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Giant cell pneumonia associated with parainfluenza virus type 3 infection and chronic poliovirus type 2 meningoencephalomyelitis are documented in an infant with combined immunologic deficiency (Swiss type). Caution should be exercised in attributing cases of giant cell pneumonia to measles virus without serologic or virologic evidence.
The effect of some factors on in vitro transcription of human influenza and parainfluenza viruses was studied. Dinucleotide AfG was shown to stimulate transcription of RNP of human parainfluenza type 3 virus and Sendai virus in the presence of magnesium but not manganese ions same as in the case of influenza viruses transcription. Among two monoclonal antibodies to NP protein of influenza virus, clone F 81 inhibited transcription of RNP of human influenza A viruses, and its influence on transcription of animal influenza viruses was weak. Another clone, H 12, had a low effect. The M protein isolated from influenza and parainfluenza virions inhibited in vitro transcription in the corresponding homologous systems. The inhibiting effect was exerted also by the M protein heterologous to HPIV-3 (M protein of Sendai virus) which suggests the nonspecificity of M protein interaction with transcriptive complexes in large RNA viruses.
Recombinant parainfluenza virus 3 (rPIV3) is being developed as a vector to express foreign genes as a bivalent or multivalent live attenuated virus vaccine. In the present study, we examined the effect of inserted foreign sequence on virus replication in vitro and in vivo, focusing on the parameter of insert length. In one type of construct, foreign sequence of increasing length was flanked by PIV3 transcription signals and inserted as an additional gene unit (GU insert) between the HN and L genes, so that one additional mRNA would be made. In a second type of construct, foreign sequence was inserted into the downstream NCR (NCR insert) of the HN gene, so that the number of encoded mRNAs remained unchanged. In each case, the foreign sequence was designed to lack any significant open reading frame, which permitted an evaluation of the effect of insert length on replication independent of an effect of an expressed protein. The GU or NCR insert sizes ranged from 168 nucleotides (nt) to 3918 nt. rPIV3s containing GU insertions of up to 3918 nt in length, the largest size tested, were viable and replicated efficiently at permissive temperatures in vitro, but a reduction in plaque size was seen at 39 degrees C and 40 degrees C. The rPIV3 with a 3918-nt GU insertion was restricted in replication in the upper (fivefold) and lower (25-fold) respiratory tracts of hamsters. Although a 1908-nt GU insertion did not significantly modify replication of wild-type PIV3 in vitro or in vivo, its introduction significantly augmented the level of temperature sensitivity (ts) and attenuation (att) specified by three mutations in the L protein of a cold-passaged attenuated PIV3 vaccine virus. rPIV3s bearing a 3126- or 3894-nt NCR insertion exhibited in vitro and in vivo phenotypes like those of the rPIV3s bearing similar-sized GU insertions. These findings indicate that rPIV3s whose genome length has been increased by more than 3000 nt by either a GU or an NCR insertion exhibit an unexpected host-range phenotype, that is, efficient replication in vitro but restricted replication in hamsters, especially in the lower respiratory tract. Furthermore, these effects were greatly enhanced when the rPIV3 backbone contained other ts or att mutations. The implications of these findings for the use of single-stranded, negative-sense RNA viruses as vectors for vaccines are discussed.
The Kansas strain of bovine parainfluenza virus type 3 (BPIV3) is 100- to 1,000-fold restricted in replication in the respiratory tracts of nonhuman primates compared to human PIV3 (HPIV3), an important pathogen of infants and young children. BPIV3 is also restricted in replication in human infants and children, yet it is immunogenic and is currently being evaluated in clinical trials as a vaccine candidate to protect against illness caused by HPIV3. We have examined the genetic basis for the host range attenuation phenotype of BPIV3 by exchanging each open reading frame (ORF) of a recombinant wild-type HPIV3 with the analogous ORF from BPIV3, with the caveats that the multiple ORFs of the P gene were exchanged as a single unit and that the HN and F genes were exchanged as a single unit. Recombinant chimeric bovine-human PIV3s were recovered from cDNA, and the levels of viral replication in vitro and in the respiratory tract of rhesus monkeys were determined. Recombinant chimeric HPIV3s bearing the BPIV3 N or P ORF were highly attenuated in the upper and lower respiratory tracts of monkeys, whereas those bearing the BPIV3 M or L ORF or the F and HN genes were only moderately attenuated. This indicates that the genetic determinants of the host range restriction of replication of BPIV3 for primates are polygenic, with the major determinants being the N and P ORFs. Monkeys immunized with these bovine-human chimeric viruses, including the more highly attenuated ones, developed higher levels of HPIV3 hemagglutination-inhibiting serum antibodies than did monkeys immunized with BPIV3 and were protected from challenge with wild-type HPIV3. Furthermore, host range determinants could be combined with attenuating point mutations to achieve an increased level of attenuation. Thus, chimeric recombinant bovine-human PIV3 viruses that manifest different levels of attenuation in rhesus monkeys are available for evaluation as vaccine candidates to protect infants from the severe lower respiratory tract disease caused by HPIV3.
