Growth Characteristics and Intraspecies Host Specificity of a Large Virus Infecting the Dinoflagellate Heterocapsa circularisquama

The growth characteristics and intraspecies host specificity of Heterocapsa circularisquama virus (HcV), a large icosahedral virus specifically infecting the bivalve-killing dinoflagellate H. circularisquama, were examined. Exponentially growing host cells were more sensitive to HcV than those in the stationary phase, and host cells were more susceptible to HcV infection in the culture when a higher percent of the culture was replaced with fresh medium each day, suggesting an intimate relationship between virus sensitivity and the physiological condition of the host cells. HcV was infective over a wide range of temperatures, 15 to 30°C, and the latent period and burst size were estimated at 40 to 56 h and 1,800 to 2,440 infective particles, respectively. Transmission electron microscopy revealed that capsid formation began within 16 h postinfection, and mature virus particles appeared within 24 h postinfection at 20°C. Compared to Heterosigma akashiwo virus, HcV was more widely infectious to H. circularisquama strains that had been independently isolated in the western part of Japan, and only 5.3% of the host-virus combinations (53 host and 10 viral strains) showed resistance to viral infection. The present results are helpful in understanding the ecology of algal host-virus systems in nature.     

Bunyamwera Virus Replication in Cultured Aedes albopictus (Mosquito) Cells: Establishment of a Persistent Viral Infection

Bunyamwera virus replication was examined in Aedes albopictus (mosquito) cell cultures in which a persistent infection is established and in cytopathically infected BHK cells. During primary infection of A. albopictus cells, Bunyamwera virus reached relatively high titers (10⁷ PFU/ml), and autointerference was not observed. Three virus-specific RNAs (L, M, and S) and two virion proteins (N and G1) were detected in infected cells. Maximum rates of viral RNA synthesis and viral protein synthesis were extremely low, corresponding to <2% of the synthetic capacities of uninfected control cells. Viral protein synthesis was maximal at 12 h postinfection and was shut down to barely detectable levels at 24 h postinfection. Virus-specific RNA and nucleocapsid syntheses showed similar patterns of change, but later in infection. The proportions of cells able to release a single PFU at 3, 6, and 54 days postinfection were 100, 50, and 1.5%, respectively. Titers fell to 10³ to 10⁵ PFU/ml in carrier cultures. Persistently infected cultures were resistant to superinfection with homologous virus but not with heterologous virus. No changes in host cell protein synthesis or other cytopathic effects were observed at any stage of infection. Small-plaque variants of Bunyamwera virus appeared at approximately 7 days postinfection and increased gradually until they were 75 to 95% of the total infectious virus at 66 days postinfection. Temperature-sensitive mutants appeared between 23 and 49 days postinfection. No antiviral activity similar to that reported in A. albopictus cell cultures persistently infected with Sindbis virus (R. Riedel and D. T. Brown, J. Virol. 29: 51-60, 1979) was detected in culture fluids by 3 months after infection. Bunyamwera virus replicated more rapidly in BHK cells than in mosquito cells but reached lower titers. Autointerference occurred at multiplicities of infection of 10. Virus-specific RNA and protein syntheses were at least 20% of the levels in uninfected control cells. Host cell protein synthesis was completely shut down, and nucleocapsid protein accumulated until it was 4% of the total cell protein. We discuss these results in relation to possible mechanisms involved in determining the outcome of arbovirus infection of vertebrate and mosquito cells.     

Hybridization Selection and In Vitro Translation of Autographa californica Nuclear Polyhedrosis Virus mRNA

We isolated polyadenylated RNA from the cytoplasm of cells infected with Autographa californica nuclear polyhedrosis virus late after infection (21 h postinfection). At that time intracellular protein synthesis was directed almost exclusively toward infected cell-specific proteins. The polyadenylic acid-containing RNA sequences in the cytoplasm at 21 h postinfection were radiolabeled in vitro and hybridized to A. californica nuclear polyhedrosis virus DNA restriction fragments. The polyadenylic acid-containing RNA was derived from regions representing the entire viral genome. Translation in a reticulocyte cell-free protein-synthesizing system of cytoplasmic RNA selected by hybridization to viral DNA and polyadenylic acid-containing RNA produced almost identical polypeptide patterns, suggesting that late after infection almost all of the cytoplasmic polyadenylic acid-containing RNA present in infected cells was of viral origin. Polyhedrin protein (molecular weight, 33,000) and a number of virion structural proteins were among the translation products which were identified by immunoprecipitation and by comparing molecular weights. In addition, some tentative nonstructural infected cell-specific proteins were also detected. Using the hybridization selection technique, we determined that sequences complementary to the message coding for polyhedrin were located on EcoRI fragment I of A. californica nuclear polyhedrosis virus DNA, whereas sequences coding for a putative nonstructural protein (molecular weight, 39,000) were on EcoRI fragment J.     

