Some properties of the virus causing the mosaic of sweet peas in Czechoslovakia

Identification trials were carried out to determine what virus causes a mosaic disease of sweet peas in Czechoslovakia. The found properties of the identified sweet pea mosaic virus, the character of its transmission and its host range prove that sweet peas in Czechoslovakia are attacked by common pea mosaic virus (CPMV). Some insignificant differences in properties between our virus isolate and CPMV were observed in the course of the determination of properties of our virus isolate on various host plants. The possible existence of more strains of CPMV is discussed. The transmission of sweet pea mosaic to the plants ofPhaseolus vulgaris L. was negative. For this reason bean yellow mosaic virus (BYMV) was eliminated as a possible pathogen of our virus isolate. At the same time the indicator plantsChenopodium giganteum Don. andChenopodium guinoa Willd. with eight developed leaves were established to be most suitable for the determination of the properties of the isolate by the half-leaf test. The transmission of the virus isolate by seeds was not proved.     

Effect of the Deletion of Genes Encoding Proteins of the Extracellular Virion Form of Vaccinia Virus on Vaccine Immunogenicity and Protective Effectiveness in the Mouse Model

Antibodies to both infectious forms of vaccinia virus, the mature virion (MV) and the enveloped virion (EV), as well as cell-mediated immune response appear to be important for protection against smallpox. EV virus particles, although more labile and less numerous than MV, are important for dissemination and spread of virus in infected hosts and thus important in virus pathogenesis. The importance of the EV A33 and B5 proteins for vaccine induced immunity and protection in a murine intranasal challenge model was evaluated by deletion of both the A33R and B5R genes in a vaccine-derived strain of vaccinia virus. Deletion of either A33R or B5R resulted in viruses with a small plaque phenotype and reduced virus yields, as reported previously, whereas deletion of both EV protein-encoding genes resulted in a virus that formed small infection foci that were detectable and quantifiable only by immunostaining and an even more dramatic decrease in total virus yield in cell culture. Deletion of B5R, either as a single gene knockout or in the double EV gene knockout virus, resulted in a loss of EV neutralizing activity, but all EV gene knockout viruses still induced a robust neutralizing activity against the vaccinia MV form of the virus. The effect of elimination of A33 and/or B5 on the protection afforded by vaccination was evaluated by intranasal challenge with a lethal dose of either vaccinia virus WR or IHD-J, a strain of vaccinia virus that produces relatively higher amounts of EV virus. The results from multiple experiments, using a range of vaccination doses and virus challenge doses, and using mortality, morbidity, and virus dissemination as endpoints, indicate that the absence of A33 and B5 have little effect on the ability of a vaccinia vaccine virus to provide protection against a lethal intranasal challenge in a mouse model.     

A baculovirus from the mosquito Wyeomyia smithii

A Baculovirus was found in larvae of the pitcher plant mosquito Wyeomyia smithii. This virus is similar in size and appearance to Baculoviruses from other mosquitoes. A unique feature of the virus is the formation of polymorphic inclusions. The prepatent period for this virus in W. smithii is 3–5 days.     

Molecular Engineering of the Autographa californica Nuclear Polyhedrosis Virus Genome: Deletion Mutations Within the Polyhedrin Gene

We describe a method to introduce site-specific mutations into the genome of Autographa californica nuclear polyhedrosis virus. Specifically, the A. californica nuclear polyhedrosis virus gene for polyhedrin, the major protein that forms viral occlusions in infected cells, was mutagenized by introducing deletions into the cloned DNA fragment containing the gene. The mutagenized polyhedrin gene was transferred to the intact viral DNA by mixing fragment and viral DNAs, cotransfecting Spodoptera frugiperda cells, and screening for viral recombinants that had undergone allelic exchange. Recombinant viruses with mutant polyhedrin genes were obtained by selecting the progeny virus that did not produce viral occlusions in infected cells (occlusion-negative mutants). Analyses of occlusion-negative mutants demonstrated that the polyhedrin gene was not essential for the production of infectious virus and that deletion of certain sequences within the gene did not alter the control, or decrease the level of expression, of polyhedrin. An early viral protein of 25,000 molecular weight was apparently not essential for virus replication in vitro, as the synthesis of this protein was not detected in cells infected with a mutant virus.     

