.i. detection rates reached 90%, and even though the tested shrimp failed to show visible evidence of disease, they nonetheless suffered 33% mortality. The appearance of WSBV in experimentally infected P. penicillatus postlarvae was detected at 24 h p.i. and reached 100% by 72 h p.i. Healthy P. monodon fed with diseased P. japonicus as well as those fed with diseased P. monodon became 80–90% WSBV-positive 24 h p.i. by PCR and all of the tested shrimp died within 5 d. Obvious white spots appeared on the exoskeleton of shrimp whether they were infected by waterborne contact or orally. WSBV was found highly pathogenic to the three tested shrimp species and was readily transmitted across different penaeid shrimp.

Nested polymerase chain reaction and in situ hybridization for detection of nucleopolyhedrosis

A nested polymerase chain reaction (PCR) and in situ hybridization were developed for detection of baculoviruses in insects or other arthropods with nucleopolyhedrosis. The nested PCR was based on the sequences of polyhedrin genes from baculoviruses. Two sets of primers were designed, primers set, 35/36, was for the first step of amplification and yielded a product of around 680 bp, the second primer, 35-1/36-1, was designed to yield a product of around 335bp from the fragment amplified by the first primer set. The sensitivity of this two-step amplification was 100 to 1000 times higher than that of the one-step amplification by primer set (35/36). Samples which contained baculovirus DNA yielded an amplification product showing the expected DNA fragment mobility, whereas nucleic acid extracted from tissue samples of clinically healthy insects or uninfected cells showed no such DNA fragment, thereby confirming the specificity of the primers. Using the 35/36 amplicon as a probe, the PenuNPV-infected cells show positive reaction by in situ hybridization. Two-step DNA amplification and in situ hybridization with the DNA probe developed in the present paper provide effective detection and diagnostic tools for screening insects or other arthropods, especially crustacean species, crabs and shrimps, for baculovirus infections, and may be important in preventing (and/or controlling/enhancing) the infection of baculoviruses.

Purification and immunological characterization of color carp (Cyprinus carpio) fibroblast heat-shock proteins

Abstract Eighty-seven, 72 and 30 kDa heat-shock proteins from color carp testis cell line, CCT, were purified, and rabbit antibodies were raised against them. After heat shock at 37°C, hsp87 appeared mainly in the cytoplasm and hsp72 and hsp30 appeared in the nucleus. When cells were restressed at 37°C for 8 hr after three recovery periods, the intensity and localization of the three anti-CCT hsps staining changed from those following an initial stress for 8 hr. The anti-CCT hsp87 and hsp72 antibodies crossreacted with proteins of similar molecular weights in all tested fish, lizard, mouse and human cell lines, but they showed various degrees in antigenic relevance.