Identification of insertion and deletion genes in Autographa californica nucleopolyhedrovirus variants isolated from Galleria mellonella, Spodoptera exigua, Spodoptera litura and Xestia c-nigrum.

The genomic DNA of four Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) variants isolated from Galleria mellonella, Spodoptera exigua, Spodoptera litura and Xestia c-nigrum was analyzed in comparison with the AcMNPV E2 strain. Restriction endonuclease analysis revealed a deletion and an insertion in collinear regions of the four variants. Polymerase chain reaction analysis indicated that, in the four variants, the deletion occurred in the region corresponding to AcMNPV C6 ORF86 (pnk/pnl). Also the insertion, with a length of approximately 1.1 kb, was commonly identified in the fragments corresponding to the PstI-J fragment (18.5 m.u.-21.2 m.u.) of AcMNPV E2. Sequencing analysis of the variant from S. litura showed that the insertion contains an additional open reading frame encoding 322 amino acids between homologues of AcMNPV ORF30 and ORF31 (the superoxide dismutase gene). This ORF has 82.8% amino acid identity to Bombyx mori NPV T3 ORF 22 (bro-a, one of the baculovirus repeated ORFs) and thus, it was named Splt-bro-a. Southern blot hybridization study indicated that the other three variants also contain Splt-bro-a homologue. In addition, the labeled Splt-bro-a gene weakly hybridized to the PstI-D fragment (99.0 m.u.-8.0 m.u.) of AcMNPV E2. This fragment contains AcMNPV ORF2, a member of bro family. The signal was also observed on the corresponding fragment of the four variants. This result suggested that two bro genes are present in the four variants, although AcMNPV E2 and C6 are known to contain a single bro gene.

Red swamp crawfish (Procambarus clarkii): an alternative experimental host in the study of white spot syndrome virus.

The pathogenicity of white spot syndrome virus (WSSV) for the red swamp crawfish (Procambarus clarkii) was investigated after infection by intramuscular (i.m.) injection and oral route. The cumulative mortality of crawfish injected i.m. with WSSV reached 100% in 5 days. After oral feeding WSSV-infected kuruma shrimp (Penaeus japonicus) muscle tissues to the crawfish the cumulative mortality of this host reached 100% in 11 days. On reinfection trials, all the crawfish fed WSSV-infected crawfish muscle tissues died in 9 days. All the shrimp injected with a filtrate of infected crawfish heart tissues died in 12 days with typical signs of white spot syndrome (WSS). Electron microscopy clearly demonstrated that WSSV propagated in the cells of the crawfish midgut. This study showed that the red swamp crawfish can be used as alternative experimental host in the study of WSSV.

Spherical parasporal inclusions of the lepidoptera-specific and coleoptera-specific Bacillus thuringiensis strains: a comparative electron microscopic study.

Four Lepidoptera-specific Bacillus thuringiensis strains that belong to the four H serogroups (serovars sumiyoshiensis, fukuokaensis, darmstadiensis, and japonensis) and a Coleoptera (Scarabaeidae)-specific strain belonging to serovar japonensis were examined for comparative ultrastructure of spherical parasporal inclusions. The prominent feature of the inclusions of the Lepidoptera-specific strains was the existence of thick, highly electron-dense envelopes surrounding a homogeneous protein matrix. The envelopes were 15.0-66.7 nm thick and consisted of 5-12 layers of membrane. This is also the case with inclusions of a Coleoptera-specific strain. The ultrastructure of inclusions from the five strains was in marked contrast to that of the bipyramidal parasporal inclusions produced by a Lepidoptera-specific serovar sotto strain.

Geographic dimorphism of the wild silkworm, Bombyx mandarina, in the chromosome number and the occurrence of a retroposon-like insertion in the arylphorin gene.

Individuals of the wild silkworm, Bombyx mandarina, collected in South Korea (Taegu City) and Japan (Tsushima Islands and Fukuoka City) had the chromosome number of 2n = 54, while those collected in China (Hangzhou City) had the chromosome number of 2n = 56. Analysis by PCR (polymerase chain reaction) showed that the 66-bp-long retroposon-like insertion known in the arylphorin gene was present in the B. mandarina specimens with 2n = 54, but not in those with 2n = 56. Thus, dimorphism in the chromosome number coincided with the occurrence of the insertion. It is likely that the boundary dividing the two geographic B. mandarina populations lies somewhere in the northern part of the Korean Peninsula.

Replication of Spodoptera exigua nucleopolyhedrovirus in permissive and non-permissive lepidopteran cell lines.

The Spodoptera exigua multinucleocapsid nucleopolyhedrovirus (SeMNPV) was inoculated to eight lepidopteran cell lines derived from Spodoptera exigua (Se301), Spodoptera frugiperda (SF21AEII), Spodoptera littoralis (CLS-79), Spodoptera litura (SpLi-221), Pseudaletia separata (LeSe-11), Trichoplusia ni (hi-5), Plutella xylostella (PXL/C) and Bombyx mori (BmN4). The productive infection of SeMNPV was observed only in Se301 cells. However, a dot-blot hybridization analysis revealed that SeMNPV DNA replicated in five non-permissive cell lines: SF21AEII, CLS-79, SpLi-221, hi-5 and BmN4. In addition, the virus-infected hi-5 and BmN4 cells displayed morphological changes. In contrast, CLS-79 cells inoculated with SeMNPV showed membrane blebbing at 20 hrs post inoculation (p.i.) and fragmentation of genomic DNA. All that indicated that the infected CLS-79 cells underwent apoptosis. These findings indicate that the SeMNPV replication was restricted at various points in dependence upon each cell line.

