Monoclonal antibodies against Aleutian disease virus distinguish virus strains and differentiate sites of virus replication from sites of viral antigen sequestration.

Monoclonal antibodies (mAbs) were used to study antigenic differences among strains of Aleutian disease virus (ADV) and to characterize viral proteins in vitro and in vivo. A number of ADV field strains could be discriminated, and highly virulent Utah I ADV was clearly delineated from the tissue culture-adapted avirulent ADV-G strain. This specificity could be demonstrated by indirect immunofluorescence against infected cultures of Crandell feline kidney cells or against tissues of Utah I ADV-infected mink. Viral antigens were demonstrated in both the nuclei and the cytoplasm of infected tissue culture cells. However, in mink mesenteric lymph node, spleen, and liver, viral antigen was observed only in the cytoplasm. Absence of nuclear fluorescence suggested that the detected antigen represented phagocytized viral antigens rather than replicating virus. This conclusion was supported by the finding that mAbs reactive only against low-molecular-weight polypeptides derived from intact viral proteins gave the same pattern of in vivo fluorescence as mAbs with broad reactivity for large or small (or both) viral polypeptides. The distribution of infected cells was the same as that described for macrophages in these tissues and suggested that cells of the reticuloendothelial system had sequestered viral antigens.     

Cell Fusion by Newcastle Disease Virus in the Absence of RNA Synthesis

OUR studies on the cytopathic effects of Newcastle disease virus (NDV) grown in chick embryo fibroblast cell cultures have shown that the principal cytopathic effect involves the formation of polykaryocytes by cell fusion (our unpublished work). This ability is related directly to the virulence of the infecting strain; those pathogenic for chick embryos readily induce cell fusion whereas avirulent strains induce little or no fusion¹. We now report that, although protein synthesis is required for NDV-induced cell fusion, RNA synthesis is not. Furthermore, blocking of RNA synthesis significantly increases cell fusion by avirulent strains.     

Isolation and characterization of heat-resistant (HR) mutants of Newcastle disease virus

Heat-resistant (HR) mutants (MR 70 and HR 74) of Newcastle disease virus (NDV) which exhibited significantly higher thermostability in their infectivity than wild-type virus were isolated and characterized. They differ from each other in their plaque morphology; HR 70 produces small turbid plaques, whereas those of HR 74 are large and clear. Cytopathogenicity of these mutants is much lower than that of the wild-type virus in cultured cells such as CEF, LLCMK2 and HeLa cells. Moreover, these HR mutants exhibited extended mean embryo survival times. Synthesis of cellular RNA's and proteins in cells infected with HR mutants was not significantly reduced under conditions in which synthesis of these macromolecules was strongly reduced in cells infected with wild-type virus. No significant differences were observed between HR mutants and wild-type virus in their other phenotypic characteristics such as the capacity for interferon production, growth characteristics at a low multiplicity of infection, and cleavage of viral glycoproteins in infected cells. From these findings, it was suggested that the inhibitory effect of virus infection on cellular macromolecular synthesis is a possible determinant of cytopathogenicity of NDV.     

Complementation between avirulent Newcastle disease virus and a fusion protein gene expressed from a retrovirus vector: requirements for membrane fusion.

The cDNA derived from the fusion gene of the virulent AV strain of Newcastle disease virus (NDV) was expressed in chicken embryo cells by using a retrovirus vector. The fusion protein expressed in this system was transported to the cell surface and was efficiently cleaved into the disulfide-linked F1-F2 form found in infectious virions. The cells expressing the fusion gene grew normally and could be passaged many times. Monolayers of these cells would plaque, in the absence of trypsin, avirulent NDV strains (strains which encode a fusion protein which is not cleaved in tissue culture). Fusion protein-expressing cells would not fuse if mixed with uninfected cells or uninfected cells expressing the hemagglutinin-neuraminidase (HN) protein. However, the fusion protein-expressing cells, if infected with avirulent strains of NDV, would fuse with uninfected cells, suggesting that fusion requires both the fusion protein and another viral protein expressed in the same cell. Fusion was also seen after transfection of the HN protein gene into fusion protein-expressing cells. Thus, the expressed fusion protein gene is capable of complementing the virus infection, providing an active cleaved fusion protein required for the spread of infection. However, the fusion protein does not mediate cell fusion unless the cell also expresses the HN protein. Fusion protein-expressing cells would not plaque influenza virus in the absence of trypsin, nor would influenza virus-infected fusion protein-expressing cells fuse with uninfected cells. Thus, the influenza virus HA protein will not substitute for the NDV HN protein in cell-to-cell fusion.     

