Structural components of influenza C virions.

The genome RNA species of influenza type C virions were analyzed by polyacrylamide gel electrophoresis. The pattern obtained was found to resemble those of other influenza viruses. Six RNA species were resolved, with estimated sizes ranging from 0.37 X 10(6) to 1.25 X 10(6) daltons. The internal ribonucleoproteins of influenza C virions were found to sediment heterogeneously in glycerol velocity gradients as demonstrated previously with influenza A/WSN virus. The ribonucleoproteins possessed diameters of 12 to 15 nm, with lengths ranging from 30 to 100 nm. Of the three major virion polypeptides (molecular weights, 88,000, 66,000, and 26,000), only the largest is glycosylated. Similar polypeptide species were present in influenza C virions of five different strains. All three major proteins of influenza C virions possess electrophoretic mobilities distinguishable from those of the major polypeptides of influenza A/WSN. The 66,000-dalton protein is associated with the ribonucleoprotein components. Two additional glycosylated polypeptides, with estimated molecular weights of 65,000 and 30,000, were detected in virions grown in embryonated eggs, but not in virus particles obtained from chicken embryo fibroblasts.     

Generation of defective interfering particles of Semliki Forest virus in a clone of Aedes albopictus (mosquito) cells.

Serial undiluted passage of Semliki Forest virus in a clone of Aedes albopictus cells resulted in a marked decrease in infectious virus yields due to the generation and accumulation of defective interfering particles. Virus from the third passage had a high particle/infectivity ratio and interfered specifically with homologous but not heterologous standard virus replication. Two RNA species of molecular weights 0.78 X 10(6) and 0.61 X 10(6) were the major RNA components of purified passage 4 virus. These RNA species were also the predominant virus RNA species detected in cells infected with passage 3 virus. Synthesis of standard virus RNA and virus-specified protein was much reduced in passage 3 virus-infected cells. Interference with standard virus replication and the synthesis of large amounts of defective interfering RNA were also observed in chicken embryo cells infected with passage 3 virus from mosquito cells.     

Defective Interfering Passages of Sindbis Virus: Chemical Composition, Biological Activity, and Mode of Interference

Defective interfering (DI) particles of Sindbis virus, appearing between the eighth and fourteenth passages, cosediment with and have the same buoyant density as standard virus. Virion RNA from such late passages is heterogeneous by polyacrylamide gel electrophoresis, whereas early passage RNA is homogeneous. No differences were found in the virion proteins from such passages. Cells co-infected with early and late passage virus synthesize as much intracellular viral-specific RNA and protein as is made after infection with early passage virus alone, although virus production is inhibited by 90% or more. Such cells synthesize two new intracellular species of RNA with molecular weights of 2.2 x 10(6) and 0.86 x 10(6). Nucleocapsid assembly is blocked in these cells, and the amount of intracellular capsid protein made is reduced by 50%. The presence of a new intracellular protein in late passage infection was detected by polyacrylamide gel electrophoresis.     

Messenger RNA species synthesized in vitro by the virion-associated RNA polymerase of vesicular stomatitis virus.

The virion-associated RNA-dependent RNA polymerase of vesicular stomatitis virus (VSV) synthesizes in vitro two size classes of RNA products similar to those observed in VSV-infected cells. One RNA product sediments at 31S with an approximate molecular weight of 2.1 X 106. The smaller products consist of at least three classes of RNA sedimenting at 17S, 14.5S, and 12S with molecular weights of 0.7 X 106, 0.52 X 106, and 0.37 X 106, respectively. Hybridization experiments show that both the 31S and 12-18S RNA products are complementary to the genome RNA, and that each class is transcribed from different nucleotide sequences. From the molecular weights of the RNA species and the hybridization experiments, it seems that almost the entire VSV genome RNA is transcribed in vitro.     

