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Although maternally transmitted microorganisms such as Wolbachia are well known to have a variety of effects on the reproduction of diverse insect species, little is known about the underlying mechanisms of actions. Artificial transfer of Wolbachia between taxonomically distant host species may provide insights into Wolbachia-induced manipulations of hosts. Here we performed a cross-order transfer of feminizing Wolbachia derived from a butterfly, Eurema mandarina. The Wolbachia were propagated in the Eurema hecabe cell line, called NTU-YB, and then used to inject prepupal/pupal females of a Wolbachia-free hymenopteran sawfly, Athalia rosae. The 14 females that emerged as adults looked morphologically and behaviorally healthy, and ovarian development appeared normal on dissection. However, in contrast to the control, none of the 333 eggs harbored by the seven Wolbachia-injected females developed successfully. Similarly, none of the 140 eggs laid on host plant by the four Wolbachia-injected females, which were mated with males, showed any signs of development. Wolbachia infection was detected from whole-body samples of the inoculated individuals, but not from the eggs they produced. Disruption of embryonic development despite the absence of Wolbachia in the egg cytoplasm may represent a new phenotype involving maternal effects that result in female sterility.

We briefly described the morphology and transmission pathway of a Spiroplasma sp. isolated from the field cricket, Gryllus bimaculatus in Taiwan, followed by the phylogenetic analysis based on the 16S rRNA gene sequence. The cricket spiroplasma infected the hemolymph, gut, muscle tissues and tracheal cells; therefore we suggest that the pathogen invaded tissues and organs from the hemolymph through the tracheal system and the endoplasmic reticular system. Based on 16S rRNA gene sequences and the phylogeny, this spiroplasma was most closely related to Spiroplasma platyhelix (Identity=95%) isolated from the dragonfly Pachydiplax longipennis and belongs to the Ixodetis clade.

This report shows how the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (rDNA) can be used to determine the species identity of insect cell lines and to distinguish between cell lines derived from closely related insect species. A PCR-RFLP method with the endonucleases HincII and PstI produces restriction fragment profiles that could distinguish between insect cell lines at the species level. Another PCR-based method used three species-specific primer sets, Ly-ITS1/Ly-ITS2, ITS1-1/Ld-ITS1 and Sf9-F2/ITS4, to identify the cell lines from Lymantria xylina, L. dispar and Spodoptera frugiperda, respectively. This method also detected cell-line cross-contaminations (CLCC) with contamination levels as low as 1% (10 cells in a population of 1000 cells) even when the contaminating cells were from a closely related species. Compared with conventional methods used for cell-line identification and CLCC detection, the methods presented here are fast and sensitive and could easily be applied to other cell culture laboratories.

The complete genome of the Maruca vitrata nucleopolyhedrovirus (MaviNPV) isolated from the legume pod borer, Maruca vitrata (Lepidoptera: Pyralidae), was sequenced. It was found to be 111 953 bp in length, with an overall 39 % G+C content, and contained 126 open reading frames (ORFs) encoding predicted proteins of over 50 aa. The gene content and gene order of MaviNPV have the highest similarity to those of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and their shared homologous genes are 100 % collinear. In fact, MaviNPV seems to be a mini-AcMNPV that is native to Taiwan and possesses a smaller genome with fewer auxiliary genes than the AcMNPV type species. Except for one ORF (Mv74), all of the MaviNPV ORFs have homologues in the AcMNPV genome. MaviNPV is the first lepidopteran-specific baculovirus to lack homologues of vfgf and odv-e66. In addition, MaviNPV lacks the baculovirus repeat ORF (bro) gene that corresponds to AcMNPV ORF2. Five homologous regions (hrs) were located within the MaviNPV genome, and these contained a total of 44 imperfect palindromes. Phylogenetic analysis of the whole genome revealed that MaviNPV was separated from the common ancestor of AcMNPV and Bombyx mori nucleopolyhedrovirus before these two viral species diverged from each other. Moreover, replication of MaviNPV in several cell lines and an egfp–MaviNPV infection assay revealed that IPLB-LD-652Y cells are only partially permissive to MaviNPV, which supports our conclusion that MaviNPV is a distinct species of the group I lepidopteran NPVs.

Nosema ceranae is a common microsporidian pathogen, one of two Nosema species that cause "nosema disease" in honeybees, Apis cerana and Apis mellifera. Samples of N. ceranae rDNA from isolates collected in different locations were sequenced and one 5S rRNA was found to be upstream of SSUrRNA. The rDNA arrangement, 5'-5S rRNA-IGS-SSUrRNA-ITS-LSUrRNA-3', was found in all isolates. In order to better understand the distribution relationship between N. ceranae isolates from A. cerana and A. mellifera, their rRNA spacer regions were also sequenced for analysis. Results showed that there are no significant differences between the IGS sequences of the isolates and no difference in the ITS sequence with the exception of one transition found in an isolate from Martinique. These isolates showed consistency in the IGS phylogenic analysis suggesting that no transmission barrier exists between A. mellifera and A. cerana and there is no difference between isolates from geography separated areas.

Here we describe the establishment of a new cell line, NTU-MV, derived from pupal tissues of an economically important pest, the legume pod borer Maruca vitrata. This cell line contained four major cell types: polymorphic cells, round cells, spindle-shaped cells, and comma cells. The doubling time of MV cells in TNM-FH medium supplemented with 8% FBS at 28 °C was 27 h. The chromosome numbers of MV cells varied widely from 16 to 268. Compared to other insect cell lines, the MV cell line produced distinct isozyme patterns with esterase, malate dehydrogenase (MDH), and lactate dehydrogenase (LDH). Confirmation that NTU-MV was derived from M. vitrata was demonstrated by showing that the sequence of the internal transcribed spacer regions (ITS) of the MV cells was 98% identical to that of M. vitrata larvae. Two NTU-MV cell strains, NTU-MV1 and NTU-MV56, were selected based on susceptibility to MaviMNPV (M. vitrata multiple nucleopolyhedrovirus). NTU-MV, MV1, and MV56 cells showed a high susceptibility to MaviMNPV and produced high yields of polyhedra (47–50 OBs/cell, 4 × 107–5.96 × 107 OBs/ml) after 2 weeks of MaviMNPV infection. We conclude that the NTU-MV cell line will be a useful tool for studying MaviMNPV as well as for the mass production of MaviMNPV polyhedra for the biocontrol of M. vitrata.