The effect of the transmission of the beet mosaic virus on the variability of its incubation period

The variability of the incubation period of the beet mosaic virus [Beta virus 2(Lind) Smith] was investigated in a three year experiment. The virus was transmitted by the black bean aphid (Aphis fabae Scop.) or by a mechanical inoculation onAmaranthus caudatus L. Variability characterized by the standard deviation indicates a closer distribution of measurements when the virus was transmitted by mechanical inoculation. The differences in variability of the incubation period between the two methods of virus tramission are statistically significant; dispersion and standard deviation are lower after mechanical inoculation. The average length of the incubation period after virus transmission by aphids and by mechanical inoculation was 10.8 and 7.7 days, respectively. The difference was found statistically significant and may be explained by the higher number of viral particles transmitted by mechanical inoculation which may accelerate the process of infection. A significant correlation between the temperature of the environment and the length of the incubation period was also found in some experiments.     

The effect of soil pH on the persistence of cabbage looper nuclear polyhedrosis virus in soil

The persistence of singly embedded Trichoplusia ni nuclear polyhedrosis virus in Norfolk A loamy sand of various pH was studied under laboratory conditions. Virus extracts of the treated soils were bioassayed at three monthly intervals and showed that virus residues are affected by soil pH. Within the range of soil pH tested (4.83-7.17), the lower the pH, the more rapidly the virus was inactivated. The practice of liming fields for maintaining favorable physiological characteristics as well as for nutritional reasons, enhances the persistence of the virus in the soil.     

Can VHS Virus Bypass the Protective Immunity Induced by DNA Vaccination in Rainbow Trout?

DNA vaccines encoding viral glycoproteins have been very successful for induction of protective immunity against diseases caused by rhabdoviruses in cultured fish species. However, the vaccine concept is based on a single viral gene and since RNA viruses are known to possess high variability and adaptation capacity, this work aimed at evaluating whether viral haemorrhagic septicaemia virus (VHSV), an RNA virus and member of Rhabdoviridae family, was able to evade the protective immune response induced by the DNA vaccination of rainbow trout. The experiments comprised repeated passages of a highly pathogenic VHSV isolate in a fish cell line in the presence of neutralizing fish serum (in vitro approach), and in rainbow trout immunized with the VHS DNA vaccine (in vivo approach). For the in vitro approach, the virus collected from the last passage (passaged virus) was as sensitive as the parental virus to serum neutralization, suggesting that the passaging did not promote the selection of virus populations able to bypass the neutralization by serum antibodies. Also, in the in vivo approach, where virus was passaged several times in vaccinated fish, no increased virulence nor increased persistence in vaccinated fish was observed in comparison with the parental virus. However, some of the vaccinated fish did get infected and could transmit the infection to naïve cohabitant fish. The results demonstrated that the DNA vaccine induced a robust protection, but also that the immunity was non-sterile. It is consequently important not to consider vaccinated fish as virus free in veterinary terms.     

Physical Map of the DNA Genome of Autographa californica Nuclear Polyhedrosis Virus

A physical map of the 88 × 10⁶ dalton, circular DNA genome of Autographa californica nuclear polyhedrosis virus was constructed. The complete order of BamHI and XmaI restriction enzyme sites was determined. The EcoRI and HindIII fragments were partially ordered, and their general locations, relative to the BamHI and XmaI maps, were determined. Alterations in the restriction endonuclease fragment patterns of natural genotypic variants of A. californica nuclear polyhedrosis virus, including Trichoplusia ni MEV nuclear polyhedrosis virus, were located on the physical map. Alterations were found throughout the A. californica nuclear polyhedrosis virus DNA genome.     

SiRNA Inhibits Replication of Langat Virus, a Member of the Tick-Borne Encephalitis Virus Complex in Organotypic Rat Brain Slices

Tick-borne encephalitis virus is the causative agent of tick-borne encephalitis, a potentially fatal neurological infection. Tick-borne encephalitis virus belongs to the family of flaviviruses and is transmitted by infected ticks. Despite the availability of vaccines, approximately 2000–3000 cases of tick-borne encephalitis occur annually in Europe for which no curative therapy is available. The antiviral effects of RNA mediated interference by small interfering RNA (siRNA) was evaluated in cell culture and organotypic hippocampal cultures. Langat virus, a flavivirus highly related to Tick-borne encephalitis virus exhibits low pathogenicity for humans but retains neurovirulence for rodents. Langat virus was used for the establishment of an in vitro model of tick-borne encephalitis. We analyzed the efficacy of 19 siRNA sequences targeting different regions of the Langat genome to inhibit virus replication in the two in vitro systems. The most efficient suppression of virus replication was achieved by siRNA sequences targeting structural genes and the 3′ untranslated region. When siRNA was administered to HeLa cells before the infection with Langat virus, a 96.5% reduction of viral RNA and more than 98% reduction of infectious virus particles was observed on day 6 post infection, while treatment after infection decreased the viral replication by more than 98%. In organotypic hippocampal cultures the replication of Langat virus was reduced by 99.7% by siRNA sequence D3. Organotypic hippocampal cultures represent a suitable in vitro model to investigate neuronal infection mechanisms and treatment strategies in a preserved three-dimensional tissue architecture. Our results demonstrate that siRNA is an efficient approach to limit Langat virus replication in vitro.     

