ac18 is not essential for the propagation of Autographa californica multiple nucleopolyhedrovirus

orf18 (ac18) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved gene in lepidopteran nucleopolyhedroviruses, but its function remains unknown. In this study, an ac18 knockout AcMNPV bacmid was generated to determine the role of ac18 in baculovirus life cycle. After transfection of Sf-9 cells, the ac18-null mutant showed similar infection pattern to the parent virus and the ac18 repair virus with respect to the production of infectious budded virus, occlusion bodies, or the formation of nucleocapsids as visualized by electron microscopy. The deletion mutant did not reduce AcMNPV infectivity for Trichoplusia ni in LD(50) bioassay; however, it did take 24 h longer for deleted mutant to kill T. ni larvae than wild-type virus in LT(50) bioassay. Our results demonstrate that ac18 is not essential for viral propagation both in vitro and in vivo, but it may play a role in efficient virus infection in T. ni larvae.     

Autographa californica multiple nucleopolyhedrovirus odv-e66 is an essential gene required for oral infectivity

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) odv-e66 is a core gene and encodes an occlusion-derived virus (ODV)-specific envelope protein, ODV-E66. The N-terminal 23 amino acid of the envelope protein ODV-E66 are sufficient to direct native and fusion proteins to induced membrane microvesicles and the viral envelope during infection with AcMNPV. In this study, an odv-e66-knockout bacmid can not express N-terminal hydrophobic domains was constructed via homologous recombination in Escherichia coli. The odv-e66 deletion had no effect on budded virus (BV) production and viral DNA replication in infected Sf9 cells. Larval bioassays demonstrated that injection of odv-e66 deletion BV into the hemocoel could kill P. xylostella larvae as efficiently as repaired and control viruses; however, odv-e66 deletion mutant resulted in a 50% lethal dose that was 10(3) higher than that of the repaired and control viruses when inoculated per os. These results indicated that ODV-E66 envelope protein most likely played an important role in the oral infectivity of AcMNPV, but is not essential for virus replication.     

The RAD50 gene of Saccharomyces cerevisiae is not essential for vegetative growth

Summary The gene RAD50 was located by the ability of subclones to restore the Rad⁺ phenotype following transformation of a rad50-1 mutant. Disruption of the gene was achieved by directed integration of a plasmid carrying a fragment internal to RAD50. Haploids with the disrupted gene are viable and do not differ in growth rate or plating efficiency from isogenic rad50-1 or Rad⁺ strains.     

IFN-gamma is not an essential mediator of murine antibody responses in vitro

We have used purified murine gamma-interferon (Mu IFN-gamma) and anti-Mu IFN-gamma monoclonal antibody to study the participation of gamma-interferon (IFN-gamma) in in vitro plaque-forming cell (PFC) responses to antigen. We have studied several supernatants with T-cell replacing factor (TRF) activity as well as helper T-cell dependent anti-sheep red blood cells (SRBC) PFC responses. Our findings demonstrate that neither TRF- nor T-cell mediated responses are critically dependent upon IFN-gamma.     

Identification of an intergenic region that is not essential for replication of goatpox virus

A recombinant goatpox virus was constructed in which an enhanced green fluorescent protein gene was inserted under the control of the 11K late promoter, a guanine phosphoribosyltransferase gene was inserted under the control of the 7.5K early/late promoter, and exogenous genes were inserted into an intergenic region between loci gp_24 and gp_24.5 of the recombinant genome. Analysis of protein expression showed that LT cells infected with only the recombinant virus produced specific fluorescence. A comparative growth assay demonstrated the stability of the recombinant virus at the replication level. These results suggest that the intergenic region is not essential for replication of goatpox virus.     