Characterization of EV-2, a Virus Isolated from European Eels (Anguilla anguilla) with Stomatopapilloma

A virus designated EV-2 has been isolated from external tumor tissue and internal organs of European eels (Anguilla anguilla) with stomatopapilloma. It contains RNA and is ether, acid, and temperature labile above 4°C, and concentrated preparations agglutinate chicken and sheep erythrocytes. The addition of actinomycin D during the first 2.75 h of infection inhibits viral replication. As determined in sucrose gradients, the buoyant density of the virus is 1.19 g/cm³. EV-2 has a moderately pleomorphic spherical morphology; its diameter ranges from 80 to 140 nm. The virion has narrow, regularly spaced surface projections about 10 nm long. Replication in FHM cells at 15°C shows new infectivity appearing at 10 h postinfection and reaching a plateau at 20 h. Cytopathic effects consist of cell fusion, syncytia, and irregularly rounded cell masses. Viral antigen was detected in the cytoplasm of infected cells by specific immunofluorescence.     

Protein Synthesis in a Lymantria dispar Cell Line Infected by Cytoplasmic Polyhedrosis Virus

The efficiency of replication of a cytoplasmic polyhedrosis virus isolated from a member of the order Lepidoptera, Euxoa scandens, was studied in eight different lepidopterean cell lines. Lymantria dispar cells, which were found to support viral replication, more efficiently, were used to follow the kinetics of appearance of viral-specific polypeptides by a 2-h pulse with [³⁵S]methionine. Five polypeptides (ca. 120,000 molecular weight [120K], 105K, 66K, 46K, and 28K) were identified as components of the polyhedral inclusion bodies, and two polypeptides (112K and 39K) were assigned as viral-particle polypeptides. All these polypeptides were present after 24 h and were still being produced 96 h after infection. The rate of synthesis of the major polyhedral polypeptide (28K) increased in the time course of infection, whereas the background of cellular polypeptides seemed to be unaffected. An indirect immunoperoxidase technique, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis was blotted to a nitrocellulose membrane, showed that traces of the major polyhedral polypeptide were found from 8 h postinfection.     

Analysis of the Spodoptera frugiperda Nuclear Polyhedrosis Virus Genome by Restriction Endonucleases and Electron Microscopy

Restriction endonuclease analysis was used to differentiate between four strains of Spodoptera frugiperda nuclear polyhedrosis virus from different geographical areas. In addition, partial denaturation was performed, and a partial denaturation map was constructed for the Ohio strain of this virus.     

Establishment of a persistent baculovirus infection in a lepidopteran cell line

The paper describes the first overt attempt to establish an insect cell line (Spodoptera frugiperda), persistently infected with its homologous baculovirus. The persistently infected cells were morphologically different and grew to a higher density than the noninfected parent line. The parent line, however, had a shorter doubling time. Persistently infected cells were passaged 40 times over 10 months; they still continued to produce infectious virus and polyhedral inclusion bodies. However, the infectious viral titer was ca. 100 times lower in the persistently infected line than in the parent line; also, the number of inclusion bodies was reduced ca. 98%. Interference with both homologous and heterologous baculoviruses was demonstrated in the persistently infected cell line. Sevently percent of the persistently infected cells contained antigens for S. frugiperda nuclear polyhedrosis virus, ca. 1% of the cells showed infectious viral centers, and ca. 3% of the cells contained inclusion bodies. Although the inclusion bodies from the persistently infected cells were infectious for S. frugiperda larvae, they were about 3 times less infectious than the inclusion bodies produced in the parent line.     