A self-excisable infectious bacterial artificial chromosome clone of varicella-zoster virus allows analysis of the essential tegument protein encoded by ORF9

In order to facilitate the generation of mutant viruses of varicella-zoster virus (VZV), the agent causing varicella (chicken pox) and herpes zoster (shingles), we generated a full-length infectious bacterial artificial chromosome (BAC) clone of the P-Oka strain. First, mini-F sequences were inserted into a preexisting VZV cosmid, and the SuperCos replicon was removed. Subsequently, mini-F-containing recombinant virus was generated from overlapping cosmid clones, and full-length VZV DNA recovered from the recombinant virus was established in Escherichia coli as an infectious BAC. An inverted duplication of VZV genomic sequences within the mini-F replicon resulted in markerless excision of vector sequences upon virus reconstitution in eukaryotic cells. Using the novel tool, the role in VZV replication of the major tegument protein encoded by ORF9 was investigated. A markerless point mutation introduced in the start codon by two-step en passant Red mutagenesis abrogated ORF9 expression and resulted in a dramatic growth defect that was not observed in a revertant virus. The essential nature of ORF9 for VZV replication was ultimately confirmed by restoration of the growth of the ORF9-deficient mutant virus using trans-complementation via baculovirus-mediated gene transfer.     

Use of primary fat body cultures for the study of baculovirus replication

Fat body cultures of Trichoplusia ni and Estigmene acrea were established for use in the study of the two baculoviruses Autographa californica nuclear polyhedrosis virus (AcNPV) and Estigmene acrea granulosis virus (EaGV), respectively. Multiplication of AcNPV observed by phase and electron microscopy was correlated with an increase in viral specific proteins as determined by indirect enzyme-linked immunosorbent assay (ELISA). Although EaGV morphogenesis was not observed in fat body cultures, an increase in specific proteins of this virus could be detected with the ELISA.     

Generation of Influenza Virus from Avian Cells Infected by Salmonella Carrying the Viral Genome

Domestic poultry serve as intermediates for transmission of influenza A virus from the wild aquatic bird reservoir to humans, resulting in influenza outbreaks in poultry and potential epidemics/pandemics among human beings. To combat emerging avian influenza virus, an inexpensive, heat-stable, and orally administered influenza vaccine would be useful to vaccinate large commercial poultry flocks and even migratory birds. Our hypothesized vaccine is a recombinant attenuated bacterial strain able to mediate production of attenuated influenza virus in vivo to induce protective immunity against influenza. Here we report the feasibility and technical limitations toward such an ideal vaccine based on our exploratory study. Five 8-unit plasmids carrying a chloramphenicol resistance gene or free of an antibiotic resistance marker were constructed. Influenza virus was successfully generated in avian cells transfected by each of the plasmids. The Salmonella carrier was engineered to allow stable maintenance and conditional release of the 8-unit plasmid into the avian cells for recovery of influenza virus. Influenza A virus up to 10⁷ 50% tissue culture infective doses (TCID₅₀)/ml were recovered from 11 out of 26 co-cultures of chicken embryonic fibroblasts (CEF) and Madin-Darby canine kidney (MDCK) cells upon infection by the recombinant Salmonella carrying the 8-unit plasmid. Our data prove that a bacterial carrier can mediate generation of influenza virus by delivering its DNA cargoes into permissive host cells. Although we have made progress in developing this Salmonella influenza virus vaccine delivery system, further improvements are necessary to achieve efficient virus production, especially in vivo.     

