scholarly journals Behavior of a Recombinant Baculovirus in Lepidopteran Hosts with Different Susceptibilities

2001 ◽  
Vol 67 (3) ◽  
pp. 1140-1146 ◽  
Author(s):  
Pedro Hernández-Crespo ◽  
Steven M. Sait ◽  
Rosemary S. Hails ◽  
Jenny S. Cory
Keyword(s):  
Trichoplusia Ni ◽  
Recombinant Virus ◽  
Genetically Modified ◽  
Recombinant Baculovirus ◽  
Virus Yield ◽  
Wild Type Virus ◽  
Mamestra Brassicae ◽  
Speed Of Kill ◽  
Wild Type ◽  
Susceptible Host

ABSTRACT Insect pathogens, such as baculoviruses, that are used as microbial insecticides have been genetically modified to increase their speed of action. Nontarget species will often be exposed to these pathogens, and it is important to know the consequences of infection in hosts across the whole spectrum of susceptibility. Two key parameters, speed of kill and pathogen yield, are compared here for two baculoviruses, a wild-type Autographa californica nucleopolyhedrovirus (AcNPV), AcNPV clone C6, and a genetically modified AcNPV which expresses an insect-selective toxin, AcNPV-ST3, for two lepidopteran hosts which differ in susceptibility. The pathogenicity of the two viruses was equal in the less-susceptible host, Mamestra brassicae, but the recombinant was more pathogenic than the wild-type virus in the susceptible species, Trichoplusia ni. Both viruses took longer to kill the larvae of M. brassicae than to kill those of T. ni. However, whereas the larvae of T. ni were killed more quickly by the recombinant virus, the reverse was found to be true for the larvae ofM. brassicae. Both viruses produced a greater yield inM. brassicae, and the yield of the recombinant was significantly lower than that of the wild type in both species. The virus yield increased linearly with the time taken for the insects to die. However, despite the more rapid speed of kill of the wild-type AcNPV in M. brassicae, the yield was significantly lower for the recombinant virus at any given time to death. A lower yield for the recombinant virus could be the result of a reduction in replication rate. This was investigated by comparing determinations of the virus yield per unit of weight of insect cadaver. The response of the two species (to both viruses) was very different: the yield per unit of weight decreased over time for M. brassicae but increased for T. ni. The implications of these data for risk assessment of wild-type and genetically modified baculoviruses are discussed.

scholarly journals Effects of Ac150 on virulence and pathogenesis of Autographa californica multiple nucleopolyhedrovirus in noctuid hosts

2005 ◽  
Vol 86 (6) ◽  
pp. 1619-1627 ◽  
Author(s):  
Ji-Hong Zhang ◽  
Taro Ohkawa ◽  
Jan O. Washburn ◽  
Loy E. Volkman
Keyword(s):  
Trichoplusia Ni ◽  
Autographa Californica ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Lepidopteran Insect ◽  
Charged Amino Acids ◽  
Multiple Nucleopolyhedrovirus ◽  
Budded Virus ◽  
Autographa Californica Multiple Nucleopolyhedrovirus

Ac150 is expressed late during infection of cultured lepidopteran insect cells by Autographa californica multiple nucleopolyhedrovirus. The Ac150 gene product is predicted to have a molecular mass of 11 161 Da and consists of a hydrophobic N terminus and a single ‘peritrophin-A’-like domain, connected by a short region of charged amino acids. An Ac150 deletion mutant and its parental wild-type virus were compared for differences in virulence by both oral and intrahaemocoelic routes of infection. It was found that the mutant was significantly less virulent in larvae of all three host species tested (Heliothis virescens, Spodoptera exigua and Trichoplusia ni) when occlusions were administered orally, but not when isolated occlusion-derived virus (ODV) was administered orally or budded virus was administered intrahaemocoelically. ODV yields were the same from equal numbers of mutant and wild-type occlusions, and nucleocapsid-distribution frequencies within the two ODV populations were the same, eliminating these features as explanations for the observed differences in virulence. Comparison of pathogenesis, as revealed by lacZ expression from identical reporter-gene cassettes in the mutant and wild-type virus, indicated that the mutant was less efficient at establishing primary infection in midgut cells; otherwise, it exhibited infection kinetics identical to those of wild-type virus. Ac150, therefore, can be considered a per os infection factor that mediates, but is not essential for, oral infection.