Integrity of membrane lipid rafts is necessary for the ordered assembly and release of infectious Newcastle disease virus particles

Membrane lipid raft domains are thought to be sites of assembly for many enveloped viruses. The roles of both classical lipid rafts and lipid rafts associated with the membrane cytoskeleton in the assembly of Newcastle disease virus (NDV) were investigated. The lipid raft-associated proteins caveolin-1, flotillin-2, and actin were incorporated into virions, while the non-lipid raft-associated transferrin receptor was excluded. Kinetic analyses of the distribution of viral proteins in lipid rafts, as defined by detergent-resistant membranes (DRMs), in non-lipid raft membranes, and in virions showed an accumulation of HN, F, and NP viral proteins in lipid rafts early after synthesis. Subsequently, these proteins exited the DRMs and were recovered quantitatively in purified virions, while levels of these proteins in detergent-soluble cell fractions remained relatively constant. Cholesterol depletion of infected cells drastically altered the association of viral proteins with DRMs and resulted in an enhanced release of virus particles with reduced infectivity. Decreased infectivity was not due to effects on subsequent virus entry, since the extraction of cholesterol from intact virus did not significantly reduce infectivity. Particles released from cholesterol-depleted cells had very heterogeneous densities and altered ratios of NP and glycoproteins, demonstrating structural abnormalities which potentially contributed to their lowered infectivity. Taken together, these results indicate that lipid rafts, including cytoskeleton-associated lipid rafts, are sites of NDV assembly and that these domains are important for ordered assembly and release of infectious Newcastle disease virus particles.     

An attenuating mutation in the 2A protease of swine vesicular disease virus, a picornavirus, regulates cap- and internal ribosome entry site-dependent protein synthesis

Virulent and avirulent strains of swine vesicular disease virus (SVDV), a picornavirus, have been characterized previously. The major determinants for attenuation have been mapped to specific residues in the 1D-2A-coding region. The properties of the 2A proteases from the virulent and avirulent strains of SVDV have now been examined. Both proteases efficiently cleaved the 1D/2A junction in vitro and in vivo. However, the 2A protease of the avirulent strain of SVDV was much less effective than the virulent-virus 2A protease at inducing cleavage of translation initiation factor eIF4GI within transfected cells. Hence the virulent-virus 2A protease is much more effective at inhibiting cap-dependent protein synthesis. Furthermore, the virulent-virus 2A protease strongly stimulated the internal ribosome entry sites (IRESs) from coxsackievirus B4 and from SVDV, while the avirulent-virus 2A protease was significantly less active in these assays. Thus, the different properties of the 2A proteases from the virulent and avirulent strains of SVDV in regulating protein synthesis initiation reflect the distinct pathogenic properties of the viruses from which they are derived. A single amino acid substitution, adjacent to His21 of the catalytic triad, is sufficient to confer the characteristics of the virulent-strain 2A protease on the avirulent-strain protease. It is concluded that the efficiency of picornavirus protein synthesis, controlled directly by the IRES or indirectly by the 2A protease, can determine virus virulence.     

Homologous Interference by Incomplete Sendai Virus Particles: Changes in Virus-Specific Ribonucleic Acid Synthesis

Incomplete Sendai virus particles (I particles) interfered with the replication of several strains of infectious Sendai virions (standard virus) but not with the replication of Newcastle disease virus, mumps virus, or Sindbis virus. I particles did not induce interferon, and ultraviolet irradiation of I particles abolished their ability to interfere. Protein synthesis was not necessary to establish interference. The degree of interference depended on the interval between exposure of cells to the I particles and challenge by standard virus, and this was reflected in the degree of inhibition of virus-specific ribonucleic acid (RNA) synthesis in infected cells. The most dramatic change was decreased accumulation of 50S virus-specific RNA in infected cells. RNA species sedimenting slower than 50S were not as markedly reduced in total amount, but hybridization experiments showed that a substantial portion of these slowly sedimenting RNA species were plus strands, presumably representing replicas of the RNA species in I particles. When I particles in insufficient numbers to interfere were added to cells as late as 8 hr after standard virus, there were no obvious changes in virus-specific RNA species in the cells; however, significant amounts of 19 and 25S RNA species, representing progeny of the I particles, appeared in the culture medium. It was concluded that interference was an intracellular event affecting an early step in virus replication. Competition by I particles for cell sites or substrates needed by standard virus seemed a less likely mechanism of interference than competition for enzymes specified by standard virus.     