Microsporidiosis (nosema disease) of the honeybee, Apis mellifera, has spread worldwide and caused heavy economic losses in apiculture. We obtained a spore isolate from worker ventriculi of A. mellifera colonies kept on the campus of National Taiwan University and sequenced the ribosomal genes. The entire length of the ribosomal DNA is about 3828 bp and the organization is similar to that of Nosema apis. However, the SSUrRNA, ITS, and LSUrRNA sequences have comparatively low identities with those of N. apis (92, 52, and 89%, respectively) and the SSUrRNA has a 99% identity with Nosema ceranae. These results indicate that this isolate is not N. apis, but N. ceranae. Moreover, the morphological characteristics are identical to those of N. ceranae. These results show that nosema disease of the honeybee, A. mellifera, may not be caused solely by the infection of N. apis.

Distribution and biotypes of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) in eastern Asia are not clear. In this study, B. tabaci was collected from Taiwan and neighboring islands of eastern Asia. Host range records included 51 plant species from 15 families. The molecular marker of the mitochondrial cytochrome oxidase I (COI) gene sequence was used to distinguish biotypes. Phylogenetic analyses indicated three biotypes in eastern Asia: B, Nauru, and An. This is the first molecular evidence to demonstrate the B and An biotypes in Taiwan and that the Nauru and An biotypes are new to China. Therefore, at least the B, Q, Nauru, and An biotypes are now known from China. Phylogenetic trees revealed that the invasion event of the B biotype in Taiwan was recent, and multiple invasions may have occurred due to human trade activities. The B biotype is the most widely distributed biotype in Taiwan. Invasions by the Nauru and An biotypes in Taiwan may have included both natural movements and human trade activities. Otherwise, distributions of the Nauru and An biotypes overlap in Asia, and it is presumed they are closely related. The result supports that the Nauru biotype is of Asian origin, and the An biotype may be of Asian or Australian origin. In addition, the tree topology supports B. tabaci being differentiated into three major clades: the New World, Asia/Australia, and Mediterranean/Asia Minor/African groups. This study also provides some information on the relationships within the B. tabaci species complex. In addition, we hope this study will be helpful to crop protection and appropriate control materials of B. tabaci in eastern Asia.

A microsporidium from the Ficus pest, Ocinara lida, in Taiwan is characterized. The taxonomic position of this species was preliminarily determined by sequencing small subunit rRNA gene (SSUrRNA). Analysis of the SSUrRNA sequence indicated that this isolate from O. lida is a member of the genus Endoreticulatus and belongs to the genetic grouping containing other lepidopteran Endoreticulatus species we have analyzed phylogenetically. The taxonomic position of this isolate was also confirmed by the ultrastructural characteristics of this isolate. The congruence between SSUrRNA sequence analysis and ultrastructural characteristics shows that this isolate is more closely related to Endoreticulatus bombycis than to Endoreticulatus schubergi Zwölfer.

A baculovirus has been isolated from infected larvae of the casuarina moth, Lymantria xylina Swinehoe (Lepidoptera: Lymantriidae) in Taiwan. Ultrastructural observation revealed that this virus is a multiple nucleopolyhedrovirus (MNPV), and the name L. xylina MNPV (LyxyMNPV) was proposed. Restriction endonuclease (BamHI, EcoRI, and EcoRV) profiles of LyxyMNPV genome differed from those of other known NPVs. The size of the LyxyMNPV genome was estimated to be 154+/-1.26 kbp (mean+/-SE). The polyhedrin gene is located in the BamHI-D, EcoRI-C, and EcoRV-K fragments of LyxyMNPV genome. The gene organization of the LyxyMNPV EcoRV-K fragment and the phylogenetic analysis based on the polyhedrin gene sequences showed that LyxyMNPV is closely related to the L. dispar MNPV (LdMNPV). Furthermore, a rapid assay method was developed to distinguish LyxyMNPV from LdMNPV based on PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) analysis of polyhedrin genes.

One unique feature of the shrimp white spot syndrome virus (WSSV) genome is the presence of a giant open reading frame (ORF) of 18,234 nucleotides that encodes a long polypeptide of 6,077 amino acids with a hitherto unknown function. In the present study, by applying proteomic methodology to analyze the sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of purified WSSV virions by liquid chromatography-mass spectrometry (LC-MS/MS), we found that this giant polypeptide, designated VP664, is one of the viral structural proteins. The existence of the corresponding 18-kb transcript was confirmed by sequencing analysis of reverse transcription-PCR products, which also showed that vp664 was intron-less. A time course analysis showed that this transcript was actively transcribed at the late stage, suggesting that this gene product should contribute primarily to the assembly and morphogenesis of the virion. Several polyclonal antisera against this giant protein were prepared, and one of them was successfully used for immunoelectron microscopy analysis to localize the protein in the virion. Immunoelectron microscopy with a gold-labeled secondary antibody showed that the gold particles were regularly distributed around the periphery of the nucleocapsid with a periodicity that matched the characteristic stacked ring subunits that appear as striations. From this and other evidence, we argue that this giant ORF in fact encodes the major WSSV nucleocapsid protein.

Distinct Taura syndrome virus (TSV) isolates were found in Metapenaeus ensis (isolate Tw2KMeTSV), Penaeus monodon (isolate Tw2KPmTSV) and Litopenaeus vannamei (isolate Tw02LvTSV). Nucleotide sequence analysis of these three isolates revealed differences in the TSV structural protein (capsid protein precursor) gene orf2. TSV ORF2 amino acid sequence comparison and phylogenetic analysis suggested a comparatively close relationship between these three Taiwanese isolates and the Hawaiian isolate HI94TSV. In P. monodon specimens that were naturally and experimentally infected with the Tw2KPmTSV isolate, the virus was contained and shrimps showed no clinical signs of infection. However, when P. monodon was challenged with the Tw2KMeTSV isolate, the virus replicated freely. The ORF2 amino acid sequence of the Tw2KMeTSV isolate differed from that of isolate Tw2KPmTSV in four positions and these differences may account for their phenotypic differences, at least in terms of their ability to replicate in specific hosts.

We present here for the first time the complete DNA sequence data (4301bp) of the ribosomal RNA (rRNA) gene of the microsporidian type species, Nosema bombycis. Sequences for the large subunit gene (LSUrRNA: 2497bp, GenBank Accession No. ), the internal transcribed spacer (ITS: 179bp, GenBank Accession No. ), the small subunit gene (SSUrRNA: 1232bp), intergenic spacer (IGS: 279bp), and 5S region (114bp) are also given, and the secondary structure of the large subunit is discussed. The organization of the N. bombycis rRNA gene is LSUrRNA-ITS-SSUrRNA-IGS-5S. This novel arrangement, in which the LSU is 5' of the SSU, is the reverse of the organizational sequence (i.e., SSU-ITS-LSU) found in all previously reported microsporidian rRNAs, including Nosema apis. This unique character in the type species may have taxonomic implications for the members of the genus Nosema.

