Immunocytochemical colocalization of specific immunoglobulin A with sendai virus protein in infected polarized epithelium.

Immunoglobulin (Ig)A provides the initial immune barrier to viruses at mucosal surfaces. Specific IgA interrupts viral replication in polarized epithelium during receptor-mediated transport, probably by binding to newly synthesized viral proteins. Here, we demonstrate by immunoelectron microscopy that specific IgA monoclonal antibodies (mAbs) accumulate within Sendai virus-infected polarized cell monolayers and colocalize with the hemagglutinin- neuraminidase (HN) viral protein in a novel intracellular structure. Neither IgG specific for HN nor irrelevant IgA mAbs colocalize with viral protein. Treatment of cultures with viral-specific IgA but not with viral-specific IgG or irrelevant IgA decreases viral titers. These observations provide definitive ultrastructural evidence of a subcellular compartment in which specific IgA and viral envelope proteins interact, further strengthening our hypothesis of intracellular neutralization of virus by specific IgA antibodies. Our results have important implications for intracellular protein trafficking, viral replication, and viral vaccine development.

Virus potentiation of tumor vaccine T-cell stimulatory capacity requires cell surface binding but not infection.

This study elucidates a basically new mechanism of function of a virus-modified tumor cell vaccine which has been successful in mouse tumor models (metastatic ESb lymphoma and B16-F10 melanoma) in preventing or delaying metastatic spread and improving survival and which is being tested in clinical studies. Modification of tumor cells by a low dose of Newcastle disease virus (NDV), which caused this therapy effect, led to an augmentation of the tumor-specific cytotoxic CD8 T-cell (CTL) response and to increased CD4 T-helper activity in the absence of an antiviral T-cell response. When various noninfectious NDV preparations, which, according to newly established quantitative tests, had lost one or several of the viral functions, were tested, noninfectious virus particles with inactive fusion proteins and virus inactivated by UV light, which could fuse but could not replicate, were as active as infectious NDV in the tumor-specific CTL response. In contrast, NDV inactivated by heat treatment (NDV-HI) had no effect on the CTL response. NDV-HI, even when added to the cultures in excess, did not modulate the antitumor CTL response, which argues against a nonspecific adjuvant effect. There was no mitogenic effect of NDV. Because NDV-HI was not able to bind to the tumor cell surface and because hemagglutinin-neuraminidase c-DNA transfectants increased antigen-presenting function as virus-modified cells do, we propose that the NDV effect in the CTL response is caused by the introduction of functional viral hemagglutinin-neuraminidase molecules (1000 per virus particle) into the tumor cell surface, thereby facilitating cell-cell interactions through their cell-binding and neuraminidase activity.

Mature, cell-associated HN protein of Newcastle disease virus exists in two forms differentiated by posttranslational modifications.

Characterization of the posttranslational modifications of the mature, cell-associated hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) revealed that the HN protein exists in two forms differentiated by disulfide bonds and glycosylation. One form, HNa, contains intermolecular disulfide bonds and is endoglycosidase H partially resistant. The other form, HNb, is not linked by disulfide bonds and is endoglycosidase H sensitive. Both forms of the protein are modified with fucose indicating transport to the Golgi membranes. Both forms are detected at the cell surface by monoclonal antibody. Furthermore, both forms are transported to the cell surface with identical kinetics. HNa is incorporated into virions. HNb is not incorporated into virions and is presumably degraded. The cDNA derived from the HN gene was expressed from a retrovirus vector. The majority of the protein expressed was in the nonvirion-associated form b. Evidence is presented that the level of gene expression determines the ratio of the two forms of HN protein. At high levels of expression, the virion-associated form is favored while at low levels of expression the nonvirion-associated form is favored. The results presented have implications for persistent infections as well as expression of viral genes from different vectors.

Paramyxovirus mediated cell fusion requires co-expression of both the fusion and hemagglutinin-neuraminidase glycoproteins.