scholarly journals Cytokine Imbalance after Measles Virus Infection Has No Correlation with Immune Suppression

2009 ◽  
Vol 83 (14) ◽  
pp. 7244-7251 ◽  
Author(s):  
Mary Carsillo ◽  
Kay Klapproth ◽  
Stefan Niewiesk
Keyword(s):  
Virus Infection ◽  
Immune Suppression ◽  
Recombinant Virus ◽  
Measles Virus ◽  
Vaccine Virus ◽  
Gamma Interferon ◽  
Wild Type Virus ◽  
Wild Type ◽  
Spleen Cells ◽  
Th2 Response

ABSTRACT Measles virus infection leads to immune suppression. A potential mechanism is the reduction of interleukin 12 (IL-12) secretion during acute measles, resulting in a TH2 response. Studies in humans have reported conflicting results, detecting either a TH2 or a TH1 response. We have investigated the correlation between a TH2 response and immune suppression in specific-pathogen-free inbred cotton rats which were infected with measles vaccine and wild-type viruses. After infection of bone marrow-derived macrophages with wild-type virus, IL-12 secretion was reduced in contrast to the level for vaccine virus infection. In bronchoalveolar lavage cells, IL-12 secretion was suppressed after infection with both wild-type and vaccine virus on days 2, 4, and 6 and was detectable on days 8 and 10. After stimulation of mediastinal lymph node and spleen cells with UV-inactivated measles virus at various time points after infection, gamma interferon but no IL-4 was found. After stimulation with phorbol myristate acetate-ionomycin, high gamma interferon and low IL-4 levels were detected. To investigate whether the secretion of IL-4 contributes to immune suppression, a recombinant vaccine virus was created which secretes cotton rat IL-4. After infection with this recombinant virus, IL-4 secretion was enhanced. However, neither inhibition of concanavalin A-stimulated spleen cells nor keyhole limpet hemocyanin-specific proliferation of spleen cells was altered after infection with the recombinant virus in comparison to the levels with the parental virus. Our data indicate that measles virus infection leads to a decrease in IL-12 secretion and an increase in IL-4 secretion, but this does not seem to correlate with immune suppression.

scholarly journals The Human Cytomegalovirus UL97 Protein Kinase, an Antiviral Drug Target, Is Required at the Stage of Nuclear Egress

2003 ◽  
Vol 77 (2) ◽  
pp. 905-914 ◽  
Author(s):  
Paula M. Krosky ◽  
Moon-Chang Baek ◽  
Donald M. Coen
Keyword(s):  
Gene Expression ◽  
Protein Kinase ◽  
Virus Infection ◽  
Human Cytomegalovirus ◽  
Antiviral Drug ◽  
Virus Yield ◽  
Wild Type Virus ◽  
Wild Type ◽  
Viral Dna ◽  
Nuclear Egress

ABSTRACT Human cytomegalovirus encodes an unusual protein kinase, UL97, that activates the established antiviral drug ganciclovir and is specifically inhibited by a new antiviral drug, maribavir. We used maribavir and a UL97 null mutant, which is severely deficient in viral replication, to determine what stage of virus infection critically requires UL97. Compared with wild-type virus, there was little or no decrease in immediate-early gene expression, viral DNA synthesis, late gene expression, or packaging of viral DNA into nuclease-resistant structures in mutant-infected or maribavir-treated cells under conditions where the virus yield was severely impaired. Electron microscopy studies revealed similar proportions of various capsid forms, including DNA-containing capsids, in the nuclei of wild-type- and mutant-infected cells. However, capsids were rare in the cytoplasm of mutant-infected or maribavir-treated cells; the magnitudes of these decreases in cytoplasmic capsids were similar to those for virus yield. Thus, genetic and pharmacological evidence indicates that UL97 is required at the stage of infection when nucleocapsids exit from the nucleus (nuclear egress), and this poorly understood stage of virus infection can be targeted by antiviral drugs. Understanding UL97 function and maribavir action should help elucidate this interesting biological process and help identify new antiviral drug targets for an important pathogen in immunocompromised patients.

scholarly journals Development of genetic markers in the non-structural protein 2 region of a US type 1 porcine reproductive and respiratory syndrome virus: implications for future recombinant marker vaccine development

