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PubMed2003

Bovine parainfluenza virus type 3 (PIV3) expressing the respiratory syncytial virus (RSV) attachment and fusion proteins protects hamsters from challenge with human PIV3 and RSV.

Parainfluenza virus type 3 (PIV3) and respiratory syncytial virus (RSV) are the main causes of ubiquitous acute respiratory diseases of infancy and early childhood, causing 20-25 % of pneumonia and 45-50 % of bronchiolitis in hospitalized children. The primary goal of this study was to create an effective and safe RSV vaccine based on utilizing attenuated bovine PIV3 (bPIV3) as a virus vector backbone. bPIV3 had been evaluated in human clinical trials and was shown to be attenuated and immunogenic in children as young as 2 months of age. The ability of bPIV3 to function as a virus vaccine vector was explored further by introducing the RSV attachment (G) and fusion (F) genes into the bPIV3 RNA genome. The resulting virus, bPIV3/RSV(I), contained an insert of 2900 nt, comprising two translationally competent transcription units. Despite this increase in genetic material, the virus replicated to high titres in Vero cells. This recombinant virus expressed the RSV G and F proteins sufficiently to evoke a protective immune response in hamsters upon challenge with RSV or human PIV3 and to elicit RSV neutralizing and PIV3 haemagglutinin inhibition serum antibodies. In effect, a bivalent vaccine was produced that could protect vaccinees from RSV as well as PIV3. Such a vaccine would vastly reduce the respiratory disease burden, the associated hospitalization costs and, most importantly, decrease morbidity and mortality of infants, immunocompromised individuals and the elderly.

Animals↗
PubMed2003

Triggering of human parainfluenza virus 3 fusion protein (F) by the hemagglutinin-neuraminidase (HN) protein: an HN mutation diminishes the rate of F activation and fusion.

For human parainfluenza virus type 3 and many other paramyxoviruses, membrane fusion mediated by the fusion protein (F) has a stringent requirement for the presence of the homotypic hemagglutinin-neuraminidase protein (HN). With the goal of gaining further insight into the role of HN in the fusion process, we developed a simple method for quantitative comparison of the ability of wild-type and variant HNs to activate F. In this method, HN/F-coexpressing cells with red blood cells (RBC) bound to them at 4 degrees C are transferred to 22 degrees C, and at different times after transfer 4-guanidino-neu5Ac2en (4-GU-DANA) is added; this inhibitor of the HN-receptor interaction then releases all reversibly bound RBC but not those in which F insertion in the target membrane or fusion has occurred. Thus, the amount of irreversibly bound (nonreleased) RBC provides a measure of F activation, and the use of fluorescently labeled RBC permits microscopic assessment of the extent to which F insertion has progressed to fusion. We studied two neuraminidase-deficient HN variants, C28a, which has two mutations, P111S and D216N, and C28, which possesses the D216N mutation only. C28a but not C28 exhibits a slow fusion phenotype, although determination of the HNs' receptor-binding avidity (with our sensitive method, employing RBC with different degrees of receptor depletion) showed that the receptor-binding avidity of C28a or C28 HN was not lower than that of the wild type. The F activation assay, however, revealed fusion-triggering defects in C28a HN. After 10 and also 20 min at 22 degrees C, irreversible RBC binding was significantly less for cells coexpressing wild-type F with C28a HN than for cells coexpressing wild-type F with wild-type HN. In addition, F insertion progressed to fusion more slowly in the case of C28a HN-expressing cells than of wild-type HN-expressing cells. Identical defects were found for P111S HN, whereas for C28 HN, representing the 216 mutation of C28a, F activation and fusion were as rapid as for wild-type HN. The diminished fusion promotion capacity of C28a HN is therefore attributable to P111S, a mutation in the stalk region of the molecule that causes no decrease in receptor-binding avidity. C28a HN is the first parainfluenza virus variant found so far to be specifically defective in HN's F-triggering and fusion promotion functions and may contribute to our understanding of transmission of the activating signal from HN to F.

