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1.  Human PIV-2 Recombinant Sendai Virus (rSeV) Elicits Durable Immunity and combines with two additional rSeVs to protect against hPIV-1, hPIV-2, hPIV-3, and RSV 
Vaccine  2009;27(12):1848-1857.
The human parainfluenza viruses (hPIVs) and respiratory syncytial viruses (RSV) are the leading causes of hospitalizations due to respiratory viral disease in infants and young children, but no vaccines are yet available. Here we describe the use of recombinant Sendai viruses (rSeVs) as candidate vaccine vectors for these respiratory viruses in a cotton rat model. Two new SeV-based hPIV-2 vaccine constructs were generated by inserting the fusion (F) gene or the hemagglutinin-neuraminidase (HN) gene from hPIV-2 into the rSeV genome. The inoculation of either vaccine into cotton rats elicited neutralizing antibodies toward both homologous and heterologous hPIV-2 virus isolates. The vaccines elicited robust and durable antibodies toward hPIV-2, and cotton rats immunized with individual or mixed vaccines were fully protected against hPIV-2 infections of the lower respiratory tract. The immune responses toward a single inoculation with rSeV vaccines were long-lasting and cotton rats were protected against viral challenge for as long as 11 months after vaccination. One inoculation with a mixture of the hPIV2-HN-expressing construct and two additional rSeVs (expressing the F protein of RSV and the HN protein of hPIV-3) resulted in protection against challenge viruses hPIV-1, hPIV-2, hPIV-3, and RSV. Results identify SeV vectors as promising vaccine candidates for four different paramyxoviruses, each responsible for serious respiratory infections in children.
doi:10.1016/j.vaccine.2009.01.041
PMCID: PMC2744503  PMID: 19200447
respiratory syncytial virus; parainfluenza virus; protective immunity
2.  Recovery of a Fully Viable Chimeric Human Parainfluenza Virus (PIV) Type 3 in Which the Hemagglutinin-Neuraminidase and Fusion Glycoproteins Have Been Replaced by Those of PIV Type 1 
Journal of Virology  1998;72(4):2955-2961.
The recent recovery of human parainfluenza virus type 3 (PIV3) from cDNA, together with the availability of a promising, highly characterized live attenuated PIV3 vaccine virus, suggested a novel strategy for the rapid development of comparable recombinant vaccine viruses for human PIV1 and PIV2. The strategy, illustrated here for PIV1, is to create chimeric viruses in which the two protective antigens, the hemagglutinin-neuraminidase (HN) and fusion (F) envelope glycoproteins, of an attenuated PIV3 variant are replaced by those of PIV1 or PIV2. As a first step, this has been achieved by using recombinant wild-type (wt) PIV3 as the recipient for PIV1 HN and F, engineered so that each PIV1 open reading frame is flanked by the existing PIV3 nontranslated regions and transcription signals. This yielded a viable chimeric recombinant virus, designated rPIV3-1, that encodes the PIV1 HN and F glycoproteins in the background of the wt PIV3 internal proteins. There were three noteworthy findings. First, in contrast to recently reported glycoprotein replacement chimeras of vesicular somatitis virus or measles virus, the PIV3-1 chimera replicates in LLC-MK2 cells and in the respiratory tract of hamsters as efficiently as its PIV1 and PIV3 parents. This is remarkable because the HN and F glycoproteins share only 43 and 47%, respectively, overall amino acid sequence identity between serotypes. In particular, the cytoplasmic tails share only 9 to 11% identity, suggesting that their presumed role in virion morphogenesis does not involve sequence-specific contacts. Second, rPIV3-1 was found to possess biological properties derived from each of its parent viruses. Specifically, it requires trypsin for efficient plaque formation in tissue culture, like its PIV1 parent but unlike PIV3. On the other hand, it causes an extensive cytopathic effect (CPE) in LLC-MK2 cultures which resembles that of its PIV3 parent but differs from that of its noncytopathic PIV1 parent. This latter finding indicates that the genetic basis for the CPE of PIV3 in tissue culture lies outside regions encoding the HN or F glycoprotein. Third, it should now be possible to rapidly develop a live attenuated PIV1 vaccine by the staged introduction of known, characterized attenuating mutations present in a live attenuated PIV3 vaccine candidate into the PIV3-1 cDNA followed by recovery of attenuated derivatives of rPIV3-1.
PMCID: PMC109741  PMID: 9525616
3.  Receptor Specificities of Human Respiroviruses 
Journal of Virology  2001;75(10):4604-4613.
Through their hemagglutinin-neuraminidase glycoprotein, parainfluenza viruses bind to sialic acid-containing glycoconjugates to initiate infection. Although the virus-receptor interaction is a key factor of infection, the exact nature of the receptors that human parainfluenza viruses recognize has not been determined. We evaluated the abilities of human parainfluenza virus types 1 (hPIV-1) and 3 (hPIV-3) to bind to different types of gangliosides. Both hPIV-1 and hPIV-3 preferentially bound to neolacto-series gangliosides containing a terminal N-acetylneuraminic acid (NeuAc) linked to N-acetyllactosamine (Galβ1-4GlcNAc) by the α2-3 linkage (NeuAcα2-3Galβ1-4GlcNAc). Unlike hPIV-1, hPIV-3 bound to gangliosides with a terminal NeuAc linked to Galβ1-4GlcNAc through an α2-6 linkage (NeuAcα2-6Galβ1-4GlcNAc) or to gangliosides with a different sialic acid, N-glycolylneuraminic acid (NeuGc), linked to Galβ1-4GlcNAc (NeuGcα2-3Galβ1-4GlcNAc). These results indicate that the molecular species of glycoconjugate that hPIV-1 recognizes are more limited than those recognized by hPIV-3. Further analysis using purified gangliosides revealed that the oligosaccharide core structure is also an important element for binding. Gangliosides that contain branched N-acetyllactosaminoglycans in their core structure showed higher avidity than those without them. Agglutination of human, cow, and guinea pig erythrocytes but not equine erythrocytes by hPIV-1 and hPIV-3 correlated well with the presence or the absence of sialic acid-linked branched N-acetyllactosaminoglycans on the cell surface. Finally, NeuAcα2-3I, which bound to both viruses, inhibited virus infection of Lewis lung carcinoma-monkey kidney cells in a dose-dependent manner. We conclude that hPIV-1 and hPIV-3 preferentially recognize oligosaccharides containing branched N-acetyllactosaminoglycans with terminal NeuAcα2-3Gal as receptors and that hPIV-3 also recognizes NeuAcα2-6Gal- or NeuGcα2-3Gal-containing receptors. These findings provide important information that can be used to develop inhibitors that prevent human parainfluenza virus infection.