鸭胚成纤维细胞中鸭瘟强毒的增殖特性研究

通过细胞培养物光学显微观察、细胞超薄切片研究、定量PCR检测技术对鸭胚成纤维细胞中鸭瘟强毒的增殖特性进行了研究.结果表明:鸭瘟强毒接种鸭胚成纤维细胞后42 h,可使细胞培养物出现大量明显的蚀斑病变.细胞培养物的超薄切片电镜观察研究表明,病毒核酸在胞核内常集中分布,呈直径35～45 nm的圆形颗粒状;核衣壳在胞核和胞浆内都有分布,呈直径90～100 nm的网形颗粒状;成熟病毒位于胞浆空泡内,呈直径150～300 nm的圆形,有囊膜和皮层结构;病毒通过囊膜与胞膜融合入侵细胞,在核内生成核酸、装配核衣壳,在胞浆中得到皮层,出芽到胞浆空泡内获得囊膜,通过胞吐作用释放到胞外.定量PCR研究表明:鸭瘟强毒接种细胞后10 h开始明显复制,接毒后30 h时含量趋于稳定,接毒后22 h时开始向胞外释放,50 h时达最大值,细胞和上清中病毒含量的增幅均为103左右,病毒主要存在于细胞中,其含量为上清的102～103倍.     

Baculoviruses Modulate a Proapoptotic DNA Damage Response To Promote Virus Multiplication

The baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) initiates apoptosis in diverse insects through events triggered by virus DNA (vDNA) replication. To define the proapoptotic pathway and its role in antivirus defense, we investigated the link between the host''s DNA damage response (DDR) and apoptosis. We report here that AcMNPV elicits a DDR in the model insect Drosophila melanogaster. Replication of vDNA activated DDR kinases, as evidenced by ATM-driven phosphorylation of the Drosophila histone H2AX homolog (H2Av), a critical regulator of the DDR. Ablation or inhibition of ATM repressed H2Av phosphorylation and blocked virus-induced apoptosis. The DDR kinase inhibitors caffeine and KU55933 also prevented virus-induced apoptosis in cells derived from the permissive AcMNPV host, Spodoptera frugiperda. This block occurred at a step upstream of virus-mediated depletion of the cellular inhibitor-of-apoptosis protein, an event that initiates apoptosis in Spodoptera and Drosophila. Thus, the DDR is a conserved, proapoptotic response to baculovirus infection. DDR inhibition also repressed vDNA replication and reduced virus yields 100,000-fold, demonstrating that the DDR contributes to virus production, despite its recognized antivirus role. In contrast to virus-induced phosphorylation of Drosophila H2Av, AcMNPV blocked phosphorylation of the Spodoptera H2AX homolog (SfH2AX). Remarkably, AcMNPV also suppressed SfH2AX phosphorylation following pharmacologically induced DNA damage. These findings indicate that AcMNPV alters canonical DDR signaling in permissive cells. We conclude that AcMNPV triggers a proapoptotic DDR that is subsequently modified, presumably to stimulate vDNA replication. Thus, manipulation of the DDR to facilitate multiplication is an evolutionarily conserved strategy among DNA viruses of insects and mammals.     

Characterization of a Variant ofAutographa californicaNuclear Polyhedrosis Virus with a Nonfunctional ORF 603

A new genotypic variant ofAutographa californicanuclear polyhedrosis virus (AcMNPV), the V8 variant, was originally identified by an additionalHindIII site in theHindIII–F fragment. Insect bioassays of this variant displayed a decreased time of mortality compared with the L1 variant of AcMNPV inSpodoptera frugiperdalarvae but not inTrichoplusia nilarvae. A 1.8-kb region containing the 3′ end of ORF 5,lef-2,ORF 603, and the 5′ end of the polyhedrin gene (polh) of both L1 and V8 was sequenced. V8 exhibited extensive sequence variation in the region between the 3′ end oflef-2and the 5′ end ofpolh; V8 had six amino acid substitutions in thelef-2gene product and a nonfunctional ORF 603. A site-specific frameshift mutation in ORF 603 of the L1 variant was constructed to determine the effect of ORF 603 inS. frugiperdalarvae. Truncation of ORF 603 was found to decrease the time of mortality inS. frugiperdalarvae. The insect-selective toxin gene,tox34, was inserted into the V8 variant by direct cloning. The efficacy of this recombinant as a biopesticide was equivalent to similar L1 recombinants.     

Sedimentation Changes of L Cells in a Density Gradient Early After Infection with Vaccinia Virus

By 2 h postinfection, LM cells infected with vaccinia virus show a shift in their distribution when separated on a Ficoll density gradient. This shift is dependent on both time and the multiplicity of infection and is due, at least in part, to an increase in cell size. Those cells which do shift in position in the gradient represent infected members of the population. Physical changes induced in virus-infected cells can be utilized for studying early events in virus replication.     