Cucumber mosaic virus, a model for RNA virus evolution

Taxonomic relationships: Cucumber mosaic virus (CMV) is the type member of the Cucumovirus genus, in the family Bromoviridae . Additional members of the genus are Peanut stunt virus (PSV) and Tomato aspermy virus (TAV). The RNAs 3 of all members of the genus can be exchanged and still yield a viable virus, while the RNAs 1 and 2 can only be exchanged within a species.
Physical properties: The virus particles are about 29 nm in diameter, and are composed of 180 subunits (T = 3 icosahedral symmetry). The particles sediment with an s value of approximately 98. The virions contain 18% RNA, and are highly labile, relying on RNA–protein interactions for their integrity. The three genomic RNAs, designated RNA 1 (3.3 kb in length), RNA 2 (3.0 kb) and RNA 3 (2.2 kb) are packaged in individual particles; a subgenomic RNA, RNA 4 (1.0 kb), is packaged with the genomic RNA 3, making all the particles roughly equivalent in composition. In some strains an additional subgenomic RNA, RNA 4A is also encapsidated at low levels. The genomic RNAs are single stranded, plus sense RNAs with 5' cap structures, and 3' conserved regions that can be folded into tRNA-like structures.
Satellite RNAs: CMV can harbour molecular parasites known as satellite RNAs (satRNAs) that can dramatically alter the symptom phenotype induced by the virus. The CMV satRNAs do not encode any proteins but rely on the RNA for their biological activity.
Hosts: CMV infects over 1000 species of hosts, including members of 85 plant families, making it the broadest host range virus known. The virus is transmitted from host to host by aphid vectors, in a nonpersistent manner.
Useful web sites: http://mmtsb.scripps.edu/viper/1f15.html (structure); http://www.ncbi.nlm.nih.gov/ICTVdb/ICTVdB/10040001.htm (general information)     

Effect of Ultraviolet Light on Mengovirus: Formation of Uracil Dimers, Instability and Degradation of Capsid, and Covalent Linkage of Protein to Viral RNA

UV irradiation of purified mengovirus resulted in a very rapid inactivation of the infectivity of the virions (D(37) [37% survival dose] = 700 ergs/mm(2)) which correlated in time with the formation of uracil dimers in the viral RNA. During the first 2 min of irradiation, an average of 1.7 uracil dimers were formed per PFU of virus inactivated. Hemagglutination activity of the virions began to decrease only after a lag period of about 5 min and at a much lower rate (D(37) = 84,000 ergs/mm(2)). This decrease coincided in time with the appearance of altered proteins in the capsid and a structural change in the capsid. Although 10- to 20-min irradiated virions appeared intact in the electron microscope and sedimented at 150S in sucrose density gradients, the RNA of the virions became accessible to RNase and extractable by low concentrations of sodium dodecyl sulfate, and the virions broke down upon equilibrium centrifugation in CsCl gradients. During longer periods of irradiation (30 to 60 min), a progressively greater proportion of the virions were converted to 14S protein particles and 80S ribonucleoprotein particles composed of intact viral RNA and about 30% of the capsid proteins, alpha, beta, and gamma. Empty capsids were not detectable at any time during 60 min of irradiation, by which time disruption of the virions was complete. Irradiation of complete virions also resulted in an increased sedimentation rate of the viral RNA and in the covalent linkage to the viral RNA of about 1% of the total capsid protein in the form of heterogeneous low-molecular-weight polypeptides. The two observations seem to be causally related, since irradiation of isolated viral RNA did not result in an increase in sedimentation rate of the RNA, even though uracil dimer formation in viral RNA occurred at about the same rate and to the same extent whether intact virions or viral RNA were irradiated.     

