Identification of a phosphorylation site within the P protein important for mRNA transcription and growth of parainfluenza virus 5

The viral RNA-dependent RNA polymerase (vRdRp) of paramyxovirus consists of the large (L) protein and the phosphoprotein (P). P is heavily phosphorylated, and it is thought that the phosphorylation of P plays a role in regulating viral RNA synthesis. However, no phosphorylation site within the P protein in paramyxovirus has been identified as playing a positive role in viral RNA synthesis in virus infection. Using mass spectrometry analysis, the threonine residue at position 286 of P of parainfluenza virus 5 (PIV5) was found phosphorylated. Mutation of T286 to alanine (T286A), aspartic acid (T286D), or glutamic acid (T286E) reduced minigenome activity. Recombinant virus containing a mutation at the T286 position (rPIV5-P-T286A) grew slower than wild-type virus; viral mRNA synthesis and protein expression of rPIV5-P-T286A were delayed. Biochemical studies showed that the binding of NP or L protein with the P mutants or tetramer formation by the mutant P proteins was unaltered from that for wild-type P. While we failed to rescue rPIV5-P-T286E virus, several revertant viruses were obtained. All non-wild-type revertants had mutations at T286 and showed defects in both minigenome activity and viral growth. This is the first time that a phosphorylation site within the P protein in paramyxovirus has been found to play a positive role in viral mRNA synthesis and virus growth.     

Evaluating a Parainfluenza Virus 5-Based Vaccine in a Host with Pre-Existing Immunity against Parainfluenza Virus 5

Parainfluenza virus 5 (PIV5), formerly known as simian virus 5 (SV5), is a paramyxovirus often referred to as canine parainfluenza virus (CPI) in the veterinary field. PIV5 is thought to be a contributing factor to kennel cough. Kennel cough vaccines containing live PIV5 have been used in dogs for many decades. PIV5 is not known to cause any diseases in humans or other animals. PIV5 has been used as a vector for vaccine development for humans and animals. One critical question concerning the use of PIV5 as a vector is whether prior exposure to PIV5 would prevent the use of PIV5-based vaccines. In this work, we have examined immunogenicity of a recombinant PIV5 expressing hemagglutinin (HA) of influenza A virus subtype 3 (rPIV5-H3) in dogs that were immunized against PIV5. We found that vaccination of the dogs containing neutralizing antibodies against PIV5 with rPIV5-H3 generated immunity against influenza A virus, indicting that PIV5-based vaccine is immunogenic in dogs with prior exposure. Furthermore, we have examined exposure of PIV5 in human populations. We have detected neutralizing antibody (nAb) against PIV5 in 13 out of 45 human serum samples (about 29 percent). The nAb titers in humans were lower than that in vaccinated dogs, suggesting that nAb in humans is unlikely to prevent PIV5 from being an efficacious vector in humans.     

Viral infections of little blue penguins (Eudyptula minor) along the southern coast of Australia

Serum antibodies to strains of avian paramyxovirus and flavivirus were detected in little blue penguins sampled at Port Campbell and Phillip Island, Victoria, Australia. No antibody to Newcastle disease virus (NDV) was detected in 267 sera collected, although one penguin captured for experimental studies had a hemagglutination-inhibition antibody titer of 2(4) to this virus. Experimental studies showed that the avian paramyxovirus designated APMV-IM and strain V4 of NDV were non-pathogenic for penguins, although the penguins could have been previously infected with these or similar virus strains. A flavivirus designated Saumarez reef virus, and an unnamed virus isolated from ticks on Macquarie Island, Southern Ocean were pathogenic causing disease and mortality in penguins inoculated with the viruses.     

Nucleotide sequence of the bovine parainfluenza 3 virus genome: the genes of the F and HN glycoproteins.

By analysing complementary DNA clones constructed from genomic RNA of bovine parainfluenza 3 virus (BPIV3), we determined the nucleotide sequence of the region containing the entire F and HN genes. Their deduced amino acid sequences showed about 80% homologies with those of human parainfluenza 3 virus (HPIV3), about 45% with those of Sendai virus, and about 20% with those of SV5 and Newcastle disease virus (NDV), indicating, together with the results described in the preceding paper on the NP, P, C and M proteins of BPIV3, that BPIV3, HPIV3 and Sendai virus constitute a paramyxovirus subgroup, and that BPIV3 and HPIV3 are very closely related. The F and HN proteins of all these viruses, including SV5 and NDV, however, were shown to have protein-specific structures as well as short but well-conserved amino acid sequences, suggesting that these structures and sequences are related to the activities of these glycoproteins.     

