Features of Circulating Parainfluenza Virus Required for Growth in Human Airway.

UNLABELLED: Respiratory paramyxoviruses, including the highly prevalent human parainfluenza viruses, cause the majority of childhood croup, bronchiolitis, and pneumonia, yet there are currently no vaccines or effective treatments. Paramyxovirus research has relied on the study of laboratory-adapted strains of virus in immortalized cultured cell lines. We show that findings made in such systems about the receptor interaction and viral fusion requirements for entry and fitness-mediated by the receptor binding protein and the fusion protein-can be drastically different from the requirements for infection in vivo. Here we carried out whole-genome sequencing and genomic analysis of circulating human parainfluenza virus field strains to define functional and structural properties of proteins of circulating strains and to identify the genetic basis for properties that confer fitness in the field. The analysis of clinical strains suggests that the receptor binding-fusion molecule pairs of circulating viruses maintain a balance of properties that result in an inverse correlation between fusion in cultured cells and growth in vivo. Future analysis of entry mechanisms and inhibitory strategies for paramyxoviruses will benefit from considering the properties of viruses that are fit to infect humans, since a focus on viruses that have adapted to laboratory work provides a distinctly different picture of the requirements for the entry step of infection. IMPORTANCE: Mechanistic information about viral infection-information that impacts antiviral and vaccine development-is generally derived from viral strains grown under laboratory conditions in immortalized cells. This study uses whole-genome sequencing of clinical strains of human parainfluenza virus 3-a globally important respiratory paramyxovirus-in cell systems that mimic the natural human host and in animal models. By examining the differences between clinical isolates and laboratory-adapted strains, the sequence differences are correlated to mechanistic differences in viral entry. For this ubiquitous and pathogenic respiratory virus to infect the human lung, modulation of the processes of receptor engagement and fusion activation occur in a manner quite different from that carried out by the entry glycoprotein-expressing pair of laboratory strains. These marked contrasts in the viral properties necessary for infection in cultured immortalized cells and in natural host tissues and animals will influence future basic and clinical studies.

Infection with Menangle virus in flying foxes (Pteropus spp.) in Australia.

OBJECTIVE: To examine flying foxes (Pteropus spp.) for evidence of infection with Menangle virus. DESIGN: Clustered non-random sampling for serology, virus isolation and electron microscopy (EM). PROCEDURE: Serum samples were collected from 306 Pteropus spp. in northern and eastern Australia and tested for antibodies against Menangle virus (MenV) using a virus neutralisation test (VNT). Virus isolation was attempted from tissues and faeces collected from 215 Pteropus spp. in New South Wales. Faecal samples from 68 individual Pteropus spp. and four pools of faeces were examined by transmission EM following routine negative staining and immunogold labelling. RESULTS: Neutralising antibodies (VNT titres > or = 8) against MenV were detected in 46% of black flying foxes (P. alecto), 41% of grey-headed flying foxes (P. poliocephalus), 25% of spectacled flying foxes (P. conspicillatus) and 1% of little red flying foxes (P. scapulatus) in Australia. Positive sera included samples collected from P. poliocephalus in a colony adjacent to a piggery that had experienced reproductive disease caused by MenV. Virus-like particles were observed by EM in faeces from Pteropus spp. and reactivity was detected in pooled faeces and urine by immunogold EM using sera from sows that had been exposed to MenV. Attempts to isolate the virus from the faeces and tissues from Pteropus spp. were unsuccessful. CONCLUSION: Serological evidence of infection with MenV was detected in Pteropus spp. in Australia. Although virus-like particles were detected in faeces, no viruses were isolated from faeces, urine or tissues of Pteropus spp.

Isolation and partial characterization of a novel paramyxovirus from the gills of diseased seawater-reared Atlantic salmon (Salmo salar L).

