Structures of the mumps virus polymerase complex via cryo-electron microscopy.

The viral polymerase complex, comprising the large protein (L) and phosphoprotein (P), is crucial for both genome replication and transcription in non-segmented negative-strand RNA viruses (nsNSVs), while structures corresponding to these activities remain obscure. Here, we resolved two L-P complex conformations from the mumps virus (MuV), a typical member of nsNSVs, via cryogenic-electron microscopy. One conformation presents all five domains of L forming a continuous RNA tunnel to the methyltransferase domain (MTase), preferably as a transcription state. The other conformation has the appendage averaged out, which is inaccessible to MTase. In both conformations, parallel P tetramers are revealed around MuV L, which, together with structures of other nsNSVs, demonstrates the diverse origins of the L-binding X domain of P. Our study links varying structures of nsNSV polymerase complexes with genome replication and transcription and points to a sliding model for polymerase complexes to advance along the RNA templates.

Purification of mumps virus particles of high viability.

Mumps is a highly infectious viral disease of humans with a wide array of clinical manifestations ranging from painful swelling of the salivary glands to meningitis and encephalitis. Despite the clinical importance of mumps virus, most of what is known of its biological properties comes from studies using supernatants from virus infected cell cultures, which contain substantial levels of host cell derived debris and biologically active substances such as cytokines, transcription factors and secreted virus proteins. These contaminants complicate interpretation of studies of virus replication, virus-host interactions and in vivo virulence. Here we describe a protocol for concentration of the virus from cell culture supernatants followed by gradient purification, resulting in attainment of high titer live virus of high purity.

Virion-associated complement regulator CD55 is more potent than CD46 in mediating resistance of mumps virus and vesicular stomatitis virus to neutralization.

Enveloped viruses can incorporate host cell membrane proteins during the budding process. Here we demonstrate that mumps virus (MuV) and vesicular stomatitis virus (VSV) assemble to include CD46 and CD55, two host cell regulators which inhibit propagation of complement pathways through distinct mechanisms. Using viruses which incorporated CD46 alone, CD55 alone, or both CD46 and CD55, we have tested the relative contribution of these regulators in resistance to complement-mediated neutralization. Virion-associated CD46 and CD55 were biologically active, with VSV showing higher levels of activity of both cofactors, which promoted factor I-mediated cleavage of C3b into iC3b as well as decay-accelerating factor (DAF) activity against the C3 convertase, than MuV. Time courses of in vitro neutralization with normal human serum (NHS) showed that both regulators could delay neutralization, but viruses containing CD46 alone were neutralized faster and more completely than viruses containing CD55 alone. A dominant inhibitory role for CD55 was most evident for VSV, where virus containing CD55 alone was not substantially different in neutralization kinetics from virus harboring both regulators. Electron microscopy showed that VSV neutralization proceeded through virion aggregation followed by lysis, with virion-associated CD55 providing a delay in both aggregation and lysis more substantial than that conferred by CD46. Our results demonstrate the functional significance of incorporation of host cell factors during virion envelope assembly. They also define pathways of virus complement-mediated neutralization and suggest the design of more effective viral vectors.

[A new method for quantitative estimation of the number of virus particles].

[In the present work virus particles of live mumps virus vaccine widely used for vaccination in Russia have been detected and visualized by atomic force microscopy. For quantitative estimation of the number of observed virus particles the special method has been proposed. The presence of protein component of the virus in vaccine was tested by ELISA and dot-blot analysis. Using quantitative real-time PCR assay the number of copies of viral RNA was estimated. The results of quantitative estimation obtained by real-time PCR corresponded with atomic force microscopy data.

Characterization of a mumps virus nucleocapsidlike particle.

The nucleocapsid protein (NP) of mumps virus (MuV), a paramyxovirus, was coexpressed with the phosphoprotein (P) in Escherichia coli. The NP and P proteins form a soluble complex containing RNA. Under a transmission electron microscope, the NP-RNA complex appears as a nucleocapsidlike ring that has a diameter of approximately 20 nm with 13 subunits. There is a piece of single-stranded RNA with a length of 78 nucleotides in the NP-RNA ring. Shorter RNA pieces are also visible. The MuV NP protein may provide weaker protection of the RNA than the NP protein of some other negative-strand RNA viruses, reflecting the degree of NP protein association.

Mumps virus matrix, fusion, and nucleocapsid proteins cooperate for efficient production of virus-like particles.