Analysis of the pH requirement for membrane fusion of different isolates of the paramyxovirus parainfluenza virus 5

Paramyxoviruses enter cells by fusing their envelopes with the plasma membrane, a process that occurs at neutral pH. Recently, it has been found that there is an exception to this dogma in that a porcine isolate of the paramyxovirus parainfluenza virus 5 (PIV5), known as SER, requires a low-pH step for fusion (S. Seth, A. Vincent, and R. W. Compans, J. Virol. 77: 6520-6527, 2003). As a low-pH activation mechanism for fusion would greatly facilitate biophysical studies of paramyxovirus-mediated membrane fusion, we have reexamined the triggering of the PIV5 SER fusion protein. Using multiple assays, we could not find a requirement for low-pH triggering of PIV5 SER fusion. The challenge of discovering how the paramyxovirus receptor binding protein (HN, H, or G) activates the metastable fusion protein to cause membrane fusion at neutral pH remains.     

Conserved glycine residues in the fusion peptide of the paramyxovirus fusion protein regulate activation of the native state

Hydrophobic fusion peptides (FPs) are the most highly conserved regions of class I viral fusion-mediating glycoproteins (vFGPs). FPs often contain conserved glycine residues thought to be critical for forming structures that destabilize target membranes. Unexpectedly, a mutation of glycine residues in the FP of the fusion (F) protein from the paramyxovirus simian parainfluenza virus 5 (SV5) resulted in mutant F proteins with hyperactive fusion phenotypes (C. M. Horvath and R. A. Lamb, J. Virol. 66:2443-2455, 1992). Here, we constructed G3A and G7A mutations into the F proteins of SV5 (W3A and WR isolates), Newcastle disease virus (NDV), and human parainfluenza virus type 3 (HPIV3). All of the mutant F proteins, except NDV G7A, caused increased cell-cell fusion despite having slight to moderate reductions in cell surface expression compared to those of wild-type F proteins. The G3A and G7A mutations cause SV5 WR F, but not NDV F or HPIV3 F, to be triggered to cause fusion in the absence of coexpression of its homotypic receptor-binding protein hemagglutinin-neuraminidase (HN), suggesting that NDV and HPIV3 F have stricter requirements for homotypic HN for fusion activation. Dye transfer assays show that the G3A and G7A mutations decrease the energy required to activate F at a step in the fusion cascade preceding prehairpin intermediate formation and hemifusion. Conserved glycine residues in the FP of paramyxovirus F appear to have a primary role in regulating the activation of the metastable native form of F. Glycine residues in the FPs of other class I vFGPs may also regulate fusion activation.     

人3型副流感病毒疫苗的研究进展

人3型副流感病毒是一种主要感染人类肺部上皮细胞的副黏病毒,可引起肺炎和支气管炎,在婴幼儿和免疫力低下的成人中有较高的发病率。经过多年的研究,对人3型副流感病毒疫苗的研究取得了重要的进展,但还没有有效的抗病毒药物和批准的疫苗上市。目前研究主要集中在减毒活疫苗及亚单位疫苗等,对人3型副流感病毒当前疫苗的研究情况做简要的综述。     

Recombinant newcastle disease virus expressing a foreign viral antigen is attenuated and highly immunogenic in primates

Paramyxoviruses such as human parainfluenza viruses that bear inserts encoding protective antigens of heterologous viruses can induce an effective immunity against the heterologous viruses in experimental animals. However, vectors based on common human pathogens would be expected to be restricted in replication in the adult human population due to high seroprevalence, an effect that would reduce vector immunogenicity. To address this issue, we evaluated Newcastle disease virus (NDV), an avian paramyxovirus that is serotypically distinct from common human pathogens, as a vaccine vector. Two strains were evaluated: the attenuated vaccine strain LaSota (NDV-LS) that replicates mostly in the chicken respiratory tract and the Beaudette C (NDV-BC) strain of intermediate virulence that produces mild systemic infection in chickens. A recombinant version of each virus was modified by the insertion, between the P and M genes, of a gene cassette encoding the human parainfluenza virus type 3 (HPIV3) hemagglutinin-neuraminidase (HN) protein, a test antigen with considerable historic data. The recombinant viruses were administered to African green monkeys (NDV-BC and NDV-LS) and rhesus monkeys (NDV-BC only) by combined intranasal and intratracheal routes at a dose of 10(6.5) PFU per site, with a second equivalent dose administered 28 days later. Little or no virus shedding was detected in nose-throat swabs or tracheal lavages following immunization with either strain. In a separate experiment, direct examination of lung tissue confirmed a highly attenuated, restricted pattern of replication by parental NDV-BC. The serum antibody response to the foreign HN protein induced by the first immunization with either NDV vector was somewhat less than that observed following a wild-type HPIV3 infection; however, the titer following the second dose exceeded that observed with HPIV3 infection, even though HPIV3 replicates much more efficiently than NDV in these animals. NDV appears to be a promising vector for the development of vaccines for humans; one application would be in controlling localized outbreaks of emerging pathogens.     

