Relationships Among Virus Spread, Cytopathogenicity, and Virulence as Revealed by the Noncytopathic Mutants of Newcastle Disease Virus

We have studied protein synthesis in cultured cells infected with the six noncytopathic (nc) mutants of the Australia-Victoria strain (AV-WT) of Newcastle disease virus and their plaque-forming revertants. Virus-specific polypeptides accumulated at 30 to 63% of wild-type levels in nc mutant-infected cells and between 66 and 175% of wild-type levels in revertant-infected cells. An exception was the L polypeptide, which accumulated in nc mutant-infected cells at only 5 to 20% of the levels found in wild-type infection. The reduced accumulation of the L polypeptide did not appear to be due to increased degradation of that polypeptide. A new polypeptide (X) accumulated instead of polypeptide P in cells infected with mutants nc4 or nc16 and in virions released from them. Peptide mapping identified X as an altered form of P. A revertant of mutant nc4 (nc4S1), which forms larger hemadsorbing spots, but still does not form plaques, accumulated P instead of the X polypeptide. Thus, a lesion in P can affect virus spread without affecting cytopathogenicity. Virions of mutant nc7 and two naturally occurring avirulent strains of Newcastle disease virus (NJ LaSota and B1-Hitchner) contained polypeptides (F₇ and F_A, respectively) related to, but migrating more rapidly than, F₀ in sodium dodecyl sulfate-polyacrylamide gels. As previously reported for avirulent strains, a brief treatment of nc7 virions with trypsin converted F₇ to F and increased infectivity. Similarly, culturing nc7-infected cells in the presence of trypsin facilitated fusion from within and viral spread from cell to cell. A plaque-forming revertant of nc7 still accumulated F₇ in virions, indicating that the lesions responsible for the F₇ and noncytopathic phenotypes are genetically separable. The virulent parental strain, AV-WT, exhibited a mean embryo death time of 42 h. Both the larger-spot-forming revertant of nc4 (nc4S1) and the small-plaque-forming revertant of nc7 exhibited a decrease in mean embryo death time (increase in virulence) from 74 to 63 h. A second-step, plaque-forming revertant derived from nc4S1 (nc4S1R1) exhibited a further decrease in mean embryo death time from 63 to 44 h. The results suggest that the F_A-F₇ and X lesions affect the ability of virus to spread from cell to cell. In addition, these lesions appear to be genetically separable from those responsible for the noncytopathic phenotype. However, both types of lesions cause an extension of mean embryo death time and, thus, may be relevant to virulence in vivo.     

利用寡核苷酸微阵列技术对HBV、HCV、HIV、HAV、GBV-C/HGV和B19进行同时监测的初步研究

探讨研制能同时检测HBV、HCV、HIV、HAV、GBV-C/HGV和B19的微阵列监控芯片。根据病毒公开发表序列,序列比对,得出保守区域,设计病毒的特异性检测探针,同时设置阴性、阳性参照探针,制备监控微阵列。利用随机引物PCR方法标记样品中的病毒靶序列,标记产物与微阵列上的探针杂交,清洗、扫描后进行结果分析。通过对质粒或模式分子的检测以及经HBV、HCV、HIV临床标本的验证,发现该微阵列监控芯片具有良好的特异性。其对质粒的检测灵敏度可达102病毒拷贝数,对临床标本的检测灵敏度可达103病毒拷贝数。此外,该微阵列监控芯片可检测出病毒混合感染血清。为微阵列监控芯片应用于此六种血液病毒的检测打下一定的基础。     

Innovative Toolbox for the Quantification of Drosophila C Virus,Drosophila A Virus,and Nora Virus

Quantification of viral replication underlies investigations into host-virus interactions. In Drosophila melanogaster, persistent infections with Drosophila C virus, Drosophila A virus, and Nora virus are commonly observed in nature and in laboratory fly stocks. However, traditional endpoint dilution assays to quantify infectious titers are not compatible with persistently infecting isolates of these viruses that do not cause cytopathic effects in cell culture. Here we present a novel assay based on immunological detection of Drosophila C virus infection that allows quantification of infectious titers for a wider range of Drosophila C virus isolates. We also describe strand specific RT-qPCR assays for quantification of viral negative strand RNA produced during Drosophila C virus, Drosophila A virus, and Nora virus infection. Finally, we demonstrate the utility of these assays for quantification of viral replication during oral infections and persistent infections with each virus.     