Syncytia formation in either CV-1 or HeLa T4+ cells required recombinant expression of both fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins from the human parainfluenza virus type 3 (HPIV3), human parainfluenza virus type 2 (HPIV2), and simian virus 5 (SV5). In this system, recombinant T7 transcription vectors (pT7-5 or pGEM) containing F or HN, were transfected individually or in combination into cells previously infected with a recombinant vaccinia virus expressing T7 RNA polymerase (vTF7-3). While both proteins were processed and expressed at the cell surface, syncytia formation occurred only when both glycoproteins were co-expressed. The function of HN in the fusion process could not be replaced using lectins or by co-expression of heterologous F and HN proteins. Further, cell fusion was not observed when experiments were performed using individually expressed F and HN proteins in adjacent cells. The data presented in this report support the notion that a specific interaction between both paramyxoviral glycoproteins is required for the formation of syncytia in tissue culture monolayers.

Uptake of porcine rubulavirus (LPMV) by PK-15 cells.

BACKGROUND: The porcine virus denominated La Piedad Michoacan Virus (LPMV) is a member of the family Paramyxoviridae and is the cause of a disease in pigs present only in Mexico. The disease is characterized by meningoencephalitis and respiratory distress in young pigs, epididymitis and orchitis in boars, and reproductive failure and abortion in sows. METHODS: The cytopathology, morphology, and distribution of the hemagglutination neuraminidase (HN) and nucleoprotein (NP) proteins of LPMV were investigated following inoculation into PK-15 cells. The cytopathic effect was characterized by cytoplasmic vacuolation and the formation of syncytia and cytoplasmic inclusion bodies. RESULTS: In immunofluorescence assays using a monoclonal antibody (MAb) against the HN protein at 5-60 min post-infection (early infection), a diffuse immunofluorescence was observed near the cell membrane and adjacent to the nuclear membrane. At 24 h post-infection (late infection), a dust-like immunofluorescence was observed throughout the cytoplasm. LPMV-infected cells incubated with the MAb against the NP protein showed punctate cytoplasmic fluorescence during the early stages of infection. At the late infection stage, these fluorescent particles became larger and were seen predominantly in the cytoplasm of syncytia. This pattern was also apparent by immunohistochemical labeling and immunogold electron microscopy. The latter technique revealed that HN protein was diffusely distributed throughout the cytoplasm. When using the MAb against the NP protein, nucleocapsid organization was the most prominent feature and resulted in the formation of cytoplasmic inclusion bodies visible by light and electron microscopy. Immunogold labeling of purified nucleocapsids was shown by electron microscopy. Virus particles and nucleocapsids were morphologically similar to members of the Paramyxoviridae family. CONCLUSIONS: The morphologic characteristics of the virions and the distribution patterns of the HN and NP proteins in PK-15 infected cells indicate that the mechanisms of LPMV replication are generally similar to those of the members of the Paramyxoviridae family.

Characterization of field isolates of Newcastle disease virus using antipeptide antibodies.

A recent Australian field isolate of Newcastle disease virus (NDV) was analyzed with antipeptide antibodies capable of differentiating between the sequences at the cleavage activation sites of fusion proteins of different NDV pathotypes. The isolate was found to have the same fusion protein cleavage activation signal as the V4 isolate of the Queensland strain of NDV. However, the isolate failed to react with an antibody specific for the carboxyl-terminal extension on the hemagglutinin/neuraminidase (HN)-protein precursor (HNo-protein) of the V4 isolate and other similar strains (e.g., Ulster and D26). Identity of the fusion protein cleavage activation motif of the isolate was confirmed by chemical analysis of purified fusion protein subunits of the isolate. The combination of a V4-like fusion protein cleavage activation motif but lack of an HNo-protein has not been described before, and the findings indicate that the isolate is a distinct strain of NDV. Analysis of a range of isolates from the state of Victoria, Australia, indicated that similar strains have been present in Australia since at least 1976. Other isolates examined appeared to have fusion protein cleavage activation motifs that could not be defined with the fusion-protein-targeted antibodies. These isolates also appeared to lack the HNo-protein characteristic of the Queensland strain.

Onset of protective immunity in chicks after vaccination with a recombinant herpesvirus of turkeys vaccine expressing Newcastle disease virus fusion and hemagglutinin-neuraminidase antigens.