The pyrH gene of Vibrio vulnificus is an essential in vivo survival factor

We have suggested an important role of the pyrH gene during the infectious process of Vibrio vulnificus. Previously, we have identified 12 genes expressed preferentially during human infections by using in vivo-induced antigen technology. Among the in vivo-expressed genes, pyrH encodes UMP kinase catalyzing UMP phosphorylation. Introduction of a deletion mutation to the pyrH gene was lethal to V. vulnificus, and an insertional mutant showed a high frequency of curing. We constructed a site-directed mutant strain (R62H/D77N) on Arg-62 and Asp-77, both predicted to be involved in UMP binding, and characterized the R62H/D77N strain compared with the previously reported insertional mutant. We further investigated the essential role of the pyrH gene in the establishment of infection using the R62H/D77N strain. Cytotoxicity was decreased in the R62H/D77N strain, and the defect was restored by an in trans complementation. The intraperitoneal 50% lethal dose of the R62H/D77N strain increased by 26- and 238,000-fold in normal and iron-overloaded mice, respectively. The growth of the R62H/D77N strain in 50% HeLa cell lysate, 100% human ascitic fluid, and 50% human serum was significantly retarded compared to that of the isogenic wild-type strain. The R62H/D77N mutant also had a critical defect in the ability to survive and replicate even in iron-overloaded mice. These results demonstrate that pyrH is essential for the in vivo survival and growth of V. vulnificus and should be an attractive new target for the development of antibacterial drugs and replication-controllable live attenuated vaccines.     

Autographa californica multiple nucleopolyhedrovirus ODV-E56 is a per os infectivity factor, but is not essential for binding and fusion of occlusion-derived virus to the host midgut

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) occlusion-derived virus (ODV) envelope protein ODV-E56 is essential for oral infection of larvae of Heliothis virescens. Bioassays with recombinant clones of AcMNPV lacking a functional odv-e56 gene showed that ODV-E56 was required for infectivity of both polyhedra and to a lesser extent, purified ODV. However, binding and fusion assays showed that ODV lacking ODV-E56 bound and fused to midgut cells at levels similar to ODV of wild-type virus. Fluorescence microscopy of midguts from larvae inoculated with ODV-E56-positive and -negative viruses that express GFP indicated that ODV-E56 was required for infection of the midgut epithelium. Purified ODV-E56 bound to several proteins in midgut-derived brush border membrane vesicles, but failed to rescue infectivity of ODV-E56-negative viruses in trans. These results indicate that ODV-E56 is a per os infectivity factor (pif-5) required for primary midgut infection at a point before or after virion binding and fusion.     

Efficacy of spiracular infection of Helicoverpa armigera with its nucleopolyhedrovirus and its role in virus production

Baculoviruses are important microbial control agents of insects, with per os mode of infectivity. However, recently the spiracular infection of this virus group was suggested as an optimum method for virus production in grown up larvae. In this regard, a detailed evaluation of the spiracular infection with intact polyhedral inclusion bodies (PIB), alkali liberated virions and alkali liberated filtered virions of Helicoverpa armigera (Hubner) nucleopolyhedrovirus at 1 x 10(8), 1 x 10(7) and 2 x 10(6)PIB/ml concentrations was undertaken and compared with the standard diet surface treatment method. All the spiracle treatments resulted in larval death due to virus infection with alkali liberated virions causing higher mortality of larvae than alkali liberated filtered virions and intact PIB. Diet surface treatment method resulted in very high mortality as compared to spiracle treatment and among the different inoculum tested the intact PIB resulted in higher larval mortality. The PIB yield/larva in spiracle treatment was comparable with the diet surface treatment method, but due to very low larval mortality it resulted in low virus yield/100 inoculated larvae. Diet surface treatment with 5 x 10(7)PIB/ml concentration of virus resulted in the maximum yield of PIB/100 inoculated larvae. Low mortality, higher labour requirement and low amenability for mechanization for spiracle treatment method make it unviable for mass production of the virus in large scale compared to the standard diet surface treatment method.     