A multiple-nucleocapsid nucleopolyhedrovirus (MNPV), named Perina nuda MNPV (or PenuNPV), isolated from P. nuda (Fabricius) (Lepidoptera: Lymantriidae)—a major banyan pest—was studied in terms of its main morphological, biochemical, and biological properties. The pleiomorphic occlusion bodies were 1.7‐5.4 mm in diameter with a mean

Microsporidian isolates from five lepidopteran pests— Spodoptera litura , Spodoptera exigua , Helicoverpa armigera , Plutella xylostella , and Pieris spp.—were compared by spore morphology, infectivity to S. litura , Western-blot banding patterns, the sequences of small subunit rRNA gene (SSUrRNA sequence), and random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR). All the isolates could infect experimentally and multiply in the larvae of S. litura . The S. exigua isolate showed the highest virulence to the larvae of S. litura while the P. xylostella isolate showed the lowest. No significant differences either in spore morphology or in SSUrRNA sequences of these isolates were found. The SSUrRNA sequences of these isolates confirmed they are members of the genus Nosema . Based on the result of Western-blot hybridization with the rabbit anti- Nosema spodopterae spore antiserum, three serotypes could be distinguished: N. spodopterae ( S. litura isolate) and Pi . spp. isolate; S. exigua and H. armigera isolates; and P. xylostella isolate. The amplicons of RAPD-PCR with 60 primers yielded clear patterns that were selected and used for identification and also for phylogenic analysis of these five isolates. Based on analysis by the computer, isolates could be clearly divided into three groups that were coincident with the serotypes; therefore we suggest that N. spodopterae and isolates of Pi. spp., S. exigua , and H. armigera are more closely related in phylogenesis. In addition, in the amplification with the Nosema bombycis specific primer set, only DNAs from P. xylostella isolate and N. bombycis yielded amplicons. Therefore, we suggest that four isolates, excluding the P. xylostella isolate, are N. spodopterae , and the taxonomic position of P. xylostella isolate needs to be elucidated.

DNA amplification fingerprinting (DAF) and nuclear ribosomal DNA (nrDNA) amplification were used to discriminate between two laboratory colonies of two closely related species of weevils: the rice weevil, Sitophilus oryzae (L.), and the maize weevil, S. zeamais Motschulsky. For DAF, three sets of primers (aldolase, prolactin receptor, and interleukin-1 β ) were used for identification by polymerase chain reaction (PCR) and agarose gel electrophoresis, and the highly similar patterns of the resultant amplicons reconfirmed that the two weevils are closely related. The fragments of nrDNA amplification showed that for S. oryzae and S. zeamais , the homologies of the nucleotide sequences of the internal transcribed spacer regions, ITS-1 and ITS-2, were unusually high, at 96% and 97%, respectively. Based on the ITS-1 and ITS-2 sequences, two species-specific primer sets were designed: with the primer set ITS3/So , the predicted 450 bp DNA fragment was yielded with S. oryzae genomic DNA after PCR amplification ( n =10), but no PCR product was obtained with S. zeamais ( n =10); with the primer set ITS3/Sz , the same 10 S. zeamais specimens yielded a 550 bp DNA fragment, but S. oryzae yielded no amplicons. In view of the difficulty of distinguishing between these two closely related species, the specificity and availability of these two primer sets might prove to be a useful tool for distinguishing between them. However, the nrDNA sequences of the ITS-1 and ITS-2 regions of geographically isolated populations of both weevils still need to be elucidated, and the applicability of this technique to different geographical populations will need to be confirmed by further study.

Infection of shrimp cells with white spot syndrome virus (WSSV) results in an increase in ribonucleotide reductase (RR) expression at the RNA level. In this article we further express and characterize the induction of a novel ribonucleotide reductase after WSSV infection of shrimp cells. A baculovirus/insect system was used to express the two recombinant protein subunits RR1 and RR2, and a DNA polymerase coupled RR activity assay showed a marked increase in ribonucleotide reductase activity when cell extracts containing recombinant RR1 and RR2 were combined. The same assay revealed that RR activity increased as infection advanced in the gills of experimentally infected shrimp. An increase in RR expression was also detected at the protein level in WSSV-infected shrimp cells. An immunocytochemistry assay by confocal laser scanning microscopy showed that in hemocytes collected from WSSV-infected shrimp, both of the subunit proteins (RR1 and RR2) were concentrated mainly around the nucleus, but only RR1 was detected inside it. All of these results suggest that WSSV RR is functionally involved during WSSV infection.

Positive results were obtained with nested white spot syndrome virus (WSSV) diagnos- tic PCR performed on 5 commercial brands of dry-packed Artemia cysts using several WSSV genomic sequence-specific primers. In 2 brands, PCR and nucleotide sequence analysis found C→ T and C→ G point mutations in the pms 146 WSSV amplicon, but in all 5 brands, the nucleotide sequences that were successfully amplified by the rr1, rr2 and tk-tmk gene-specific primer sets were identical to those of Penaeus monodon WSSV. However, despite the inarguable presence of WSSV or WSSV-like template DNA, we were unable to detect WSSV by PCR in hatched nauplii derived from PCR-positive cysts or in P. monodon postlarvae fed Artemia nauplii hatched from such cysts. Most simply, these results suggested that the cysts were externally contaminated with WSSV or WSSV-like template material that was removed during hatching and washing of the nauplii. Given the small sequence variations found, it may also have been a variety of WSSV non-infectious for P. monodon or Artemia and derived from other crustaceans or arthropods in the Artemia environment. However, we could not establish this conclusively and a small possibility remained that the PCR template in these tests was derived from WSSV template present internally in the cysts and derived from infected Artemia adults. However small, this possibility must be vigorously tested, given the impact that a positive outcome could have on the shrimp industry.

An open reading frame (ORF) that encodes a 715-amino-acid polypeptide was found in an 8421-bp Eco RI fragment of the shrimp white spot syndrome virus (WSSV) genome. The polypeptide shows significant homology to eukaryotic serine/threonine protein kinase (PK) and contains the major conserved subdomains for eukaryotic protein kinases. Coupled in vitro transcription and translation generated a protein having an apparent molecular mass of about 87 kDa according to sodium dodecyl sulfate–polyacrylamide gel electrophoresis. For transcriptional analysis of the pk gene, total RNA was isolated from WSSV-infected shrimp at different times after infection. Northern blot analysis with pk -specific riboprobe found a major and a minor transcript of 2.7 and 5.7 kb, respectively. Rapid amplification of the 5′ cDNA ends of the major 2.7-kb pk transcript showed that there were two transcriptional initiation sites located at nucleotide residues -38(G) and -39(G) relative to the ATG translational start codon. Temporal expression analysis by RT-PCR indicated that the transcription of the pk gene started 2 h after infection and continued for at least 60 h. Phylogenetic analysis showed that WSSV protein kinase does not have any close relatives and does not fall into any of the major protein kinase groups.