Paramyxovirus particles, like other enveloped virus particles, are formed by budding from membranes of infected cells. To define mumps virus (MuV) proteins important for this process, viral proteins were expressed either singly or in combination in mammalian cells to produce virus-like particles (VLPs). Only the MuV matrix (M) protein when expressed by itself was capable of inducing particle release, but the quantity of these M-alone particles was very small. Efficient production of mumps VLPs occurred only when the M protein was coexpressed together with other viral proteins, with maximum production achieved upon coexpression of the viral M, nucleocapsid (NP), and fusion (F) proteins together. Electron microscopy analysis confirmed that VLPs were morphologically similar to MuV virions. The two MuV glycoproteins were not equal contributors to particle formation. The F protein was a major contributor to VLP production, while the hemagglutinin-neuraminidase protein made a smaller contribution. Evidence for the involvement of class E protein machinery in VLP budding was obtained, with mumps VLP production inhibited upon expression of dominant-negative versions of the class E proteins Vps4A and Chmp4b. Disruption of the sequence 24-FPVI-27 within the MuV M protein led to poor VLP production, consistent with findings of earlier studies of a related sequence, FPIV, important for the budding of parainfluenza virus 5. Together, these results demonstrate that different MuV structural proteins cooperate together for efficient particle production and that particle budding likely involves host class E protein machinery.

Assessment of mumps virus growth on various continuous cell lines by virological, immunological, molecular and morphological investigations.

Vero cells have been used as a convenient laboratory substrate for the isolation of mumps virus but may not be very sensitive and may select for particular adapted variants from clinical specimens. Continuous cell lines were evaluated for their ability to support the replication of mumps virus. Criteria included the production of infectious virus, detection of intracellular mumps proteins by immunofluorescence and electron microscopy and detection of specific nucleic acid by RT-PCR. Of the cells tested, CaCo-2, PLC/PRF/5, and Vero cells produced infectious virus, with Vero and CaCo-2 being the most permissive. The other substrates tested included cells of murine, canine and human origin showed signs of intracellular proteins and RNA but the amounts produced were much lower, and no infectious virus was detected in some cases. The virus use was a low passage of a Vero derived wild type strain, and it will ultimately be necessary to continue the studies with an unpassaged clinical specimen to identify a cell line able to isolate mumps virus at high efficiency and in unmodified form.

A search for evidence of viral infection in pancreases of newly diagnosed patients with IDDM.

Techniques were developed to look for evidence of viral infection in formalin-fixed paraffin-embedded autopsy pancreatic tissues from patients who had died of recent-onset insulin-dependent diabetes mellitus. DNA extracted from 47 pancreases in which good DNA preservation was confirmed was analysed by a polymerase chain reaction for Epstein-Barr virus and by a nested polymerase chain reaction for cytomegalovirus. Histological sections from 29 pancreases in which there was good RNA preservation were tested for the presence of enterovirus and Epstein-Barr virus using in situ hybridization techniques. Seventy-five pancreases were analysed immunohistochemically for the presence of mumps virus. None of these viruses could be detected in any of the diabetic pancreases studied. Control studies suggested that the techniques employed were as sensitive as culture done at the time of autopsy. Pancreas was available for study in 9 infants who had died of myocarditis; enterovirus was demonstrable in islets in 5 of these cases. An acute or persisting infection in the pancreas at the time of clinical onset of insulin-dependent diabetes by any of the 4 virus included in this study seems unlikely.

[Characteristics of parotitis virus, isolated in Siberia]. / Kharakteristika virusa parotita, vydelennogo v Sibiri.

Two strains of parotitis virus were isolated from patients with clinical symptoms of the disease in epidemiological screening which was carried out during an outbreak of epidemic parotitis in the village of Koltsovo in 1994. The strains were isolated from the saliva of children aged 7 and 8 years vaccinated with live parotitis vaccine at the age of 1.5 years. Primers for the genome site coding for the gene F terminal and the SH gene (a total of 509 n. p.) were estimated and synthesized and the site was amplified. Electron-microscopic examination of purified virus and Vero cells infected with it and serological tests showed a similarity of the newly isolated virus with the Anders strain of parotitis virus. The Dragun-1 and Dragun-2 strains of parotitis virus have been deposited in the collection of viruses at the Vektor State Research Center of Virology and Biotechnology in the village of Koltsovo, Novosibirsk district.

Membrane fusion of mumps virus with ghost erythrocytes and CV-1 cells.

The octadecyl rhodamine (R18) fluorescent dequenching assay was used to examine membrane fusion between mumps virus and mammalian cells. Rapid fluorescent dequenching, indicative of membrane fusion, was observed when labeled mumps virus was mixed with either ghost erythrocytes or CV-1 cells. After 15 min a saturation limit of 18 virus per erythrocyte ghost and 6400 virus per CV-1 cell was observed. Fetuin was found to inhibit virus fusion, suggesting a role for sialic acid in virus binding to the cells. Two dequenching processes were observed of which the faster process is thought to be membrane fusion and the second process is thought to be probe proximal transfer.