Animals↗
PubMed2000

Rapid molecular epidemiologic studies of human parainfluenza viruses based on direct sequencing of amplified DNA from a multiplex RT-PCR assay.

Sequencing studies of limited regions of the human parainfluenza viruses (HPIVs) genomes have helped describe patterns of virus circulation and characterize institutional outbreaks of HPIVs-associated respiratory illness. In this study, we sequenced reverse transcription polymerase chain reaction (RT-PCR)-amplified HPIVs RNA obtained from a multiplex RT-PCR assay described previously for simultaneous detection of HPIV-1, 2 and 3. Differences in the nucleotide sequences of limited regions of the HN gene allowed us to distinguish temporally and geographically diverse HPIV isolates (43 HPIV-1, 7 HPIV-2, 12 HPIV-3 isolates from this and previously published studies). In addition, an outbreak of HPIV-3-associated illness among infants on a pediatric ward was investigated by comparing sequences of three ward isolates with three matched community controls. Sequences of all ward isolates were identical and differed from those of the community controls, suggesting a single introduction and nosocomial transmission of the virus. Combining multiplex reverse transcription polymerase chain reaction (RT-PCR) assays with direct sequencing of the PCR products can provide an integrated system for rapid diagnosis and characterization of HPIVs.

Child↗
PubMed2000

A recombinant human parainfluenza virus type 3 (PIV3) in which the nucleocapsid N protein has been replaced by that of bovine PIV3 is attenuated in primates.

The shipping fever (SF) and Kansas (Ka) strains of bovine parainfluenza virus type 3 (BPIV3) are restricted in their replication in rhesus monkeys 100- to 1,000-fold compared to human parainfluenza virus type 3 (HPIV3), and the Ka strain also was shown to be attenuated in humans. To initiate an investigation of the genetic basis of the attenuation of BPIV3 in primates, we produced viable chimeric HPIV3 recombinants containing the nucleoprotein (N) open reading frame (ORF) from either BPIV3 Ka or SF in place of the HPIV3 N ORF. These chimeric recombinants were designated cKa-N and cSF-N, respectively. Remarkably, cKa-N and cSF-N grew to titers comparable to those of their HPIV3 and BPIV3 parents in LLC-MK2 monkey kidney and Madin-Darby bovine kidney cells. Thus, the heterologous nature of the N protein did not impede replication in vitro. However, cKa-N and cSF-N were each restricted in replication in rhesus monkeys to a similar extent as Ka and SF, respectively. This identified the BPIV3 N protein as a determinant of the host range restriction of BPIV3 in primates. These chimeras thus combine the antigenic determinants of HPIV3 with the host range restriction and attenuation phenotype of BPIV3. Despite their restricted replication in rhesus monkeys, the chimeric viruses induced a level of resistance to HPIV3 challenge in these animals which was indistinguishable from that conferred by immunization with HPIV3. The infectivity, attenuation, and immunogenicity of these BPIV3/HPIV3 chimeras suggest that the modified Jennerian approach described in the present report represents a novel method to design vaccines to protect against HPIV3-induced disease in humans.

Animals↗
PubMed1992

Evaluation of a live attenuated, cold-adapted parainfluenza virus type 3 vaccine in children.

Cold passage 18 (CP18) parainfluenza virus type 3 (PIV-3) vaccine was evaluated in a double-blind, randomized, placebo-controlled study of 95 infants and young children. None of 19 seropositive older children 41 to 124 months old became infected when 10(6) 50% tissue culture infective doses (TCID50) of vaccine virus was administered intranasally. Two of nine and seven of twenty-four young seropositive children given 10(5) or 10(6) TCID50 of CP18 PIV-3, respectively, became infected. Each of four seronegative young children became infected, as indicated by virus shedding and antibody response, when given 10(6) TCID50 of CP18 PIV-3 intranasally. Illness was not observed in seropositive children. Two of the four seronegative children developed a mild illness characterized by rhinorrhea and wheezing on auscultation; none had fever. In one case, vaccine virus spread from a vaccine to a sibling control but did not cause illness. The vaccine is attenuated relative to wild-type PIV-3, but additional attenuation will be required to achieve a satisfactory PIV-3 vaccine.