一株传染性软化病病毒的分离和鉴定

病毒性软化病是一种严重影响蚕业生产的流行病,其病原为家蚕传染性软化病病毒(Bombyx moriinfectious flacherie virus,BmIFV)。本实验室在浙江省收集到具有软化病症状的家蚕幼虫,并分离纯化获得一株病毒。电镜观察到病毒粒子为直径26nm左右、无包涵体的球状颗粒。病毒添食四龄起蚕和五龄起蚕,分别在3~4d和5~7d出现明显症状和病变。纯化病毒SDS-PAGE条带与BmIFV蛋白相仿。病毒核酸鉴定为RNA,并可用套式RT-PCR扩增出特异性片段,测序发现片段序列和BmIFV日本坂城株的同源性为99.5%。由此可确定该病毒为BmI-FV,这是我国首次分离到该病毒,暂命名为BmIFV-CHN001。     

P3HR-1 Epstein-Barr virus with heterogeneous DNA is an independent replicon maintained by cell-to-cell spread.

We present results of biological experiments which indicate that the subpopulation of Epstein-Barr virus strain P3HR-1 with heterogeneous (het) DNA consists of self-contained replicons which multiply alongside, but independently of, Epstein-Barr virus strain HR-1 containing standard DNA. When a population of HR-1 virions containing het DNA was introduced into X50-7 cells, the input heterogeneous DNA increased in abundance, as did the DNA of the endogenous virus of X50-7 cells. The input standard HR-1 viral DNA, however, was not amplified. When parental HR-1 cells or a cellular subclone containing het DNA were grown for several weeks in the presence of human serum with neutralizing antibody, the het DNA was lost from the culture; standard HR-1 DNA, however, was not affected by antiserum. Furthermore, virions containing het DNA could be serially propagated through cellular subclones of HR-1 cells which lack het DNA. After each serial passage, cells which acquired het DNA released virions with the ability to induce early antigens in Raji cells. These experiments define a novel in vitro life cycle of an Epstein-Barr virus variant which is maintained, not vertically by partitioning to daughter cells in cell division, but horizontally by cell-to-cell spread.     

Comparative ultrastructural study of four reticuloendothelias viruses.

The morphology and development of four members of the reticuloendotheliosis virus group were studied by transmission electron microscopy. Virions of duck spleen necrosis virus, duck infectious anemia virus, chicken syncytial virus, and reticuloendotheliosis virus strain T are sperical with a diameter of approximately 110 nm. They are covered with surface projections about 6 nm long and 10 nm in diameter. The center-to-center distance of surface projections is about 14 nm. The budding virions contain crescent-shaped electron-dense cores 73 nm in diameter with electron-lucent centers. After release of the virions the cores apparently become condensed to 67 nm in diameter. Virions were found budding at the plasma membrane and into smooth-walled, intracytoplasmic vesicles of productively infected cells. The distribution of budding reticuloendotheliosis viruses on cells appeared random over the cell surface, and occasionally aberrant multiple forms of budding virions were observed. The virions appear to resemble mammalian leukemia and sarcoma viruses more closely than avian leukosis-sarcoma viruses.     

Factors involved in the generation and replication of rhabdovirus defective T particles.

Previous indications that cloned B virions might be genetically predisposed to generate a particular defective T particle are shown to be inaccurate. T particle generation was found to be a much more random process than was previously believed. We show that the previously observed generation of particular sizes of T particles by B virion pools is due to the random generation of T particles during preparation of first-passage pools of cloned B virions, and these breed true during the additional passages needed to produce visible quantities of T particles. It is also shown that different host cell lines selectively amplify different T particles, suggesting a strong role of host cell factors in T particle replication. Surprisingly, our line of HeLa cells did not generate or replicate detectable T particles of vesicular stomatitis virus (VSV) Indiana after either serial undiluted passage or direct addition of T particles, even though the added T particles strongly interfered with B virion replication. In contrast to VSV, rabies virus generates large amounts of T particles during the first passage of cloned B virions, and every rabies-infected baby hamster kidney-21 cell culture evolves into a persistent carrier state. We find that T particle RNA is biologically inactive although T particle nucleocapsid ribonucleoprotein replicates and interferes in cells coinfected with B virions. Efforts to study the mechanism of T particle generation by in vitro attempts to generate T particles or modify their size (using sheared ribonucleoprotein or chemical or UV mutagenesis) were unsuccessful. The kinetics of UV and nitrous acid inactivation of T particles indicate a smaller target size relative to B virions, even after correcting for lengths of RNA molecules. The intercalating dye proflavine does not photosensitize VSV B virions or T particles when present during replication, indicating that there is little or no RNA base pairing in the helical nucleocapsids of either.     