Experimental mumps virus-induced labyrinthitis. Immunohistochemical and ultrastructural studies.

Guinea pigs were inoculated with mumps virus (Torii strain) by the intralabyrinthine or intravascular route and their cochleas were examined by immunofluorescence and electron microscopy. In these animals, viral antigen was detected in the cochleas, most often in the stria vascularis. However, viral infection was produced only in those with intralabyrinthine inoculation. The cochlear lesion commonly observed in this study was severe degeneration of the organ of Corti, which was usually found in the basal turn. Morphological evidence of viral infection, as depicted by intracellular strands of nucleocapsids and budding of the virus at the endolymphatic surface, was prominent in both the stria vascularis and Reissner's membrane. The cochlear location of mumps infection correlated closely with that of the viral antigen formerly reported in newborn hamsters.

[Physicochemical characteristics of a vaccinal strain of the Leningrad-3 mumps virus (L-3)]. / Fiziko-khimicheskaia kharakteristika vaktsinnogo shtamma virusa parotita Leningrada-3 (L-3).

Purified virions of the vaccine strain Leningrad-3 of mumps virus propagated in Japanese quail embryo cell cultures had a buoyant density 1.18-1.19 g/ml in sucrose gradient, contained 50 S RNA and showed variable sizes in electron microscopy as manifested by heterogeneity of the virus zone in sedimentation analysis. Purified L-3 virus contained 5 major polypeptides with molecular weights of 74,000, 68,000, 58,000, 45,000, and 39,000 daltons. Each polypeptide had an individual oligopeptide composition.

Intrinsic fluorescence of some myxo- and paramyxoviruses and of their subviral fractions.

Fluorescence spectra of Sendai and influenza A(H1N1) viruses have different emission maxima; their quantum yield is much lower than that of tobacco mosaic virus. Solubilized envelopes and nucleoproteins of Sendai, influenza and mumps virus have different half-bandwidth and relative quantum yield values of emission, according to the agent used for disruption. Thus emission (as well as excitation and absorption) spectra of Triton X-100-solubilized envelopes show a marked hypsochromic shift as compared with the envelopes obtained by Tween20-disruption. The results are correlated with the different disruption extent achieved with the two agents.

Intrauterine infection with mumps virus.

The histopathologic study of 3 cases of gestational mumps is presented. The tissue studies was obtained from a spontaneous abortion (case 1) and from 2 therapeutic abortions (cases 2 and 3). Severe placental and fetal lesions were observed, indicating a probable association with maternal mumps. The main placental lesion was a diffuse proliferative necrotic villitis with severe lesions in the fetal vascular circuit, probably the cause of death. In the fetal viscera, areas of necrosis and mineralization were observed. Viral inclusions identical to those described in mumps infection were observed in the chorionic and fetal tissues.

Morphological heterogeneity in relation to structural and functional properties of mumps virus.

The morphological heterogeneity of the chick embryo-adampted Enders strain of mumps virus was examined in relation to biological and biochemical properties of the virus. The range of virion sizes increased as multiplicity of infection (m.o.i.) increased, with virions of 800 to 1000 nm in diam. occurring in preparations grown at the higher multiplicities. There was no evidence for a distinct class of noninfectious haemagglutinating particles. Dense fractions from isopycnic gradients of virus were enriched in larger virions, but virus RNA was predominantly a single 50S species. Despite evidence that many virions were multiploid, u.v. inactivation kinetics were single-hit, suggesting only a single biologically active genome per virion.

Polypeptide composition of mumps virus.

The Enders strain of mumps virus grown in ovo was purified by differential and equilibrium sucrose gradient sedimentation. Purified virus contained seven polypeptides of mol. wts 68,000, 66,000, 61,000, 54,000, 52,000, 49,000, 47,000. Nucleocapsids isolated from DOC-treated virus contained two polypeptides of mol. wts 66,000 and 61,000.

Ultrastructure of mumps virus replication in organotypic cultures of hamster choroid plexus.

Organotypic cultures of newborn hamster choroid plexus were inoculated with equal titre doses of newly isolated or hamster adapted strains of mumps virus. The ultrastructure of virus replication in choroid epithelial cells of the cultures was compared. No qualitative differences were observed; however, the adapted strain produced significantly greater numbers of virions and earlier destruction of the cultures. These findings are consistent with previous in vivo observations of the ultrastructure of the replication of these strains in the newborn hamster central nervous system. This in vitro study leads further support to the hypothesis that differences in the in vivo biological effects of the virus strains are primarily the result of virus-cell rather than virus-host interactions.