The onset of protective immunity from lethal Newcastle disease virus (NDV) challenge of chicks was determined after vaccination with a recombinant herpes virus of turkeys (HVT) expressing the fusion and hemagglutinin-neuraminidase proteins of NDV. One-day-old specific-pathogen-free chicks devoid of maternal antibodies to NDV were vaccinated with 130 to 3300 plaque forming units of HVT (depending on the trial) and then challenged at 4, 7, 10, and 14 days postvaccination (DPV) with a neurotropic velogenic strain of NDV (GB Texas). The recombinant vaccine afforded 0%, 35-75%, 85%, and 94-100% protection when the vaccinated birds were challenged at 4, 7, 10, and 14 DPV, respectively. In all trials, challenge caused 100% mortality in unvaccinated control chicks. Newcastle disease virus was reisolated from the lung, liver, spleen, and brain of birds dying in all trials regardless of vaccine dosage or time of challenge, except when challenge occurred at 14 DPV.

Brain lesions in chickens experimentally infected with a neuroadapted strain of mesogenic Newcastle disease virus.

Neuroadapted Newcastle disease virus (Q10) was selected by tenth serial passage, in the chicken brain of a mesogenic strain (Q0) originally isolated from quails. Specific pathogen-free birds were inoculated intranasally with one of these viruses. At daily intervals for 7 days and then at 10, 14, and 21 days post-inoculation (PI), two birds from each group were killed and samples of the brain were collected for histopathological and virological examination. Q10 caused severe nonsuppurative encephalitis with nervous signs and high mortality. Lesions characterized by neuronal degeneration and necrosis, perivascular lymphocytic infiltration, and focal or diffuse astrogliosis occurred mainly in the parahippocampal cortex, hippocampus, hyperstriatum, neostriatum, subleptomeningeal and periventricular regions of the cerebrum. Spongy changes with neuronal degeneration and axonal spheroids were also observed in the brain stem of a few cases. The amount of virus in the brain reached a peak on day 4 PI and virus could not be recovered from the brain after 6 days PI. In contrast, Q0 caused nonfatal asymptomatic disease and virus could not be isolated from the brain, sections of which showed only minimal inflammatory changes. This difference in the lesions of the brain might be related to neurovirulence and, neuroadaptation by serial passage may occur by increased efficiency of viral replication in neurons.

Antigen quantification as in vitro alternative for potency testing of inactivated viral poultry vaccines.

Routine batch control of licensed inactivated viral vaccines for poultry usually includes a potency assay as a measure of vaccine efficacy. Potency assays often consist of vaccination-challenge experiments in the target species or in laboratory animals. Instead of measuring the protection of vaccinated animals against virulent pathogens, the serological response after vaccination can be quantified for some vaccines. In vitro antigen quantification assays would be attractive alternatives for the current potency assays because the time and costs involved could be greatly reduced and animal use could be avoided. Such in vitro assays will only be acceptable when the correlation between results and efficacy or potency has been demonstrated convincingly. The results of our studies on antigen quantification assays indicate that, in principle, quantification of viral antigens from inactivated oil-adjuvanted vaccines is feasible and reproducible using specially developed antigen capture ELISAs in combination with specific software for statistical analysis of the ELISA data. We have developed methods to quantify the haemagglutination-neuraminidase (HN) and fusion (F) proteins of Newcastle disease virus (NDV), the viral protein 3 (VP3) of the infectious bursal disease virus (IBDV), and the spike-1 (S1) protein of the infectious bronchitis virus (IBV). Vaccination experiments with inactivated ND vaccines indicate that the in vitro quantified HN- or F-proteins of NDV are reliable indicators of the serological response after vaccination.

The source of avian paramyxoviruses isolated during an outbreak of influenza among children.

In 1986 five avian paramyxovirus (PMV) strains were isolated in embryonated chicken eggs from sick children with influenza. The strains were identified as PMV-2 serotype due to the close antigenic relationships between their HN-proteins and of the reference PMV-2 strains isolated from different birds all over the world. No seroconversion to the isolates was found in the sick children, however, HI-antibodies were detected in hen's sera, eggs of which were used for the new strains isolation. The possible origin of isolated PMV-2 viruses is discussed.

Establishment and characterisation of a porcine rubulavirus (LPMV) persistent infection in porcine kidney cells.