Coinfection of Spodoptera exigua and Spodoptera frugiperda cell lines with the nuclear polyhedrosis viruses of Autographa californica and Spodoptera exigua.

The Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) has a broad host range among Lepidoptera. In contrast, the Spodoptera exigua MNPV (SeMNPV) can replicate efficiently only in S. exigua larvae or S. exigua-derived cell lines. In this study, we examined the coinfection of S. exigua Se301 and Spodoptera frugiperda IPLB-SF21AEII (Sf21) cell lines with SeMNPV and AcMNPV recombinant (Ac360-501beta-gal) which was constructed for expression of beta-galactosidase under control of the polyhedrin promoter. Coinfection led to the restriction as the level of late gene expression, nonoccluded virus production, and DNA replication of Ac360-501beta-gal in both Se301 and Sf21 cell lines. In contrast, Ac360-501beta-gal supported the SeMNPV replication in Sf21 cells. Occurrence of recombinants, between Ac360-501beta-gal and SeMNPV, with expanded host range was not observed in coinfected Sf21 cells. This suggests that Ac360-501beta-gal supports the SeMNPV replication through trans-activation.

In vivo and in vitro characterization of several isolates of spodoptera exigua nuclear polyhedrosis virus.

Spodoptera exigua nuclear polyhedrosis viruses (SeNPVs), isolated form five geographically distinct regions of Japan and Thailand, were characterized by their DNA restriction endonuclease pattern, level of virus production in a continuous cell line of S. exigua and biological activity to S. exigua larvae. The EcoRI and PstI fragments exhibited similar overall patterns with minor differences. Digestion of virus DNA from a plaque-purified isolate, SeNPV-I1, with PstI yielded 14 fragments and the estimated genome size was approximately 123 kbp. The SeNPV wild isolate from Kagoshima, SeNPV-KW, showed the highest yield of extracellular virus (ECV) in the Se301 cell line of S. exigua among five wild isolates, but there was no significant difference in the level of polyhedral inclusion body (PIB) formation. In comparative studies of biological activity using 2nd-instar S. exigua larvae, SeNPV-KW had the highest virulence with an LD50 value of 3.0 PIBs per larva. When 16 clones, plaque-purified from the Isahaya isolate, SeNPV-IW, were examined for genetic relatedness, seven distinct EcoRI patterns were observed, indicating that SeNPV-IW wild isolate consisted of a mixture of different genotypes.

Replication of a Mamestra brassicae nuclear polyhedrosis virus in a newly established Mamestra brassicae cell line.

A continuous cell line, designated MbL-3, was newly established from minced neonate larvae of the cabbage armyworm, Mamestra brassicae. This new cell line is heteroploid, containing triploid cells predominantly at the frequency of about 50%. Doubling time of the cell population is 33 hrs. The Akutsu isolate of a Mamestra brassicae nuclear polyhedrosis virus (MbNPV) was examined for replication in 16 continuous cell lines, including the cell line MbL-3, from seven lepidoptera species: Mamestra brassicae, Pseudaletia separata, Spodoptera exigua, Spodoptera frugiperda, Spodoptera littoralis, Spodoptera litura and Plutella xylostella. Of these cell lines, only the MbL-3 cells supported a high replication of the virus. Four other Mamestra cell lines and a P. separata cell line showed a low virus-susceptibility, while the other cell lines were not permissive for the virus infection. Maximum infection rate of MbL-3 cells to the Akutsu isolate was 23.4%. The growth kinetics of this virus isolate in MbL-3 cells showed that the virus was released from infected cells 24 hrs p.i. and reached a maximal titer 96 hrs p.i. The number of polyhedral inclusion bodies (PIBs) reached a maximum of 10(7.5) PIBs/ml 120 hrs p.i.

Plaque assay for Spodoptera exigua and Autographa californica nuclear polyhedrosis viruses in a newly established cell line of the beet armyworm, Spodoptera exigua.

The nuclear polyhedrosis viruses of Spodoptera exigua (SeNPV) and Autographa californica (AcNPV) produced plaques in a newly established cell line of the beet armyworm, Spodoptera exigua. Plaques were composed of infected cells containing many polyhedra and were visible without any staining procedure. Dose-response assays showed a direct correlation between the number of plaques and the inoculum size. Growth kinetic studies of the two viruses, using the developed plaque assay system, revealed that the release of extracellular viruses began 6 hrs post infection (p.i.) and the titer reached a plateau 48 hrs p.i. The sensitivity of this plaque assay system was 100 times greater for the heterologous AcNPV than for the homologous SeNPV.