The Orf virus E3L homologue is able to complement deletion of the vaccinia virus E3L gene in vitro but not in vivo

Orf virus (OV), the prototypic parapoxvirus, is resistant to the effects of interferon (IFN) and this function of OV has been mapped to the OV20.0L gene. The protein product of this gene shares 31% amino acid identity to the E3L-encoded protein of vaccinia virus (VV) that is required for the broad host range and IFN-resistant phenotype of VV in cells in culture and for virulence of the virus in vivo. In this study we investigated whether the distantly related OV E3L homologue could complement the deletion of E3L in VV. The recombinant VV (VV/ORF-E3L) expressing the OV E3L homologue in place of VV E3L was indistinguishable from wt VV in its cell-culture phenotype. But VV/ORF-E3L was over a 1000-fold less pathogenic than wt VV (LD(50) > 5 x 10(6) PFU, compared to LD(50) of wtVV = 4 x 10(3) PFU) following intranasal infection of mice. While wt VV spread to the lungs and brain and replicated to high titers in the brain of infected mice, VV/ORF-E3L could not be detected in the lungs or brain following intranasal infection. VV/ORF-E3L was at least 100,000-fold less pathogenic than wt VV on intracranial injection. Domain swap experiments demonstrate that the difference in pathogenesis maps to the C-terminal domain of these proteins. This domain has been shown to be required for the dsRNA binding function of the VV E3L.     

Self-interaction of ORF II protein through the leucine zipper is essential for Soybean chlorotic mottle virus infectivity

The ORF II protein (PII) of Soybean chlorotic mottle virus (SbCMV) is essential for the virus life cycle. We investigated the interactions of SbCMV PII with itself and with other essential virus proteins using a Gal4-based yeast two-hybrid system. PII interacted only with itself and not with any other virus proteins. The PII-PII interaction was confirmed by a Sos-based yeast two-hybrid system and a far-western analysis. Deletion mutagenesis mapped the self-interacting domain to the C-terminal 48 amino acids (amino acids 154-201), which contain two putative leucine zipper motifs. Introduction of amino acid substitutions to leucine/isoleucine in zipper sequences prevented the PII-PII interaction and abolished the infectivity of SbCMV. These results revealed that the self-interaction of PII through a leucine zipper is necessary for virus infection.     

Autographa californica multiple nucleopolyhedrovirus ac142, a core gene that is essential for BV production and ODV envelopment

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac142 is a baculovirus core gene and encodes a protein previously shown to associate with occlusion-derived virus (ODV). To determine its role in the baculovirus life cycle, we used the AcMNPV bacmid system to generate an ac142 deletion virus (AcBAC(ac142KO-PH-GFP)). Fluorescence and light microscopy revealed that AcBAC(ac142KO-PH-GFP) exhibits a single-cell infection phenotype. Titration assays and Western blot confirmed that AcBAC(ac142KO-PH-GFP) is unable to produce budded virus (BV). However, viral DNA replication is unaffected and the development of occlusion bodies in AcBAC(ac142KO-PH-GFP)-transfected cells evidenced progression to very late phases of the viral infection. Western blot analysis showed that AC142 is expressed in the cytoplasm and nucleus throughout infection and that it is a structural component of BV and ODV which localizes to nucleocapsids. Electron microscopy indicates that ac142 is required for nucleocapsid envelopment to form ODV and their subsequent occlusion, a fundamental process to all baculoviruses.     

A caspase-like gene from Heliothis virescens ascovirus (HvAV-3e) is not involved in apoptosis but is essential for virus replication

Ascoviruses (AVs) are double-stranded DNA viruses causing a fatal disease in lepidopteran host larvae. A unique feature of AV infection is cleavage of host cells into membrane bound vesicles containing the virions. A recent study showed that a caspase from Spodoptera frugiperda AV (SfAV) is directly involved in initiation of apoptosis and eventually cell cleavage. Results shown here indicate that Heliothis virescens AV does not induce apoptosis in host cells. HvAV codes for a caspase-like protein but no apoptosis was observed when the gene was expressed in vitro. RNAi studies indicated that the gene is essential for virus replication.