Instillation of parainfluenza virus type 2 in the mammary gland of the goat resulted in the appearance of specific and high titered antibodies in the milk. The globulin fraction of the milk was separated and conjugated with fluorescein isothiocyanate. These conjugated antibodies could be successfully used in the specific staining of the homologous virus in infected L-132 cells. Therefore, milk from such immunized animals represents a rich and convenient source of specific antibodies for use in immunofluorescence and other serological tests.
A parainfluenza virus type 3 (PIV3) subunit vaccine consisting of detergent-solubilized, affinity-purified haemagglutinin-neuraminidase (HN) and fusion (F) surface glycoproteins was tested in cotton rats for immunogenicity, short-term effects on virus-induced immunopathology and protective efficacy. Groups of animals were immunized twice, 4 weeks apart, with graded doses of vaccine administered either alone or with aluminium phosphate (AlPO4). The minimum immunogenic dose of vaccine was 0.1 microgram HN and F when the vaccine was given alone and 0.01 microgram when the vaccine was administered with AlPO4 adjuvant. Antibody responses in animals immunized with 1 microgram HN and F mixed with adjuvant were similar to those in control animals infected with live PIV3 intranasally. Pulmonary and nasal wash PIV3 titres generally were inversely correlated with serum antibody levels. Virus titres were significantly reduced in all groups of animals immunized with greater than or equal to 0.1 microgram HN and F compared with control animals immunized with vehicle only. Four days after virus challenge, there was no evidence of enhanced histopathology in lung sections from animals immunized with the candidate vaccine.
The immunogenicity of a parainfluenza virus type 3 (PIV-3) subunit vaccine consisting of affinity-purified haemagglutinin-neuraminidase (HN) and fusion (F) surface glycoproteins was tested in guinea-pigs and hamsters. The ability of several different immunopotentiating agents to enhance the antibody response of animals to the PIV-3 surface glycoproteins was evaluated. The immunity induced by HN and F alone was compared with the response elicited by purified proteins combined with Freund's complete adjuvant, aluminium phosphate, Syntex's threonyl-muramyl dipeptide (MDP) SAF-MF formulation, or Ribi's adjuvant formulation containing BCG cell wall skeleton (CWS), trehalose dimycolate (TDM) and monophosphoryl lipid A (MPL) in a 2% squalene-in-water emulsion. Purified proteins were also incorporated into three different liposome formulations prepared by the detergent dialysis procedure. Immunization of guinea-pigs and hamsters with two 15 micrograms doses of the PIV-3 surface glycoproteins administered in the absence of adjuvant elicited high haemagglutination inhibition, neutralization and anti-fusion titres. The liposome preparations failed to enhance the antibody titres. Ribi's adjuvant formulation was effective at inducing a good secondary response to the purified proteins while the immunostimulatory effects of aluminium phosphate, Syntex and Freund's adjuvants were clearly demonstrated in both primary and secondary responses. When administered without adjuvant, a 15 microgram dose of the HN and F mixture was capable of protecting hamsters against live virus challenge. The immunoprotective dose of the purified proteins could be reduced to at least 0.1 microgram by the addition of aluminium phosphate, Syntex or Freund's adjuvants.
Human parainfluenza virus 3 replicates well in the noses and lungs of two species of cotton rats, Sigmodon hispidus and Sigmodon fulviventer. Peak viral titers of nearly 10(6) PFU/g are reached 2 days after infection in both tissues, are maintained through day 5, and are equivalent in the two species. Infectious virus is eliminated by day 8 after infection. Both species produce a strong neutralizing antibody response with titers of 1:10,000 4 weeks after infection. Viral replication in the nasal epithelium results in only minor histological changes, and viral antigen is found only in the apical portion of epithelial cells. Infection of S. hispidus causes a bronchiolitis with a peribronchiolar lymphoid cell infiltration that reaches a peak 6 days after infection, and there is only a minor component of interstitial pneumonia. In contrast, infection of S. fulviventer causes an interstitial pneumonia, and this lesion reaches its maximal extent by 6 days after infection. There is minimal peribronchiolar lymphoid cell infiltration in infected S. fulviventer. Lung lesions in both species of cotton rats are largely healed 9 days after infection, and the lungs are indistinguishable from those of uninfected controls 16 days after infection. These species of cotton rats offer separate models for the two major pulmonary manifestations of human parainfluenza virus 3 infection. The models may be useful for basic studies of the pathogenesis of this infection and for initial evaluation of candidate vaccines.
