New endogenous herpesvirus of guinea pigs: biological and molecular characterization.

Two known guinea pig herpesviruses, guinea pig cytomegalovirus (GPCMV) and guinea pig herpes-like virus (GPHLV), and well characterized. A third herpesvirus (GPXV) was originally isolated from leukocytes of healthy strain 2 guinea pigs. Growth of GPXV in guinea pig embryo fibroblastic cells produced a characteristic cytopathic effect. Electron microscopy of guinea pig cells infected with GPXV revealed the morphological development of a herpesvirus. Cross-neutralization tests and immunoferritin electron microscopy demonstrated that GPXV, GPCMV, and GPHLV were serologically distinct herpeviruses of guinea pigs. To confirm the distinction between these three herpesviruses, DNA genomes were compared by CsCl equilibrium buoyant density measurements and restriction endonuclease cleavage analysis. 32P-labeled viral DNA ws obtained from nucleocapsids isolated from virus-infected cells, and the buoyant density of GPXV DNA differed from that of GPCMV and GPHLV. Cleavage of viral DNAs with restriction endonucleases followed by gel electrophoresis revealed distinct patterns for each virus.     

Characterization of the guinea pig cytomegalovirus genome by molecular cloning and physical mapping.

Fragments of guinea pig cytomegalovirus (GPCMV) DNA produced by HindIII or EcoRI restriction endonuclease digestion were cloned into vectors pBR322 and pACYC184, and recombinant fragments representing ca. 97% of the genome were constructed. Hybridization of 32P-labeled cloned and gel-purified HindIII, EcoRI, and XbaI fragments to Southern blots of HindIII-, EcoRI-, and XbaI-cleaved GPCMV DNA verified the viral origin of cloned fragments and allowed construction of HindIII, EcoRI, and XbaI restriction maps. On the basis of the cloning and mapping experiments, the size of GPCMV DNA was calculated to include 239 kilobase pairs, corresponding to a molecular weight of 158 X 10(6). No cross-hybridization between any internal fragments was seen. We conclude that the GPCMV genome consists of a long unique sequence with terminal repeat sequences but without internal repeat regions. In addition, GPCMV DNA molecules exist in two forms. In the predominant form, the molecules demonstrate sequence homology between the terminal fragments; in the minor population, one terminal fragment is smaller by 0.7 X 10(6) daltons and is not homologous with the fragment at the other end of the physical map. The structural organization of GPCMV DNA is unique for a herpesvirus DNA, similar in its simplicity to the structure reported for murine cytomegalovirus DNA and quite dissimilar from that of human cytomegalovirus DNA.     

Deoxyribonucleic Acid Replication in Simian Virus 40-Infected Cells: II. Detection and Characterization of Simian Virus 40 Pseudovirions

Purified simian virus 40 (SV40) virions, grown in primary African green monkey kidney cells labeled prior to infection with (3)H-thymidine, contain a variable quantity of (3)H-labeled deoxyribonucleic acid (DNA). This DNA is resistant to deoxyribonuclease, sediments at 250S, and is enclosed in a particle that can be precipitated with SV40-specific antiserum. DNA-DNA hybridization experiments demonstrate that this (3)H-labeled component in purified SV40 virions is cellular DNA. When this (3)H-labeled DNA is released from purified virus with sodium dodecyl sulfate, it has an average sedimentation constant of 14S. Sedimentation through neutral and alkaline sucrose gradients shows that this 14S DNA is composed of a collection of different sizes of DNA molecules that sediment between 11 and 15S. As a result of this size heterogeneity, SV40 virions containing cellular DNA (pseudovirions) have a variable DNA to capsid protein ratio and exhibit a spectrum of buoyant densities in a CsCl equilibrium gradient. Pseudovirions are enriched, relative to true virions, on the lighter density side of infectious SV40 virus banded to equilibrium in a CsCl gradient. Little or no cellular DNA was found in purified SV40 virus preparations grown in BSC-1 or CV-1 cells.     

Analysis of Euglena gracilis chloroplast deoxyribonucleic acid with a restriction endonuclease, EcoRI.