doi:10.1128/JVI.75.10.4604-4613.2001
PMCID: PMC114213  PMID: 11312330
4.  Sendai virus recombinant vaccine expressing hPIV-3 HN or F elicits protective immunity and combines with a second recombinant to prevent hPIV-1, hPIV-3 and RSV infections 
Vaccine  2008;26(27-28):3480-3488.
The human parainfluenza viruses (hPIVs) and Respiratory Syncytial Virus (RSV) are the leading causes of serious respiratory illness in the human pediatric population. Despite decades of research, there are currently no licensed vaccines for either the hPIV or RSV pathogens. Here we describe the testing of hPIV-3 and RSV candidate vaccines using Sendai virus (SeV, murine PIV-1) as a vector. SeV was selected as the vaccine backbone, because it has been shown to elicit robust and durable immune activities in animal studies, and has already advanced to human safety trials as a xenogenic vaccine for hPIV-1. Two new SeV-based hPIV-3 vaccine candidates were first generated by inserting either the fusion (F) gene or hemagglutinin-neuraminidase (HN) gene from hPIV-3 into SeV. The resultant rSeV-hPIV3-F and rSeV-hPIV3-HN vaccines expressed their inserted hPIV-3 genes upon infection. The inoculation of either vaccine into cotton rats elicited binding and neutralizing antibody activities, as well as interferon-γ-producing T-cells. Vaccination of cotton rats resulted in protection against subsequent challenges with either homologous or heterologous hPIV-3. Furthermore, vaccination of cotton rats with a mixture of rSeV-hPIV3-HN and a previously described recombinant SeV expressing the F protein of RSV resulted in protection against three different challenge viruses: hPIV-3, hPIV-1 and RSV. Results encourage the continued development of the candidate recombinant SeV vaccines to combat serious respiratory infections of children.
doi:10.1016/j.vaccine.2008.04.022
PMCID: PMC2728217  PMID: 18499307
respiratory syncytial virus; parainfluenza virus; protective immunity
5.  Replacement of the Ectodomains of the Hemagglutinin-Neuraminidase and Fusion Glycoproteins of Recombinant Parainfluenza Virus Type 3 (PIV3) with Their Counterparts from PIV2 Yields Attenuated PIV2 Vaccine Candidates 
Journal of Virology  2000;74(14):6448-6458.
We sought to develop a live attenuated parainfluenza virus type 2 (PIV2) vaccine strain for use in infants and young children, using reverse genetic techniques that previously were used to rapidly produce a live attenuated PIV1 vaccine candidate. The PIV1 vaccine candidate, designated rPIV3-1cp45, was generated by substituting the full-length HN and F proteins of PIV1 for those of PIV3 in the attenuated cp45 PIV3 vaccine candidate (T. Tao et al., J. Virol. 72:2955–2961, 1998; M. H. Skiadopoulos et al., Vaccine 18:503–510, 1999). However, using the same strategy, we failed to recover recombinant chimeric PIV3-PIV2 isolate carrying the full-length PIV2 glycoproteins in a wild-type PIV3 backbone. Viable PIV3-PIV2 chimeras were recovered when chimeric HN and F open reading frames (ORFs) rather than complete PIV2 F and HN ORFs were used to construct the full-length cDNA. The recovered viruses, designated rPIV3-2CT, in which the PIV2 ectodomain and transmembrane domain were fused to the PIV3 cytoplasmic domain, and rPIV3-2TM, in which the PIV2 ectodomain was fused to the PIV3 transmembrane and cytoplasmic tail domain, possessed similar in vitro and in vivo phenotypes. Thus, it appeared that only the cytoplasmic tail of the HN or F glycoprotein of PIV3 was required for successful recovery of PIV3-PIV2 chimeras. Although rPIV3-2CT and rPIV3-2TM replicated efficiently in vitro, they were moderately to highly attenuated for replication in the respiratory tracts of hamsters, African green monkeys (AGMs), and chimpanzees. This unexpected finding indicated that chimerization of the HN and F proteins of PIV2 and PIV3 itself specified an attenuation phenotype in vivo. Despite this attenuation, these viruses were highly immunogenic and protective against challenge with wild-type PIV2 in hamsters and AGMs, and they represent promising candidates for clinical evaluation as a vaccine against PIV2. These chimeric viruses were further attenuated by the addition of 12 mutations of PIV3cp45 which lie outside of the HN and F genes. The attenuating effects of these mutations were additive with that of the chimerization, and thus inclusion of all or some of the cp45 mutations provides a means to further attenuate the PIV3-PIV2 chimeric vaccine candidates if necessary.
PMCID: PMC112153  PMID: 10864657
6.  Expression of the Surface Glycoproteins of Human Parainfluenza Virus Type 3 by Bovine Parainfluenza Virus Type 3, a Novel Attenuated Virus Vaccine Vector 
Journal of Virology  2000;74(24):11626-11635.
Bovine parainfluenza virus type 3 (bPIV3) is being evaluated as an intranasal vaccine for protection against human PIV3 (hPIV3). In young infants, the bPIV3 vaccine appears to be infectious, attenuated, immunogenic, and genetically stable, which are desirable characteristics for an RNA virus vector. To test the potential of the bPIV3 vaccine strain as a vector, an infectious DNA clone of bPIV3 was assembled and recombinant bPIV3 (r-bPIV3) was rescued. r-bPIV3 displayed a temperature-sensitive phenotype for growth in tissue culture at 39°C and was attenuated in the lungs of Syrian golden hamsters. In order to test whether r-bPIV3 could serve as a vector, the fusion and hemagglutinin-neuraminidase genes of bPIV3 were replaced with those of hPIV3. The resulting bovine/human PIV3 was temperature sensitive for growth in Vero cells at 37°C. The replication of bovine/human PIV3 was also restricted in the lungs of hamsters, albeit not as severely as was observed for r-bPIV3. Despite the attenuation phenotypes observed for r-bPIV3 and bovine/human PIV3, both of these viruses protected hamsters completely upon challenge with hPIV3. In summary, bPIV3 was shown to function as a virus vector that may be especially suitable for vaccination of infants and children against PIV3 and other viruses.