Three human and six bovine parainfluenza virus type 3 (PIV3) strains were examined by the use of 60 monoclonal antibodies (MAbs). Fifty-three MAbs to the human C243 strain were directed against six, four, nine and seven epitopes of the haemagglutinin-neuraminidase (HN), fusion (F), nucleocapsid (N) and matrix proteins, respectively. Seven MAbs to the bovine strain were directed against three epitopes of the HN protein and three epitopes of the F protein. Each strain was characterized in ELISA and immunofluorescence tests with all MAbs and in a haemagglutination inhibition assay with the anti-HN MAbs. There were marked differences between human and bovine viruses, primarily in the HN protein where five epitopes differed. One epitope of the F and one of the N protein also differed. Bovine PIV3 was found to be a homogeneous subtype and distinct from human PIV3.
Human parainfluenza virus type 3 (HPIV-3) can be responsible for mild to severe respiratory infections and hospital epidemics. We investigated an outbreak in a hematology unit. Respiratory viruses were screened using multiplex PCR. HPIV-3 quantification and whole-genome sequencing were performed on HPIV-3 positive respiratory samples. Clinical characteristics, infection progression, incidence rates of respiratory viruses within the hospital and detection of respiratory viruses were documented, along with the reinforcement of infection prevention and control (IPC) measures implemented. Between November 2022, and January 2023, HPIV-3 was identified in 20 of 113 hematology patients (17.7%), of whom 80% had multiple myeloma. A majority of HPIV-3-positive patients developed pneumonia (60%), and mortality was notably higher (35%) compared to patients who were negative (3%, p < 0.0001). Respiratory HPIV-3 viral loads were similar between patients with and without pneumonia. In parallel, HPIV-3 incidence in the hospital overall was lower than in the hematology unit (p < 0.0001). Air virus screening showed the detection of HPIV-3 in the air in different areas, and whole-genome sequencing confirmed the circulation of a single HPIV-3 strain. Strengthened IPC measures were associated with the containment of the outbreak. HPIV-3 has high epidemic potential in patients with multiple myeloma and causes severe infections. Our findings highlight the need for routine HPIV-3 testing in hematology units.
Zanamivir (4-guanidino-Neu5Ac2en [4-GU-DANA]) inhibits not only the neuraminidase activity but also the receptor interaction of the human parainfluenza virus type 3 (HPIV3) hemagglutinin-neuraminidase (HN), blocking receptor binding and subsequent fusion promotion. All activities of the HPIV3 variant ZM1 HN (T193I/I567V) are less sensitive to 4-GU-DANA's effects. The T193I mutation in HN confers both increased receptor binding and increased neuraminidase activity, as well as reduced sensitivities of both activities to 4-GU-DANA inhibition, consistent with a single site on the HN molecule carrying out both catalysis and binding. We now provide evidence that the HPIV3 variant's resistance to receptor-binding inhibition by 4-GU-DANA is related to a reduced affinity of the HN receptor-binding site for this compound as well as to an increase in the avidity of HN for the receptor. Newcastle disease virus (NDV) HN and HPIV3 HN respond differently to inhibition in ways that suggest a fundamental distinction between them. NDV HN-receptor binding is less sensitive than HPIV3 HN-receptor binding to 4-GU-DANA, while its neuraminidase activity is highly sensitive. Both HPIV3 and NDV HNs are sensitive to receptor-binding inhibition by the smaller molecule DANA. However, for NDV HN, some receptor binding cannot be inhibited. These data are consistent with the presence in NDV HN of a second receptor-binding site that is devoid of enzyme activity and has a negligible, if any, affinity for 4-GU-DANA. Avidity for the receptor contributes to resistance by allowing the receptor to compete effectively with inhibitors for interaction with HN, while the further determinant of resistance is the reduced binding of the inhibitor molecule to the binding pocket on HN. Based upon our data and recent three-dimensional structural information on the HPIV3 and NDV HNs, we propose mechanisms for the observed sensitivity and resistance of HN to receptor-binding inhibition and discuss the implications of these mechanisms for the distribution of HN functions.