Cleavage of chloroplast deoxyribonucleic acid (DNA) of Euglena gracilis Z with restriction endonuclease RI from Escherichia coli (EcoRI) yielded 23 bands upon electrophoresis in gels of agarose. Four of the bands contained twice the stoichiometric amount of DNA. One of these bands contained two similarly sized fragments. The sum of the molecular weight of the 24 different fragments equaled the molecular weight of the circular molecule. The restriction fragments had different buoyant densities, with four having distinctly heavy densities in CsCl. Restriction fragments with a high buoyant density were preferentially lost when broken chloroplast DNA was purified by equilibrium density gradient centrifugation. Hybridization of chloroplast ribosomal ribonucleic acid to intact chloroplast DNA determined that there are two cistrons for 16S and 23S ribosomal ribonucleic acid. These two cistrons are located on six restriction fragments, all of which have buoyant densities greater than the intact molecule of chloroplast DNA.     

Frequency of replication from alternative origins in the composite R plasmid NR1.

The relative frequency of initiation of DNA replication within the RTF-Tc and r-determinant components of the composite drug resistance plasmid NR1 in Proteus mirabilis was evaluated. Using fractionated radioactively labeled plasmid DNA, analytical procedures that distinguished between the two components of the composite plasmid were carried out. A mixture of uniformly 14C-labeled and 3H-pulse-labeled plasmid DNA (pulse-labeled origin[s] of replication) was used in each of three experiments. First, shear products of the DNA were analyzed using CsCl density gradient centrifugation. Second, fragmented DNA was hybridized to nonradioactive RTF-Tc and r-determinant DNAs immobilized on nitrocellulose filters. Third, the radioactive plasmid DNAs immobilized on nitrocellulose filters. Third, the radioactive plasmid DNA was digested with restriction enzyme (EcoRI), producing a set of RTF-Tc and r-determinant fragments with differing 3H/14C isotpe ratios. The three experiments suggested that under the conditions used to accumulate replicating plasmid DNA molecules (DNA substrate limitation), the r-determinant origin of replication was preferentially utilized in the composite plasmid.     

A Neutralizing Anti-gH/gL Monoclonal Antibody Is Protective in the Guinea Pig Model of Congenital CMV Infection

Human cytomegalovirus (HCMV) is the most common cause of congenital virus infection. Congenital HCMV infection occurs in 0.2–1% of all births, and causes birth defects and developmental abnormalities, including sensorineural hearing loss and developmental delay. Several key studies have established the guinea pig as a tractable model for the study of congenital HCMV infection and have shown that polyclonal antibodies can be protective [1]–[3]. In this study, we demonstrate that an anti-guinea pig CMV (GPCMV) glycoprotein H/glycoprotein L neutralizing monoclonal antibody protects against fetal infection and loss in the guinea pig. Furthermore, we have delineated the kinetics of GPCMV congenital infection, from maternal infection (salivary glands, seroconversion, placenta) to fetal infection (fetus and amniotic fluid). Our studies support the hypothesis that a neutralizing monoclonal antibody targeting an envelope GPCMV glycoprotein can protect the fetus from infection and may shed light on the therapeutic intervention of HCMV congenital infection in humans.     

Restriction analysis of the DNA of aleutian mink disease virus strain P-1

The II-1 strain of the Aleutian disease virus (ADV-II-1) was isolated from experimentally infected mink organs. The viral particles were isolated having 23 to 24 nm in diameter with the buoyant density of the virions in CsCl gradient being 1.41 g.ml-1. The single stranded ADV DNA extracted from the purified virus particles had the molecular mass about 1.4 . 10(6) (4800 bases). The double-stranded replicative form of ADV DNA has been synthesized in vitro with the use of a large "Klenow" fragment of DNA-polymerase I. A restriction endonuclease map of ADV-II-1 DNA has been constructed with the use of in vitro synthesized double-stranded DNA.     

Viral Glycoprotein Complex Formation,Essential Function and Immunogenicity in the Guinea Pig Model for Cytomegalovirus