PMCID: PMC112444  PMID: 11090161
7.  Human Parainfluenza Virus Type 2 Vector Induces Dendritic Cell Maturation Without Viral RNA Replication/Transcription 
Human Gene Therapy  2013;24(7):683-691.
Abstract
The dendritic cell (DC), a most potent antigen-presenting cell, plays a key role in vaccine therapy against infectious diseases and malignant tumors. Although advantages of viral vectors for vaccine therapy have been reported, potential risks for adverse effects prevent them from being licensed for clinical use. Human parainfluenza virus type 2 (hPIV2), one of the members of the Paramyxoviridae family, is a nonsegmented and negative-stranded RNA virus. We have developed a reverse genetics system for the production of infectious hPIV2 lacking the F gene (hPIV2ΔF), wherein various advantages for vaccine therapy exist, such as cytoplasmic replication/transcription, nontransmissible infectivity, and extremely high transduction efficacy in various types of target cells. Here we demonstrate that hPIV2ΔF shows high transduction efficiency in human DCs, while not so high in mouse DCs. In addition, hPIV2ΔF sufficiently induces maturation of both human and murine DCs, and the maturation state of both human and murine DCs is almost equivalent to that induced by lipopolysaccharide. Moreover, alkylating agent β-propiolactone-inactivated hPIV2ΔF (BPL-hPIV2ΔF) elicits DC maturation without viral replication/transcription. These results suggest that hPIV2ΔF may be a useful tool for vaccine therapy as a novel type of paramyxoviral vector, which is single-round infectious vector and has potential adjuvant activity.
Using a reverse genetics approach, Hara and colleagues generate a human parainfluenza vector that lacks the F gene. These investigators show that the vector has high transduction efficiency in both mouse and human dendritic cells (DCs) and can trigger DC maturation in the absence of viral replication/transcription.
doi:10.1089/hum.2013.024
PMCID: PMC3719437  PMID: 23790317
8.  Stability of the Parainfluenza Virus 5 Genome Revealed by Deep Sequencing of Strains Isolated from Different Hosts and following Passage in Cell Culture 
Journal of Virology  2014;88(7):3826-3836.
ABSTRACT
The strain diversity of a rubulavirus, parainfluenza virus 5 (PIV5), was investigated by comparing 11 newly determined and 6 previously published genome sequences. These sequences represent 15 PIV5 strains, of which 6 were isolated from humans, 1 was from monkeys, 2 were from pigs, and 6 were from dogs. Strain diversity is remarkably low, regardless of host, year of isolation, or geographical origin; a total of 7.8% of nucleotides are variable, and the average pairwise difference between strains is 2.1%. Variation is distributed unevenly across the PIV5 genome, but no convincing evidence of selection for antibody-mediated evasion in hemagglutinin-neuraminidase was found. The finding that some canine and porcine, but not primate, strains are mutated in the SH gene, and do not produce SH, raised the possibility that dogs (or pigs) may not be the natural host of PIV5. The genetic stability of PIV5 was also demonstrated during serial passage of one strain (W3) in Vero cells at a high multiplicity of infection, under conditions of competition with large proportions of defective interfering genomes. A similar observation was made for a strain W3 mutant (PIV5VΔC) lacking V gene function, in which the dominant changes were related to pseudoreversion in this gene. The mutations detected in PIV5VΔC during pseudoreversion, and also those characterizing the SH gene in canine and porcine strains, predominantly involved U-to-C transitions. This suggests an important role for biased hypermutation via an adenosine deaminase, RNA-specific (ADAR)-like activity.
IMPORTANCE Here we report the sequence variation of 16 different isolates of parainfluenza virus 5 (PIV5) that were isolated from a number of species, including humans, monkeys, dogs, and pigs, over 4 decades. Surprisingly, strain diversity was remarkably low, regardless of host, year of isolation, or geographical origin. Variation was distributed unevenly across the PIV5 genome, but no convincing evidence of immune or host selection was found. This overall genome stability of PIV5 was also observed when the virus was grown in the laboratory, and the genome stayed remarkably constant even during the selection of virus mutants. Some of the canine isolates had lost their ability to encode one of the viral proteins, termed SH, suggesting that although PIV5 commonly infects dogs, dogs may not be the natural host for PIV5.
doi:10.1128/JVI.03351-13
PMCID: PMC3993540  PMID: 24453358
9.  Mucosal Immunization of Rhesus Monkeys against Respiratory Syncytial Virus Subgroups A and B and Human Parainfluenza Virus Type 3 by Using a Live cDNA-Derived Vaccine Based on a Host Range-Attenuated Bovine Parainfluenza Virus Type 3 Vector Backbone 
Journal of Virology  2002;76(3):1089-1099.