Ribonucleic Acid Species of Intracellular Nucleocapsids and Released Virions of Vesicular Stomatitis Virus

Infection of chicken embryo cells with vesicular stomatitis (VS) virus resulted in variable production of three classes of intracellular viral ribonucleocapsids with sedimentation coefficients of approximately 140S, 110S, and 80S, as well as three corresponding classes of released virions designated B, LT, and T. Intracellular nucleocapsids of each class contained three proteins of which the major N protein was firmly bound, and the minor L and NS1 proteins were readily dissociated with 0.5 m NaCl. The ribonucleic acid (RNA) species extracted from B, LT, and T virions, and from corresponding intracellular nucleocapsids, contained RNA species with approximate molecular weights of 3.2 x 10(6), 2.0 x 10(6), and 10(6), respectively, as determined by polyacrylamide gel electrophoresis. These values are roughly equivalent to sedimentation coefficients of 42S, 28S, and 23S for each of the virion and nucleocapsid RNA species. Cells infected at high multiplicity with undiluted passage VS virus gave rise primarily to virions and nucleocapsids containing 23S RNA, whereas cells productively infected with purified B virions produced predominantly B and LT virions and nucleocapsids. At late stages in the productive cycle of infection, more virions containing 42S RNA were produced, but the intracellular pool of nucleocapsids containing 28S and 23S RNA remained relatively constant. Additional studies by more refined techniques are required to test the hypothesis that nucleocapsids containing 28S and 23S RNA are precursors of the 42S RNA in infectious VS-B virions and that production of defective T and LT virions results from failure of ligation of the RNA precursors.     

Cucumber mosaic virus: viral genes as virulence determinants

TAXONOMIC RELATIONSHIPS: Cucumber mosaic virus (CMV) is the type species of the genus Cucumovirus in the family Bromoviridae, which also encompasses the Peanut stunt virus (PSV) and the Tomato aspermy virus (TAV). Nucleotide sequence similarity among these three cucumoviruses is 60%-65%. CMV strains are divided into three subgroups, IA, IB and II, based on the sequence of the 5' untranslated region of the genomic RNA 3. Overall nucleotide sequence similarity among CMV strains is approximately 70%-98%. GEOGRAPHICAL DISTRIBUTION, HOST RANGE AND SYMPTOMATOLOGY: CMV is distributed worldwide, primarily in temperate to tropical climate zones. CMV infects more than 1200 species of 100 plant families, including monocot and dicot plants. Symptoms caused by CMV infection vary with the host species and/or CMV strain, and include mosaic, stunt, chlorosis, dwarfing, leaf malformation and systemic necrosis. CMV disease is spread primarily by aphid transmission in a nonpersistent manner. PHYSICAL PROPERTIES: In tobacco sap, the thermal inactivation point of the viral infectivity is approximately 70 °C (10 min), the dilution end-point is approximately 10(-4) and viral infectivity is lost after a few days of exposure to 20 °C. Viral infectivity can be retained in freeze-dried tissues and in the form of virions purified using 5 mm sodium borate, 0.5 mm ethylenediaminetetraacetic acid and 50% glycerol (pH 9.0) at -20 °C. CMV particles are isometric, approximately 28-30 nm in diameter and are composed of 180 capsid subunits arranged in pentamer-hexamer clusters with T= 3 symmetry. The sedimentation coefficient (s(20) ,(w) ) is c. 98 S and the particle weight is (5.8-6.7) × 10(6) Da. The virions contain 18% RNA. The RNA-protein interactions that stabilize the CMV virions are readily disrupted by sodium dodecylsulphate or neutral chloride salts. GENOMIC PROPERTIES: The genomic RNAs are single-stranded messenger sense RNAs with 5' cap and 3' tRNA-like structures containing at least five open reading frames. The viral RNA consists of three genomic RNAs, RNA 1 (c. 3.3 kb), RNA 2 (c. 3.0 kb) and RNA 3 (c. 2.2 kb), and two subgenomic RNAs, RNA 4 (c. 1.0 kb) and RNA 4A (c. 0.7 kb). The 3' untranslated regions are conserved across all viral RNAs. CMV is often accompanied by satellite, noncoding, small, linear RNA that is nonhomologous to the helper CMV.     