The causative agent of white spot syndrome (WSS) is a large double-stranded DNA virus, WSSV, which is probably a representative of a new genus, provisionally called Whispovirus. From previously constructed WSSV genomic libraries of a Taiwan WSSV isolate, clones with open reading frames (ORFs) that encode proteins with significant homology to the class I ribonucleotide reductase large (RR1) and small (RR2) subunits were identified. WSSV rr1 and rr2 potentially encode 848 and 413 amino acids, respectively. RNA was isolated from WSSV-infected shrimp at different times after infection and Northern blot analysis with rr1 - and rr2 -specific riboprobes found major transcripts of 2.8 and 1.4 kb, respectively. 5′ RACE showed that the major rr1 transcript started at a position of −84 (C) relative to the ATG translational start, while transcription of the rr2 gene started at nucleotide residue −68 (T). A consensus motif containing the transcriptional start sites for rr1 and rr2 was observed (TCAc/tTC). Northern blotting and RT-PCR showed that the transcription of rr1 and rr2 started 4–6 h after infection and continued for at least 60 h. The rr1 and rr2 genes thus appear to be WSSV “early genes.”

SUMMARYAmerican foulbrood (Paenibacillus larvae larvae), is a major concern of the beekeeping industry in Taiwan. However, disease signs have never been encountered during hive inspections of local colonies of the Asian honey bee, Apis cerana. To study the susceptibility of A. cerana larvae to AFB, various doses of P. I. larvae spores were added to larval food and disease development was monitored. Results showed that 1-day-old larvae were most susceptible, next were 2-day-old larvae, while 3-day-old larvae showed no signs of disease even when fed a large dose (4.5 × 104 spores/larva). The negative correlation between susceptibility and larval age was similar to that found for A. mellifera. Further, at the susceptible age, A. cerana larvae showed higher resistance than A. mellifera larvae when fed the same dose of spores. The dose of spores that would cause 95% mortality of A. mellifera larvae only led to 47.1 % mortality of A. cerana larvae of the same age. This resistance by A. cerana larvae apparently ...

This study presents the ultrastructure of the microsporidian infecting the trunk musculature of Anguilla japonica and originally described as Pleistophora anguillarum Hoshina, 1959. All stages develop within a special structure, the sporophorocyst (SPC), which is equipped with a thick dense wall. This wall grows along with the growth of the parasites within it. Meronts are uni- to binucleate, which divide and steadily give rise to sporonts. During transition to sporonts the cell coat of the meronts increases its thickness, temporarily featuring thick irregular projections. Eventually a uniformly thick sporont wall is formed, then the sporont cells detach themselves from the wall (= future wall of the sporophorous vesicle, SPV) and start a series of divisions to produce sporoblasts. The SPV wall is compact, has no pores and consists of 2 layers. The presence of the SPC justifies the transfer of the species into the genus Heterosporis. Spores from disrupted SPCs are ingested by macrophages and within them are spread into various body tissues including the outermost layers of the epidermis. From here, they can easily be released to the outside and can contaminate the environment while the host is still alive.

PCR and in situ hybridization analysis were used for detection of white spot syndrome virus (WSSV) in an infected, cultured shrimp population over a long period in the absence of disease outbreaks. The shrimp were derived from a single WSSV-carrier brooder and cultured first in a tank and then in outdoor ponds. Prior to harvest at 13 mo, no l-step PCR-positive specimens were found, even though most tested specimens were found to be 2-step PCR-positive. At 7 mo, 2-step PCR-positive tissues were found in 5 sampled shrimp. Heart, gill, integument, muscle and stomach tissues best supported viral replication At 13 mo several shrimp died, and l-step PCR-positive individuals were found for the first time Although superficially healthy, 10% of the surviving adults had tiny white spots on their carapace, and In sjtu hybridization analysis revealed WSSV-positive cells in 40% of the specimens examined. As before, most were found in the stomach, integument and gills, and only very few in the lymphoid organ and other organs. These observations contrasted to those for experimentally infected shrimp with gross signs of terminal WSSV infection, where strong positive signals were also observed in the lymphoid organ and in other organs of ectodermal or mesodermal origin. Our results showed clearly that whatever the source, WSSV was carried in the shrimp population at a low intensity (i.e. nested PCR was required for detection) for a very long time in the absence of massive mortality. We hypothesize that disease outbreaks do not occur if shrimp defense mechanisms manage to contain lowintensity viral infections under low-stress culture conditions. Conversely, outbreaks may occur under stressful conditions.

By sequencing the entire ribosomal RNA (rRNA) gene of Nosema spodopterae, we show here that its gene organization follows a pattern similar to the Nosema type species, Nosema bombycis, i.e. 5'-large subunit rRNA (2,497 bp)-internal transcribed spacer (185 bp)-small subunit rRNA (1,232 bp)-intergenic spacer (277 bp)-5S rRNA (114 bp)-3'. Gene sequences and the secondary structures of large subunit rRNA, small subunit rRNA, and 5S rRNA are compared with the known corresponding sequences and structures of closely related microsporidia. The results suggest that the Nosema genus may be heterogeneous and that the rRNA gene organization may be a useful characteristic for determining which species are closely related to the type species.

The Perina nuda nucleopolyhedrovirus (PenuNPV) polyhedrin gene was located in EcoRI-G (6.3 kilobase pairs; kbp) and PstI-G (4.3 kbp) fragments of its genomic DNA. A portion of 1333 nucleotides (nt) containing this gene was sequenced. An open reading frame of 735 nt encoded a 245-amino-acid-long polyhedrin. A conserved TAAG motif which is associated with transcriptional start sites was identified 51 nt upstream of the translation initiation codon of PenuNPV polyhedrin gene. A putative polyadenylation signal, AATAAA, was found 116 nt downstream of the termination codon (TAA). Comparison of the amino acid sequences of PenuNPV polyhedrin with those of other NPVs showed that PenuNPV polyhedrin was most closely related to Orgyia pseudotsugata multiple NPV (OpMNPV) polyhedrin.

A continuous cell line, designated NTU-PN-HH, was established from the pupal ovary ofPerina nudaFabricius (Lepidoptera: Lymantriidae). The cells have been through more than 300 passages during 4 years in TNM-FH medium supplemented with 10% FBS, at a constant temperature of 28°C. The cell line consists of four major morphologic types: polymorphic cells, spindle-shaped cells, round cells, and squamous cells. The characterization of this cell line showed that NTU-PN-HH is a newly established cell line. It is the first cell line that is permissive to PnNPV (P. nudamultiple nuclear polyhedrosis virus).