Vaccine potential of Nipah virus-like particles

Nipah virus (NiV) was first recognized in 1998 in a zoonotic disease outbreak associated with highly lethal febrile encephalitis in humans and a predominantly respiratory disease in pigs. Periodic deadly outbreaks, documentation of person-to-person transmission, and the potential of this virus as an agent of agroterror reinforce the need for effective means of therapy and prevention. In this report, we describe the vaccine potential of NiV virus-like particles (NiV VLPs) composed of three NiV proteins G, F and M. Co-expression of these proteins under optimized conditions resulted in quantifiable amounts of VLPs with many virus-like/vaccine desirable properties including some not previously described for VLPs of any paramyxovirus: The particles were fusogenic, inducing syncytia formation; PCR array analysis showed NiV VLP-induced activation of innate immune defense pathways; the surface structure of NiV VLPs imaged by cryoelectron microscopy was dense, ordered, and repetitive, and consistent with similarly derived structure of paramyxovirus measles virus. The VLPs were composed of all the three viral proteins as designed, and their intracellular processing also appeared similar to NiV virions. The size, morphology and surface composition of the VLPs were consistent with the parental virus, and importantly, they retained their antigenic potential. Finally, these particles, formulated without adjuvant, were able to induce neutralizing antibody response in Balb/c mice. These findings indicate vaccine potential of these particles and will be the basis for undertaking future protective efficacy studies in animal models of NiV disease.     

Advances in diagnostics,vaccines and therapeutics for Nipah virus

Nipah virus is an emerging zoonotic paramyxovirus that causes severe and often fatal respiratory and neurological disease in humans. The virus was first discovered after an outbreak of encephalitis in pig farmers in Malaysia and Singapore with subsequent outbreaks in Bangladesh or India occurring almost annually. Due to the highly pathogenic nature of NiV, its pandemic potential, and the lack of licensed vaccines or therapeutics, there is a requirement for research and development into highly sensitive and specific diagnostic tools as well as antivirals and vaccines to help prevent and control future outbreak situations.     

Antibody Response of Guinea Pigs to Trivalent Parainfluenza Virus Vaccine Prepared from Embryonated Eggs

A trivalent parainfluenza virus vaccine has been tested in guinea pigs. The parainfluenza 2 virus vaccine component was superior in the magnitude of antibody titers, and in the ability to convert animals serologically after two doses of an undiluted or a 10-fold diluted vaccine. The parainfluenza 1 virus vaccine gave a higher percentage of conversion than parainfluenza 3 virus vaccine after administration of two doses of either undiluted or 10-fold diluted vaccine.     

Down-regulation of paramyxovirus hemagglutinin-neuraminidase glycoprotein surface expression by a mutant fusion protein containing a retention signal for the endoplasmic reticulum.

The human parainfluenza virus type 3 (HPIV3) fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins are the principal components involved in virion receptor binding, membrane penetration, and ultimately, syncytium formation. While the requirement for both F and HN in this process has been determined from recombinant expression studies, stable physical association of these proteins in coimmunoprecipitation studies has not been observed. In addition, coexpression of other heterologous paramyxovirus F or HN glycoproteins with either HPIV3 F or HN does not result in the formation of syncytia, suggesting serotype-specific protein differences. In this study, we report that simian virus 5 and Sendai virus heterologous HN proteins and measles virus hemagglutinin (H) were found to be down-regulated when coexpressed with HPIV3 F. As an alternative to detecting physical associations of these proteins by coimmunoprecipitation, further studies were performed with a mutant HPIV3 F protein (F-KDEL) lacking a transmembrane anchor and cytoplasmic tail and containing a carboxyl-terminal retention signal for the endoplasmic reticulum (ER). F-KDEL was defective for transport to the cell surface and could down-regulate surface expression of HPIV3 HN and heterologous HN/H proteins from simian virus 5, Sendai virus, and measles virus in coexpression experiments. HN/H down-regulation appeared to result, in part, from an early block to HPIV3 HN synthesis, as well as an instability of the heterologous HN/H proteins within the ER. In contrast, coexpression of F-KDEL with HPIV3 wild-type F or the heterologous receptor-binding proteins, respiratory syncytial virus glycoprotein (G) and vesicular stomatitis virus glycoprotein (G), were not affected in transport to the cell surface. Together, these results support the notion that the reported serotype-specific restriction of syncytium formation may involve, in part, down-regulation of heterologous HN expression.     