Adaptation, Physiological↗
PubMed2024

Human parainfluenza virus 3 vaccine candidates attenuated by codon-pair deoptimization are immunogenic and protective in hamsters.

Human parainfluenza virus type 3 (HPIV3) is a major pediatric respiratory pathogen lacking available vaccines or antiviral drugs. We generated live-attenuated HPIV3 vaccine candidates by codon-pair deoptimization (CPD). HPIV3 open reading frames (ORFs) encoding the nucleoprotein (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin-neuraminidase (HN), and polymerase (L) were modified singly or in combination to generate 12 viruses designated Min-N, Min-P, Min-M, Min-FHN, Min-L, Min-NP, Min-NPM, Min-NPL, Min-PM, Min-PFHN, Min-MFHN, and Min-PMFHN. CPD of N or L severely reduced growth in vitro and was not further evaluated. CPD of P or M was associated with increased and decreased interferon (IFN) response in vitro, respectively, but had little effect on virus replication. In Vero cells, CPD of F and HN delayed virus replication, but final titers were comparable to wild-type (wt) HPIV3. In human lung epithelial A549 cells, CPD F and HN induced a stronger IFN response, viral titers were reduced 100-fold, and the expression of F and HN proteins was significantly reduced without affecting N or P or the relative packaging of proteins into virions. Following intranasal infection in hamsters, replication in the nasal turbinates and lungs tended to be the most reduced for viruses bearing CPD F and HN, with maximum reductions of approximately 10-fold. Despite decreased in vivo replication (and lower expression of CPD F and HN in vitro), all viruses induced titers of serum HPIV3-neutralizing antibodies similar to wt and provided complete protection against HPIV3 challenge. In summary, CPD of HPIV3 yielded promising vaccine candidates suitable for further development.

Animals↗
PubMed1975

An animal model for studying infection and immunity to and attenuation of human parainfluenza viruses.

Neonatal ferrets were found to be highly via the intranasal route of inoculation to infection with wild type isolates of parainfluenza viruses. Wild type infection consistently resulted in deaths of these animals in 48 to 72 hours. Autopsy and histopathological findings in lung tissue were consistent with those of viral infection. Virus could consistently be isolated from lung tissues. Using wild green monkey kidney grown viruses and selected passage levels of egg adapted viruses, virus attenuation markers were observed by comparing infectivity in neonatal ferrets, propagation in cell cultures of human, primate, rabbit and chick embryo tissues, and in chick embryos and interferon induction. Adult pregant dams could be immunized (silently infected) and circulating hemagglutinin-inhibiting antibody could be detected within two weeks of intranasal instillation of wild and low egg passage types of virus. The animal model described could be valuable in studying immunity to parainfluenza virus infections.

Aerosols↗
PubMed1987

Nucleotide sequence of the coding and flanking regions of the human parainfluenza virus 3 hemagglutinin-neuraminidase gene: comparison with other paramyxoviruses.

The nucleotide sequence of the human parainfluenza virus 3 (HPIV3) hemagglutinin-neuraminidase (HN) gene has been determined using cDNA clones derived from both HPIV3 genomic RNA and mRNA. The HN mRNA contains 1,882 nucleotides, not including the poly(A) tail. Primer extension experiments were carried out to locate the 5' terminal nucleotide of the HN mRNA. The 3' end of the mRNA was located at a putative polyadenylation signal. The HPIV3 HN mRNA has one large open reading frame that codes for 572 amino acids with a deduced molecular weight of 64,178. Potential polymerase recognition signals for the HN and L genes were located in the flanking regions. The HN protein of HPIV3 shares some common features with the previously sequenced HN proteins of Sendai virus and Simian virus 5. The features include: an N-terminal membrane anchor, two regions of highly conserved amino acid sequence and strong conservation in the positions of the cysteine residues. The relationship is closest between Sendai virus and HPIV3.