Porcine rubulavirus (LPMV) can establish persistent infections in porcine kidney cells. Cell cultures characterised at passages 25 and 65 demonstrated haemadsorption, formation of syncytia, and a slower growth rate. The nucleoprotein (NP) and haemagglutinin-neuraminidase (HN) protein were present in all cells, although not to the same extent as in wild type infected cells. Incubation of the cell cultures with virus neutralising antibodies could not cure them from the infection. The cells were resistant to LPMV high multiplicity superinfection, but lysed rapidly upon infection with VSV. These cells thus fulfilled the criteria of a true persistent infection. Viral particles were released into the medium from the persistently infected cells as measured by HA and infection of PK-15 cells with medium from the persistently infected cells. The infectious titer of the virus released from the persistently infected cells was 3 logs lower compared to wild type virus, the HN titer still being comparable. Virus released from the persistently infected cells was unable to cause a lytic infection in PK-15 cells, and showed a reduced ability to spread when compared to a LPMV lytic infection.

Incorporation of functional HN-F glycoprotein-containing complexes into newcastle disease virus is dependent on cholesterol and membrane lipid raft integrity.

Newcastle disease virus assembles in plasma membrane domains with properties of membrane lipid rafts, and disruption of these domains by cholesterol extraction with methyl-beta-cyclodextrin resulted in the release of virions with irregular protein composition, abnormal particle density, and reduced infectivity (J. P. Laliberte, L. W. McGinnes, M. E. Peeples, and T. G. Morrison, J. Virol. 80:10652-10662, 2006). In the present study, these results were confirmed using Niemann-Pick syndrome type C cells, which are deficient in normal membrane rafts due to mutations affecting cholesterol transport. Furthermore, cholesterol extraction of infected cells resulted in the release of virions that attached to target cells at normal levels but were defective in virus-cell membrane fusion. The reduced fusion capacity of particles released from cholesterol-extracted cells correlated with significant loss of HN-F glycoprotein-containing complexes detected in the virion envelopes of these particles and with detection of cell-associated HN-F protein-containing complexes in extracts of cholesterol-extracted cells. Extraction of cholesterol from purified virions had no effect on virus-cell attachment, virus-cell fusion, particle infectivity, or the levels of glycoprotein-containing complexes. Taken together, these results suggest that cholesterol and membrane rafts are required for the formation or maintenance of HN-F glycoprotein-containing complexes in cells but not the stability of preformed glycoprotein complexes once assembled into virions.

Vaccination of chicken embryos with escape mutants of La Sota Newcastle disease virus induces a protective immune response.

To reduce the embryonic pathogenicity of Newcastle disease virus (NDV), escape mutants of the La Sota strain were produced with selected monoclonal antibodies. Immunoselection resulted in the elimination of an epitope by single amino acid substitution (F and HN molecule) or in a conformational change (HN molecule). The embryonic pathogenicity of these escape mutants was reduced and their dose was optimised for in ovo vaccination. Because antibody responses and protection of in ovo vaccinated chicks were similar to controls vaccinated at hatch with the La Sota strain, immunoselection appears a valuable technique to produce attenuated NDV strains, which are candidate in ovo vaccines.

Antibody response in children to antigen sites on human PIV-3 HN: correlation with known epitopes mapped by monoclonal antibodies.

The antibody response in children to known epitopes on the HN of human parainfluenza virus type 3 was investigated. Children's sera with Haemagglutination-Inhibition titres between 1/480 to 1/1280 were used. When tested by ELISA, this high-titre serum from each of five children blocked 7 of 17 specific anti-HN murine monoclonal antibodies by greater than 75% at 1 micrograms well-1 of antigen. However, four monoclonal antibodies were blocked less than or equal to 30%, while six were partially blocked between 50% and 75%. Antigen concentrations of 0.5, 1.5 and 2.0 micrograms well-1 did not substantially change this pattern. Comparison of our results with published antigenic maps indicated that antigenic site A on the HN protein was the site with the most significant antibody representation in the children's sera. These findings suggest that antigenic maps deduced using monoclonal antibodies need to be carefully interpreted before they are used in vaccine development. Murine monoclonal antibodies may not fully represent either qualitatively or quantitatively important antibody components of the human or murine immune response to human PIV-3 HN.