Whlte spot syndrome baculovirus (WSBV) has been found across ddferent shrimp species and in different Asian countries. The detection of WSBV in shrimp with white spot syndrome has already been achieved by means of l-step polymerase chain reaction (PCR). In an attempt to establish a more sensitive assay, we evaluated the effect of 2-step amplification with nested primers on the sensitivity of WSBV diagnostic PCR. The sensitivity of the 2-step amplification was 10" to 10"imes higher than that of l-step amplification. Using both techniques, we successfully detected WSBV DNA in cultured and captured shrimp, crabs and other arthropods. Cultured Penaeus monodon (black tiger shrimp), P. japonicus (kuruma shrimp), P penicjllatus (red tail shrimp), and Metapenaeus ensis (sand shrimp) displaying white spot syndrome were collected from farms at different localities. One-step amplification of the DNA extracted from these shrimps consistently yielded an expected 1447 bp PCR product. Some of the tested specimens of cultured Scylla serrata (mud crab) that exhibited white spot syndrome were positive in l-step WSBV diagnostic PCR, while others were positive only in 2-step WSBV diagnostic PCR. Use of the 2-step amplification protocol also detected a WSBV-specific DNA fragment in Macrobrachium rosenbergii (the giant freshwater prawn) exhibiting white spot syndrome. We also confirmed that WSBV exists in wild-caught shrimp (P monodon, f! japonicus, P semisulcatus and P penicillatus) and crabs (Charybdisferiatus. Portunuspelagicus and P. sanguinolentus) collected from the natural environment in coastal waters around southern Taiwan. Detection of WSBV in non-cultured arthropods collected from WSBV-affected shrimp farms revealed that copepods, the pest crab Hehce tndens, small pest Palaemonidae prawn and the larvae of an Ephydridae insect were reservoir hosts of WSBV. The relatedness between WSBV and Thailand's systemc ectodermal and mesodermal baculovirus (SEMBV) is d~scussed in this paper.

White spot syndrome associated baculovlrus (WSBV) is the causative agent of a dlsease which has recently caused high shrimp mortahties and severe damage to shrimp cultures. In thls study, a strain of WSBV from black tiger shrimp Penaeus monodon was used to develop a diagnostlc tool for the detection of WSBV and related agent lnfect~ons in shnmp The vlnons were punfied from P monodon Infected with LVSBV V~ral genomlc DNA was extracted from purlfled vinons by treatlng the vlnons \n th proteinase K dnd cetyltnmethylammonium bromlde (CTAB) followed by phenol-chloroform extraction and ethanol precipitation A qualitative assessment Ivas performed using polymerase chain reaction (PCR) analys~s on the viral DNA and primers specif~c to shrimp genomic DNA in order to mon~ t o r shrimp DNA contamination In the viral genomic DNA preparations A WSBV genomlc DNA llbrary was constructed and based upon the sequence of the cloned WSBV DNA fragment, we deslgned a LVSBV-specific prlmer set for PCR to detect WSBV Infection in penaeld shrimp Samples which contained WSBV DNA yielded an evident ampl~f~cat lon product showing the expected moblllty of a 1447-bp DNA fragment whereas nuc le~c aclds extracted from tissue samples of clln~cally healthy shnmp showed no such DNA fragment, thereby confirming the speclficity of our pnmers By PCR with thls prlmer set, ~t was demonstrated that the causative agents of white spot syndrome in different shnmp specles are closely related An effective diagnostlc tool is thus provided for screening shnmp for \.VSBV infections, and may be important In preventing the further spread of this d~sease

Clam virus (CV HB-1) isolated from hard clam Meretrix lusoria , typed as infectious pancreatic necrosis virus (IPNV) Ab serotype, was employed for the present study. The structural polypeptides of purified virus generated by serial undiluted and diluted passaging were analyzed using sodiumdodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The results indicated that in VP2, the virus particles of serial undiluted passaging had viral polypeptides smaller than those of serial diluted passaging. Furthermore, a polypeptide with a molecular mass of 105 kDa was found in the serial undiluted virus preparation. Further analysis by monoclonal antibodies showed that VP105 might be a polyprotein produced by genomic RNA segment A. To investigate the sequential changes of viral polypeptides of the virus preparation at high multiplicity infection, the viral polypeptides at each undiluted passage were analyzed by SDS-PAGE followed by immunoblotting. The results indicated that VP105 was present by undiluted passage 7, while the smaller VP2 was present by undiluted passage 10. The amount of smaller VP2 and VP105 increased as the number of passages increased.

Tissues collected from Japanese eel elver ( Anguilla japonica ) experimentally infected with Pleistophora anguillarum were cultivated in Leiboritz's L-15 medium. A cell line designated as EP-1 (eel cells persistently infected with P. anguillarum) was established, and was found to be persistently infected with P. anguillarum . With this culture system, we first described the merogonial reproduction of P. anguillarum . Using histochemical and indirect immunofluorescent technique, it was revealed that there were 3–4 merozoites of different sizes residing in the cytoplasm of EP-1 cells. Electron micrographs of EP-1 cells showed that the merogonial stages had isolated nuclei (monokaryotic). The merozoite plasmalemma was separated from host cytoplasm by a membrane of host origin. The results of the infection experiment revealed that the present in vitro cultivation system could provide infective merozoites which were capable of proceeding to further sporogonial reproduction in the muscle of their natural host, eels.

The causative viral agent was purified from diseased shrimp Penaeus monodon with white spot syndrome. Negatively stained preparations show that the virus is pleiomorphic. It is fusiform or rod-shaped. In negatively stained preparations, the virion measures 70 to 150 nm at its broadest point and is 250 to 380 nm long. In some virions, a tail-like projection extends from one end. The capsid is apparently composed of rings of subunits in a stacked series. The rings are aligned perpendicular to the longitudinal axis of the capsid. The genome of the virus is a double-stranded DNA molecule which produces at least 22 Hind 111 fragments. The full length of the DNA is estimated to be longer than 150 kbp. Based on the morphological characteristics and genomic structures of the virus, we confirm that white spot syndrome associated virus (MJSSV) is a member of genus NOB (Non-Occluded Baculovirus) of the subfamily Nudibaculovirinae of Baculoviridae, name the present isolate PmNOBIII, and propose the use of WSBV (Baculovirus associated with White Spot syndrome) to indicate PmNOBIII related agents.