Serp2, an Inhibitor of the Interleukin-1β-Converting Enzyme, Is Critical in the Pathobiology of Myxoma Virus

Recently, myxoma virus was shown to encode an additional member of the serpin superfamily. The viral gene, called serp2, was cloned, and the Serp2 protein was shown to specifically bind to interleukin-1β (IL-1β)-converting enzyme (ICE), thus inhibiting the cleavage of pro-IL-1β by the protease (F. Petit, S. Bertagnoli, J. Gelfi, F. Fassy, C. Boucraut-Baralon, and A. Milon, J. Virol. 70:5860–5866, 1996). Here, we address the role of Serp2 in the development of myxomatosis, a lethal infectious disease of the European rabbit. A Serp2 mutant myxoma virus was constructed by disruption of the single-copy serp2 gene and insertion of the Escherichia coli gpt gene serving as the selectable marker. A revertant virus was obtained by replacing the E. coli gpt gene by the intact serp2 open reading frame. The Serp2⁻ mutant virus replicated with wild-type kinetics both in rabbit fibroblasts and a rabbit CD4⁺ T-cell line (RL5). Moderate reduction of cell surface levels of major histocompatibility complex I was observed after infection with wild-type or Serp2⁻ mutant myxoma virus, and both produced white pocks on the chorioallantoic membrane of the chick embryo. After the infection of European rabbits, the Serp2⁻ mutant virus proved to be highly attenuated compared to wild-type myxoma virus, as demonstrated by the clinical course of myxomatosis and the survival rates of infected animals. Pathohistological examinations revealed that infection with wild-type myxoma virus resulted in a blockade of the inflammatory response at the vascular level. In contrast, rapid inflammatory reactions occurred upon infection with the Serp2⁻ mutant virus. Furthermore, lymphocytes in lymph nodes derived from animals inoculated with Serp2 mutant virus were shown to rapidly undergo apoptosis. We postulate that the virulence of myxoma virus in the European rabbit can be partially attributed to an impairment of host inflammatory processes and to the prevention of apoptosis in lymphocytes. The weakening of host defense is directly linked to serp2 gene function and is likely to involve the inhibition of IL-1β-converting-enzyme-dependent pathways.     

Pathology and Properties of the Tetravirus Helicoverpa armigera Stunt Virus

A quantitative study of the pathogenicity of Helicoverpa armigera stunt virus (HaSV) (Tetraviridae) isolates toward larvae of several heliothine species was conducted along with studies on the stability of the virus to a variety of chemical, enzymic, and temperature treatments. Surface contamination bioassays of several HaSV isolates against H. armigera produced 50% effective concentration (EC₅₀) estimates ranging between 568 and 9244 virus particles (vp)/mm². Against mid 1st instar larvae of H. armigera, H. punctigera, and Heliothis punctifera, EC₅₀ estimates for one isolate were 1288, 16,137, and 2667 vp/mm², respectively. The virulence of HaSV infection varied markedly with the age at which larvae were exposed to the virus. Presentation of the virus to the first three instars of H. armigera was accompanied by cessation of feeding, growth retardation, and eventual lethality, whereas no adverse effects were observed when later instars were exposed to the virus, even at very high concentrations. Active HaSV was recovered from frass of larvae exposed to the virus as 1st instars. Household bleach (1% v/v; 0.04% w/v available chlorine, 0.004% w/v NaOH), formaldehyde (1% w/v), and temperatures ≥65°C completely inactivated HaSV in suspension. Treatments with ether, proteinase K (1 mg/ml), H. armigera gut contents, and temperatures between 22 and 55°C partially inactivated virus activity. No observable inactivation was observed after treatment with chloroform, chymotrypsin (1 mg/ml), trypsin (1 mg/ml), or RNase A (1 mg/ml). The virus was stable between pH 2.8 and pH 10.0 with around 60% loss of activity observed at pH 11.4. The pattern of pathogenic effects seen in several other insect species challenged by high concentrations of HaSV indicated that the host range of the virus is limited to species within the lepidopteran family Noctuidae. The apparently restricted host range of HaSV along with a number of other features indicate that this virus has considerable potential for the development of novel control agents for use against heliothine pests.     

Attenuation of Mouse Hepatitis Virus by Deletion of the LLRKxGxKG Region of Nsp1

Coronaviruses are a family of large positive-sense RNA viruses that are responsible for a wide range of important veterinary and human diseases. Nsp1 has been shown to have an important role in the pathogenetic mechanisms of coronaviruses in vivo. To assess the function of a relatively conserved domain (LLRKxGxKG) of MHV nsp1, a mutant virus, MHV-nsp1-27D, with a 27 nts (LLRKxGxKG) deletion in nsp1, was constructed using a reverse genetic system with a vaccinia virus vector. The mutant virus had similar growth kinetics to MHV-A59 wild-type virus in 17CI-1 cells, but was highly attenuated in vivo. Moreover, the mutant virus completely protected C57BL/6 mice from a lethal MHV-A59 challenge. To further analyze the mechanism of the attenuation of the mutant virus, changes in reporter gene expression were measured in nsp1- or nsp1-27D-expressing cells; the results showed that nsp1 inhibited reporter gene expression controlled by different promoters, but that this inhibition was reduced for nsp1-27D. The research in vivo and in vitro suggests that the LLRKxGxKG region of nsp1 may play an important role in this process.     