A formerly undescribed virus has been isolated from the gills of farmed Atlantic salmon post-smolts in Norway suffering from gill disease. Cytopathic effects appeared in RTgill-W1 cells 9 weeks post-inoculation with gill tissue material. Virus production continued for an extended period thereafter. Light and electron microscopic examination revealed inclusions and replication in the cytoplasm. The viral nucleocapsid consisted of approximately 17 nm thick filaments in a herringbone pattern. Certain areas of the plasma membrane were thickened by the alignment of nucleocapsids on the internal surface and projections of 10 nm long viral glycoprotein spikes on the external surface. Virus assembly and release was achieved by budding through the modified plasma membrane. Negatively stained virions were spherical and partly pleomorphic with a diameter of 150-300 nm as seen by electron microscopy. The virus was sensitive to chloroform, heat and low and high pH, and replication was not inhibited by Br-dU or IdU indicating an RNA genome. Both haemagglutination and receptor-destroying enzyme activity were associated with the virions and the formation of syncytia in infected cultures indicated fusion activity. The receptor-destroying enzyme was identified as neuraminidase. The virus contained five major structural polypeptides with estimated molecular masses of 70, 62, 60, 48 and 37 kDa. Its buoyant density was 1.18-1.19 g ml(-1) in CsCl gradients. From the observed properties we conclude that this new virus belongs to the Paramyxoviridae and suggest the name Atlantic salmon paramyxovirus (ASPV). Furthermore, replication occurred at 6-21 degrees C, suggesting a host range confined to cold-blooded animals.

Tioman virus, a novel paramyxovirus isolated from fruit bats in Malaysia.

A search for the natural host of Nipah virus has led to the isolation of a previously unknown member of the family Paramyxoviridae. Tioman virus (TiV) was isolated from the urine of fruit bats (Pteropus hypomelanus) found on the island of the same name off the eastern coast of peninsular Malaysia. An electron microscopic study of TiV-infected cells revealed spherical and pleomorphic-enveloped viral particles (100--500 nm in size) with a single fringe of embedded peplomers. Virus morphogenesis occurred at the plasma membrane of infected cells and morphological features of negative-stained ribonucleoprotein complexes were compatible with that of viruses in the family Paramyxoviridae. Serological studies revealed no cross-reactivity with antibodies against a number of known Paramyxoviridae members except for the newly described Menangle virus (MenV), isolated in Australia in 1997. Failure of PCR amplification using MenV-specific primers suggested that this new virus is related to but different from MenV. For molecular characterization of the virus, a cDNA subtraction strategy was employed to isolate virus-specific cDNA from virus-infected cells. Complete gene sequences for the nucleocapsid protein (N) and phosphoprotein (P/V) have been determined and recombinant N and V proteins produced in baculovirus. The recombinant N and V proteins reacted with porcine anti-MenV sera in Western blot, confirming the serological cross-reactivity observed during initial virus characterization. The lack of a C protein-coding region in the P/V gene, the creation of P mRNA by insertion of 2-G residues, and the results of phylogenetic analyses all indicated that TiV is a novel member of the genus Rubulavirus.

Paramyxovirus infection in caiman lizards (Draecena guianensis).

Three separate epidemics occurred in caiman lizards (Dracaena guianensis) that were imported into the USA from Peru in late 1998 and early 1999. Histologic evaluation of tissues from necropsied lizards demonstrated a proliferative pneumonia. Electron microscopic examination of lung tissue revealed a virus that was consistent with members of the family Paramyxoviridae. Using a rabbit polyclonal antibody against an isolate of ophidian (snake) paramyxovirus, an immunoperoxidase staining technique demonstrated immunoreactivity within pulmonary epithelial cells of 1 lizard. Homogenates of lung, brain, liver, or kidney from affected lizards were placed in flasks containing monolayers of either terrapene heart cells or viper heart cells. Five to 10 days later, syncytial cells formed. When Vero cells were inoculated with supernatant of infected terrapene heart cells, similar syncytial cells developed. Electron microscopic evaluation of infected terrapene heart cells revealed intracytoplasmic inclusions consisting of nucleocapsid strands. Using negative-staining electron microscopy, abundant filamentous nucleocapsid material with a herringbone structure typical of the Paramyxoviridae was observed in culture medium of infected viper heart cells. Seven months following the initial epizootic, blood samples were collected from surviving group 1 lizards, and a hemagglutination inhibition assay was performed to determine presence of specific antibody against the caiman lizard isolate. Of the 17 lizards sampled, 7 had titers of < or =1:20 and 10 had titers of >1:20 and < or =1:80. This report is only the second of a paramyxovirus identified in a lizard and is the first to snow the relationship between histologic and ultrastructural findings and virus isolation.