Characterization of Newcastle Disease Viruses in Wild and Domestic Birds in Luxembourg from 2006 to 2008

Newcastle disease virus (NDV) is one of the most important viral diseases of birds. Wild birds constitute a natural reservoir of low-virulence viruses, while poultry are the main reservoir of virulent strains. Exchange of virus between these reservoirs represents a risk for both bird populations. Samples from wild and domestic birds collected between 2006 and 2010 in Luxembourg were analyzed for NDV. Three similar avirulent genotype I strains were found in ducks during consecutive years, suggesting that the virus may have survived and spread locally. However, separate introductions cannot be excluded, because no recent complete F gene sequences of genotype I from other European countries are available. Detection of vaccine-like strains in wild waterbirds suggested the spread of vaccine strains, despite the nonvaccination policy in Luxembourg. Among domestic birds, only one chicken was positive for a genotype II strain differing from the LaSota vaccine and exhibiting a so-far-unrecognized fusion protein cleavage site of predicted low virulence. Three genotype VI strains from pigeons were the only virulent strains found. The circulation of NDV in wild and free-ranging domestic birds warrants continuous surveillance because of increased concern that low-virulence wild-bird viruses could become more virulent in domestic populations.     

Newcastle Disease Virus Fusion Protein Is the Major Contributor to Protective Immunity of Genotype-Matched Vaccine

Virulent strains of Newcastle disease virus (NDV) can cause devastating disease in chickens worldwide. Although the current vaccines are substantially effective, they do not completely prevent infection, virus shedding and disease. To produce genotype-matched vaccines, a full-genome reverse genetics system has been used to generate a recombinant virus in which the F protein cleavage site has been changed to that of avirulent vaccine virus. In the other strategy, the vaccines have been generated by replacing the F and HN genes of a commercial vaccine strain with those from a genotype-matched virus. However, the protective efficacy of a chimeric virus vaccine has not been directly compared with that of a full-genome virus vaccine developed by reverse genetics. Therefore, in this study, we evaluated the protective efficacy of genotype VII matched chimeric vaccines by generating three recombinant viruses based on avirulent LaSota (genotype II) strain in which the open reading frames (ORFs) encoding the F and HN proteins were replaced, individually or together, with those of the circulating and highly virulent Indonesian NDV strain Ban/010. The cleavage site of the Ban/010 F protein was mutated to the avirulent motif found in strain LaSota. In vitro growth characteristics and a pathogenicity test indicated that all three chimeric viruses retained the highly attenuated phenotype of the parental viruses. Immunization of chickens with chimeric and full-length genome VII vaccines followed by challenge with virulent Ban/010 or Texas GB (genotype II) virus demonstrated protection against clinical disease and death. However, only those chickens immunized with chimeric rLaSota expressing the F or F plus HN proteins of the Indonesian strain were efficiently protected against shedding of Ban/010 virus. Our findings showed that genotype-matched vaccines can provide protection to chickens by efficiently preventing spread of virus, primarily due to the F protein.     

Intrinsic resistance of feline peritoneal macrophages to coronavirus infection correlates with in vivo virulence.

Cats infected with virulent feline coronavirus strains develop feline infectious peritonitis, an invariably fatal, immunologically mediated disease; avirulent strains cause either clinically inapparent infection or mild enteritis. Four virulent coronavirus isolates and five avirulent isolates were assessed by immunofluorescence and virus titration for their ability to infect and replicate in feline peritoneal macrophages in vitro. The avirulent coronaviruses infected fewer macrophages, produced lower virus titers, were less able to sustain viral replication, and spread less efficiently to other susceptible macrophages than the virulent coronaviruses. Thus, the intrinsic resistance of feline macrophages may play a pivotal role in the outcome of coronavirus infection in vivo.     