The second receptor binding site of the globular head of the Newcastle disease virus hemagglutinin-neuraminidase activates the stalk of multiple paramyxovirus receptor binding proteins to trigger fusion

The hemagglutinin-neuraminidase (HN) protein of paramyxoviruses carries out three distinct activities contributing to the ability of HN to promote viral fusion and entry: receptor binding, receptor cleavage (neuraminidase), and activation of the fusion protein. The relationship between receptor binding and fusion triggering functions of HN are not fully understood. For Newcastle disease virus (NDV), one bifunctional site (site I) on HN's globular head can mediate both receptor binding and neuraminidase activities, and a second site (site II) in the globular head is also capable of mediating receptor binding. The receptor analog, zanamivir, blocks receptor binding and cleavage activities of NDV HN's site I while activating receptor binding by site II. Comparison of chimeric proteins in which the globular head of NDV HN is connected to the stalk region of either human parainfluenza virus type 3 (HPIV3) or Nipah virus receptor binding proteins indicates that receptor binding to NDV HN site II not only can activate its own fusion (F) protein but can also activate the heterotypic fusion proteins. We suggest a general model for paramyxovirus fusion activation in which receptor engagement at site II plays an active role in F activation.     

Structural studies of the parainfluenza virus 5 hemagglutinin-neuraminidase tetramer in complex with its receptor, sialyllactose

The paramyxovirus hemagglutinin-neuraminidase (HN) functions in virus attachment to cells, cleavage of sialic acid from oligosaccharides, and stimulating membrane fusion during virus entry into cells. The structural basis for these diverse functions remains to be fully understood. We report the crystal structures of the parainfluenza virus 5 (SV5) HN and its complexes with sialic acid, the inhibitor DANA, and the receptor sialyllactose. SV5 HN shares common structural features with HN of Newcastle disease virus (NDV) and human parainfluenza 3 (HPIV3), but unlike the previously determined HN structures, the SV5 HN forms a tetramer in solution, which is thought to be the physiological oligomer. The sialyllactose complex reveals intact receptor within the active site, but no major conformational changes in the protein. The SV5 HN structures do not support previously proposed models for HN action in membrane fusion and suggest alternative mechanisms by which HN may promote virus entry into cells.     

Postembedding immunocytochemical localization of paramyxovirus antigens by light and electron microscopy

A postembedding method is described to localize antigens specific for various paramyxoviruses in sections of cells and tissues that have been fixed and embedded in epoxy resins for conventional electron microscopy. Viral antigens were localized in CV-1 cell cultures infected with simian virus 5 (SV5), brains of suckling hamsters inoculated with either neuroadapted mumps virus or hamster-adapted measles virus, and brains of adult mice infected with Sendai (parainfluenza I) virus. Both 1-micrometer-thick and thin (gold) tissue sections were etched with alcoholic sodium hydroxide-solution and then treated following either the unlabeled antibody peroxidase-antiperoxidase or the biotinylated protein A:avidin peroxidase procedure. Primary reagents included immunoglobulin isolated from hyperimmune rabbit sera with specificity to the major viral components of SV5 or SV5 hemagglutinin-neuraminidase, to whole mumps virus or mumps virus nucleocapsids, and to whole Sendai virus. Crude rabbit anti-Sendai virus antiserum and whole human subacute sclerosing panencephalitis (SSPE) sera were used in parallel. The results indicate that tissues processed for conventional evaluation by electron microscopy may be suitable, within limits, for postembedding immunocytochemical staining of paramyxovirus antigens.     

Protection against canine distemper virus in dogs after immunization with isolated fusion protein.

Canine distemper virus attachment (hemagglutinin [H] equivalent) and fusion (F) antigens were purified by affinity chromatography with monoclonal antibodies. The purified antigens were used to immunize groups of three dogs. Radioimmune precipitation assays with sera from these animals showed that the F antigen preparation was pure and induced only an F polypeptide-specific antibody response but that the H antigen preparation had a slight contamination by the F antigen. Immunized animals were challenged with virulent canine distemper virus. Two animals in each group developed pronounced humoral and cellular immune responses after challenge. Among these infected animals, only the dogs immunized with H antigen developed symptoms, albeit mild. In contrast, three nonimmunized control animals developed severe disease, with a fatal outcome in two cases. The complete resistance against challenge in two dogs was interpreted to reflect in one case anti-F immunity and in the other case most likely a high level of anti-H immunity. It is suggested that the F antigen may be of particular interest for the development of morbillivirus and possibly other paramyxovirus subunit or synthetic vaccines, because it can induce immunity capable of blocking virus infection and in situations of virus replication prevent the emergence of symptoms.     