Neonatal syncytial giant cell hepatitis with paramyxoviral-like inclusions.

Syncytial giant cell hepatitis in the neonatal period has been associated with many different etiologic agents and may present initially as cholestasis. Infectious causes are most common and include: (1 ) generalized bacterial sepsis, (2) viral agents, (3) toxoplasmosis, (4) syphilis, (5) listeriosis, and (6) tuberculosis. Viral hepatitis may be due to cytomegalovirus, rubella virus, herpes simplex, HHV-6, varicella, coxsackievirus, echovirus, reovirus 3, parvovirus B19, HIV, enteroviruses, paramyxovirus, and hepatitis A, B, or C (rare). Giant cell hepatitis may result in fulminant liver failure with massive hepatocyte necrosis and severe liver dysfunction leading to death, resolution with severely compromised liver function, or liver transplantation. The authors report a 6-week-old male who had an unremarkable perinatal period, became jaundiced after developing diarrhea, and subsequently developed liver dysfunction with massively increased liver enzymes and a coagulopathy. Open wedge and core liver biopsies were performed to determine if the patient should be listed for liver transplantation. Giant cell hepatitis with a significant mixed lymphocytic and neutrophilic infiltrate was present on both the wedge and core biopsies. The residual 60% of hepatocytes had ballooning degeneration and many possessed pyknotic nuclei. The hepatocytes were arranged in a pseudoacinar pattern. Electron microscopy showed paramyxoviral-like inclusions in the giant cells, characterized as large inclusions with fine filamentous, beaded substructures (18-20 nm). Paramyxoviridae are nonsegmented, negative-sense, single-stranded RNA viruses. This family is divided into the Paramyxovirinae subfamily containing respirovirus (Sendai virus, parainfluenza virus type 3), rubulavirus (mumps, parainfluenza virus type 2), and morbillivirus genera (measles); and Pneumovirinae subfamily (pneumovirus genus [respiratory syncytial virus]). Supportive care to determine if hepatic function resolves following the viral episode, liver transplantation with fulminant liver failure, and ongoing evaluation in those who recover to assess chronic liver disease are necessary. Ultrastructural evaluation may unmask the etiologic agent for hepatitis and direct therapy.

Meningoencephalitis in a Boelen's python (Morelia boeleni) associated with paramyxovirus infection.

An adult male Boelen's python, Morelia boeleni, presented with acute neurologic disease and was euthanatized. Histologic examination revealed nonsuppurative meningoencephalitis. Occasional eosinophilic intracytoplasmic inclusions were noted in glial cells. On the basis of clinical signs and histopathology, inclusion body disease of boid snakes was suspected, but inclusions were not seen in other organs commonly affected with the disease. Moreover, electron microscopy revealed that the inclusions contained stacks of filaments 13-14 nm wide. With the use of a generic paramyxovirus cDNA probe, sections of brain and esophageal ganglion demonstrated hybridization. The findings indicate that paramyxovirus was the likely cause of the encephalomyelitis in this python, and this virus should be included in the differential diagnosis of pythons exhibiting central nervous system disease.

Fusion of Sendai virus with vesicles of oligomerizable lipids: a microcalorimetric analysis of membrane fusion.