Newcastle Disease Virus Infection of L Cells

Newcastle disease virus (NDV) California strain reportedly grows poorly in L cells but replicates very well in chicken embryo cells. NDV-infected L cell cultures show a characteristic virus growth curve with respect to uridine incorporation, but plaque assays of the virus produced 24 h postinfection (PI) show no infectious particles when assayed on L cell monolayers and only a very low titer on chick cell monolayers. Plasma membranes isolated and purified from infected L cells 8 h PI contain all of the major virion proteins. In addition, NDV-infected L cells show a 50% loss of H-2 antigenic activity, a phenomenon previously observed in cells productively infected with vesicular stomatitis virus. These results suggest that at least part of the normal process of NDV maturation occurs in NDV-infected L cells. Sodium dodecyl sulfate-polyacrylamide gel patterns of supernatant virus purified from cells radiolabeled with amino acids from 3 to 24 h PI in the presence of actinomycin D show that all the major NDV structural proteins are present. Electron micrographs of NDV-infected L cells show extensive virus maturation at cell membranes. It can be concluded that infection of L cells with NDV results in a normal production of virus-specific RNA, synthesis of all the major structural proteins, association of the viral envelope proteins with the L cell plasma membrane, and the loss of cell surface H-2 antigenic activity. However, most of the virus particles produced are noninfectious.     

Inhibition of cellular protein synthesis by simultaneous pretreatment of host cells with fowl plague virus and actinomycin D: a method for studying early protein synthesis of several RNA viruses.

A method is described for analysis of viral protein synthesis early after infection when minute amounts of viral proteins are effectively concealed by large amounts of produced host-specific proteins. The method is superior to a radioimmune assay, since all virus-induced proteins can be measured independent of their immunological reactivity. Host-specific protein synthesis can be suppressed by infection with fowl plague virus. Addition of actinomycin C 1.25 h postinfection does not prevent this suppression, but it does block effectively the formation of fowl plague virus-specific proteins. Such cells synthesize only small amounts of cellular proteins, as revealed by polyacrylamide electrophoresis. They can be superinfected with several different enveloped viruses, however, without significant diminution of virus yeilds. In pretreated cells the eclipse is shortened for Semliki Forest virus, Sindbis virus, and vesicular stomatitis virus, but prolonged for Newcastle disease virus. The onset of protein synthesis, specific for the superinfecting virus, could be clearly demonstrated within 1 h after superinfection. At this time, in cells superinfected with Semliki Forest virus, great amounts of NSP 75 (nonstructural protein; molecular weight, 75 X 10(3)) and reduced amounts of the core protein C could be deomonstrated. The precursor glycoprotein NSP 68 is followed by a new polypeptide, NSP 65: three proteins with molecular weights exceeding 100 X 10(3) were observed which are missing later in the infectious cycle. Similar results were obtained after superinfection with Sindbis virus. The formation of a new polypeptide with a molecular weight of about 80 X 10(3) was detected. After superinfection with vesicular stomatis virus or Newcastle disease virus the formation of new proteins, characteristic for the early stage of infeciton, was not observed.     

In vitro secondary generation of cytotoxic T lymphocytes in mice with mumps virus and their mumps-specific cytotoxicity among paramyxoviruses.

Murine cells (L929, MC57G, and P815 mastocytoma) defectively infected with the egg-adapted vaccine strain of mumps virus were found to be susceptible to cytotoxic T-lymphocyte (CTL)-mediated lysis. In vitro secondary, but not in vivo primary, generated CTL caused cytolysis of these targets in an H-2-restricted manner. UV-inactivated-mumps virus-coated murine cells were also found to be susceptible to CTL-mediated lysis. Comparisons of murine CTL-mediated lysis by three paramyxoviruses (mumps, Sendai, and Newcastle disease viruses) indicated that no cross-reactivity occurred. The CTL response with mumps virus exhibited specific unresponsiveness patterns, as influenced by the H-2 K/D regions of the mouse strains, that were partially different from those of Sendai virus and Newcastle disease virus.     

Persistent Infection of Cells in Culture by Measles Virus III. Comparison of Virus-Specific RNA Synthesized in Primary and Persistent Infection in HeLa Cells

The pattern of actinomycin D-resistant RNA synthesis was examined during primary infection of HeLa cells by virulent Edmonston measles virus and in two HeLa clones persistently infected by the same strain of virus. One of these clones, K11, produces infectious virus of low virulence for HeLa cells, and the other, K11A-HG-1, has thus far failed to yield infectious virus. The patterns of virus-specific RNA synthesized in these three types of infection are qualitatively similar to each other and to the patterns of virus-specific RNA synthesis in other paramyxovirus infections. There were, however, quantitative differences. In addition, virions of the virulent Edmonston strain of measles virus were found to contain high-molecular-weight RNA with a sedimentation constant identical to that of Newcastle disease virus.     