2008 ◽  
Vol 89 (12) ◽  
pp. 3086-3096 ◽  
Author(s):  
Ying Fang ◽  
Jane Christopher-Hennings ◽  
Elizabeth Brown ◽  
Haixia Liu ◽  
Zhenhai Chen ◽  
...  
Keyword(s):  
Recombinant Virus ◽  
Fluorescent Protein ◽  
Vaccine Development ◽  
Infectious Clone ◽  
Wild Type Virus ◽  
Respiratory Syndrome Virus ◽  
Type Virus ◽  
Wild Type ◽  
Marker Virus

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be a major problem in the pork industry worldwide. The limitations of current PRRSV vaccines require the development of a new generation of vaccines. One of the key steps in future vaccine development is to include markers for diagnostic differentiation of vaccinated animals from those naturally infected with wild-type virus. Using a cDNA infectious clone of type 1 PRRSV, this study constructed a recombinant green fluorescent protein (GFP)-tagged PRRSV containing a deletion of an immunogenic epitope, ES4, in the nsp2 region. In a nursery pig disease model, the recombinant virus was attenuated with a lower level of viraemia in comparison with that of the parental virus. To complement the marker identification, GFP and ES4 epitope-based ELISAs were developed. Pigs immunized with the recombinant virus lacked antibodies directed against the corresponding deleted epitope, but generated a high-level antibody response to GFP by 14 days post-infection. These results demonstrated that this recombinant marker virus, in conjunction with the diagnostic tests, enables serological differentiation between marker virus-infected animals and those infected with the wild-type virus. This rationally designed marker virus will provide a basis for further development of PRRSV marker vaccines to assist with the control of PRRS.

scholarly journals Biological Significance of a Human Enterovirus B-Specific RNA Element in the 3′ Nontranslated Region

2002 ◽  
Vol 76 (19) ◽  
pp. 9900-9909 ◽  
Author(s):  
Ingrid Merkle ◽  
Mark J. M. van Ooij ◽  
Frank J. M. van Kuppeveld ◽  
Dirk H. R. F. Glaudemans ◽  
Jochem M. D. Galama ◽  
...  
Keyword(s):  
Recombinant Virus ◽  
Biological Significance ◽  
Secondary Structures ◽  
Human Enterovirus ◽  
Wild Type Virus ◽  
Wild Type ◽  
Nontranslated Region ◽  
Z Domain ◽  
Functional Features ◽  
Hairpin Structures

ABSTRACT The secondary structures predicted for the enteroviral 3′ nontranslated region (3′NTR) all seem to indicate a conformation consisting of two (X and Y) hairpin structures. The higher-order RNA structure of the 3′NTR appears to exist as an intramolecular kissing interaction between the loops of these two hairpin structures. The enterovirus B-like subgroup possesses an additional stem-loop structure, domain Z, which is not present in the poliovirus-like enteroviruses. It has been suggested that the Z domain originated from a burst of short sequence repetitions (E. V. Pilipenko, S. V. Maslova, A. N. Sinyakov, and V. I. Agol, Nucleic Acids Res. 20:1739-1745, 1992). However, no functional features have yet been ascribed to this enterovirus B-like-specific RNA element in the 3′NTR. In this study, we tested the functional characteristics and biological significance of domain Z. A mutant of the cardiovirulent coxsackievirus group B3 strain Nancy which completely lacked the Z domain and which therefore acquired enterovirus C-like secondary structures exhibited a wild-type growth phenotype, as determined by single-cycle growth analysis with BGM cells. This result proves that the Z domain is virtually dispensable for viral growth in tissue cultures. Partial distortion of the Z domain structure resulted in a disabled virus with reduced growth kinetics, probably due to alternative conformations of the overall structure of the domain. Infection of mice showed that the recombinant coxsackievirus group B3 mutant which completely lacked the Z domain was less virulent. Pancreatic tissues from mice infected with wild-type virus and recombinant virus were equally affected. However, the heart tissue from mice infected with the recombinant virus showed only slight signs of myocarditis. These results suggest that the enterovirus B-like-specific Z domain plays a role in coxsackievirus-induced pathogenesis.

scholarly journals Both Lymantria disparNucleopolyhedrovirus Enhancin Genes Contribute to Viral Potency