Amino Acid Sequence↗
PubMed2004

Role of nucleolin in human parainfluenza virus type 3 infection of human lung epithelial cells.

Human parainfluenza virus type 3 (HPIV-3) is an airborne pathogen that infects human lung epithelial cells from the apical (luminal) plasma membrane domain. In the present study, we have identified cell surface-expressed nucleolin as a cellular cofactor required for the efficient cellular entry of HPIV-3 into human lung epithelial A549 cells. Nucleolin was enriched on the apical cell surface domain of A549 cells, and HPIV-3 interacted with nucleolin during entry. The importance of nucleolin during HPIV-3 replication was borne out by the observation that HPIV-3 replication was significantly inhibited following (i). pretreatment of cells with antinucleolin antibodies and (ii). preincubation of HPIV-3 with purified nucleolin prior to its addition to the cells. Moreover, HPIV-3 cellular internalization and attachment assays performed in the presence of antinucleolin antibodies and purified nucleolin revealed the requirement of nucleolin during HPIV-3 internalization but not during attachment. Thus, these results suggest that nucleolin expressed on the surfaces of human lung epithelial A549 cells plays an important role during HPIV-3 cellular entry.

Epithelial Cells↗
PubMed1988

Molecular cloning and sequence determination of the fusion protein gene of human parainfluenza virus type 1.

Undegraded mRNA transcripts were isolated from human parainfluenza virus type 1 (hPIV-1)-infected LLC-MK2 cells and their size was determined through denaturing agarose electrophoresis. The two predominantly represented mRNA species (1.65 and 1.87 kb) are similar in size to other paramyxoviral mRNAs that encode their respective glycoproteins. The cDNA transcripts corresponding to these two mRNAs were used to construct two size-restricted cDNA libraries. A cDNA clone, containing a 1.87-kb insert, was identified as encoding the hPIV-1 fusion protein by positively hybridizing with a synthetic oligonucleotide mix whose sequence was derived from the conserved sequences of other paramyxoviral F0 genes. The nucleotide sequence of the cDNA insert was determined and found to contain a single, large open reading frame encoding a putative protein of 60,795 Da consisting of 556 amino acids. Comparison of the amino acid sequence with the fusion proteins of other paramyxoviruses enabled the identification of the highly conserved amino acids of the F1 N-terminus. In addition, the positions of the hydrophobic signal and transmembrane regions, cysteine, and proline residues are all conserved. These analyses confirm that the cDNA sequence is that of the F0 protein. The 5' end of the fusion protein mRNA was determined by primer extension to lie 155 bases beyond the 5' end of the cDNA insert.

Amino Acid Sequence↗
PubMed1986

Sequence analysis of the P and C protein genes of human parainfluenza virus type 3: patterns of amino acid sequence homology among paramyxovirus proteins.

The complete nucleotide sequence of the P + C mRNA of human parainfluenza virus type 3 (PF3) was determined by sequencing cDNA, viral genomic RNA and mRNA. The P + C mRNA is 2009 nucleotides in length, exclusive of poly(A), and contains two overlapping open reading frames (ORFs). The P + C mRNA encodes two proteins, the 602 amino acid nucleocapsid phosphoprotein P and the 199 amino acid non-structural protein C. Peptide mapping confirmed that the two proteins are unrelated. Hybrid-arrest translation experiments assigned each of the two proteins to its respective ORF. These studies showed that the coding strategy of the PF3 P + C mRNA is similar to that of Sendai virus. Amino acid sequence alignment showed that the P and C proteins of PF3 and Sendai virus represent homologous pairs. However, these homologies are represented by high contents of accepted amino acid substitutions and by similarity in hydropathy profiles rather than by high contents of exact amino acid matches. Homology with the P and C proteins of measles, canine distemper and respiratory syncytial viruses was at the threshold of significance. The patterns of amino acid sequence homology among the paramyxovirus HN, F, NP, P and C proteins are compared.

