Insecticidal properties and microbial contaminants in a Spodoptera exigua multiple nucleopolyhedrovirus (Baculoviridae) formulation stored at different temperatures.

The Spodoptera exigua (Hübner) multiple nucleopolyhedrovirus (SeMNPV) is currently being tested as a biological insecticide for use in greenhouse crops in southern Spain. We performed a study in which semipurified SeMNPV occlusion bodies (OBs) were formulated in phosphate-buffered saline, pH 6.5, with 5% (vol:vol) glycerol and 0.15% (wt:vol) sorbic acid, and they were stored at -20, 4, or 25 degrees C during 18 mo. Initial aerobic counts (+/-SE) averaged 1.4 (+/-0.17) x 10(7) colony-forming units/ml after 17-h incubation at 37 degrees C. Aerobic counts of microorganisms that contaminated OB formulations stored at 250C decreased markedly over the period of the study, whereas only small decreases were observed in counts from OBs stored at 4 or -20 degrees C. The principal microbial contaminants of OB suspensions were Enterococcus spp., Enterobacteriaceae, and yeasts. Potential human pathogens (Salmonella, Shigella, and Vibrio species) were not detected, and populations of Staphylococcus aureus and Bacillus cereus were extremely low. Compared with newly formulated OBs, the estimated LD50 values of OBs stored at 25 degrees C increased by >16,666-fold over the 18 mo of storage, whereas LD50 values were not greatly affected by storage at 4 or -20 degrees C. Significant changes over time in OB concentrations were only observed in the 25 degrees C treatment. Complete degradation of viral DNA was observed at 25 degrees C but not in refrigerated or frozen OBs. We conclude that OB formulation with bacteriostatic or antioxidant additives, together with storage and distribution in refrigerated conditions, will likely result in an SeMNPV biopesticide shelf life that exceeds 18 mo.

Preexisting nuclear architecture defines the intranuclear location of herpesvirus DNA replication structures.

Herpes simplex virus DNA replication proteins localize in characteristic patterns corresponding to viral DNA replication structures in the infected cell nucleus. The intranuclear spatial organization of the HSV DNA replication structures and the factors regulating their nuclear location remain to be defined. We have used the HSV ICP8 DNA-binding protein and bromodeoxyuridine labeling as markers for sites of herpesviral DNA synthesis to examine the spatial organization of these structures within the cell nucleus. Confocal microscopy and three-dimensional computer graphics reconstruction of optical series through infected cells indicated that viral DNA replication structures extend through the interior of the cell nucleus and appear to be spatially separate from the nuclear lamina. Examination of viral DNA replication structures in infected, binucleate cells showed similar or virtually identical patterns of DNA replication structures oriented along a twofold axis of symmetry between many of the sister nuclei. These results demonstrate that HSV DNA replication structures are organized in the interior of the nucleus and that their location is defined by preexisting host cell nuclear architecture, probably the internal nuclear matrix.

Physicochemical properties and cytopathogenicity of an adenovirus-like agent isolated from corn snake (Elaphe guttata).

A virus isolated from the internal organs of a moribund corn snake (Elaphe guttata) replicated in reptilian cell cultures (IgH-2, TH-1 cells) between 10 and 30 degrees C. Highest infectivity titers of 10(5.5) TCID50/ml were obtained in IgH-2 cells at 25 degrees C. Infected IgH-2 cells showed the development of three morphologically different intranuclear inclusion bodies. During viral assembly the particles formed typical crystalline aggregates in the nucleus. About 64 h after infection progressive desintegration of the nuclear membrane was evident and virus particles were released into the cytoplasm. Different fish cell lines (CLC, CHSE-214, BF-2, PG, RTG-2) were not capable of propagating the virus. The DNA containing agent proved to be stable at pH3, more or less at pH 12 and to treatment with chloroform, but it was rapidly inactivated at 56 degrees C. Electron microscopy revealed nonenveloped icosahedral particles with a diameter of 65-70 nm.

Fatal disseminated infection with human herpesvirus-6.

A 13-month-old immunocompetent girl had fever, rash, and multisystem disease, and she eventually died of cardiac failure. Autopsy revealed intracellular viral inclusions of the herpesvirus group, with results of in situ hybridization positive for human herpesvirus-6. This is apparently the first case of fatal disseminated herpesvirus-6 infection.

Recurrent herpes simplex keratitis with concurrent epithelial and stromal involvement. Immunohistochemical and ultrastructural observations.