Chandipura Virus Induces Neuronal Death through Fas-Mediated Extrinsic Apoptotic Pathway

Chandipura virus (CHPV; genus Vesiculovirus, family Rhabdoviridae) is an emerging tropical pathogen with a case fatality rate of 55 to 75% that predominantly affects children in the age group of 2 to 16 years. Although it has been established as a neurotropic virus causing encephalitis, the molecular pathology leading to neuronal death is unknown. The present study elucidates for the first time the mechanism of cell death in neurons after CHPV infection that answers the basic cause of CHPV-mediated neurodegeneration. Through various cell death assays in vitro and in vivo, a relationship between viral replication within neuron and neuronal apoptosis has been established. We report that expression of CHPV phosphoprotein increases up to 6 h postinfection and diminishes thereafter in neuronal cell lines, signifying the replicative phase of CHPV. Various analyses conducted during the investigation established that CHPV-infected neurons are undergoing apoptosis through an extrinsic pathway mediated through the Fas-associated death domain (FADD) following activation of caspase-8 and -3 and prominent cleavage of poly(ADP-ribose) polymerase (PARP). Knocking down the expression of caspase-3, the final executioner of apoptosis, in a neuronal cell line by endoribonuclease-prepared small interfering RNA (siRNA) validated its pivotal role in CHPV-mediated neurodegeneration by showing reduction in apoptosis after CHPV infection.     

Isolation and Characterization of a Single-Stranded DNA Virus Infecting the Marine Diatom Chaetoceros sp. Strain SS628-11 Isolated from Western JAPAN

Diatoms are significant organisms for primary production in the earth''s aquatic environment. Hence, their dynamics are an important focus area in current studies. Viruses are a great concern as potential factors of diatom mortality, along with other physical, chemical, and biological factors. We isolated and characterized a new diatom virus (Csp07DNAV) that lyses the marine planktonic diatom Chaetoceros sp. strain SS628-11. This paper examines the physiological, morphological, and genomic characteristics of Csp07DNAV. The virus was isolated from a surface water sample that was collected at Hiroshima Bay, Japan. It was icosahedral, had a diameter of 34 nm, and accumulated in the nuclei of host cells. Rod-shaped virus particles also coexisted in the host nuclei. The latent period and burst size were estimated to be <12 h and 29 infectious units per host cell, respectively. Csp07DNAV had a closed circular single-stranded DNA genome (5,552 nucleotides), which included a double-stranded region and 3 open reading frames. The monophyly of Csp07DNAV and other Bacilladnavirus group single-stranded DNA viruses was supported by phylogenetic analysis that was based on the amino acid sequence of each virus protein. On the basis of these results, we considered Csp07DNAV to be a new member of the genus Bacilladnavirus.     

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.     

Mouse Hepatitis Virus (MHV-2)

Various factor sinfluencing the plaque formation of mouse hepatitis virus (MHV-2) in DBT cell monolayers were studied and a practical method for plaque assay was developed. Infected DBT cells yielded high-titered virus and were a satisfactory source of complement-fixing viral antigen. The predominant cytopathic effect of MHV-2 in DBT cells was cell rounding and detachment, but no syncytial formation was observed. Fluorescent antibody staining revealed specific fluorescence only in the cytoplasm of infected DBT cells. In one-step growth experiment, newly formed virus was first recognized within 4-hr postinfection and showed subsequently a rapid exponential increase. Release of newly formed virus from the cell was rapid, and a continuous release lasted for a certain period of time. The average per-cell yield of active virus was estimated to be about 6–7 × 10² plaque-forming units.     

Two tissue culture media for production of lepidopteran cells and nuclear polyhedrosis viruses

Two media supporting the growth of several established lepidopteran cell lines in monolayer and suspension culture are described. The medium designated $\text{BML-TC}\text{10}$ was developed specifically as an inexpensive medium for production of cells of Spodoptera frugiperda and the homologous nuclear polyhedrosis virus (NPV) of this species. Simultaneously, a second medium was formulated in which the amino acid requirements were provided by enzymatic protein hydrolysates, one of which was termed $\text{BML-TC}\text{7A}$. Several cell lines could be adapted easily to this medium. $\text{BML-TC}\text{10}$ supported growth of S. frugiperda cells and production of the NPV's of S. frugiperda and Autographa californica. $\text{BML-TC}\text{7A}$ supported the growth of cells of S. frugiperda. Carpocapsa pomonella, Heliothis zea, and Trichoplusia ni. Cells of the latter produced the polyhedra of T. ni and A. californica NPV's in this medium.     