Reverse genetics was used to develop a two-component, trivalent live attenuated vaccine against human parainfluenza virus type 3 (HPIV3) and respiratory syncytial virus (RSV) subgroups A and B. The backbone for each of the two components of this vaccine was the attenuated recombinant bovine/human PIV3 (rB/HPIV3), a recombinant BPIV3 in which the bovine HN and F protective antigens are replaced by their HPIV3 counterparts (48). This chimera retains the well-characterized host range attenuation phenotype of BPIV3, which appears to be appropriate for immunization of young infants. The open reading frames (ORFs) for the G and F major protective antigens of RSV subgroup A and B were each placed under the control of PIV3 transcription signals and inserted individually or in homologous pairs as supernumerary genes in the promoter proximal position of rB/HPIV3. The level of replication of rB/HPIV3-RSV chimeric viruses in the respiratory tract of rhesus monkeys was similar to that of their parent virus rB/HPIV3, and each of the chimeras induced a robust immune response to both RSV and HPIV3. RSV-neutralizing antibody titers induced by rB/HPIV3-RSV chimeric viruses were equivalent to those induced by infection with wild-type RSV, and HPIV3-specific antibody responses were similar to, or slightly less than, after infection with the rB/HPIV3 vector itself. This study describes a novel vaccine strategy against RSV in which vaccine viruses with a common attenuated backbone, specifically rB/HPIV3 derivatives expressing the G and/or F major protective antigens of RSV subgroup A and of RSV subgroup B, are used to immunize by the intranasal route against RSV and HPIV3, which are the first and second most important viral agents of pediatric respiratory tract disease worldwide.
doi:10.1128/JVI.76.3.1089-1099.2002
PMCID: PMC135799  PMID: 11773385
10.  Comparative Analysis of the Full-Length Genome Sequence of a Clinical Isolate of Human Parainfluenza Virus 4B 
Scientifica  2012;2012:871201.
We are engaged in airborne transmission and epidemiology studies of respiratory pathogens, with particular interest in human parainfluenza virus type 4 (hPIV-4) and other lesser studied viruses. In this paper, hPIV-4 was detected in primary rhesus monkey kidney (PRMK) cells that had been inoculated with nasopharyngeal swab material obtained from a child with a mild upper respiratory tract illness. Attempts to isolate the virus in pure culture were hampered by the presence of a fast-growing simian spumavirus that was a contaminant of the PRMK cells. Total RNA was extracted from the PRMK cell culture, and PCR followed by sequencing of a subgenomic section of the fusion protein gene suggested the hPIV-4 was subtype 4B. At the time of this work, two complete but dissimilar hPIV-4B genomes had been deposited by others in GenBank. To gain better insights on hPIV-4B, and to test methods that we are developing for viral forensics, the entire genomic sequence of our virus was determined from archived RNA. The hPIV-4B genomic sequence that we determined conforms to the paramyxovirus “rule of six.” Here, we compare and contrast the genetic features of the three completely sequenced hPIV-4B genomes currently present in GenBank.
doi:10.6064/2012/871201
PMCID: PMC3820592  PMID: 24278751
11.  Determinants of the Host Range Restriction of Replication of Bovine Parainfluenza Virus Type 3 in Rhesus Monkeys Are Polygenic 
Journal of Virology  2003;77(2):1141-1148.
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.
doi:10.1128/JVI.77.2.1141-1148.2003
PMCID: PMC140817  PMID: 12502830
12.  Generation of Recombinant Human Parainfluenza Virus Type 1 Vaccine Candidates by Importation of Temperature-Sensitive and Attenuating Mutations from Heterologous Paramyxoviruses 
Journal of Virology  2004;78(4):2017-2028.
Human parainfluenza virus type 1 (HPIV1) is a significant cause of respiratory tract disease in infants and young children for which a vaccine is needed. In the present study, we sought to attenuate HPIV1 by the importation of one or more known attenuating point mutations from heterologous paramyxoviruses into homologous sites in HPIV1. The introduced mutations were derived from three attenuated paramyxoviruses: (i) HPIV3cp45, a live-attenuated HPIV3 vaccine candidate containing multiple attenuating mutations; (ii) the respiratory syncytial virus cpts530 with an attenuating mutation in the L polymerase protein; and (iii) a murine PIV1 (MPIV1) attenuated by a mutation in the accessory C protein. Recombinant HPIV1 (rHPIV1) mutants bearing a single imported mutation in C, any of three different mutations in L, or a pair of mutations in F exhibited a 100-fold or greater reduction in replication in the upper or lower respiratory tract of hamsters. Both temperature-sensitive (ts) (mutations in the L and F proteins) and non-ts (the mutation in the C protein) attenuating mutations were identified. rHPIV1 mutants containing a combination of mutations in L were generated that were more attenuated than viruses bearing the individual mutations, showing that the systematic accretion of mutations can yield progressive increases in attenuation. Hamsters immunized with rHPIV1 mutants bearing one or two mutations developed neutralizing antibodies and were resistant to challenge with wild-type HPIV1. Thus, importation of attenuating mutations from heterologous viruses is an effective means for rapidly identifying mutations that attenuate HPIV1 and for generating live-attenuated HPIV1 vaccine candidates.
doi:10.1128/JVI.78.4.2017-2028.2004
PMCID: PMC369514  PMID: 14747566
13.  Mutation at Residue 523 Creates a Second Receptor Binding Site on Human Parainfluenza Virus Type 1 Hemagglutinin-Neuraminidase Protein 
Journal of Virology  2006;80(18):9009-9016.
The paramyxovirus hemagglutinin-neuraminidase (HN) is a multifunctional protein mediating hemagglutination (HA), neuraminidase (NA), and fusion promotion activities. It has been a matter of debate whether HN contains combined or separate sites for HA and NA activities. To clear the issue, we determined the presence of the second binding site on human parainfluenza virus (hPIV) type 1, 2, and 3 and Sendai virus (SeV) HN proteins. Results of virus elution from erythrocytes at an elevated temperature and HA inhibition by NA inhibitor BCX-2798 suggest that all hPIVs bind to the receptor only through the NA catalytic site, while SeV HN has an additional receptor binding site. Comparison of SeV and hPIV1 HN sequences revealed two amino acid differences at residues 521 and 523 in the region close to the second binding site identified in Newcastle disease virus HN. We mutated hPIV1 HN at position 523 from Asn to the residue of SeV HN, Asp, and rescued a recombinant SeV that carries the mutated hPIV1 HN by a reverse genetics system. The hPIV1 HN with Asp at position 523 hemagglutinated in the presence of BCX-2798, suggesting that the amino acid difference at position 523 is critical for the formation of a second binding site. Creation of the second binding site on hPIV1 HN, however, did not significantly affect the growth or fusion activity of the recombinant virus. Our study indicates that the presence and requirement of a second binding site vary among paramyxoviruses.
doi:10.1128/JVI.00969-06
PMCID: PMC1563932  PMID: 16940513
14.  Identification of Paramyxovirus V Protein Residues Essential for STAT Protein Degradation and Promotion of Virus Replication 
Journal of Virology  2005;79(13):8591-8601.