Intracellular maturation of the Newcastle disease virus fusion protein is affected by strain differences in the predicted amphipathic alpha-helix adjacent to the fusion domain.

The fusion (F) glycoprotein of Newcastle disease virus (NDV) contains a predicted amphipathic alpha-helix C-terminal to its fusion domain. Of the 13 available NDV F protein sequences, only the Australia-Victoria (AV) strain alpha-helix is weakened, by the replacement of Ala159 with Thr. In this report, we demonstrate that the efficiency of cleavage and virion incorporation of the AV F protein, unlike that of other strains, is temperature sensitive. Pulse/chase experiments at 42 degrees revealed disulfide-linked aggregates containing both the F and hemagglutinin-neuraminidase glycoproteins in strain AV, but not in strain Beaudette C. Furthermore, a revertant derived from AV, whose helix-weakening Thr159 has been replaced with the consensus Ala, produced fewer F protein aggregates, confirming the structural importance of this region in maturation. In addition, a novel disulfide-defined folding intermediate of the F protein was detected.

Rotational mobility of Sendai virus glycoproteins in membranes of fused human erythrocytes and in the envelopes of cell-bound virions.

The rotational mobility of Sendai virus envelope glycoproteins (F, the fusion protein, and HN, the hemagglutinin/neuraminidase) was determined by using erythrosin (ER)-labeled monovalent Fab' antibody fragments directed specifically against either F or HN. By use of time-resolved phosphorescence anisotropy, the rotational mobility of Er-Fab'-viral glycoprotein complexes was studied both in the envelopes of unfused virions bound to erythrocyte ghosts and in the target cell membrane after fusion had occurred. The rotational correlation times (phi) of Er-Fab'-labeled F and HN were rather similar in the envelopes of bound unfused virions, but highly different in membranes of fused cells. The different phi values indicate that F and HN diffuse separately in the target cell membrane and for the major part are not complexed together. The temperature dependence of the phi values of the Er-Fab'-viral glycoprotein complexes revealed a breakpoint at 22 degrees C for the F protein both in bound virions and in the membranes of fused cells, and for the HN proteins in the envelopes of bound virions. In all these cases, the phi values increased between 4 and 22 degrees C, demonstrating a reduction in the rate of rotational diffusion. Further elevation of the temperature reversed the direction of the change in phi. This phenomenon may reflect a temperature-dependent microaggregation of F and HN saturating at ca. 22 degrees C and presumably related to the fusion mechanism since the breakpoint temperature correlates closely with the threshold temperature for virus-cell and cell-cell fusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of amino acid residues important to the neuraminidase activity of the HN glycoprotein of Newcastle disease virus.

Monoclonal antibodies (MAbs) to three overlapping antigenic sites (designated 12, 2, and 23) on the hemagglutinin-neuraminidase glycoprotein (HN) of Newcastle disease virus (NDV) were previously shown to inhibit neuraminidase activity (NA) on neuraminlactose (R. M. Iorio and M. A. Bratt, 1984a, J. Immunol. 133, 2215-2219; R. M. Iorio et al., 1989, Virus Res. 13, 245-262). However, a competitive inhibitor of NA blocks the binding of only MAbs to site 23, suggesting that the domain they recognize may be closely related to the NA site. Antigenic variants selected with site 23 MAbs have single amino acid substitutions at HN residues 192, 193, or 200. Virions of variants, which have a substitution at residue 193 or 200, have alterations in NA which are not attributable to a commensurate change in HN content. A revertant of a temperature-sensitive mutant, which has markedly diminished NA relative to the wild type, has an amino acid substitution at residue 175. A second step revertant having partially restored NA has an additional substitution at residue 192 identical to that in one of the site 23 variants, which, in turn, also makes the revertant resistant to neutralization by site 23 MAbs. Thus, an amino acid substitution at residue 175, 193, or 200 of the HN of NDV can have marked effects on the NA of the protein. The amino acids in the region around residue 175 are highly conserved between the HNs of NDV and other paramyxoviruses, suggesting that this domain is important to the integrity of the NA site in this group of viruses.