Development of a cytomegalovirus (CMV) vaccine is a major public health priority due to the risk of congenital infection. A key component of a vaccine is thought to be an effective neutralizing antibody response against the viral glycoproteins necessary for cell entry. Species specificity of human CMV (HCMV) precludes direct studies in an animal model. The guinea pig is the only small animal model for congenital cytomegalovirus infection. Analysis of the guinea pig CMV (GPCMV) genome indicates that it potentially encodes homologs to the HCMV glycoproteins (including gB, gH, gL, gM, gN and gO) that form various cell entry complexes on the outside of the virus: gCI (gB); gCII (gH/gL/gO); gCIII (gM/gN). The gB homolog (GP55) has been investigated as a candidate subunit vaccine but little is known about the other homolog proteins. GPCMV glycoproteins were investigated by transient expression studies which indicated that homolog glycoproteins to gN and gM, or gH, gL and gO were able to co-localize in cells and generate respective homolog complexes which could be verified by immunoprecipitation assays. ELISA studies demonstrated that the individual complexes were highly immunogenic in guinea pigs. The gO (GP74) homolog protein has 13 conserved N-glycosylation sites found in HCMV gO. In transient expression studies, only the glycosylated protein is detected but in virus infected cells both N-glycosylated and non-glycosylated gO protein were detected. In protein interaction studies, a mutant gO that lacked N-glycosylation sites had no impact on the ability of the protein to interact with gH/gL which indicated a potential alternative function associated with these sites. Knockout GPCMV BAC mutagenesis of the respective glycoprotein genes (GP55 for gB, GP75 for gH, GP115 for gL, GP100 for gM, GP73 for gN and GP74 for gO) in separate reactions was lethal for virus regeneration on fibroblast cells which demonstrated the essential nature of the GPCMV glycoproteins. The gene knockout results were similar to HCMV, except in the case of the gO homolog, which was non-essential in epithelial tropic virus but essential in lab adapted GPCMV. Overall, the findings demonstrate the similarity between HCMV and GPCMV glycoproteins and strengthen the relevance of this model for development of CMV intervention strategies.     

Dramatic effects of 2-bromo-5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole riboside on the genome structure, packaging, and egress of guinea pig cytomegalovirus

The halogenated benzimidazoles BDCRB (2-bromo-5,6-dichloro-1-beta-D-riborfuranosyl benzimidazole riboside) and TCRB (2,5,6-trichloro-1-beta-D-riborfuranosyl benzimidazole riboside) were the first compounds shown to inhibit cleavage and packaging of herpesvirus genomes. Both inhibit the formation of unit length human cytomegalovirus (HCMV) genomes by a poorly understood mechanism (M. R. Underwood et al., J. Virol. 72:717-715, 1998; P. M. Krosky et al., J. Virol. 72:4721-4728, 1998). Because the simple genome structure of guinea pig cytomegalovirus (GPCMV) provides a useful model for the study of herpesvirus DNA packaging, we investigated the effects of BDCRB on GPCMV. GPCMV proved to be sensitive to BDCRB (50% inhibitory concentration = 4.7 microM), although somewhat less so than HCMV. In striking contrast to HCMV, however, a dose of BDCRB sufficient to reduce GPCMV titers by 3 logs (50 microM) had no effect on the quantity of GPCMV genomic DNA that was formed in infected cells. Electron microscopy revealed that this DNA was in fact packaged within intranuclear capsids, but these capsids failed to egress from the nucleus and failed to protect the DNA from nuclease digestion. The terminal structure of genomes formed in the presence of BDCRB was also altered. Genomes with ends lacking a terminal repeat at the right end, which normally exist in an equimolar ratio with those having one copy of the repeat at the right end, were selectively eliminated by BDCRB treatment. At the left end, BDCRB treatment appeared to induce heterogeneous truncations such that 2.7 to 4.9 kb of left-end-terminal sequences were missing. These findings suggest that BDCRB induces premature cleavage events that result in truncated genomes packaged within capsids that are permeable to nuclease. Based on these and other observations, we propose a model for duplication of herpesvirus terminal repeats during the cleavage and packaging process that is similar to one proposed for bacteriophage T7 (Y. B. Chung, C. Nardone, and D. C. Hinkle, J. Mol. Biol. 216:939-948, 1990).     

Structure of varicella-zoster virus DNA

Varicella-zoster virus (VZV) DNA was prepared from nucleocapsids and from enveloped virions of a laboratory strain (Ellen) and directly from the vesicle fluids of patients with zoster infections. VZV Ellen nucleocapsid DNA was cleaved with 11 different restriction endonucleases and electrophoresed in agarose gels. The restriction profiles of the nucleocapsid DNA were identical to those of the DNA recovered from purified virions, but differed from those of another VZV strain (KM). In vitro-labeled VZV K.M. DNA purified directly from vesicle fluid yielded a distinct restriction pattern which appeared to be unchanged after several tissue culture passages of the isolate from that fluid. Restriction endonuclease analysis (EcoRI or BglII) of VZV DNA revealed the presence of four cleavage fragments with a molar ratio of approximately 0.5. No individual fragments with molar ratios of 0.25 were noted. This observation suggests that the VZV genome may contain one invertible segment. Comparison of the electrophoretic migrations of VZV DNA fragments relative to those of DNAs of known size permitted calculation of the VZV genome size to be 72 X 10(6) to 80 X 10(6) daltons. These results were confirmed by electron microscopy which demonstrated a genome size of about 76 X 10(6) daltons for passaged and unpassaged VZV DNA. Electron microscopy also revealed that some of the DNA molecules recovered from nucleocapsids or directly from vesicle fluids were superhelical circles.     