Vaccinia Virus Induces Rapid Necrosis in Keratinocytes by a STAT3-Dependent Mechanism

Rationale

Humans with a dominant negative mutation in STAT3 are susceptible to severe skin infections, suggesting an essential role for STAT3 signaling in defense against cutaneous pathogens.

Methods

To focus on innate antiviral defenses in keratinocytes, we used a standard model of cutaneous infection of severe combined immunodeficient mice with the current smallpox vaccine, ACAM-2000. In parallel, early events post-infection with the smallpox vaccine ACAM-2000 were investigated in cultured keratinocytes of human and mouse origin.

Results

Mice treated topically with a STAT3 inhibitor (Stattic) developed larger vaccinia lesions with higher virus titers and died more rapidly than untreated controls. Cultured human and murine keratinocytes infected with ACAM-2000 underwent rapid necrosis, but when treated with Stattic or with inhibitors of RIP1 kinase or caspase-1, they survived longer, produced higher titers of virus, and showed reduced activation of type I interferon responses and inflammatory cytokines release. Treatment with inhibitors of RIP1 kinase and STAT3, but not caspase-1, also reduced the inflammatory response of keratinocytes to TLR ligands. Vaccinia growth properties in Vero cells, which are known to be defective in some antiviral responses, were unaffected by inhibition of RIP1K, caspase-1, or STAT3.

Conclusions

Our findings indicate that keratinocytes suppress the replication and spread of vaccinia virus by undergoing rapid programmed cell death, in a process requiring STAT3. These data offer a new framework for understanding susceptibility to skin infection in patients with STAT3 mutations. Interventions which promote prompt necroptosis/pyroptosis of infected keratinocytes may reduce risks associated with vaccination with live vaccinia virus.     

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.     

Conditional Dependence of Fusion from Within and Other Cell Membrane Alterations by Newcastle Disease Virus

Fusion from within (FFWI) by Newcastle disease virus occurs optimally in medium maintained at pH 8.2, whereas fusion from without is relatively insensitive to the pH of the medium in the range of 7.0 to 8.3. The pH-sensitive events in FFWI take place in the synthesis of the hypothetical fusion factor rather than in the response to it. pH pulse and pH shift experiments have localized the pH-sensitive events between 4 and 6.5 h postinfection (a period of synthesis of proteins required for FFWI), but before the fusion process. The pH sensitivity is not due to a pH-sensitive interference phenomenon. Virus production and the appearance of hemadsorbing cell surfaces are also pH sensitive, but for these functions the pH optima depend upon the virus strains tested. The independence of FFWI, hemadsorption, and virus production is discussed. Also discussed are the possible roles of virus-specific proteins in the fusion process.     

The Genome Sequence of Lone Star Virus,a Highly Divergent Bunyavirus Found in the Amblyomma americanum Tick

Viruses in the family Bunyaviridae infect a wide range of plant, insect, and animal hosts. Tick-borne bunyaviruses in the Phlebovirus genus, including Severe Fever with Thrombocytopenia Syndrome virus (SFTSV) in China, Heartland virus (HRTV) in the United States, and Bhanja virus in Eurasia and Africa have been associated with acute febrile illness in humans. Here we sought to characterize the growth characteristics and genome of Lone Star virus (LSV), an unclassified bunyavirus originally isolated from the lone star tick Amblyomma americanum. LSV was able to infect both human (HeLa) and monkey (Vero) cells. Cytopathic effects were seen within 72 h in both cell lines; vacuolization was observed in infected Vero, but not HeLa, cells. Viral culture supernatants were examined by unbiased deep sequencing and analysis using an in-house developed rapid computational pipeline for viral discovery, which definitively identified LSV as a phlebovirus. De novo assembly of the full genome revealed that LSV is highly divergent, sharing <61% overall amino acid identity with any other bunyavirus. Despite this sequence diversity, LSV was found by phylogenetic analysis to be part of a well-supported clade that includes members of the Bhanja group viruses, which are most closely related to SFSTV/HRTV. The genome sequencing of LSV is a critical first step in developing diagnostic tools to determine the risk of arbovirus transmission by A. americanum, a tick of growing importance given its expanding geographic range and competence as a disease vector. This study also underscores the power of deep sequencing analysis in rapidly identifying and sequencing the genomes of viruses of potential clinical and public health significance.