Some paramyxovirus V proteins induce STAT protein degradation, and the amino acids essential for this process in the human parainfluenza virus type 2 (hPIV2) V protein have been studied. Various recombinant hPIV2s and cell lines constitutively expressing various mutant V proteins were generated. We found that V proteins with replacement of Cys residues of the Cys cluster were still able to bind STATs but were unable to induce their degradation. The hPIV2 V protein binds STATs via a W-(X)3-W-(X)9-W Trp motif located just upstream of the Cys cluster. Replacements of two or more Trp residues in this motif resulted in a failure to form a V/STAT2 complex. We have also identified two Phe residues of the hPIV2 V protein that are essential for STAT degradation, namely, Phe207, lying within the Cys cluster, and Phe143, in the P/V common region of the protein. Interestingly, infection of BHK cells with hPIV2 led to the specific degradation of STAT1 and not STAT2. Other evidence for the cell species specificity of hPIV2-induced STAT degradation is presented. Finally, a V-minus hPIV2, which can express only the P protein from its P gene, was generated and partially characterized. In contrast to V-minus viruses of other paramyxovirus genera, this V-minus rubulavirus was highly debilitated, and its growth even in Vero cells was very limited. The structural rubulavirus V proteins, as expected, are thus clearly important in promoting virus growth, independent of their anti-interferon (IFN) activity. Interestingly, many of the residues that are essential for anti-IFN activity, e.g., the Cys of this cluster and Phe207 within this cluster, as well as the Trp of this motif, are also essential for promoting virus growth.
doi:10.1128/JVI.79.13.8591-8601.2005
PMCID: PMC1143765  PMID: 15956600
15.  Differential Regulation of Type I Interferon and Epidermal Growth Factor Pathways by a Human Respirovirus Virulence Factor 
PLoS Pathogens  2009;5(9):e1000587.
A number of paramyxoviruses are responsible for acute respiratory infections in children, elderly and immuno-compromised individuals, resulting in airway inflammation and exacerbation of chronic diseases like asthma. To understand the molecular pathogenesis of these infections, we searched for cellular targets of the virulence protein C of human parainfluenza virus type 3 (hPIV3-C). We found that hPIV3-C interacts directly through its C-terminal domain with STAT1 and GRB2, whereas C proteins from measles or Nipah viruses failed to do so. Binding to STAT1 explains the previously reported capacity of hPIV3-C to block type I interferon signaling, but the interaction with GRB2 was unexpected. This adaptor protein bridges Epidermal Growth Factor (EGF) receptor to MAPK/ERK pathway, a signaling cascade recently found to be involved in airway inflammatory response. We report that either hPIV3 infection or transient expression of hPIV3-C both increase cellular response to EGF, as assessed by Elk1 transactivation and phosphorylation levels of ERK1/2, 40S ribosomal subunit protein S6 and translation initiation factor 4E (eIF4E). Furthermore, inhibition of MAPK/ERK pathway with U0126 prevented viral protein expression in infected cells. Altogether, our data provide molecular basis to explain the role of hPIV3-C as a virulence factor and determinant of pathogenesis and demonstrate that Paramyxoviridae have evolved a single virulence factor to block type I interferon signaling and to boost simultaneous cellular response to growth factors.
Author Summary
Respiroviruses are important pathogens responsible for acute respiratory tract infections associated with severe airway inflammation in children, elderly and immuno-compromised individuals. Their RNA genome encodes for structural proteins that compose viral particles, but also for virulence factors that alter cell biology to enhance virus replication and spreading. Among them, the C protein plays a critical role by blocking cellular response to type I interferons, the main antiviral cytokines secreted during virus infections. To provide molecular basis to this activity, we found that the C protein of human parainfluenza virus type 3 (hPIV3-C), the most frequent human Respirovirus, interacts with STAT1, a key component of type I interferon receptor complex. But hPIV3-C was also found to interact with GRB2, an adaptor molecule involved in cellular response to Epidermal Growth Factor (EGF), and to enhance cell response to this cytokine. This pathway increases protein translation, promotes cell survival and contributes to airway inflammation and mucus secretion. Thus, our findings show that hPIV3-C not only inhibits the antiviral response but also stimulates cellular response to EGF, which benefits virus replication and induces an excessive inflammation of airways during infection.
doi:10.1371/journal.ppat.1000587
PMCID: PMC2736567  PMID: 19806178
16.  Human Parainfluenza Virus Type 3 (PIV3) Expressing the Hemagglutinin Protein of Measles Virus Provides a Potential Method for Immunization against Measles Virus and PIV3 in Early Infancy 
Journal of Virology  2000;74(15):6821-6831.
Recombinant human parainfluenza virus type 3 (PIV3) was used as a vector to express the major protective antigen of measles virus, the hemagglutinin (HA) glycoprotein, in order to create a bivalent PIV3-measles virus that can be administered intranasally. The measles virus HA open reading frame (ORF) was inserted as an additional transcriptional unit into the N-P, P-M, or HA-neuraminidase (HN)-L gene junction of wild-type PIV3 or into the N-P or P-M gene junction of an attenuated derivative of PIV3, termed rcp45L. The recombinant PIV3 (rPIV3) viruses bearing the HA inserts replicated more slowly in vitro than their parental viruses but reached comparable peak titers of ≥107.5 50% tissue culture infective doses per ml. Each of the wild-type or cold-passaged 45L (cp45L) PIV3(HA) chimeric viruses replicated 5- to 10-fold less well than its respective parent virus in the upper respiratory tract of hamsters. Thus, insertion of the ∼2-kb ORF itself conferred attenuation, and this attenuation was additive to that conferred by the cp45L mutations. The attenuated cp45L PIV3(HA) recombinants induced a high level of resistance to replication of PIV3 challenge virus in hamsters and induced very high levels of measles virus neutralizing antibodies (>1:8,000) that are well in excess of those known to be protective in humans. rPIV3s expressing the HA gene in the N-P or P-M junction induced about 400-fold more measles virus-neutralizing antibody than did the rPIV3 with the HA gene in the HN-L junction, indicating that the N-P or P-M junction appears to be the preferred insertion site. Previous studies indicated that the PIV3 cp45 virus, a more attenuated version of rcp45L, replicates efficiently in the respiratory tract of monkeys and is immunogenic and protective even when administered in the presence of very high titers of passively transferred PIV3 antibodies (A. P. Durbin, C. J. Cho, W. R. Elkins, L. S. Wyatt, B. Moss, and B. R. Murphy, J. Infect. Dis. 179:1345–1351, 1999). This suggests that this intranasally administered PIV3(HA) chimeric virus can be used to immunize infants with maternally acquired measles virus antibodies in whom the current parenterally administered live measles virus vaccine is ineffective.