Bovine Diarrhea Virus

Marked cytopathic changes were induced by challenge with Newcastle disease (ND) virus in bovine testicle or kidney cell cultures which were previously infected with non-cytopathogenic strains of bovine diarrhea (BD) virus. No cytopathic changes were induced by ND virus in similar cells not infected with BD virus. The development of cytopathic effect was shown to be associated with enhancement of ND virus replication. This exalting effect of BD virus appears to be dependent on infectivity, since the effect was inhibited when infection of the cells with BD virus was blocked by specific antiserum. Various factors involved in the phenomenon were investigated and an in vitro method (END) for the assay of BD virus and its antibodies was developed. The use of this method eliminates the difficulties in recognizing non-cytopathogenic strains of BD virus which hampered systematic investigations of the nature and behavior of BD virus as well as of the natural history and pathogenesis of the infection in cattle.     

Design and characterization of viral polypeptide inhibitors targeting Newcastle disease virus fusion

Paramyxovirus infections can be detected worldwide with some emerging zoonotic viruses and currently there are no specific therapeutic treatments or vaccines available for many of these diseases. Recent studies have demonstrated that peptides derived from the two heptad repeat regions (HR1 and HR2) of paramyxovirus fusion proteins could be used as inhibitors of virus fusion. The mechanism underlying this activity is in accordance with that of class I virus fusion proteins, of which human immunodeficiency virus (HIV) and influenza virus fusion proteins are members. For class I virus fusion proteins, the HR1 fragment binds to HR2 to form a six-helix bundle with three HR1 fragments forming the central coiled bundle surrounded by three coiled HR2 fragments in the post fusion conformational state (fusion core). It is hypothesized that the introduced exogenous HR1 or HR2 can compete against their endogenous counterparts, which results in fusion inhibition. Using Newcastle disease virus (NDV) as a model, we designed several protein inhibitors, denoted HR212 as well asHR121 and 5-Helix, which could bind the HR1 or HR2 region of fusion protein, respectively. All the proteins were expressed and purified using a GST-fusion expression system in Escherichia coli. The HR212 or GST-HR212 protein, which binds the HR1 peptide in vitro, displayed inhibitory activity against NDV-mediated cell fusion, while the HR121 and 5-Helix proteins, which bind the HR2 peptide in vitro, inhibited virus fusion from the avirulent NDV strain when added before the cleavage of the fusion protein. These results showed that the designed HR212, HR121 or 5-Helix protein could serve as specific antiviral agents. These data provide additional insight into the difference between the virulent and avirulent strains of NDV.     

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.     

Biochemical Consequences of Type 2 Adenovirus and Simian Virus 40 Double Infections of African Green Monkey Kidney Cells

African green monkey kidney (AGMK) cells were nonpermissive hosts for type 2 adenovirus although the restriction was not complete; when only 3 plaque-forming units/cell was employed as the inoculum, the viral yield was about 0.1% of the maximum virus produced when simian virus 40 (SV40) enhanced adenovirus multiplication. The viral yield of cells infected only with type 2 adenovirus increased as the multiplicity of infection was increased. Type 2 adenovirus could infect almost all AGMK cells in culture; adenovirus-specific early proteins and DNA were synthesized in most cells, but small amounts of late proteins were made in relatively few cells. Even when cells were infected with both SV40 and adenovirus, only about 50% were permissive for synthesis of adenovirus capsid proteins. Approximately the same quantity of adenovirus deoxyribonucleic acid (DNA) was synthesized in the restricted as in the SV40-enhanced infection. However, in cells infected with SV40 and type 2 adenovirus, replication of SV40 DNA was blocked, multiplication of SV40 was accordingly inhibited, and synthesis of host DNA was not stimulated. To enhance propagation of type 2 adenovirus, synthesis of an early SV40 protein was essential; 50 mug of cycloheximide per ml prevented the SV40-induced enhancement of adenovirus multiplication, whereas 5 x 10(-6)m 5-fluoro-2-deoxyuridine did not abrogate the enhancing phenomenon.