An attenuation mechanism of Newcastle disease vaccine strain TCND.

To provide information on the mechanism of attenuation of a Newcastle disease vaccine strain, TCND, we compared it with the parental virulent strain California 11,914 (CAL) biologically and genetically. It was found that TCND bore the fusion protein of virulent type, consisting of a pair of dibasic amino acid residues at the cleavage site and was a temperature sensitive (ts) mutant restricted to grow at 41.5 degrees C. Revertants were obtained by prolonged incubation of chicken embryos inoculated with TCND at the nonpermissive temperature. In cultured cells, viral gene transcription and protein synthesis of TCND occurred similarly to those of CAL and the revertants at 41.5 degrees C. Hemadsorption and immunofluorescence assays revealed that cell surface expression of functional hemagglutinin-neuraminidase (HN) of TCND at 41.5 degrees C was lower than that at 35 degrees C. The revertants exhibited lower activity in fusion assay than CAL and recovered virulence to chicken only in part. The results indicate that the ts mutation of TCND in association with the defect of HN glycoprotein transport is a mechanism of the attenuation, and in addition, some other factors such as fusion activity should be involved in the loss of virulence of CAL to chickens.

Possible involvement of microtubule disruption in bipolar budding of a Sendai virus mutant, F1-R, in epithelial MDCK cells.

Envelope glycoproteins F and HN of wild-type Sendai virus are transported to the apical plasma membrane domain of polarized epithelial MDCK cells, where budding of progeny virus occurs. On the other hand, a pantropic mutant, F1-R, buds bipolarly at both the apical and basolateral domains, and the viral glycoproteins have also been shown to be transported to both of these domains (M. Tashiro, M. Yamakawa, K. Tobita, H.-D. Klenk, R. Rott, and J.T. Seto, J. Virol. 64:4672-4677, 1990). MDCK cells were infected with wild-type virus and treated with the microtubule-depolymerizing drugs colchicine and nocodazole. Budding of the virus and surface expression of the glycoproteins were found to occur in a nonpolarized fashion similar to that found in cells infected with F1-R. In uninfected cells, the drugs were shown to interfere with apical transport of a secretory cellular glycoprotein, gp80, and basolateral uptake of [35S]methionine as well as to disrupt microtubule structure, indicating that cellular polarity of MDCK cells depends on the presence of intact microtubules. Infection by the F1-R mutant partially affected the transport of gp80, uptake of [35S]methionine, and the microtubule network, whereas wild-type virus had a marginal effect. These results suggest that apical transport of the glycoproteins of wild-type Sendai virus in MDCK cells depends on intact microtubules and that bipolar budding by F1-R is possibly due, at least in part, to the disruption of microtubules. Nucleotide sequence analyses of the viral genes suggest that the mutated M protein of F1-R might be involved in the alteration of microtubules.

The paramyxovirus SV5 small hydrophobic (SH) protein is not essential for virus growth in tissue culture cells.

The SH gene of the paramyxovirus SV5 is located between the genes for the glycoproteins, fusion protein (F) and hemagglutinin-neuraminidase (HN), and the SH gene encodes a small 44-residue hydrophobic integral membrane protein (SH). The SH protein is expressed in SV5-infected cells and is oriented in membranes with its N terminus in the cytoplasm. To study the function of the SH protein in the SV5 virus life cycle, the SH gene was deleted from the infectious cDNA clone of the SV5 genome. By using the recently developed reverse genetics system for SV5, it was found that an SH-deleted SV5 (rSV5DeltaSH) could be recovered, indicating the SH protein was not essential for virus viability in tissue culture. Analysis of properties of rSV5DeltaSH indicated that lack of expression of SH protein did not alter the expression level of the other virus proteins, the subcellular localization of F and HN, or fusion competency as measured by lipid mixing assays and a new content mixing assay that did not require the use of vaccinia virus. The growth rate, infectivity, and plaque size of rSV5 and rSV5DeltaSH were found to be very similar. Although SH is shown to be a component of purified virions by immunoblotting, examination of purified rSV5DeltaSH by electron microscopy did not show an altered morphology from SV5. Thus in tissue culture cells the lack of the SV5 SH protein does not confer a recognizable phenotype.