The Lipidomes of Vesicular Stomatitis Virus,Semliki Forest Virus,and the Host Plasma Membrane Analyzed by Quantitative Shotgun Mass Spectrometry

Although enveloped virus assembly in the host cell is a crucial step in the virus life cycle, it remains poorly understood. One issue is how viruses include lipids in their membranes during budding from infected host cells. To analyze this issue, we took advantage of the fact that baby hamster kidney cells can be infected by two different viruses, namely, vesicular stomatitis virus and Semliki Forest virus, from the Rhabdoviridae and Togaviridae families, respectively. We purified the host plasma membrane and the two different viruses after exit from the host cells and analyzed the lipid compositions of the membranes by quantitative shotgun mass spectrometry. We observed that the lipid compositions of these otherwise structurally different viruses are virtually indistinguishable, and only slight differences were detected between the viral lipid composition and that of the plasma membrane. Taken together, the facts that the lipid compositions of the two viruses are so similar and that they strongly resemble the composition of the plasma membrane suggest that these viruses exert little selection in including lipids in their envelopes.Enveloped viruses acquire their lipid envelope from the membranes of host cells (43). In this process, the nucleocapsid or the nucleocapsid-matrix complex of the viruses buds out of the cell and becomes enveloped by a segment of the host membrane. This membrane segment is modified during the budding process, such that virally encoded membrane proteins are included in the viral envelope, while most host proteins are excluded. Since viruses usually do not carry lipid-synthesizing enzymes, the lipids in the viral envelope are derived from the host membrane. The lipid compositions of enveloped viruses have been studied for years (2, 15, 17, 18, 23, 25, 34, 36, 38, 40). One question that remains to be answered is whether the lipids are included passively, and thus the lipid composition of the envelope reflects the lipid composition of the host membrane, or whether lipid sorting occurs, leading to selective inclusion of some lipids and exclusion of others. This issue has been complicated by the fact that the lipid bilayer is no longer considered a homogenous liquid but contains fluctuating nanoscale assemblies of sphingolipids, saturated phospholipids, cholesterol (Chol), and proteins, called lipid rafts (13, 44). Lipid rafts can be induced to coalesce—usually by protein-protein interactions—into larger, dynamic platforms that function in signal transduction, intracellular membrane transport, and other membrane functions (45). It was also proposed that viruses make use of these membrane domains during their exit from cells (29, 32).A major complication in comparing viral envelopes with host cell membranes is the difficulty in obtaining host cell membranes of purity similar to that of the easily purified viruses. Many studies are faulted by the impurity of the cell membranes analyzed. Moreover, the early work in this field employed conventional analytical methods (such as thin-layer chromatography) that provide only semiquantitative estimates of the total abundance of the major lipid classes. Most importantly, lipid species diversity could not be analyzed. Recent developments in mass spectrometry (MS) have enabled comprehensive and quantitative analyses of lipidomes at the level of individual molecular species. The lipidomes of human immunodeficiency virus (HIV), murine leukemia virus (6, 7), and several bacteriophages (20, 21) were recently analyzed by these new methods.This paper focuses on two well-characterized enveloped viruses, Semliki Forest virus (SFV) and vesicular stomatitis virus (VSV). SFV is an RNA virus belonging to the Togaviridae family of the Alphaviridae that acquires its envelope by budding from the host cell plasma membrane (PM) (46). Early studies analyzed the lipid composition of the viral envelope and also that of the host cell PM (39, 40). These studies revealed strong similarity between the envelope of SFV and the host PM, but one important discrepancy was the higher Chol-to-phospholipid ratio in the virus.VSV is an RNA virus belonging to the Rhabdoviridae family and also hijacks its envelope from the host cell PM (35), but the lipid specificity of the budding process remains controversial. The most recent studies claim that VSV buds from localized regions that do not reflect the average composition of the PM (23, 36). It has also been claimed that lipid rafts are involved in VSV envelope assembly during budding (37).We used BHK-21 cells as host cells to purify SFV and VSV. The purposes of this study were (i) to establish a robust, comprehensive, and quantitative method to analyze lipidomes, including the full complement of glycerolipid, glycerophospholipid, and sphingolipid species as well as Chol; (ii) to establish a protocol for purification of PM suitable for MS analysis; and (iii) to analyze and compare the lipidomes of SFV, VSV, and the BHK-21 PM.We found that the lipidomes of SFV and VSV are similar in molecular composition and are closely related to that of the BHK-21 PM. The small differences observed could be explained by the high degrees of curvature generated during the viral budding process.     