Generation of capsids from unstable polyoma virions.

Polyomavirus was purified from infected mouse cell lysates under mild physiological conditions. When analyzed in a sucrose gradient, a major virus peak (240S) was identified. This sucrose-isolated virus could be divided into two populations based on its stability to CsCl gradient centrifugation. Members of the unstable population were shown to eject their DNA cores when subjected to CsCl gradient centrifugation, forming empty capsids, whereas the stable population was unaffected by the same CsCl treatment. Formaldehyde fixation of the 240S virus particles stabilized the virions and prevented ejection of DNA and generation of empty capsids. When formaldehyde-fixed 240S virus was examined with the electron microscope, only full virions were observed. These results indicate that polyoma capsids are not preformed in vivo, but instead are generated when infected cell lysates are subjected to harsh CsCl purification procedures.     

Purification and Biophysical Properties of Acute Haemorrhagic Conjunctivitis Virus

The buoyant density of acute haemorrhagic conjunctivitis virions labeled with either [(3)H]uridine or [(3)H]leucine was 1.34 g/ml in CsCl and 1.25 g/ml in sucrose. RNA extracted from the virions gave a sedimentation coefficient of approximately 34S in sucrose, and was found to be sensitive to RNase. Molecular weight of RNA was calculated to be 2.5 x 10(6) using poliovirus RNA for reference.     

Characterization of Aleutian disease virus as a parvovirus.

We characterized a strain of Aleutian disease virus adapted to growth in Crandall feline kidney cells at 31.8 degrees C. When purified from infected cells, Aleutian disease virus had a density in CsCl of 1.42 to 1.44 g/ml and was 24 to 26 nm in diameter. [3H]thymidine could be incorporated into the viral genome, and the viral DNA was then studied. In alkaline sucrose gradients, Aleutian disease virus DNA was a single species that cosedimented at 15.5S with single-stranded DNA from adeno-associated virus. When the DNA was analyzed on neutral sucrose gradients, a single species was again observed, which sedimented at 21S and was clearly distinct from 16S duplex adeno-associated virus DNA. A similar result was obtained even after incubation under annealing conditions, implying that the bulk of Aleutian disease virus virions contained a single non-complementary strand with a molecular weight of about 1.4 X 10(6). In addition, two major virus-associated polypeptides with molecular weights of 89,100 and 77,600 were demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of virus purified from infected cultures labeled with [35S]methionine. These data suggest that Aleutian disease virus is a nondefective parvovirus.     

Human cytomegalovirus DNA replicates after early circularization by concatemer formation, and inversion occurs within the concatemer.

To determine the replicative mechanism for human cytomegalovirus (HCMV) DNA, field inversion gel electrophoresis was used to separate HCMV replicative DNAs during lytic infection. Unit-length circular HCMV genomes lacking terminal restriction fragments were detected starting 4 h after infection even when cells were treated with aphidicolin, phosphonoacetic acid, or cycloheximide. Viral DNA synthesis began 24 h after infection and produced large amounts of high-molecular-weight replicative DNA that was a precursor of progeny genomes. Replicative DNA contained rare terminal restriction fragments, and long-arm termini were much less frequent than short-arm termini. Replicative DNA was not composed of unit-length circles because low-dose gamma irradiation of replicative DNA generated numerous random high-molecular-weight fragments rather than unit-length molecules. PacI digestion of replicative DNA from a recombinant HCMV with two closely spaced PacI sites revealed that replicative DNA is concatemeric and genome segment inversion occurs after concatemer synthesis. These results show that after circularization of the parental genome, DNA synthesis produces concatemers and genomic inversion occurs within concatemeric DNA. The results further suggest that concatemers acquire genomic termini during the cleavage/packaging process which preferentially inserts short-arm termini into empty capsids, causing a predominance of short-arm termini on the concatemer.     