PMCID: PMC112199  PMID: 10888621
17.  Illumination of Parainfluenza Virus Infection and Transmission in Living Animals Reveals a Tissue-Specific Dichotomy 
PLoS Pathogens  2011;7(7):e1002134.
The parainfluenza viruses (PIVs) are highly contagious respiratory paramyxoviruses and a leading cause of lower respiratory tract (LRT) disease. Since no vaccines or antivirals exist, non-pharmaceutical interventions are the only means of control for these pathogens. Here we used bioluminescence imaging to visualize the spatial and temporal progression of murine PIV1 (Sendai virus) infection in living mice after intranasal inoculation or exposure by contact. A non-attenuated luciferase reporter virus (rSeV-luc(M-F*)) that expressed high levels of luciferase yet was phenotypically similar to wild-type Sendai virus in vitro and in vivo was generated to allow visualization. After direct intranasal inoculation, we unexpectedly observed that the upper respiratory tract (URT) and trachea supported robust infection under conditions that result in little infection or pathology in the lungs including a low inoculum of virus, an attenuated virus, and strains of mice genetically resistant to lung infection. The high permissivity of the URT and trachea to infection resulted in 100% transmission to naïve contact recipients, even after low-dose (70 PFU) inoculation of genetically resistant BALB/c donor mice. The timing of transmission was consistent with the timing of high viral titers in the URT and trachea of donor animals but was independent of the levels of infection in the lungs of donors. The data therefore reveals a disconnect between transmissibility, which is associated with infection in the URT, and pathogenesis, which arises from infection in the lungs and the immune response. Natural infection after transmission was universally robust in the URT and trachea yet limited in the lungs, inducing protective immunity without weight loss even in genetically susceptible 129/SvJ mice. Overall, these results reveal a dichotomy between PIV infection in the URT and trachea versus the lungs and define a new model for studies of pathogenesis, development of live virus vaccines, and testing of antiviral therapies.
Author Summary
Human parainfluenza viruses (HPIVs) are a leading cause of pediatric hospitalization for lower respiratory tract infection, yet it is unknown why primary infection typically induces immunity without causing severe pathology. To study the determinants of PIV spread within the respiratory tracts of living animals, we developed a model for non-invasive imaging of living mice infected with Sendai virus, the murine counterpart of HPIV1. This system allowed us to measure the temporal and spatial dynamics of paramyxovirus infection throughout the respiratory tracts of living animals after direct inoculation or transmission. We found that the upper respiratory tract and trachea were highly permissive to infection, even under conditions that limit lower respiratory infection and pathogenesis. The timing of transmission coincided with high virus growth in the upper respiratory tracts and trachea of donor mice independent of the extent of infection in the lungs. After transmission, infection spread preferentially in the upper respiratory tract and trachea, inducing protective immunity without weight loss. Our work reveals a disconnect between Sendai virus transmissibility and pathogenicity, and the experimental model developed here will be instrumental in studying PIV pathogenesis.
doi:10.1371/journal.ppat.1002134
PMCID: PMC3131265  PMID: 21750677
18.  Efficacy of the Novel Parainfluenza Virus Hemagglutinin-Neuraminidase Inhibitor BCX 2798 in Mice—Further Evaluation 
Antiviral therapy  2009;14(7):891-898.
Background
Human parainfluenza virus type 1 (hPIV-1) causes serious respiratory tract infections, especially in children. This study investigated the efficacy of the novel hemagglutinin-neuraminidase (HN) inhibitor BCX 2798 in the prophylaxis of lethal and the treatment of non-lethal parainfluenza virus infection in mice.
Methods
In the prophylaxis model, 129x1/SvJ mice were inoculated with a 90% lethal dose of a recombinant Sendai virus, in which the HN gene was replaced with that of hPIV-1 (rSeV[hPIV-1HN]). The mice were intranasally treated either once or for 5 d with 1 or 10 mg/kg/d of BCX 2798, starting 4 h before infection. In the therapeutic model, mice were infected with 100 PFU per mouse of rSeV(hPIV-1HN) and treated intranasally with 0.1, 1 or 10 mg/kg/d of BCX 2798 for 5 d, starting 24 or 48 h after infection, or for 4 d starting 72 h after infection.
Results
Similar to multiple dosing, a single intranasal prophylaxis with 1 or 10 mg/kg of BCX 2798 protected approximately 40% or 90%, respectively, of mice from death by rSeV(hPIV-1HN) infection. BCX 2798 also significantly reduced virus lung titers (in a dose- and time-dependent manner) and histopathologic changes in the airways in non-lethally infected mice at multiple intranasal dosages in the therapeutic model, with the lowest effective dosage being 0.1 mg/kg/d administered 24 h after infection.
Conclusion
BCX 2798 was effective in the prophylaxis of lethal and in the therapy of non-lethal parainfluenza virus infection in mice, suggesting further consideration of BCX 2798 for clinical trials.
doi:10.3851/IMP1420
PMCID: PMC2782883  PMID: 19918093
19.  The Long Noncoding Region of the Human Parainfluenza Virus Type 1 F Gene Contributes to the Read-Through Transcription at the M-F Gene Junction 
Journal of Virology  2002;76(16):8244-8251.