A new continuous cell line, NTU-SE, was established from the pupal tissues of an economically important pest, the beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae). This cell line contains four major morphologic types: round, polymorphic, spindle-shaped, and comma-shaped cells. The population doubling time of this new line in TNM-FH medium supplemented with 8% fetal bovine serum (FBS) at 28°C is 35.5h. The chromosomal spread from NTU-SE cells is typical to the chromosomal morphology of lepidopteran cell lines. Confidently, NTU-SE cell line is a new cell line that exhibits distinct isozyme patterns of esterase, lactate dehydrogenase (LDH), and malate dehydrogenase (MDH) from those of the other insect cell lines. In addition, the DNA sequence of the nuclear ribosomal internal transcribed spacer (ITS) region of NTU-SE cells is above 96% identical to that sequence of S. exigua larvae, as compared to only 66% identical to that of S. litura larvae. The NTU-SE cell line is highly susceptible to S. exigua multiple nucleopolyhedrovirus (SeMNPV) and Autographa californica MNPV (AcMNPV). Therefore, a highly virulent SeMNPV strain, SeMNPV-1, had been successfully isolated and propagated in NTU-SE cells. We conclude that the NTU-SE cell line will be a useful tool for the selection and mass production of highly virulent SeMNPV strains for the S. exigua biocontrol and the baculovirus based recombinant protein expression systems.

Nosema ceranae, a microsporidian entomopathogen, was first reported from honey bees, Apis mellifera, in 2005 in Taiwan (Huang et al., 2007) and has become a major concern in apiculture worldwide. In Taiwan, we found one infection peak for N. ceranae during the winter months, compared to two peaks in spring and fall reported in 1980 for Nosema apis. N. ceranae infection intensity in apiaries reached a high level earlier than N. apis, a possible factor in replacement. We found a significant negative correlation of N. ceranae pathogen load with temperature; the highest spore counts were recorded at an average temperature of approximately 15 °C and infection intensity equaled the annual average at 23.8 °C. This model corresponds with published results but is most reliable for subtropical to tropical climates.

A new microsporidium was isolated from the endemic, Taiwanese shrimp, Caridina formosae (Decapoda, Atyidae) from northern Taiwan. A conspicuous symptom of infection was presence of opaque white xenomas located in the proximity of the alimentary tract, the surface of the hepatopancreas, and the gills. A fully developed xenoma consisted of a hard, thick capsule filled with sporophorous vesicles containing multiple spores. Microsporidia developed synchronously within the same sporophorous vesicle, although the stage of parasite development differed among the vesicles. Fresh spores were pyriform, mononucleated and measured 6.53 × 4.38 μm. The polar filament was anisofilar with 9–11 coils. Phylogenetic analysis based on the small subunit ribosomal DNA sequence showed that the isolate is most similar to the fish microsporidian clade containing the genera Kabatana, Microgemma, Potaspora, Spraguea, and Teramicra. The highest sequence identity, 80%, was with Spraguea spp. Based on pathogenesis, life cycle and phylogenetic analysis, we erect a new genus and species, Triwangia caridinae for the novel microsporidium.

A birnavirus was isolated from cultured loach (Misgurnus anguillicaudatus) in Taiwan with TO-2 cell line. Evident cytopathic effects of the virus, including karyopycnosis, cytoplasmic inclusion bodies and cell destruction, were observed in TO-2 cells by light microscopy. The naked icosahedral virion layered by a single capsid with a diameter of 69 ± 4 nm was observed by electron microscopy. Sometimes, complete virions were found to be enclosed by lysosomes to form cytolysome. The virus propagated in several fish cell lines and its replication cycle was completed in 24 hours at its optimum multiplication temperature of 25°C. This virus has a two segmented double-stranded RNA genome and 3 structural proteins, showing 1.33 g/cm3 buoyant density in CsCl. The chemico-physical properties of the virus together with the result of cross neutralization test indicated that it is a birnavirus and is closely related to Ab serotype of infectious pancreatic necrosis virus (IPNV).

Background Outbreaks of the casuarina moth, Lymantria xylina Swinehoe (Lepidoptera: Lymantriidae), which is a very important forest pest in Taiwan, have occurred every five to 10 years. This moth has expanded its range of host plants to include more than 65 species of broadleaf trees. LyxyMNPV (L. xylina multiple nucleopolyhedrovirus) is highly virulent to the casuarina moth and has been investigated as a possible biopesticide for controlling this moth. LdMNPV-like virus has also been isolated from Lymantria xylin a larvae but LyxyMNPV was more virulent than LdMNPV-like virus both in NTU-LY and IPLB-LD-652Y cell lines. To better understand LyxyMNPV, the nucleotide sequence of the LyxyMNPV DNA genome was determined and analysed. Results The genome of LyxyMNPV consists of 156,344 bases, has a G+C content of 53.4% and contains 157 putative open reading frames (ORFs). The gene content and gene order of LyxyMNPV were similar to those of LdMNPV, with 151 ORFs identified as homologous to those reported in the LdMNPV genome. Two genes (Lyxy49 and Lyxy123) were homologous to other baculoviruses, and four unique LyxyMNPV ORFs (Lyxy11, Lyxy19, Lyxy130 and Lyxy131) were identified in the LyxyMNPV genome, including a gag-like gene that was not reported in baculoviruses. LdMNPV contains 23 ORFs that are absent in LyxyMNPV. Readily identifiable homologues of the gene host range factor-1 (hrf-1), which appears to be involved in the susceptibility of L. dispar to NPV infection, were not present in LyxyMNPV. Additionally, two putative odv-e27 homologues were identified in LyxyMNPV. The LyxyMNPV genome encoded 14 bro genes compared with 16 in LdMNPV, which occupied more than 8% of the LyxyMNPV genome. Thirteen homologous regions (hr s) were identified containing 48 repeated sequences composed of 30-bp imperfect palindromes. However, they differed in the relative positions, number of repeats and orientation in the genome compared to LdMNPV. Conclusion The gene parity plot analysis, percent identity of the gene homologues and a phylogenetic analysis suggested that LyxyMNPV is a Group II NPV that is most closely related to LdMNPV but with a highly distinct genomic organisation.

A new lepidopteran cell line, NTU-YB, was derived from pupal tissue of Eurema hecabe (Linnaeus) (Pieridae: Lepidoptera). The doubling time of YB cells in TNM-FH medium supplemented with 8% FBS at 28 degrees C was 26.87h. The chromosome numbers of YB cells varied widely from 21 to 196 with a mean of 86. Compared to other insect cell lines, the YB cells produced distinct esterase, malate dehydrogenase, and lactate dehydrogenase isozyme patterns. Identity of the internal transcribed spacer region-I (ITS-I) of YB cells to E. hecabe larvae was 96% and to Eurema blanda larvae (tissue isolated from head) was 81%. The YB cells were permissive to Nosema sp. isolated from E. blanda and the infected YB cells showed obvious cytopathic effects after 3weeks post inoculation. The highest level of spore production was at 4weeks post inoculation when cells were infected with the Nosema isolate, and spore production was 1.34+/-0.9x10(6)spore/ml. Ultrastructrual studies showed that YB cells can host in vitro propagation of the E. blanda Nosema isolate, and developing stages were observed in the host cell nuclei as observed in the natural host, E. blanda. The NTU-YB cell line is also susceptible to Nosema bombycis.