Sendai virus fuses efficiently with small and large unilamellar vesicles of the lipid 1,2-di-n-hexadecyloxypropyl-4- (beta-nitrostyryl) phosphate (DHPBNS) at pH 7.4 and 37 degrees C, as shown by lipid mixing assays and electron microscopy. However, fusion is strongly inhibited by oligomerization of the head groups of DHPBNS in the bilayer vesicles. The enthalpy associated with fusion of Sendai virus with DHPBNS vesicles was measured by isothermal titration microcalorimetry, comparing titrations of Sendai virus into (i) solutions of DHPBNS vesicles (which fuse with the virus) and (ii) oligomerized DHPBNS vesicles (which do not fuse with the virus), respectively. The observed heat effect of fusion of Sendai virus with DHPBNS vesicles is strongly dependent on the buffer medium, reflecting a partial charge neutralization of the Sendai F and HN proteins upon insertion into the negatively-charged vesicle membrane. No buffer effect was observed for the titration of Sendai virus into oligomerized DHPBNS vesicles, indicating that inhibition of fusion is a result of inhibition of insertion of the fusion protein into the target membrane. Fusion of Sendai virus with DHPBNS vesicles is endothermic and entropy-driven. The positive enthalpy term is dominated by heat effects resulting from merging of the protein-rich viral envelope with the lipid vesicle bilayers rather than by the fusion of the viral with the vesicle bilayers per se.

Reptilian paramyxoviruses induce cytokine production in human leukocytes.

The reptilian paramyxoviruses FDLV and GOV initiated the production and release of cytokines like IL-1alpha, IL-1beta, IL-2, TNF-alpha and IFN-alpha in human peripheral blood mononuclear cells (PBMC) at 37 degrees C. The target cells produced the cytokines without replication of virus.

Gene delivery systems using the Sendai virus.

Fusogenic liposome (FL) is a delivery system that can transfer encapsulated materials into living cells directly through membrane fusion. FL is a promising approach for gene therapy because it can deliver various genetic materials much more efficiently than other non-viral vectors without damaging the cell. FL-mediated gene transfer consists of two independent membrane fusion phenomena; generation of a FL by fusing a Sendai virus (SV) particle with a simple liposome encapsulating DNA, and successive fusion of the FL with cell membrane. The former requires viral F protein but no other special molecule on the liposomal membrane, whereas the latter may require the receptor (sialic acid) and unidentified assistant molecule(s) on the cell membrane. Further analysis suggests that these assistant molecule(s), not the receptor, may control the fusion and govern the cell specificity of FL-mediated delivery. This review has described a detailed analysis of these fusion phenomena and discussed possible applications of FL-mediated gene delivery to human gene therapy.

Paramyxovirus fusion protein: characterization of the core trimer, a rod-shaped complex with helices in anti-parallel orientation.

The fusion (F) protein of the paramyxovirus SV5 contains two heptad repeat regions, HRA adjacent to the fusion peptide and HRB proximal to the transmembrane domain. Peptides, N-1 and C-1, respectively, corresponding to these heptad repeat regions form a thermostable, alpha-helical trimer of heterodimers (S. B. Joshi, R. E. Dutch, and R. A. Lamb (1998). Virology 248, 20-34). Further characterization of the N-1/C-1 complex indicated that the C-1 peptides, which are predicted to residue on the outside of the complex, are resistant to digestion by several proteases when present in the complex. Only proteinase K digested most of the C-1 peptide, though the small remaining protease protected fragment of C-1 confers extreme thermostability on the proteinase-K-resistant N-1 trimeric coiled-coil. Carboxypeptidase Y digestion of the N-1/C-1 complex indicates that the C-1 peptides associate in an antiparallel orientation relative to the N-1 peptides. Electron microscopy of the N-1/C-1 complex showed a rod-shaped complex with an average length of 9.7 nm, consistent with all of N-1 existing as an alpha helix. Mutations at heptad repeat a and d residues of N-1, positions that are predicted to point inward to the center of the N-1 trimeric coiled-coil, were found to have varying effects as analyzed by circular dichroism measurements. The mutation I137M did not affect the helical structure of the isolated N-1 peptide but did affect the thermostability of the N-1/C-1 complex. Mutations L140M and L161M perturbed the helical structure formed by N-1 in isolation but did not affect formation of a thermostable N-1/C-1 complex. Finally, a peptide, SV5 F 255-293, corresponding to a proposed leucine zipper region, was analyzed for effects on N-1, C-1, or the N-1/C-1 complex. Circular dichroism analysis demonstrated that while the presence of peptide 255-293 increased the helical signal from either N-1 or the N-1/C-1 complex, no change in thermostability was observed, indicating that this region is not a component of the final, most stable core of the F protein.