Hepatitis B virus transcripts in a human hepatoma cell line, Hep 3B

Hep 3B, a human hepatoma cell line was examined for its RNA hybridizable to the hepatitis B virus sequence. Using probes that covered different regions of the hepatitis B virus genome, five species of RNA were observed of sizes 4.0, 3.3, 2.9, 2.6 and 2.2 kilobases. The RNAs covered surface antigen gene, pre-S and X regions. None of them had a core antigen sequence. RNA with a 4.0 kilobase size was the most abundant. Using S1 nuclease analysis, its 5' end of hepatitis B virus sequence was mapped at pre-S region and its 3' end of viral sequence was mapped at DR region.     

Induction and biological properties of defective interfering particles of rabies virus.

A method for obtaining large quantities of defective interfering (DI) rabies virus particles that fulfill all the criteria delineated by Huang and Baltimore (1970) is described. The purified rabies DI virion was found to be much shorter (60 to 80 nm) than the complete virion (180 nm) and to have a viral genome of about half the size of normal rabies RNA but with all of the structural proteins of standard virions. Rabies DI virions were noninfectious for both cells in culture and for animals. As determined by in vitro and in vivo techniques, interference with the replication of standard virus was specific to rabies virus. The possible role of rabies DI virion in the pathogenicity of rabies virus infection and in the establishment of attenuated strains for use as live rabies vaccines is discussed.     

Characterization of a Small RNA-Containing Virus in Field-Collected Larvae of the Tussock Moth, Lymantria ninayi, from Papua New Guinea

Field-collected larvae of the tussock moth, Lymantria ninayi, a major pest of exotic pines in Papua New Guinea, were found to contain a small RNA-containing virus with a diameter of 29 nm and a buoyant density of 1.32 g/ml. The RNA was single stranded, had a molecular weight of 2.8 × 10⁶, and was polyadenylated. Virion RNA stimulated an in vitro translation system, and high-molecular-weight proteins were produced. Purified virions contained four structural proteins with molecular weights of 43,000, 38,000, 33,000, and 32,000. The virus reacted positively with antisera raised against a strain of Drosophila C virus. The properties of this virus indicate that it should be placed in the family Picornaviridae.     

Hemagglutinin prepared from cells of Aedes albopictus, clone C6/36, infected with type 1 dengue virus

A high titer of hemagglutinin (HAnin) was found in culture fluids of cells of Aedes albopictus, clone C6/36, infected with type 1 dengue virus. The HAnin (TC antigen) was associated with complete virions and no appreciable small-sized HAnin was produced, in contrast to the case in an infected suckling mouse brain (SMB) homogenate, in which most of the HAnin is smaller than complete virions. Extraction of the TC antigen with Tween 80-ether disrupted the HAnin into smaller-sized particles, resulting in complete loss of infectivity (TE-TC antigen), but it did not affect the titer or reactivity of the HAnin. Comparative hemagglutination-inhibition (HI) assay of human sera using TC antigen, TE-TC antigen or standard antigen extracted from infected SMB (SMB antigen) showed that TC or TE-TC antigen could be used for routine diagnostic or epidemiological HI tests instead of SMB antigen, which is rather hard to prepare.     