The live-attenuated human parainfluenza virus 3 (PIV3) cold-passage 45 (cp45) candidate vaccine was shown previously to be safe, immunogenic, and phenotypically stable in seronegative human infants. Previous findings indicated that each of the three amino acid substitutions in the L polymerase protein of cp45 independently confers the temperature-sensitive (ts) and attenuation (att) phenotypes but not the cold-adaptation (ca) phenotype (29). cp45 contains 12 additional potentially important point mutations in other proteins (N, C, M, F, and hemagglutinin-neuraminidase [HN]) or in cis-acting sequences (the leader region and the transcription gene start [GS] signal of the N gene), and their contribution to these phenotypes was undefined. To further characterize the genetic basis for the ts, ca, and att phenotypes of this promising vaccine candidate, we constructed, using a reverse genetics system, a recombinant cp45 virus that contained all 15 cp45-specific mutations mentioned above, and found that it was essentially indistinguishable from the biologically derived cp45 on the basis of plaque size, level of temperature sensitivity, cold adaptation, level of replication in the upper and lower respiratory tract of hamsters, and ability to protect hamsters from subsequent wild-type PIV3 challenge. We then constructed recombinant viruses containing the cp45 mutations in individual proteins as well as several combinations of mutations. Analysis of these recombinant viruses revealed that multiple cp45 mutations distributed throughout the genome contribute to the ts, ca, and att phenotypes. In addition to the mutations in the L gene, at least one other mutation in the 3' N region (i.e., including the leader, N GS, and N coding changes) contributes to the ts phenotype. A recombinant virus containing all the cp45 mutations except those in L was more ts than cp45, illustrating the complex nature of this phenotype. The ca phenotype of cp45 also is a complex composite phenotype, reflecting contributions of at least three separate genetic elements, namely, mutations within the 3' N region, the L protein, and the C-M-F-HN region. The att phenotype is a composite of both ts and non-ts mutations. Attenuating ts mutations are located in the L protein, and non-ts attenuating mutations are located in the C and F proteins. The presence of multiple ts and non-ts attenuating mutations in cp45 likely contributes to the high level of attenuation and phenotypic stability of this promising vaccine candidate.
The structural proteins of human parainfluenza virus 3, a member of the paramyxovirus family, were characterized by SDS-polyacrylamide gel electrophoresis of radiolabeled virus. The purified virion contains at least eight structural proteins, with estimated molecular weights of 251K, 90K, 71K, 68K, 65K, 51K, 35K, and 21K, respectively. Three of the polypeptides (71K, 65K, and 51K) were identified as glycoproteins based on their incorporation of [3H]glucosamine. Disruption of the virus by Triton X-100 in the presence of increasing salt concentrations indicated that the polypeptides of molecular weights 251K, 90K, 68K, and 21K were components of the nucleocapsid. In parainfluenza virus 3 infected BS-C-1 cells, seven virus structural polypeptides were identified. Six structural proteins (90K, 71K, 68K, 51K, 35K, and 21K) were detected in the cell lysate at 7 hr after infection, while at 10 hr an additional polypeptide (251K) was also observed. At least two nonstructural polypeptides of molecular weights 30K and 25K were also detected in infected cells. mRNAs isolated from virus-infected cells were translated in a cell-free protein-synthesizing system. The in vitro translation products were identical to the authentic virion polypeptides as determined by partial digestion with staphylococcal V8 protease.
The safety, infectivity, immunogenicity, transmissibility, and phenotypic stability of an intranasal bovine parainfluenza virus type 3 (BPIV-3) candidate vaccine was evaluated in a randomized, double-blind, placebo-controlled trial. Of human parainfluenza virus type 3 (HPIV-3)-seronegative children, 92% were infected, and 92% developed a serum hemagglutination-inhibiting (HAI) antibody response to BPIV-3 and 61% to HPIV-3. Geometric mean HAI titers were 1:40 to BPIV-3 and 1:16 to HPIV-3. In studies to evaluate vaccine transmissibility, none of 14 placebo recipients in close contact with 14 vaccinees shed BPIV-3. BPIV-3 isolates from seronegative vaccinees retained the attenuation phenotype when tested in rhesus monkeys. Although it is difficult to evaluate the safety and immunogenicity of such a vaccine in an open population of children who frequently become infected with HPIV-3, it appears that the live BPIV-3 vaccine is attenuated, infectious, immunogenic, poorly transmissible, and phenotypically stable and warrants further evaluation as a candidate vaccine in infants and children.