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

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

Pulmonary lesions in experimental ophidian paramyxovirus pneumonia of Aruba Island rattlesnakes, Crotalus unicolor.

Histologic and ultrastructural changes were observed in the respiratory portions of lung in five 29-40-month-old Aruba Island rattlesnakes, Crotalus unicolor, that were inoculated with an Aruba Island Rattlesnake virus (AIV) strain of ophidian paramyxovirus (OPMV) isolated from an Aruba Island rattlesnake. Lungs from one non-infected and three mock-infected Aruba Island rattlesnakes were examined also. From 4 to 22 days following intratracheal inoculation, progressive microscopic changes were seen in the lung. Initially, increased numbers of heterophils were observed in the interstitium followed by proliferation and vacuolation of epithelial cells lining faveoli. The changes appeared to progress from cranial to caudal portions of the respiratory lung following inoculation. Beginning at 4 days postinoculation, viral antigen was demonstrated in epithelial cells lining faveoli with an immunofluorescent technique using a rabbit anti-AIV polyclonal antibody. Electron microscopy revealed loss of type I cells, hyperplasia of type II cells, and interstitial infiltrates of heterophils and mononuclear cells. Viral nucleocapsid material was seen within the cytoplasm and mature virus was seen budding from cytoplasmic membranes of infected type I and type II cells from 8 to 19 days after infection. A virus consistent with AIV was isolated from lung tissues of infected rattlesnakes, thus fulfilling Koch's postulates.

Characterization of paramyxoviruses isolated from three snakes.

Multiple epizootics of pneumonia in captive snakes have been attributed to viruses which have been tentatively placed in the family Paramyxoviridae. Viruses isolated from an ill Neotropical rattlesnake (Crotalus durissus terrificus), from an Aruba Island rattlesnake (Crotalus unicolor), and from a bush viper (Atheris sp.) were propagated in Vero cells and characterized. Viral particles produced in Vero cells were pleomorphic, enveloped, and contained helical nucleocapsids. The viruses were sensitive to ether and to acidic and basic pH. Moreover, they had neuraminidase activity and were able to agglutinate erythrocytes from chicken and a variety of species of mammals. Hemagglutination was inhibited with rabbit antiserum raised against each virus. The buoyant densities of the three isolates ranged from 1.13/cm3 to 1.18/cm3, values consistent with that for an enveloped virus. The nucleic acid in the virion was determined to be RNA by [3H]uridine incorporation. Viral proteins characteristic of paramyxoviruses were immunoprecipitated from cells infected with each of the three isolates using rabbit anti-Neotropical virus serum. The morphologic appearance, physico- and biochemical properties, and cytopathologic effects of these snake viruses were consistent with those of certain members of the family Paramyxoviridae.

[Study of the immunobiological properties of parotitis viral strains]. / Izuchenie immunobiologichekikh svoistv shtammov virusa parotita.

The possibility of using different strains of parotitis virus (Enders, L-3, Jeryl-Leen) as antigens for enzyme immunoassay (EIA) to titer antibodies in human and animal blood sera is analyzed. Methods for preparation and purification of antigen on the basis of the said parotitis virus strains have been developed. Conditions of EIA were optimized. The sensitivity and specificity of EIA and hemagglutination inhibition test were compared.

The paramyxovirus SV5 V protein binds two atoms of zinc and is a structural component of virions.

The paramyxovirus simian virus 5 (SV5) cysteine-rich V protein has been shown to be a virus structural protein by analysis of the polypeptides of purified SV5 virions. In addition, the V protein has been identified as a component of the virus nucleocapsid core both by the analysis of the polypeptides present in radioactively labeled preparations of purified nucleocapsids and by immunoelectron microscopy. Quantitative autoradiography was used to determine that there are approximately 350 molecules of the V protein in virions. The V protein has been purified from V recombinant baculovirus-infected insect cells and by using inductively coupled argon plasma atomic emission spectroscopy it was found that each molecule of V binds two zinc atoms.