Sendai virus (SV) and human parainfluenza virus type 1 (hPIV1) have genomes consisting of nonsegmented negative-sense RNA in which the six genes are separated by well-conserved intergenic (IG) sequences and transcriptional start (S) and end signals. In hPIV1-infected cells, transcriptional termination at the M-F gene junction is ineffective; a large number of M-F read-through transcripts are produced (T. Bousse, T. Takimoto, K. G. Murti, and A. Portner, Virology 232:44-52, 1997). In contrast, few M-F read-through transcripts are detected in SV-infected cells. Sequence analysis indicated that the hPIV1 IG and S sequences in the M-F junction differ from those of SV. Furthermore, the hPIV1 F gene contains an unusually long noncoding sequence. To identify the cis-acting elements that prevent transcriptional termination at the M-F junction, we rescued recombinant SV (rSVhMFjCG) in which its M-F gene junction was replaced by that of hPIV1. Cells infected with rSVhMFjCG produced an abundance of M-F read-through transcripts; this result indicated that the hPIV1 M-F junction is responsible for inefficient termination. When one or both of the IG and S sites in rSVhMFjCG were replaced by those of SV, the efficiency of transcriptional termination increased but not to the level observed in wild-type SV-infected cells. Deletion of most of the long noncoding region of the hPIV1 F gene in rSVhMFjCG in addition to the mutations in IG and S signals resulted in efficient termination that was equivalent to the level observed in wild-type virus-infected cells. Therefore, the long noncoding sequence of the hPIV1 F gene contains cis-acting element(s) that affects transcriptional termination. Our evaluation of the effect of inefficient transcriptional termination on viral replication in culture revealed that cells infected with rSVhMFjCG produced less F protein than cells infected with wild-type SV and that assembly of the recombinant SV in culture was less efficient. These phenotypes seem to be responsible for the extended survival of mice infected with rSVhMFjCG.
doi:10.1128/JVI.76.16.8244-8251.2002
PMCID: PMC155142  PMID: 12134030
20.  Human Parainfluenza Virus Type 4 Is Incapable of Evading the Interferon-Induced Antiviral Effect 
Journal of Virology  2005;79(23):14756-14768.
The V proteins of some paramyxoviruses have developed the ability to efficiently inactivate STAT protein function as a countermeasure for evading interferon (IFN) responses. Human parainfluenza virus type 4 (hPIV4) is one of the rubulaviruses, which are members of the family Paramyxoviridae, and has a V protein with a highly conserved cysteine-rich domain that is the hallmark of paramyxovirus V proteins. In order to study the function of the hPIV4 V protein, we established HeLa cells expressing the hPIV4A V protein (HeLa/FlagPIV4V). The hPIV4 V protein had no ability to reduce the level of STAT1 or STAT2, although it associated with STAT1, STAT2, DDB1, and Cul4A. It interfered with neither STAT1 and STAT2 tyrosine phosphorylation nor IFN-induced STAT nuclear accumulation. In addition, HeLa/FlagPIV4V cells are fully sensitive to both beta interferon (IFN-β) and IFN-γ, indicating that the hPIV4 V protein has no ability to block IFN-induced signaling. We further established HeLa cells expressing various chimeric proteins between the hPIV2 and hPIV4A V proteins. The lack of IFN-antagonistic activity of the hPIV4 V protein is caused by both the P/V common and V-specific domains. At least two regions (amino acids [aa] 32 to 45 and aa 143 to 164) of hPIV4 V in the P/V common domain and one region (aa 200 to 212) of the C terminus are involved in the inability to evade the IFN-induced signaling. Moreover, we established HeLa cells persistently infected with hPIV4 to make sure of the inability to escape IFN and confirmed that hPIV4 is the only paramyxovirus analyzed to date that can't evade the IFN-induced antiviral responses.
doi:10.1128/JVI.79.23.14756-14768.2005
PMCID: PMC1287573  PMID: 16282476
21.  Loss of the N-Linked Glycan at Residue 173 of Human Parainfluenza Virus Type 1 Hemagglutinin-Neuraminidase Exposes a Second Receptor-Binding Site▿  
Journal of Virology  2008;82(17):8400-8410.
BCX 2798 (4-azido-5-isobutyrylamino-2,3-didehydro-2,3,4,5-tetradeoxy-d-glycero-d-galacto-2-nonulopyranosic acid) effectively inhibited the activities of the hemagglutinin-neuraminidase (HN) of human parainfluenza viruses (hPIV) in vitro and protected mice from lethal infection with a recombinant Sendai virus whose HN was replaced with that of hPIV-1 (rSeV[hPIV-1HN]) (I. V. Alymova, G. Taylor, T. Takimoto, T. H. Lin., P. Chand, Y. S. Babu, C. Li, X. Xiong, and A. Portner, Antimicrob. Agents Chemother. 48:1495-1502, 2004). The ability of BCX 2798 to select drug-resistant variants in vivo was examined. A variant with an Asn-to-Ser mutation at residue 173 (N173S) in HN was recovered from mice after a second passage of rSeV(hPIV-1HN) in the presence of BCX 2798 (10 mg/kg of body weight daily). The N173S mutant remained sensitive to BCX 2798 in neuraminidase inhibition assays but was more than 10,000-fold less sensitive to the compound in hemagglutination inhibition tests than rSeV(hPIV-1HN). Its susceptibility to BCX 2798 in plaque reduction assays was reduced fivefold and did not differ from that of rSeV(hPIV-1HN) in mice. The N173S mutant failed to be efficiently eluted from erythrocytes and released from cells. It demonstrated reduced growth in cell culture and superior growth in mice. The results for gel electrophoresis analysis were consistent with the loss of the N-linked glycan at residue 173 in the mutant. Sequence and structural comparisons revealed that residue 173 on hPIV-1 HN is located close to the region of the second receptor-binding site identified in Newcastle disease virus HN. Our study suggests that the N-linked glycan at residue 173 masks a second receptor-binding site on hPIV-1 HN.
doi:10.1128/JVI.00474-08
PMCID: PMC2519627  PMID: 18579600
22.  Inhibition of Primary Clinical Isolates of Human Parainfluenza Virus by DAS181 in Cell Culture and in a Cotton Rat Model 
Antiviral research  2013;100(2):10.1016/j.antiviral.2013.09.014.