Nucleocapsid Protein Subunits of Simian Virus 5, Newcastle Disease Virus, and Sendai Virus

Helical nucleocapsids of each of the paramyxoviruses simian virus 5 (SV5), Newcastle disease virus (NDV), and Sendai virus have been isolated in two different forms. One form contains larger protein subunits and is obtained from mature virions or infected cells dispersed by ethylenediaminetetraacetic acid. The other form possesses smaller subunits and is obtained from infected cells dispersed by trypsin. The estimated molecular weights of the larger subunits in the three viruses are similar: SV5, 61,000; Sendai virus, 60,000; NDV, 56,000. The smaller nucleocapsid subunits are also very similar: SV5, 43,000; Sendai virus, 46,000; NDV, 47,000. The helical nucleocapsid composed of the smaller subunit appears to be less flexible and more stable than that formed by the larger subunit. There is suggestive evidence that conversion of the larger subunit to the smaller by proteolytic cleavage may occur intracellularly. The possibility that such a mechanism could be involved in the accumulation of nucleocapsid in cells persistently infected with paramyxoviruses is discussed.     

Membrane Fusion Promoted by Increasing Surface Densities of the Paramyxovirus F and HN Proteins: Comparison of Fusion Reactions Mediated by Simian Virus 5 F, Human Parainfluenza Virus Type 3 F, and Influenza Virus HA

The membrane fusion reaction promoted by the paramyxovirus simian virus 5 (SV5) and human parainfluenza virus type 3 (HPIV-3) fusion (F) proteins and hemagglutinin-neuraminidase (HN) proteins was characterized when the surface densities of F and HN were varied. Using a quantitative content mixing assay, it was found that the extent of SV5 F-mediated fusion was dependent on the surface density of the SV5 F protein but independent of the density of SV5 HN protein, indicating that HN serves only a binding function in the reaction. However, the extent of HPIV-3 F protein promoted fusion reaction was found to be dependent on surface density of HPIV-3 HN protein, suggesting that the HPIV-3 HN protein is a direct participant in the fusion reaction. Analysis of the kinetics of lipid mixing demonstrated that both initial rates and final extents of fusion increased with rising SV5 F protein surface densities, suggesting that multiple fusion pores can be active during SV5 F protein-promoted membrane fusion. Initial rates and extent of lipid mixing were also found to increase with increasing influenza virus hemagglutinin protein surface density, suggesting parallels between the mechanism of fusion promoted by these two viral fusion proteins.     

Studies of Nondefective Adenovirus 2-Simian Virus 40 Hybrid Viruses IV. Characterization of the Simian Virus 40 Ribonucleic Acid Species Induced by Wild-Type Simian Virus 40 and by the Nondefective Hybrid Virus, Ad2+ND1

Ad2(+)ND(1), a nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid virus, has been previously shown to contain a small segment of the SV40 genome covalently linked to Ad2 deoxyribonucleic acid (DNA). The SV40 portion of this hybrid virus has been characterized by relating the SV40-specific ribonucleic acid (RNA) sequences transcribed from the Ad2(+)ND(1) DNA to those transcribed from the DNA of SV40 itself. RNA-DNA hybridization-competition studies indicate that the SV40 component of Ad2(+)ND(1) consists of some, but not all, of that part of the SV40 genome which is transcribed early, i.e., prior to viral DNA replication, in SV40 lytic infection.     

两种单负链RNA呼吸道病毒——流感病毒和呼吸道合胞病毒感染对粘蛋白1表达调控差异的研究

为进一步了解流感病毒(IFZ)是否像呼吸道合胞病毒(RSV)一样在感染过程中可以上调呼吸道上皮细胞粘蛋白1(MUC1)的表达,进而限制炎症的发展。我们利用qRT-PCR和Western Blot检测两种呼吸道单负链RNA病毒感染人呼吸道上皮细胞系——A549细胞后对MUC1的表达调控。分别利用人喉上皮细胞系——HEp-2细胞系和MDCK细胞系培养并收集RSV和IFZ。相同滴度的两种病毒分别感染A549细胞24h后,分别裂解各组细胞,收集总RNA,应用qRT-PCR检测MUC1mRNA的表达情况;或相同滴度的两种病毒分别感染A549细胞24h和48h后,裂解细胞收集总蛋白,同时收集细胞培养上清,应用Western Blot检测MUC1蛋白的表达情况,应用ELISA检测上清中TNF-α水平。结果显示,虽然两种病毒都能上调TNF-α水平,但只有RSV可以上调呼吸道上皮细胞MUC1表达,并呈剂量效应,而IFZ不能上调呼吸道上皮细胞MUC1的表达。本研究首次探讨两种常见呼吸道单负链RNA病毒感染呼吸道上皮细胞后对MUC1表达调控的差异,初步证实临床上IFZ感染后病情自限性的机制不同于RSV感染,与MUC1的表达上调无关。     