A 65-year-old man with recurrent herpetic keratitis underwent corneal transplantation for persistent nonimmunologic graft failure. Histopathologic examination of the corneal button revealed an epithelial dendrite containing Cowdry type A inclusion bodies, moderate stromal edema, and a retrocorneal fibrous membrane. Immunohistochemical studies demonstrated herpes simplex virus antigens in epithelial cells bordering the dendritic defect and in stromal keratocytes. The mean width of corneal epithelium displaying herpes simplex virus-positive epithelial cells on either side of the dendritic defect measured 200 +/- 46 microns. By electron microscopy, herpesvirus particles were identified in epithelial cells lining the dendrite as well as in stromal keratocytes. Infected keratocytes were scattered throughout the stroma but were not observed subjacent to the epithelial dendrite. This study demonstrates that a recurrent epithelial dendrite can be associated with subclinical stromal infection of the graft.

Adenoviral pancreatitis in guinea fowl (Numida meleagris).

Necrotizing pancreatitis was observed in 2-week-old Guinea fowl submitted for necropsy and histopathology. Intranuclear inclusion bodies seen histologically in acinar epithelium were examined by electron microscopy and found to contain viruses resembling adenovirus. Adenovirus was isolated in embryonated eggs from the pancreata of affected birds. The adenovirus isolated was not neutralized by chicken antisera developed against 10 known serotypes of group 1 avian adenoviruses.

Nucleotide sequence of the polyhedrin gene of Bombyx mori cytoplasmic polyhedrosis virus A strain with nuclear localization of polyhedra.

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) strain H produces many hexahedral polyhedra (inclusion bodies) in the cytoplasm of insect midgut epithelial cells. The mutant A strain, however, produces polyhedra in the nucleus. We determined cDNA sequences of the polyhedrin genes, the smallest of the 10 genome segments, of these two strains. The polyhedrin genes of the H and A strains were 944 bp long, and encoded polypeptides of 248 amino acids (Mr 28,500) and 252 amino acids (Mr 29,000), respectively. The extra four amino acid residues at the carboxy terminus of the strain A polyhedrin (Arg-Leu-Leu-Val) were the result of a single nucleotide substitution at an opal stop codon (TGA----CGA). A further amino acid substitution of the histidine residue at position 101 (His----Tyr) was seen. The carboxy-terminal extension revealed a considerable similarity to the consensus amino acid sequence of the DNA-binding domain of many DNA-binding proteins. We discuss the relationship between the intracellular localization of polyhedrins and mutations in their amino acid sequences.

[Intracellular changes induced by the canine infectious laryngotracheitis virus (CAV-2)]. / Modifications intracellulaires induites par le virus de la laryngotrachéite infectieuse canine (CAV-2).

Canine infectious laryngotracheitis virus, an adenovirus designated CAV-2, induces numerous changes in the nucleus and cytoplasm of canine kidney cells (MDCK). The following features could be observed on ultrathin sections examined in the electron microscope: a shift of the nucleolus towards the nuclear membrane and a compression of the latter by the virus particles; the release of virions into the cytoplasm following the tearing or evagination of the nuclear membrane; the attachment of virus particles onto the numerous microtubules of the infected cell. Microfilaments appear to be involved into the vectorial movement of virus particles towards the cytoplasmic membrane in the final phase of infection.

Pancreatic islet-cell damage in children with fatal viral infections.

The pancreases from 250 children with fatal infections caused by at least fourteen different viruses were examined for lesions in the islets of Langerhans. Viral cytopathology was found in 4 of 7 cases of Coxsackievirus B infection, 20 of 45 cases of cytomegalovirus infection, 2 of 14 cases of varicella-zoster infection, and 2 of 45 cases of congenital rubella. Destruction of beta cells and acute and chronic inflammatory infiltrates were found in islets from cases with Coxsackievirus B infections. Characteristic inclusion bodies were observed in islets from cases with cytomegalovirus and varicella-zoster infections. This survey provides further evidence that some viruses can infect and damage human beta cells.

Pre-lytic release of foot-and-mouth disease virus in cytoplasmic blebs.

The pre-lytic release mechanism of foot-and-mouth disease virus was investigated by immunofluorescence, acridine orange staining, and electron microscopy in infected bovine and porcine kidney coverslip cultures. Cells with cytoplasmic fluorescence and which were positive for single stranded RNA with acridine orange staining were observed at 2 h after infection. Scanning electron microscopy showed cytoplasmic blebs in all cultures examined 2 h after infection. Rounded cells with virus inclusions began to appear 3 h after infection. Rounded cells and cytoplasmic blebs were shown to have single stranded RNA by acridine orange staining. Immunofluorescence and transmission electron microscopy with immunoferritin tagging demonstrated foot-and-mouth disease virus in cytoplasmic blebs. This study presents evidence for a pre-lytic release of foot-and-mouth disease virus through virus-containing cytoplasmic blebs emerging from infected cells.