Varicella-zoster virus vaccine DNA differs from the parental virus DNA.

The DNAs of a varicella-zoster virus vaccine and its parental virus were compared by CsCl buoyant density centrifugation and restriction enzyme cleavage analysis. The varicella-zoster virus vaccine DNA showed a heterogeneous buoyant profile and altered restriction enzyme cleavage patterns. These changed properties are probably the result of the accumulation of virus containing defective varicella-zoster virus DNA during extensive cell culture passage of the vaccine virus.     

人巨细胞病毒的分子克隆及其特异性DNA探针的制备

从人巨细胞病毒（ＨＣＭＶ）培养物中提取ＨＣＭＶ并抽提其ＤＮＡ,经限制性内切酶ＢａｍＨＩ完全消化后,与质粒ｐＢｌｕｅｓｃｒｉｐｔ－ＳＫ重组建立了ＨＣＭＶ的ＤＮＡ文库,从此文库。中随机筛选出两个重组质粒（ｐＣＭＶ－１和ｐＣＭＶ－２）,用ＢａｍＨＩ分析证明其中所含的病毒ＤＮＡ片段的大小分别为１．０ｋｂ和７．５ｋｂ,将这两种质粒大量扩增纯化后,用光生物素进行标记作为探针,证明其只与ＨＣＭＶ反应,与正常人细胞ＤＮＡ及Ⅰ型和Ⅱ型单纯疤疹病毒ＤＮＡ无交叉反应。     

Analysis of the quaternary structure of the putative HCMV portal protein PUL104

In this report we analyze the UL104 open reading frame of human cytomegalovirus (HCMV) genome that encodes the putative portal protein. An affinity-purified monospecific antiserum directed against a GST-UL104 fusion protein identified proteins of approximate M(r) 73000 and 145000 in HCMV-infected cells and purified virions. Furthermore, using an in vitro assay the ability of pUL104 to bind double-stranded DNA was shown. Analysis under native conditions of pUL104 revealed that the monomeric and dimeric forms of the protein also form high molecular weight complexes upon sucrose gradient centrifugation. The protein has been purified from recombinant baculovirus UL104 infected cells. The quaternary structure of rpUL104 was investigated by gel permeation chromatography and electron microscopy. The purified rpUL104 was found to assemble into high molecular weight complexes, a prerequisite of portal proteins which form channels for DNA import into capsids.     

Rapid lysis of vaccinia virus on neutral sucrose gradients with release of intact DNA

A new and improved procedure has been developed for the isolation of intact DNA genomes from purified vaccinia virions. Purified virions are layered on a neutral sucrose gradient containing sodium dodecyl sulfate, 2-mercaptoethanol and sodium chloride at neutral pH. Intact viral DNA free from protein and fully sensitive to DNase I is rapidly released from the virions.     

水稻叶绿体DNA的提取和纯化

以籼稻品种珍讪97B为材料,采用溶液捣碎和不连续蔗糖梯度离心的方法提取了籼稻的叶绿体DNA,DNA经限制性内切酶酶解和琼脂糖胶电泳可以得到清晰的条带,来自蚕豆的核酮糖—1,5—二磷酸羧化氧合酶大亚基基因探针和23SrRNA基因探针可以与酶切条带杂交,由此确定了含这二种基因的BamHI酶切片段。     

Physical and Biological Properties of Dengue-2 Virus and Associated Antigens

Dengue virus suspensions from mouse brain and cell culture were fractionated into three components by rate zonal centrifugation in sucrose gradients. Infectious virus sedimented in a single zone and possessed hemagglutinating (HA) and complement fixing (CF) activity. Electron micrographs showed the virion to be a spherical particle 48 to 50 nm in diameter with 7-nm spherical structures on its surface. Buoyant density in CsCl of virions from mouse brain was estimated at 1.22 g/cm(3) and from cell culture at 1.24 g/cm(3). During centrifugation of virions in CsCl, an additional HA component appeared with a buoyant density of 1.18 g/cm(3). It was shown in electron micrographs to consist of virion fragments. A noninfectious component with HA and CF activity sedimented in sucrose more slowly than intact virus, had a buoyant density of 1.23 g/cm(3) in CsCl, and appeared as "doughnut" forms measuring 13.8 to 14 nm in diameter. A third component, with CF activity and no HA activity, sedimented very little in sucrose gradients. Particles of the same size and shape as the spherical subunits on the surface of the virion were observed in electron micrographs.