DAS181 is a novel drug in development for the treatment of influenza as well as human parainfluenza viruses (hPIV). Previous studies demonstrated that DAS181 inhibited laboratory strains of hPIV, but no tests were conducted with primary clinical isolates of hPIV. To fill this gap, we studied six primary isolates including hPIV-2 and hPIV-3. First tests showed that the amplification of all viruses in vitro was reproducibly inhibited with DAS181 drug concentrations ranging between 0.1 and 1 nM. An hPIV-3 primary clinical isolate was then tested in a cotton rat model for sensitivity to 0.3-1 mg/kg drug treatments. Results showed that virus amplification in the lower respiratory tract was significantly and reproducibly inhibited by drug. Together, experiments demonstrated that DAS181 inhibited primary clinical isolates of hPIV in vitro and in vivo at doses similar to those previously described for inhibition of laboratory hPIV and influenza virus isolates.
doi:10.1016/j.antiviral.2013.09.014
PMCID: PMC3871174  PMID: 24076357
23.  Biology of parainfluenza viruses. 
Clinical Microbiology Reviews  1994;7(2):265-275.
Parainfluenza virus types 1 to 4 (PIV1 to PIV4) are important human pathogens that cause upper and lower respiratory tract infections, especially in infants and children. PIV1, PIV2, and PIV3 are second only to respiratory syncytial virus as a cause of croup in young children. Although some clinical symptoms are typical of PIVs, etiologic diagnosis always requires detection of infectious virus, viral components, or an antibody response. PIVs are typical paramyxoviruses, causing a syncytial cytopathic effect in cell cultures; virus growth can be confirmed either by hemadsorption or by using immunological reagents. Currently, PIV is most often diagnosed by demonstrating viral antigens in clinical specimens by rapid and highly sensitive immunoassays. More recently, PCR has been used for the detection of PIVs. Serological diagnosis is made by detecting a rising titer of immunoglobulin G or by demonstrating immunoglobulin M antibodies. PIVs infect species other than humans, and animal models are used to study the pathogenesis of PIV infections and to test candidate vaccines. Accumulating knowledge on the molecular structure and mechanisms of replication of PIVs has accelerated research on prevention and treatment. Several strategies for vaccine development, such as the use of live attenuated, inactivated, recombinant, and subunit vaccines, have been investigated, and it may become possible to prevent PIV infections in the near future.
PMCID: PMC358320  PMID: 8055470
24.  Efficacy of Novel Hemagglutinin-Neuraminidase Inhibitors BCX 2798 and BCX 2855 against Human Parainfluenza Viruses In Vitro and In Vivo 
Human parainfluenza viruses are important respiratory tract pathogens, especially of children. However, no vaccines or specific therapies for infections caused by these viruses are currently available. In the present study we characterized the efficacy of the novel parainfluenza virus inhibitors BCX 2798 and BCX 2855, which were designed based on the three-dimensional structure of the hemagglutinin-neuraminidase (HN) protein. The compounds were highly effective in inhibiting hemagglutinin (HA) and neuraminidase (NA) activities and the growth of hPIV-1, hPIV-2, and hPIV-3 in LLC-MK2 cells. The concentrations required to reduce the activity to 50% of that of a control ranged from 0.1 to 6.0 μM in HA inhibition assays and from 0.02 to 20 μM in NA inhibition assays. The concentrations required to inhibit virus replication to 50% of the level of the control ranged from 0.7 to 11.5 μM. BCX 2798 and BCX 2855 were inactive against influenza virus HA and NA and bacterial NA. In mice infected with a recombinant Sendai virus whose HN gene was replaced with that of hPIV-1 [rSV(hHN)], intranasal administration of BCX 2798 (10 mg/kg per day) and of BCX 2855 (50 mg/kg per day) 4 h before the start of infection resulted in a significant reduction in titers of virus in the lungs and protection from death. Treatment beginning 24 h after the start of infection did not prevent death. Together, our results indicate that BCX 2798 and BCX 2855 are effective inhibitors of parainfluenza virus HN and may limit parainfluenza virus infections in humans.
doi:10.1128/AAC.48.5.1495-1502.2004
PMCID: PMC400544  PMID: 15105096
25.  Effect of Hemagglutinin-Neuraminidase Inhibitors BCX 2798 and BCX 2855 on Growth and Pathogenicity of Sendai/Human Parainfluenza Type 3 Chimera Virus in Mice▿  
Human parainfluenza virus type 3 (hPIV-3) is a major respiratory tract pathogen that affects young children, but no vaccines or antiviral drugs against it have yet been developed. We developed a mouse model to evaluate the efficacies of the novel parainfluenza virus hemagglutinin-neuraminidase (HN) inhibitors BCX 2798 and BCX 2855 against a recombinant Sendai virus (rSeV) in which the fusion (F) and HN surface glycoproteins (FHN) were replaced by those of hPIV-3 [rSeV(hPIV-3FHN)]. In the prophylaxis model, 129X1/SvJ mice were infected with a 90% or 20% lethal dose of the virus and were treated intranasally for 5 days with 10 mg/kg of body weight/day of either compound starting 4 h before infection. Prophylactic treatment of the mice with either compound did not prevent their death in a 90% lethality model of rSeV(hPIV-3FHN) infection. However, it significantly reduced the lung virus titers, the amount of weight lost, and the rate of mortality in mice infected with a 20% lethal virus dose. In the therapy model, mice were infected with a nonlethal dose of the virus (100 PFU/mouse) and were treated intranasally with 1 or 10 mg/kg/day of either compound for 5 days starting at 24 or 48 h postinfection. Treatment of the mice with either compound significantly reduced the virus titer in the lungs, subsequently causing a reduction in the number of immune cells and the levels of cytokines in the bronchoalveolar lavage fluid and histopathologic changes in the airways. Our results indicate that BCX 2798 and BCX 2855 are effective inhibitors of hPIV-3 HN in our mouse model and may be promising candidates for the prophylaxis and treatment of hPIV-3 infection in humans.
doi:10.1128/AAC.00220-09
PMCID: PMC2737897  PMID: 19564364

Results 1-25 (1355285)