Proteins and Glycoproteins of Paramyxoviruses: a Comparison of Simian Virus 5, Newcastle Disease Virus, and Sendai Virus

The polypeptides of three paramyxoviruses (simian virus 5, Newcastle disease virus, and Sendai virus) were separated by polyacrylamide gel electrophoresis. Glycoproteins were identified by the use of radioactive glucosamine as a carbohydrate precursor. The protein patterns reveal similarities among the three viruses. Each virus contains at least five or six proteins, two of which are glycoproteins. Four of the proteins found in each virus share common features with corresponding proteins in the other two viruses, including similar molecular weights. These four proteins are the nucleocapsid protein (molecular weight 56,000 to 61,000), a larger glycoprotein (molecular weight 65,000 to 74,000), a smaller glycoprotein (molecular weight 53,000 to 56,000), and a major protein which is the smallest protein in each virion (molecular weight 38,000 to 41,000).     

Transient Inhibition of Polyoma Virus Synthesis by Sendai Virus (Parainfluenza I) I. Demonstration and Nature of the Inhibition by Inactivated Virus

Superinfection of polyoma virus-infected mouse embryo cells by beta-propiolactone-inactivated Sendai virus resulted in a 90% inhibition of the synthesis of infectious polyoma progeny. The interference is dependent upon the time of superinfection and the concentration of the inactivated virus. The inhibition of polyoma virus synthesis is transient in nature since normal synthesis of polyoma progeny virus is seen upon prolonged incubation. Interferon does not appear to be implicated in the interference. Various aspects of the biological and synthetic capabilities of beta-propiolactone-inactivated Sendai virus are also described.     

Transfection of Fibroblasts by Cloned Abelson Murine Leukemia Virus DNA and Recovery of Transmissible Virus by Recombination with Helper Virus

A cloned, permuted DNA copy of the Abelson murine leukemia virus (A-MuLV) genome was capable of eliciting the morphological transformation of NIH/3T3 fibroblasts when applied to cells in a calcium phosphate precipitate. The efficiency of the process was extremely low, yielding approximately one transformant per microgram of DNA under conditions which give 10(4) transfectants per microgram of other DNAs (e.g., Moloney sarcoma virus proviral DNA). The DNA was able to induce foci, even though the 3' end of the genome was not present. The transforming gene was thus localized to the 5' portion of the genome. The transformed cells all produced viral RNA and the virus-specific P90 protein. Transmissible virus could be rescued from these cells at very low frequencies by superinfection with helper virus; the rescued A-MuLV virus had variable 3' ends apparently derived by recombination with the helper. Dimerization of the permuted A-MuLV cloned genome to reconstruct a complete provirus did not improve transformation efficiency. Virus could be rescued from these transformants, however, at a high efficiency. Cotransfection of the permuted A-MuLV DNA with proviral M-MuLV DNA yielded a significant increase in the efficiency of transformation and cotransfection of dimeric A-MuLV and proviral M-MuLV resulted in a high-efficiency transformation yielding several thousand more transformants per microgram than A-MuLV DNA alone. We propose that helper virus efficiently rescues A-MuLV from transiently transfected cells which would not otherwise have grown into foci. We hypothesize that multiple copies of A-MuLV DNA introduced into cells by transfection are toxic to cells. In support of this hypothesis, we have shown that A-MuLV DNA sequences can inhibit the stable transformation of cells by other selectable DNAs.     

Transient Inhibition of Polyoma Virus Synthesis by Sendai Virus (Parainfluenza I) II. Mechanism of the Interference by Inactivated Virus

The mechanism of the transient inhibition of polyoma virus synthesis by betapropiolactone-inactivated Sendai virus was studied. Polyoma virus early functions did not appear to be affected, although deoxyribonucleic acid (DNA) and structural protein synthesis were inhibited 60 and 35% respectively. The inhibition of macromolecular synthesis was not sufficient to account for the 90% inhibition of infectious progeny formation. Encapsidation of polyoma DNA into mature virions appears to be completely inhibited after superinfection by beta-propiolactone-inactivated Sendai virus. Ultraviolet irradiation of live or beta-propiolactone-inactivated Sendai virus preparations abolishes the interfering capacity, indicating that a functional Sendai virus ribonucleic acid molecule is the interfering component.     