Canine distemper in black-footed ferrets (Mustela nigripes) from Wyoming

In September and October 1985, six black-footed ferrets (Mustela nigripes) were captured from the only known population, located near Meeteetse, Wyoming for captive propagation. Two days following capture an adult male showed signs of canine distemper and an adult female displayed similar signs 7 days postcapture; these infections were undoubtedly acquired prior to capture. Subsequently the four remaining captive black-footed ferrets also developed canine distemper and all eventually died. Clinical signs included severe pruritus, hyperkeratosis and progressive loss of body condition. A few animals had intermittent diarrhea and respiratory disease. Intranuclear and intracytoplasmic inclusion bodies were numerous in epithelial tissues and two black-footed ferrets had a mild to moderate meningoencephalitis. Canine distemper virus was isolated from four animals and paramyxovirus nucleocapsids were observed by electron microscopy of feces from all affected black-footed ferrets. Antibodies to canine distemper virus were not detected in sera of sick black-footed ferrets. Antibodies to canine distemper virus were found in sera of badgers (Taxidea taxus) and coyotes (Canis latrans) collected in the Meeteetse area in 1986. Most free-ranging black-footed ferrets in the colony apparently died of canine distemper during the summer and fall of 1985. An attempt was made to capture all surviving animals in the affected area in order to abort the epizootic and provide black-footed ferrets for captive propagation.     

A RhoA-derived peptide inhibits syncytium formation induced by respiratory syncytial virus and parainfluenza virus type 3

The fusion glycoproteins of human respiratory syncytial virus (RSV) and human parainfluenza virus type-3 (PIV-3) mediate virus entry and syncytium formation. Interaction between the fusion protein of RSV and RhoA, a small GTPase, facilitates virus-induced syncytium formation. We show here a RhoA-derived peptide inhibits RSV and syncytium formation induced by RSV and PIV-3, both in vitro by inhibition of cell-to-cell fusion and in vivo by reduction of peak titer by 2 log10 in RSV-infected mice. These findings indicate that the interaction between these two paramyxovirus fusion proteins and RhoA is an important target for new antiviral strategies.     

Pneumonia in Ottoman vipers (Vipera xanthena xanthena) associated with a parainfluenza 2-like virus

A paramyxovirus related to parainfluenza 2 (PI2) virus was recovered from the lungs of two dead Ottoman vipers from a zoological collection. Snakes of other species in the collection were unaffected. Histologic examination of the vipers' lungs revealed interstitial pneumonia, and degeneration and hyperplasia of bronchial and atrial epithelia. Scattered vacuoles, some of which contain eosinophilic inclusion bodies, were seen in the cytoplasm of several cells of affected epithelial tissues. The virus recovered from pulmonary tissues of the snakes replicated optimally at 30 C in a variety of cell cultures and hemagglutinated chicken erythrocytes. Viral hemagglutination was inhibited by PI2 virus antiserum, but not by antisera to PI1, PI3, respiratory syncytial, and canine distemper viruses. Indirect immunofluorescence with PI2 antiserum specifically stained inclusions in the epithelial cells of respiratory tissues and infected cell cultures.     

Inhibition of hendra virus fusion

Hendra virus (HeV) is a recently identified paramyxovirus that is fatal in humans and could be used as an agent of bioterrorism. The HeV receptor-binding protein (G) is required in order for the fusion protein (F) to mediate fusion, and analysis of the triggering/activation of HeV F by G should lead to strategies for interfering with this key step in viral entry. HeV F, once triggered by the receptor-bound G, by analogy with other paramyxovirus F proteins, undergoes multistep conformational changes leading to a six-helix bundle (6HB) structure that accomplishes fusion of the viral and cellular membranes. The ectodomain of paramyxovirus F proteins contains two conserved heptad repeat regions (HRN and HRC) near the fusion peptide and the transmembrane domains, respectively. Peptides derived from the HRN and HRC regions of F are proposed to inhibit fusion by preventing F, after the initial triggering step, from forming the 6HB structure that is required for fusion. HeV peptides have previously been found to be effective at inhibiting HeV fusion. However, we found that a human parainfluenza virus 3 F-peptide is more effective at inhibiting HeV fusion than the comparable HeV-derived peptide.