Amino Acid Sequence↗
PubMed1986

Parainfluenza virus bronchiolitis. Epidemiology and pathogenesis.

An investigation of the epidemiology and pathogenesis of bronchiolitis due to parainfluenza virus (PV) was carried out. Bronchiolitis due to PV occurred most commonly in non-Caucasian males. Breast-fed infants exhibited a reduced risk of developing bronchiolitis. Once an episode of PV bronchiolitis occurred, both exposure to cigarette smoke and bottle feeding were associated with an increased frequency of recurrent wheezing, and subsequent infection with respiratory virus almost uniformly resulted in wheezing. Cell-mediated immune responses to PV antigen and titers of PV-specific IgE were greater among patients with bronchiolitis than among patients with upper respiratory tract infection. The epidemiology and pathogenesis of bronchiolitis due to PV is similar to that of respiratory syncytial virus. Lower respiratory tract infection may predispose to episodes of bronchoconstriction on subsequent exposure to cigarette smoke or other viral infections.

Antigens, Viral↗
PubMed1990

Parainfluenza virus type 1 reduces the affinity of agonists for muscarinic receptors in guinea-pig lung and heart.

Membrane preparations of guinea-pig lung (containing multiple muscarinic receptor subtypes) and heart (containing M2 receptors only) were incubated with either neuraminidase, parainfluenza virus (which contains neuraminidase), or virus plus 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, a neuraminidase inhibitor. None of these treatments affected [3H]quinuclidinyl benzilate [( 3H]QNB) binding. In the lung and heart, carbachol displaced 0.2 nM [3H]QNB from two sites. After treatment with either neuraminidase or virus the high affinity site was shifted to the right, and carbachol displaced QNB from one site with low affinity in the lung. In contrast, neuraminidase or virus decreased the affinity of carbachol for both sites in the heart. The neuraminidase inhibitor completely blocked virus-induced changes in carbachol affinity in both tissues. These results suggest that parainfluenza virus decreases the affinity of agonists for some of the muscarinic receptors in the lung, and for all of the muscarinic receptors in the heart due to its neuraminidase activity, which results in removal of sialic acid. The decreased agonist affinity in the lung may be responsible for the increased vagally induced bronchoconstriction seen in viral respiratory infections.

Animals↗
PubMedDate not supplied

Immunogenicity of inactivated vaccines prepared with parainfluenza viruses type 1 and type 3, administered experimentally by nasal or oral route.

The inactivated vaccines prepared with parainfluenza viruses type 1 and type 3, administered to mice by nasal or oral route, either as monovalent preparations in succession or as bivalent associated preparations according to the experimental models used, imparted a significant protection against the infection with the homologous active viruses. The routes of administration of vaccines, nasal and oral, which make equal demands upon the immune secretory serum and cell system, as well as the alternative of inactivated preparations for active virus vaccines are discussed.

Administration, Intranasal↗
PubMed1991

Defective interfering particles of human parainfluenza virus type 3 are associated with persistent infection in cell culture.

CV-1 cell lines persistently infected with human parainfluenza virus type 3 (HPF3) contain one or more distinct subgenomic RNAs in addition to standard viral genomes. These RNAs are shown to be the genomes of defective-interfering (DI) particles of the virus; they are present in particles in the culture fluid, and they interfere with the growth of wild-type virus. Removal of the particles from the culture fluid by ultracentrifugation yields a supernatant fluid free from inhibitory activity, demonstrating that the anti-viral effect is not mediated by soluble factors. A role for the DI particles in persistence of HPF3 is considered.

Capsid↗
PubMed2004

An oligosaccharide at the C-terminus of the F-specific domain in the stalk of the human parainfluenza virus 3 hemagglutinin-neuraminidase modulates fusion.