Barley Yellow Mosaic Virus: Purification, Electron Microscopy, Serology, and other Properties of two Types of the Virus

From naturally infected barley plants two types of barley yellow mosaic viruses have been isolated in Federal Republic of Germany. Both are identical in morphology, showing a bimodal length distribution (270–289 nm and 568–600 nm), and in symptomatology. Both induce conspicuous cytoplasmic inclusions of the pinwheel type and laminated aggregates, as well as threedimensional crystal-like arrays of membrane material. The types differ, however, in buoyant density, serology, and transmissibility. One is transmissible by soil as well as mechanically (BaYMV-M), and does not react with a Japanese antiserum to the Japanese virus (BaYMV-J). The other type (BaYMV-NM) is only transmissible by soil and reacts with BaYMV-J-antiserum. From mechanically infected plants BaYMV-M was purified, and an antiserum was produced, from soil-infected plants only mixtured BaYMV-NM and -M could be obtained. BaYMV-NM prevailed during winter, but with rising temperatures in spring BaYMV-M was predominant. BaYMV-M and the -M-NM mixture had each two species of nucleic acids (2.7–2.8 × 10⁶ and 1.4–1.5 × 10⁶ d) and two major protein subunit bands were found in SDS-PAGE (35–36 × 10³ and c. 29 × 10³ d).     

Avoiding Immunity and Apoptosis: Manipulation of the Host Environment by Herpes Simplex Virus and Epstein-Barr Virus

Herpesviruses infect their hosts in early life to establish latent or persistent infection with little damage to the host. They are able to reactivate throughout the life of the host and replicate to produce an infective innoculum in the face of a fully primed immune system. They achieve this lifestyle by careful manipulation of the host environment. Here we review two aspects of host defense—apoptosis of infected cells and cytotoxic T cell recognition of infected cells. We discuss avoidance of these host responses by an α-herpesvirus, herpes simplex virus, and a γ-herpesvirus, Epstein-Barr virus.     

Long-Term Shedding of Influenza Virus,Parainfluenza Virus,Respiratory Syncytial Virus and Nosocomial Epidemiology in Patients with Hematological Disorders

Respiratory viruses are a cause of upper respiratory tract infections (URTI), but can be associated with severe lower respiratory tract infections (LRTI) in immunocompromised patients. The objective of this study was to investigate the genetic variability of influenza virus, parainfluenza virus and respiratory syncytial virus (RSV) and the duration of viral shedding in hematological patients. Nasopharyngeal swabs from hematological patients were screened for influenza, parainfluenza and RSV on admission as well as on development of respiratory symptoms. Consecutive swabs were collected until viral clearance. Out of 672 tested patients, a total of 111 patients (17%) were infected with one of the investigated viral agents: 40 with influenza, 13 with parainfluenza and 64 with RSV; six patients had influenza/RSV or parainfluenza/RSV co-infections. The majority of infected patients (n = 75/111) underwent stem cell transplantation (42 autologous, 48 allogeneic, 15 autologous and allogeneic). LRTI was observed in 48 patients, of whom 15 patients developed severe LRTI, and 13 patients with respiratory tract infection died. Phylogenetic analysis revealed a variety of influenza A(H1N1)pdm09, A(H3N2), influenza B, parainfluenza 3 and RSV A, B viruses. RSV A was detected in 54 patients, RSV B in ten patients. The newly emerging RSV A genotype ON1 predominated in the study cohort and was found in 48 (75%) of 64 RSV-infected patients. Furthermore, two distinct clusters were detected for RSV A genotype ON1, identical RSV G gene sequences in these patients are consistent with nosocomial transmission. Long-term viral shedding for more than 30 days was significantly associated with prior allogeneic transplantation (p = 0.01) and was most pronounced in patients with RSV infection (n = 16) with a median duration of viral shedding for 80 days (range 35–334 days). Long-term shedding of respiratory viruses might be a catalyzer of nosocomial transmission and must be considered for efficient infection control in immunocompromised patients.     

Microtiter Tests for Detecting Antibody in Bovine Serum to Parainfluenza 3 Virus, Infectious Bovine Rhinotracheitis Virus, and Bovine Virus Diarrhea Virus

Microneutralization tests for detection of antibody in bovine serum to three bovine viruses are described. The Madin-Darby bovine kidney cell line was used with parainfluenza 3 virus (PI 3), whereas serially cultivated bovine embryonic kidney cells were used for infectious bovine rhinotracheitis virus and bovine virus diarrhea virus. Comparison of micro-hemagglutination-inhibition (HI) with micro-serum-neutralization (SN) tests for PI 3 showed the SN test to be more sensitive, more specific, and therefore more useful than the HI test for detecting antibody. Although the effect of trypsin-periodate treatment of serum was to reduce the HI titer of numerous sera by a twofold dilution, sufficient evidence could not be found to indicate that nonspecific HI inhibitors to PI 3 are present in bovine sera.