HB-1 virus, a strain of infectious pancreatic necrosis virus (IPNV), was isolated from hard clam, Meretrix lusoria, with TO-2 cell line derived from tilapia ovary. The defective interfering (DI) particles of HB-1 virus could be generated by serial undiluted passaging. The polypeptides and RNA in virus particles of diluted and undiluted virus preparations were compared in order to investigate the nature of defective interfering particles involved in viral interference of HB-1 virus. Cesium chloride density gradient centrifugation was applied to purify virus particles of diluted and undiluted virus preparations. By comparing the polypeptides and RNA in virus particles of diluted and undiluted virus preparations, it was revealed that (I) the virus particles of undiluted virus preparation had smaller polypeptides than normal ; (II) subgenomic RNA of large segment was found in virus particles of undiluted virus preparation. These two findings suggested that DI particles of HB-1 virus was a deletion mutant of standard virus.This is the first report indicating the polypeptides and RNA possibly related to IPN virus DI particles.

A new multiple nucleopolyhedrovirus strain was isolated from casuarina moth, Lymantria xylina Swinhoe, (Lepidoptera: Lymantriidae) in Taiwan. This Lymantria-derived virus can be propagated in IPLB-LD-652Y and NTU-LY cell lines and showed a few polyhedra (occlusion bodies) CPE in the infected cells. The restriction fragment length polymorphism (RFLP) profiles of whole genome indicated that this virus is distinct from LyxyMNPV and the virus genome size was approximately 139 kbps, which was smaller than that of LyxyMNPV. The molecular phylogenetic analyses of three important genes (polyhedrin, lef-8 and lef-9) were performed. Polyhedrin, LEF-8 and LEF-9 putative amino acid analyses of this virus revealed that this virus belongs to Group II NPV and closely related to LdMNPV than to LyxyMNPV. The phylogenetic distance analysis was further clarified the relationship to LdMNPV and this virus provisionally named LdMNPV-like virus. A significant deletion of a 44 bp sequence found in LdMNPV-like virus was noted in the fp25k sequences of LdMNPV and LyxyMNPV and may play an important role in the few polyhedra CPE. In ultrastructural observations, the nuclei of the infected LD host cells contained large occlusion bodies (OBs), and few OBs, which presented as one or two OBs in a nucleus that was otherwise filled with free nuclocapsids and virions. We concluded that this LdMNPV-like virus is a new LdMNPV strain from L. xylina.

A new microsporidium was isolated from Ocinara lida Moore (Lepidoptera: Bombycidae), a pest of Ficus microcarpa L. f. in Taiwan. The microsporidium produces systemic infections in O. lida larvae; the midgut epithelium, Malpighian tubules, and midgut muscle tissues were the target tissues for this isolate, and atrophied fat body tissues were found in heavily infected larvae. Two types of spores were observed, diplokaroytic spores with 11-13 coils of polar tube, and monokaryotic spores with 12 coils of the polar tube that developed within a sporophorous vesicle to form octospores. Electron-dense granules were abundant in the episporontal space of the sporophorous vesicles, and were similar to those of Vairimorpha invictae isolated from Solenopsis invicta, but different from granules or inclusions of other Vairimorpha species. Based on the phylogenetic analysis of the small subunit ribosomal DNA sequence, this isolate is unique within the Vairimorpha complex. Morphological and genetic characters showed this isolate to be a new species. It is placed in the genus Vairimorpha and is described as Vairimorpha ocinarae n. sp.

The viral interference exhibited by HB-1 virus, a strain of infectious pancreatic necrosis virus (IPNV) isolated from hard clam, Meretrix lusoria, was studied. The TO-2 cells infected with serial passage HB-1 virus at 100 to 10-4 dilutions showed that viral interference was related to the degree of dilution of virus inoculum. The specific viral antigens were detected with immunofluorescent antibody stain technique in TO-2 cells which survived from high multiplicity infection of HB-1 virus. The virus yield in culture fluid from a series of 20 serial undiluted or diluted passages indicated that serial undiluted or low-diluted (10-1 to 10-2) passaging of HB-1 virus could induce autointerference in TO-2 cells. The defective interfering (DI) particles in the virus samples were considered to be responsible for the interference. The SDS-PAGE analyses for the polypeptide composition of virions from serial diluted and undiluted passaging showed that there were differences in ƒA group. The truncated ƒA polypep tides were regarded to be specific polypeptides produced by DI particles generated by serial undiluted passaging. We, thus, provided new information concerning IPN DI particles by comparing the properties of the virions produced by serial diluted and undiluted passaging of HB -1 virus , a strain of IPNV.

Four new cell lines, designated as NTU-LY-1 to -4, respectively, were established from the pupal tissues of Lymantria xylina Swinhoe (Lepidoptera: Lymantriidae). These cell lines have been cultured approximately 80 passages during 2 years in TNM–FH medium supplemented with 8% fetal bovine serum, at a constant temperature of 28 °C. Each line consists of three major morphological types: round cells, spindle-shaped cells, and giant cells. The characterization of these cell lines showed that they are different from previously established lines derived from related Lepidopteran species. All new lines were susceptible to the L. xylina multiple nucleopolyhedrovirus (LyxyMNPV) and appeared to have a good potential for studying this virus.

In the present study, the existence of white spot syndrome virus (WSSV) in blue crab (Callinectes sapidus) collected from 3 different American coastal waters (New York, New Jersey, and Texas) was confirmed by 2-step diagnostic polymerase chain reaction and in situ hybridization analysis. When geographic isolates were also compared using a gene that encodes the WSSV ribonucleotide reductase large subunit RR1 (WSSV rr1), a C(1661)-to-T point mutation was found in the New Jersey WSSV isolated. This point mutation, which resulted in the creation of an additional RsaI endonuclease recognition site, was not found in the WSSV from the New York and Texas blue crab samples, or in the WSSV Taiwan isolate, or in any of the other WSSV geographical isolates for which data are available. WSSV rr1-specific RsaI amplified restriction fragment length polymorphism of an amplified 1156-bp fragment thus distinguished the New Jersey blue crab samples from the other WSSV isolates.