The promotion of membrane fusion by the fusion (F) protein of human parainfluenza virus 3 (hPIV3) is dependent on a virus-specific contribution from the hemagglutinin-neuraminidase (HN) protein. By evaluation of chimeric hPIV3-Newcastle disease virus (NDV) HN proteins, we have previously shown that hPIV3-F-specificity is determined by a domain that extends from the middle of the membrane anchor to the 82nd residue in the ectodomain [Virology 209, (1995) 457; Arch. Virol. 13 (1997) 115]. If the corresponding NDV-derived residues replace the two C-terminal residues in this domain, no fusion is detected. However, these substitutions restore a glycosylation site present in NDV HN, but not in hPIV3 HN. Deletion of this site from a nested set of chimeras with hPIV3-derived N-terminal portions of decreasing length partially restores fusion, suggesting that an oligosaccharide near the top of hPIV3 HN stalk modulates fusion. In addition, further mutational analyses show that a chimera with only 125 N-terminal hPIV3-derived residues (72 in the stalk) actually promotes fusion more efficiently than the wt protein. These findings localize the C-terminus of the F-specific domain in hPIV3 HN a full 10 residues closer to the membrane than previously shown.

Animals↗
PubMed1997

An infectious clone of human parainfluenza virus type 3.

A full-length clone of the human parainfluenza virus type 3 (HPIV-3) genome (called pHPIV-3) was constructed, and recombinant, infectious HPIV-3 was generated by transfecting pHPIV-3 and support plasmids encoding the HPIV-3 NP, P, and L proteins into HeLa cells infected with a vaccinia virus recombinant which expresses T7 RNA polymerase. T7 RNA polymerase promoters on the transfected plasmids direct the synthesis of transcripts encoding the NP, P, and L proteins and a full-length, positive-sense copy of the HPIV-3 genome. Generation of virus was dependent on transfection of pHPIV-3 and the HPIV-3 P- and L-encoding plasmids. However, a plasmid encoding the NP protein was not required since NP was expressed from pHPIV-3. Recovered virus was neutralized by anti-HPIV-3 antisera and shown to contain specific base substitutions characteristic of pHPIV-3. Recombination was shown to occur during recovery, as viruses with two distinct genotypes and phenotypes were isolated. The ability to produce infectious HPIV-3 engineered to contain specific alterations within the HPIV-3 genes and cis-acting elements expedites the study of all aspects of the virus replication cycle. Additionally, analysis of mutations may lead to the identification of attenuating genotypes, a key step in the development of a live virus vaccine.

Cloning, Molecular↗
PubMed1998

Simultaneous detection and identification of human parainfluenza viruses 1, 2, and 3 from clinical samples by multiplex PCR.

Reverse transcription (RT)-PCR assays have been widely described for use in the diagnosis of human parainfluenza viruses (HPIVs) and other respiratory virus pathogens. However, these assays are mostly monospecific, requiring separate amplifications for each HPIV type. In the present work, we describe multiplex RT-PCR assays that detect and differentiate HPIV serotypes 1, 2, and 3 in a combined reaction. Specifically, a mixture of three pairs of primers to conserved regions of the hemagglutinin-neuraminidase gene of each HPIV serotype was used for primary amplification, yielding amplicons with similar sizes. For typing, a second amplification was performed with a mixture of nested primers, yielding amplicons with sizes easily differentiated by agarose gel electrophoresis. A modified single-amplification RT-PCR assay with fluorescence-labeled nested primers, followed by analysis of the labeled products on an automated sequencing gel, was also evaluated. Fifteen temporally and geographically diverse HPIV isolates from the Centers for Disease Control and Prevention archives and 26 of 30 (87%) previously positive nasopharyngeal specimens (8 of 10 positive for HPIV serotype 1 [HPIV1], 9 of 10 positive for HPIV2, and 9 of 10 positive for HPIV3) were positive and were correctly typed by both assays. Negative results were obtained with naso- or oropharyngeal specimens and/or culture isolates of 33 unrelated respiratory tract pathogens, including HPIV4, enterovirus, rhinovirus, respiratory syncytial virus, adenovirus, influenza virus, and Streptococcus pneumoniae. Our multiplex RT-PCR assays provide sensitive, specific, and simplified tools for the rapid diagnosis of HPIV infections.

DNA Primers↗