[Interactions between Mycoplasma and human and animal viruses. Ultrastructural analysis]. / Vzaimodeistvie mikoplazm s virusami cheloveka i zhivotnykh. Ul'trastrukturnyi analiz.

Presented are the data on the ultrastructural analysis of interaction between mycoplasma and certain cancerogenic and infectious viruses in humans and animals. Revealed are spontaneous associations of mycoplasma with viruses of cattle leukemia, T-cell human leukemia and with a representative of Bunyaviruses. Immediate interaction of these agents is found possible. Simulated complexes of mycoplasma with infectious viruses are developed. Electron microscopy on supramolecular levels revealed immediate interaction of different agents in membranes. Some methodological procedures help to reveal that the interaction of M. pneumoniae and A. laidlawii with orthomyxo-, paramyxo and togavirus is of specific character and is realized as receptor ligand form due to the affinity in the receptor requirements of these pathogens. This property as well as a bequeath distribution and frequent association in respiratory infections enable one to suggest the possibility of their immediate interactions in a host body.

Parainfluenza virus infection of cultured airway epithelial cells.

Studies of the cellular effects of respiratory viruses have generally used cultures of non-airway (particularly renal) epithelial cells. This requires the assumption that, despite the marked differences between renal epithelium and airway epithelium, the virus-host cell interactions in cultures of renal epithelium will be relevant to those in airway epithelium. To study viral infection of airway epithelial cells, we removed the epithelial cells from ferret tracheas using 0.1% pronase solution, and plated them at a density of 5 X 10(5) cells/cm2 in collagen-coated plastic tissue culture wells. Cultures grew to confluence after 5-7 days. Viral inocula, consisting of supernatants from parainfluenza type 1-infected rhesus monkey kidney cell monolayers, were added to the culture medium in a concentration 10(3) times that sufficient to produce infection in 50% of rhesus monkey kidney monolayers (TCID50). Cytopathic changes, consisting of cellular elongation and detachment, became apparent after 3-6 days, at which time the medium contained 5 X 10(8) TCID50/ml. The monolayer appeared to be uniformly infected as revealed by adsorption of guinea pig erythrocytes. Specific immunofluorescence revealed uniformly positive staining for parainfluenza type 1 antigens. The ability to infect pure cultures of airway epithelial cells with viruses will allow us to examine the effects of these viruses on epithelial cell function, and to study virus-host cell interactions in cell cultures derived from the natural host cell.

Fixation procedures for retention of cellular morphologic features and for preservation of immunoreactivity of canine paramyxovirus antigens.

The effectiveness of 1% saponin at 55 C (SAP), glutaraldehyde-borohydride-saponin (GBS), and modified GBS (MGBS) as fixatives for preserving cellular morphologic features, as well as antigenicity of intracellular and membrane-bound viral proteins of canine parainfluenza virus (CPIV) and canine distemper virus (CDV) was studied. Use of the same fixatives for light and electron microscopic immunocytochemical examination also was investigated. By light microscopy, CDV inclusions were readily detected after SAP and MGBS fixation, but not after GBS fixation; CPIV inclusions were detected after GBS and MGBS fixation, but not after SAP fixation. Ultrastructurally, SAP-treated cells had moderate to severe cytoplasmic artifacts, although CDV-associated cytoplasmic and membrane viral antigens were readily labeled. The CPIV-infected cells contained only a few positively labeled membrane-associated antigens and cytoplasmic nucleocapsids (NC). Although GBS-treated cells had excellent ultrastructural preservation, immunolabeling was unsatisfactory; CPIV-NC were labeled incompletely, and CDV-NC were unlabeled. After fixation with MGBS, immunolabeling of NC and membrane-associated viral proteins for both viruses was achieved, and the architecture of infected cells was preserved.