2001 ◽  
Vol 75 (18) ◽  
pp. 8639-8648 ◽  
Author(s):  
Holly J. R. Popham ◽  
David S. Bischoff ◽  
James M. Slavicek
Keyword(s):  
Trichoplusia Ni ◽  
Amino Acid Identity ◽  
Wild Type Virus ◽  
Northern Analysis ◽  
Gene Transcript ◽  
Type Virus ◽  
Wild Type ◽  
Entire Genome ◽  
Average Decrease ◽  
Gene Transcripts

ABSTRACT Enhancins are a group of proteins first identified in granuloviruses (GV) that have the ability to enhance nuclear polyhedrosis virus potency. We had previously identified anenhancin gene (E1) in the Lymantria disparmultinucleocapsid nucleopolyhedrovirus (LdMNPV) (D. S. Bischoff and J. M. Slavicek, J. Virol. 71:8133–8140, 1997). Inactivation of the E1 gene product within the viral genome lowered viral potency by an average of 2.9-fold. A secondenhancin gene (E2) was identified when the entire genome of LdMNPV was sequenced (Kuzio et al., Virology 253:17–34, 1999). The E2 protein exhibits approximately 30% amino acid identity to the LdMNPV E1 protein as well as the enhancins fromTrichoplusia ni GV, Pseudaletia unipuncta GV,Helicoverpa armigera GV, and Xestia c-nigrumGV. Northern analysis of viral RNA indicated that the E2gene transcripts are expressed at late times postinfection from a consensus baculovirus late promoter. The effect of the enhancin proteins on viral potency was investigated through bioassay using two recombinant viruses, one with a deletion in the E2 gene (E2del) and a second with deletion mutations in bothenhancin genes (E1delE2del). Theenhancin gene viral constructs were verified by Southern analysis and shown not to produce enhancin gene transcripts by Northern analysis. The E2del virus exhibited an average decrease in viral potency of 1.8-fold compared to wild-type virus. In the same bioassays, the recombinant virus E1cat, which does not produce anE1 gene transcript, exhibited an average decrease in viral potency of 2.3-fold compared to control virus. The E1delE2del virus exhibited an average decrease in viral potency of 12-fold compared to wild-type virus. Collectively, these results suggest that both LdMNPV enhancin genes contribute to viral potency, that each enhancin protein can partially compensate for the lack of the other protein, and that both enhancin genes are necessary for wild-type viral potency.

scholarly journals Baculovirus lef-12 Is Not Required for Viral Replication

2002 ◽  
Vol 76 (23) ◽  
pp. 12032-12043 ◽  
Author(s):  
Linda A. Guarino ◽  
Toni-Ann Mistretta ◽  
Wen Dong
Keyword(s):  
Trichoplusia Ni ◽  
Late Phase ◽  
Growth Curves ◽  
Single Step ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Infected Cells ◽  
Step Growth

ABSTRACT The baculovirus lef-12 (orf41) gene is required for transient expression of baculovirus late genes. To analyze the role of LEF-12 in the context of infected cells, two mutant viruses were constructed. Both mutants were viable in Trichoplusia ni High 5 and Spodoptera frugiperda Sf9 cells. Single-step growth curves, however, indicated that virus yields were reduced approximately fivefold in the absence of LEF-12. Pulse-labeling of infected cells revealed that LEF-12 mutant viruses entered the late phase and synthesized late proteins at levels equivalent to or only twofold lower than those of wild-type virus-infected cells. Western blot analyses confirmed that LEF-12 was not synthesized in cells infected with mutant virus. In wild-type virus-infected cells, LEF-12 was not detected until 18 h postinfection, and accumulation of LEF-12 peaked at 24 to 36 h postinfection. Primer extension mapping revealed that lef-12 mRNA was synthesized by 12 h postinfection and peaked between 18 and 24 h postinfection. Furthermore, synthesis of lef-12 mRNA and LEF-12 protein were inhibited by the addition of aphidicolin, indicating that lef-12 is expressed after DNA replication.

scholarly journals Characterization of a Recombinant Herpes Simplex Virus 1 Designed To Enter Cells via the IL13Rα2 Receptor of Malignant Glioma Cells