Various extracts from the leaves of mistletoe (Viscum album L. ssp. album) were investigated for their antiviral activity on human parainfluenza virus type 2 (HPIV-2) growth in Vero cells. Plant extracts were prepared using distilled water, 50% ethanol, petroleum ether, chloroform and acetone. The 50% effective dose (ED(50)) of aqueous extract for HPIV-2 replication was 0.53 +/- 0.12 micro g/mL, and the antiviral index (AI), which was based on the ratio of the 50% inhibitory concentration (CD(50)) for host cell viability to the ED(50) for parainfluenza virus replication, was 10.05. The aqueous extract was found to be the most selective inhibitor. Furthermore, the aqueous extract at a concentration of 1 micro g/mL was found to inhibit HPIV-2 replication and the virus production was suppressed to more than 99% without any toxic effect on host cells. The chloroform extract was also found to be moderately active. In an effort to further analyse the mechanism of antiviral activity, the effectiveness of the aqueous extract on different steps of virus replication was examined. The antiviral activity could neither be attributed to the direct inactivation of the HPIV-2 nor to the inhibition of adsorption to Vero cells. The active aqueous extract has shown a dose-dependent antiviral activity on virus replication.
Human parainfluenza virus (HPIV) is a common cause of seasonal respiratory tract infections. However, little is known about the clinical presentation and impact of HPIV infections in lung transplant recipients. We reviewed HPIV infections at the University of Pittsburgh Medical Center. From January 1990 through May 2000, 32 cases of HPIV infection were identified. HPIV infection was found in 24 lung transplant recipients (75%), all of whom were included in the study group. Diagnosis was established at a median of 2.1 years after transplantation (range, 0.6-5 years). Presenting symptoms included cough (17 patients), shortness of breath (16), and temperature elevation (4). Respiratory failure occurred in 5 patients (21%). The HPIV serotypes were HPIV-1 (7 patients), HPIV-2 (2), and HPIV-3 (15 [63%]). Twenty-two patients underwent transbronchial biopsy, and 18 (82%) showed signs of acute allograft rejection. Seven patients (32%) subsequently were found to have bronchiolitis obliterans.
Etiological relationship between a bovine strain of parainfluenza virus type 3 and pneumonia in a child is suggested. Haemagglutination inhibition tests have clearly demonstrated that specific antibodies against the bovine virus were present in the patient's serum. The significance of this finding in relation to human infection is discussed.
Human parainfluenza virus type 3 (HPIV3) is a single-stranded negative-sense RNA virus belonging to the Respirovirus genus of the Paramyxoviridae family in the order Mononegavirales. The P gene encodes at least four proteins, including the C protein, which is expressed from an open reading frame (ORF) that overlaps the P ORF, and the D protein, which is encoded when the P ORF is fused to the D ORF by transcriptional editing. The P mRNA also contains a third ORF for the V protein, although it is unclear how or whether this ORF is accessed. We have used recombinant DNA technology to recover five mutant viruses that either interrupt or alter the C, D, and V ORFs. In one mutant virus, rC-KO, expression of the C protein was abrogated by changing the start codon from methionine to threonine and introducing two stop codons at amino acid positions 7 and 26 of the C ORF. In a second mutant virus, rF164S, a point mutation was introduced into the C ORF changing amino acid position 164 from phenylalanine (F) to serine (S), which corresponds to the F170S mutation described in the C protein of Sendai virus (Itoh et al., J. Gen. Virol. 78, 3207-3215). rC-KO was significantly attenuated in vitro and in vivo (rodents and primates), whereas rF164S was attenuated only in vivo. Interestingly, the rF164S mutant was more attenuated in the upper than in the lower respiratory tract of hamsters and monkeys. This pattern is the converse of that seen with temperature-sensitive attenuating mutations, and thus inclusion of this novel mutation in a recombinant live-attenuated vaccine candidate might prove useful in reducing residual virulence in the upper respiratory tract. Both rC-KO and rF164S conferred protection against challenge with wild-type HPIV3. In three other viruses, the D and V ORFs were interrupted singly or in combination. Although interruption of the D and V ORFs individually did not affect virus replication in vitro or in vivo, interruption of both together attenuated replication in vivo. These results indicate that the C, D, and V proteins of HPIV3 each has a role in virus replication in vitro, in vivo, or both, and define mutations that might be useful for the development of a vaccine against HPIV3.