Ultrastructure and Sequential Development of Infectious Pancreatic Necrosis Virus

The morphology and sequential development of infectious pancreatic necrosis (IPN) virus, a pathogen of trouts, were studied by electron microscopy. Mature virions were seen in the cytoplasm of infected cells incubated at 24 C as early as 6 hr after infection. These virions were hexagonal in profile and approximately 55 nm in diameter. Generally between 8 to 10 hr after infection, virus crystals of various sizes were occasionally observed. Although virus replication did not appear to be confined to a particular cytoplasmic locus, mature virions were sometimes seen in association with unidentified tubular structures approximately 45 nm in outside diameter. Negative stains of virus revealed unenveloped icosahedra approximately 65 nm in diameter with probably 92 capsomeres. Contrary to a previous communication which reported IPN virus to have picornavirus-like morphology, we found it to morphologically resemble members of the reovirus group.     

ULTRASTRUCTURE AND DNA RESTRICTION ENDONUCLEASE ANALYSIS OF CALLIPTAMUS ITALICUS ENTOMOPOXVIRUS

Abstract  Calliptamus italicus entomopoxvirus (CiEPV) was isolated from the grasshopper Calliptamus italicus . This virus mainly infects the host's fat body, and its development in the cytoplasm includes 4 steps: the appearance of viroplasm in the cytoplasm of fat body; the formation of immature virions by budding from viro-plasm; the differentiation of mature virions from immature virions and the accumulation of occlusion-body protein. The mature virions are oval with mulberry-like surface, measuring 190 nm X 350 nm in size. Most of the occlusion bodies are elliptical, some are square- round in shape. There are great differences in size ranging from 1.5 μm X 1.9 μm to 8.8 μm X 12.3 μm. CiEPV DNA, when cleaved with restriction endonuclease Eco R I Bg 1 II and Hin d III, produces 16–18 and 10 fragments respectively, and the molecular weight of this DNA was estimated to be 135.7 X 10⁶ daltons. Some characteristics of morphology and DNA were compared between CiEPV and Oedaleus asiaticus EPV (OaEPV) reported previously.     

Analysis of extracellular West Nile virus particles produced by cell cultures from genetically resistant and susceptible mice indicates enhanced amplification of defective interfering particles by resistant cultures

[3H]uridine-labeled extracellular West Nile virus (WNV) particles produced by cell cultures obtained from genetically resistant C3H/RV and congenic susceptible C3H/HE mice were compared by sucrose density gradient centrifugation as well as by analysis of the particle RNA. Defective interfering (DI) WNV particles were observed among progeny produced during acute infections in both C3H/RV and C3H/HE cells. Although only a partial separation of standard and DI particles was achieved, the DI particles were found to be more dense than the standard virions. Particles containing several species of small RNAs consistently constituted a major proportion of the total population of virus progeny produced by C3H/RV cells, but a minor proportion of the population produced by C3H/HE cells. Decreasing the multiplicity of infection or extensive plaque purification of the WNV inoculum decreased the proportion of small RNAs found in the progeny virus. The ratio of DI particles to standard virus observed in progeny virus was determined by the cell type used to grow the virus. The ratio could be shifted by passaging virus from one cell type to the other. Homologous interference could be demonstrated with WNV produced by C3H/RV cells but not with virus produced by C3H/HE cells. Continued passage of WNV in C3H/HE cells resulted in a cycling of infectivity. However, passage in C3H/RV cells resulted in the complete loss of infectious virus. Four size classes of small viral RNA, with sedimentation coefficients of about 8, 15, 26, and 34S, were observed in the extracellular particles. A preliminary analysis of these RNAs by oligonucleotide fingerprinting indicated that the smaller RNAs were less complex than the 40S RNA and differed from each other. The data are consistent with the conclusion that WNV DI particles interfere more effectively with standard virus replication and are amplified more efficiently in C3H/RV cells than in congenic C3H/HE cells. The relevance of these findings to the further understanding of genetically controlled resistance to flaviviruses is discussed.