2005 ◽  
Vol 79 (9) ◽  
pp. 5272-5277 ◽  
Author(s):  
Guoying Zhou ◽  
Bernard Roizman
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Malignant Glioma ◽  
Cell Line ◽  
Recombinant Virus ◽  
Wild Type Virus ◽  
Wild Type ◽  
Herpes Simplex Virus 1 ◽  
Simplex Virus

ABSTRACT Malignant glioma tumor cells in situ exhibit on their surfaces the interleukin 13 (IL-13) receptor designated IL13Rα2. To target herpes simplex virus 1 to this receptor, we constructed a recombinant virus (R5111) in which the known heparan sulfate binding sites in glycoproteins B and C were deleted and IL-13 was inserted into both glycoproteins C and D. We also transduced a baby hamster kidney cell line lacking the known viral receptors (J1-1) and Vero cells with a plasmid encoding IL13Rα2. The J1-1 derivative (J-13R) cell line is susceptible to and replicates the R5111 recombinant virus but not the wild-type parent virus. We report the following. (i) Expression of IL13Rα2 was rapidly lost from the surface of transduced cells grown in culture. The loss appeared to be related to ligands present in fetal bovine serum in the medium. None of the malignant glioma cell lines cultivated in vitro and tested to date exhibited the IL13Rα2 receptor. (ii) Soluble IL-13 but not IL-4 or IL-2 blocked the replication of R5111 recombinant virus in J-13R cells. (iii) The endocytosis inhibitor PD98059 blocked the replication in J1-1 cells of a mutant lacking glycoprotein D (gD−/−) but not the replication of R5111 in the J-13R cells. We conclude that R5111 enters cells via its interaction with the IL13Rα2 receptor in a manner that cannot be differentiated from the interaction of wild-type virus with its receptors.

scholarly journals Establishment of a Reverse Genetics System for Influenza D Virus

2020 ◽  
Vol 94 (10) ◽  
Author(s):  
Hiroho Ishida ◽  
Shin Murakami ◽  
Haruhiko Kamiki ◽  
Hiromichi Matsugo ◽  
Akiko Takenaka-Uema ◽  
...  
Keyword(s):  
Respiratory Disease ◽  
Recombinant Virus ◽  
Reverse Genetics ◽  
Molecular Mechanisms ◽  
Bovine Respiratory Disease ◽  
Wild Type Virus ◽  
Type Virus ◽  
Genomic Rna ◽  
Wild Type ◽  
Bovine Respiratory Disease Complex

ABSTRACT Influenza D virus (IDV) was initially isolated in the United States in 2011. IDV is distributed worldwide and is one of the causative agents of the bovine respiratory disease complex (BRDC), which causes high morbidity and mortality in feedlot cattle. The molecular mechanisms of IDV pathogenicity are still unknown. Reverse genetics systems are vital tools not only for studying the biology of viruses, but also for use in applications such as recombinant vaccine viruses. Here, we report the establishment of a plasmid-based reverse genetics system for IDV. We first verified that the 3′-terminal nucleotide of each 7-segmented genomic RNA contained uracil (U), contrary to previous reports, and we were then able to successfully generate recombinant IDV by cotransfecting 7 plasmids containing these genomic RNAs along with 4 plasmids expressing polymerase proteins and nucleoprotein into human rectal tumor 18G (HRT-18G) cells. The recombinant virus had a growth deficit compared to the wild-type virus, and we determined the reason for this growth difference by examining the genomic RNA content of the viral particles. We found that the recombinant virus incorporated an unbalanced ratio of viral RNA segments into particles compared to that of the wild-type virus, and thus we adjusted the amount of each plasmid used in transfection to obtain a recombinant virus with the same replicative capacity as the wild-type virus. Our work here in establishing a reverse genetics system for IDV will have a broad range of applications, including uses in studies focused on better understanding IDV replication and pathogenicity, as well as in those contributing to the development of BRDC countermeasures. IMPORTANCE The bovine respiratory disease complex (BRDC) causes high mortality and morbidity in cattle, causing economic losses worldwide. Influenza D virus (IDV) is considered to be a causative agent of the BRDC. Here, we developed a reverse genetics system that allows for the generation of IDV from cloned cDNAs and the introduction of mutations into the IDV genome. This reverse genetics system will become a powerful tool for use in studies related to understanding the molecular mechanisms of viral replication and pathogenicity and will also lead to the development of new countermeasures against the BRDC.

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