Genomic and Proteomic Analysis of Schizaphis graminum Reveals Cyclophilin Proteins Are Involved in the Transmission of Cereal Yellow Dwarf Virus

Yellow dwarf viruses cause the most economically important virus diseases of cereal crops worldwide and are transmitted by aphid vectors. The identification of aphid genes and proteins mediating virus transmission is critical to develop agriculturally sustainable virus management practices and to understand viral strategies for circulative movement in all insect vectors. Two cyclophilin B proteins, S28 and S29, were identified previously in populations of Schizaphisgraminum that differed in their ability to transmit the RPV strain of Cereal yellow dwarf virus (CYDV-RPV). The presence of S29 was correlated with F2 genotypes that were efficient virus transmitters. The present study revealed the two proteins were isoforms, and a single amino acid change distinguished S28 and S29. The distribution of the two alleles was determined in 12 F2 genotypes segregating for CYDV-RPV transmission capacity and in 11 genetically independent, field-collected S . graminum biotypes. Transmission efficiency for CYDV-RPV was determined in all genotypes and biotypes. The S29 isoform was present in all genotypes or biotypes that efficiently transmit CYDV-RPV and more specifically in genotypes that efficiently transport virus across the hindgut. We confirmed a direct interaction between CYDV-RPV and both S28 and S29 using purified virus and bacterially expressed, his-tagged S28 and S29 proteins. Importantly, S29 failed to interact with a closely related virus that is transported across the aphid midgut. We tested for in vivo interactions using an aphid-virus co-immunoprecipitation strategy coupled with a bottom-up LC-MS/MS analysis using a Q Exactive mass spectrometer. This analysis enabled us to identify a third cyclophilin protein, cyclophilin A, interacting directly or in complex with purified CYDV-RPV. Taken together, these data provide evidence that both cyclophilin A and B interact with CYDV-RPV, and these interactions may be important but not sufficient to mediate virus transport from the hindgut lumen into the hemocoel.     

The epizootiology of two pathogens of the coconut palm rhinoceros beetle

Two diseases of Oryctes rhinoceros caused by Metarrhizium anisopliae and a virus were introduced from Western Samoa into Tongatapu, Kingdom of Tonga, in 1969 and 1970, respectively. The fungus remained at a low incidence and did not significantly affect the beetle population. The virus disease developed to epizootic levels within 5 months and spread across the island in 15 months. The beetle population was monitored through palm damage surveys, which confirmed that the population was considerably reduced by the disease.The greater impact of the virus was attributed to its much higher power of dispersal through infected beetles.The experiment supports the view that the reduction in beetle numbers recorded previously in Western Samoa was caused by a virus epizootic.     

Virus Community Dynamics in the Conifer Pathogenic Fungus Heterobasidion parviporum Following an Artificial Introduction of a Partitivirus

Viruses infecting the conifer pathogenic fungus Heterobasidion annosum sensu lato are intracellular and spread via anastomosis contacts. In the laboratory, these viruses transmit readily even between somatically incompatible isolates, but their dispersal capacity in natural conditions has not been previously studied. We introduced a mycovirus to a heavily diseased forest site by inoculating Norway spruce stumps with heartrot decay using a mycelial suspension of Heterobasidion parviporum strain RT3.49C hosting the partitivirus strain HetRV4-pa1. The Heterobasidion population at the sample plot was screened for mycoviruses prior to and after the inoculation. Based on sequence analysis, the resident H. parviporum strains harbored six different strains of the virus species Heterobasidion RNA virus 6 (HetRV6) and one strain of HetRV4 prior to the inoculation. After three growth seasons, the inoculated H. parviporum host strain was not detected, but the introduced virus had infected two resident H. parviporum genets. The presence of a preexisting HetRV6 infection did not hinder spread of the introduced partitivirus but resulted in coinfections instead. The resident HetRV6 virus population seemed to be highly stable during the incubation period, while the single indigenous HetRV4 infection was not detected after the inoculation. In laboratory infection experiments, the introduced virus could be transmitted successfully into all of the resident H. parviporum genets. This study shows for the first time transmission of a Heterobasidion virus between somatically incompatible hosts in natural conditions.