The white spot syndrome virus DNA polymerase (DNA pol) gene (WSSV dnapol) has already been tentatively identified based on the presence of highly conserved motifs, but it shows low overall homology with other DNA pols and is also much larger (2351 amino acid residues vs 913-1244 aa). In the present study we perform a transcriptional analysis of the WSSV dnapol gene using the total RNA isolated from WSSV-infected shrimp at different times after infection. Northern blot analysis with a WSSV dnapol-specific riboprobe found a major transcript of 7.5 kb. 5-RACE revealed that the major transcription start point is located 27 nucleotides downstream of the TATA box, at the nucleotide residue A within a CAGT motif, one of the initiator (Inr) motifs of arthropods. In a temporal expression analysis using differential RT-PCR, WSSV dnapol transcripts were detected at low levels at 2–4 h.p.i., increased at 6 h.p.i., and remained fairly constant thereafter. This is similar to the previously reported transcription patterns for genes encoding the key enzyme of nucleotide metabolism, ribonucleotide reductase. Phylogenetic analysis showed that the DNA pols from three different WSSV isolates form an extremely tight cluster. In addition, similar to an earlier phylogenetic analysis of WSSV protein kinase, the phylogenetic tree of viral DNA pols further supports the suggestion that WSSV is a distinct virus (likely at the family level) that does not belong to any of the virus families that are currently recognized. © 2002 Elsevier Science (USA)

Perina nuda picorna-like virus (PnPV) is an insect-infecting RNA virus with morphological and physicochemical characters similar to the Picornaviridae. In this article, we determine the complete genome sequence and analyze the gene organization of PnPV. The genome of PnPV consists of 9476 nucleotides (nts) excluding the poly(A) tail and contains a single large open reading frame (ORF) of 8958 nts (2986 codons) flanked by 473 and 45 nt noncoding regions on the 5' and 3' ends, respectively. Northern blotting did not detect the presence of any subgenomic RNA. The PnPV genome codes for four structural proteins (CP1-4), and determination of their N-terminal sequences by Edman degradation, showed that all four are located in the 5' region of the genome. The 3' part of the PnPV genome contains the consensus sequence motifs for picornavirus RNA helicase, cysteine protease, and RNA-dependent RNA polymerase (RdRp) in that order from the 5' to the 3' end. In all of these characters, the genome organization of PnPV resembles the mammalian picornaviruses and two other insect picorna-like viruses, infectious flacherie virus (IFV) of the silkworm and Sacbrood virus (SBV) of the honeybee. In a phylogenetic tree based on the eight conserved domains in the RdRp sequence, PnPV formed a separate cluster with IFV and SBV, which suggests that these three insect picorna-like viruses might constitute a novel group of insect-infecting RNA viruses.

To identify the protein encoded by a 687-bp open reading frame (ORF) of a sal I genomic DNA fragment of shrimp white spot syndrome virus (WSSV), we expressed the ORF in a baculovirus/insect cell expression system. The apparent molecular mass of the recombinant protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 35 kDa in insect cells. Antibody raised against bacterially synthesized protein of the ORF identified a nucleocapsid protein (VP35) in the extracts of both the purified WSSV virions and the nucleocapsids which comigrated with the 35-kDa baculovirus-expressed recombinant protein on SDS-PAGE. We also show by transient expression in insect cells (Sf9) that VP35 targets the nucleus. Two potential nuclear localization signals (NLSs) were characterized, but only one of them was important for targeting VP35 to the nuclei of transfected insect cells. Replacement of a cluster of four positively charged residues ( 24 KRKR 27 ) at the N terminus of the protein with AAAA resulted in mutant proteins that were distributed only in the cytoplasm, thus confirming that this sequence is a critical part of the functionally active NLS of VP35.

The black tiger prawn Penaeus monodon is a valuable aquaculture product in Taiwan. Two specific diagnostic methods were established for P. monodon-type baculovirus, one using polymerase chain reaction (PCR) technology and the other enzyme-linked immunosorbent assay (ELISA) technology. Monodon-type baculovirus (MBV) was purified by sucrose gradient centrifugation from occlusion bodies of MBV-infected postlarvae of P. monodon. MBV DNA was subsequently purified from the occlusion bodies and its presence was confirmed by PCR using primers of the polyhedrin gene. Based on conserved sequences of the DNA polymerase genes of Autographa californica nuclear polyhedrosis virus (AcMNPV) and Lymantria dispar nuclear polyhedrosis virus (LdMNPV), primers were designed and synthesized to yield a 714 bp PCR fragment from MBV. However, the sequence of this fragment revealed low homology with that of LdMNPV and AcMNPV. From the DNA sequence of this fragment, a second set of primers was designed, and using these primers, a 511 bp DNA fragment was amplified only when MBV DNA was the template. DNA templates from AcMNPV, white spot syndrome diseased shrimp, or PMO cells (a cell line derived from the Oka organ of Penaeus monodon) did not give any amplified DNA fragment. Therefore, this primer pair was specific for the diagnosis of MBV. By using intraspleenic immunization of rabbits with purified MBV occlusion bodies, a polyclonal rabbit antiserum against MBV was obtained. This antiserum could detect nanogram levels of MBV, but did not cross react with white spot syndrome virus (WSSV), homogenates of PMO cells, postlarvae, hepatopancreatic tissue or intestinal tissue of black tiger prawns by competitive ELISA. This sensitive method could detect MBV even in tissue homogenates.

Two viruses, Perina nuda nucleopolyhedrovirus and a new picorna-like virus, were previously isolated from P. nuda larvae with flacherie. In this study the new picorna-like virus was characterized using physical and biochemical methods. This small virus appears to belong to the family Picornaviridae and we propose the name PnPV. PnPV can be propagated in its homogenous cell line, NTU-PN-HH. PnPV purified from the cell line resembles PnPV isolated from insects: under electron microscopy, it exhibits icosahedral symmetry, measures 30 nm in diameter, and has no envelope and no distinct surface structure in negatively stained preparations. In addition, we show here that PnPV has a buoyant density of 1.381 g/ml in cesium chloride, the viral genome was composed of one single-stranded RNA molecule with a length of 10 kb, and poly(A) tract and polyacrylamide gel electrophoresis of purified viral particles revealed three major (31.5, 29.7, and 28.4 kDa) and three minor (27. 0, 24.5, and 4.0 kDa) structural proteins.

The electron microscopic examination of the abnormally dark regions of the gill from hard clam, Meretrix lusoria, revealed that there were virus-like particles present in the cytoplasm of necrotic cells. With TO-2 cell line, the virus was isolated. Electron microscopic examinations showed that there were no detactable differences in morphology and location of virions in the gill cells of clam and TO-2 cells except that virus crystals were infrequently observed in clam gills. The subsequent serological and biochemical studies indicated that all the virus isolates from clam were similar to AB IPNV (infectious pancreatic necrosis virus). These studies represent the first complete characterizations of virus isolated from clam in Taiwan.