Pneumotropic revertants derived from a pantropic mutant, F1-R, of Sendai virus.

Revertants were isolated from the protease activation mutant of Sendai virus, F1-R, which causes a systemic infection in mice. The fusion (F) glycoprotein of F1-R is susceptible to activation cleavage by ubiquitous cellular proteases and is thus responsible for pantropism in mice (Tashiro et al., 1988. Virology 165, 577-583). The revertants regained several phenotypes of wild-type virus; they required exogenous trypsin for activation of the F protein in cell cultures and in nonpulmonary mouse tissues and they were exclusively pneumotropic in mice. On the other hand, phenotypes of F1-R that remained unchanged by the revertants were bipolar budding in polarized epithelial cells, enhanced electrophoretic migration of the matrix protein, and the lack of a glycosylation site in the F2 subunit of the F protein. Comparative RNA sequence analysis of the F gene of the revertants revealed that the reduced cleavability of the F protein of the revertants was the result of the predicted single amino acid reversion (Pro to Ser) at residue 115 adjacent to the cleavage site. Thus the sequence at the cleavage site of the revertants was Ser-Lys compared with Pro-Lys for F1-R and Ser-Arg for wild-type virus. The results indicate that enhanced cleavability of the glycoprotein, a feature often associated with multiple basic residues within the cleavage site of paramyxovirus F proteins and influenza virus hemagglutinins, can also be determined by a single basic amino acid following proline. Additionally, the revertants were less susceptible to the activator for wild-type virus present in mouse lungs and less pathogenic for this organ than wild-type virus. These results provide further evidence that proteolytic activation of the F protein by host proteases is the primary determinant for organ tropism and pathogenicity of Sendai virus in mice. One of the revertants was also temperature sensitive (ts); the ts lesion in the nucleoprotein gene was identical to that found in ts-f1, the ts host range mutant from which F1-R was derived.     

Characterization of a Sindbis virus variant with altered host range

Summary A variant of Sindbis virus which is much more infectious for mouse cells than the standard virus has been examined for biochemical properties which might be responsible for this biological difference. The variant has a much enhanced ability to adsorb to mouse plasmacytoma (MOPC 315) cells, but when these cells were pretreated with heparin, they were able to adsorb the standard virus almost as well as the variant. This suggested that there was a surface charge difference between variant and standard virus. Differential elution of the viruses from hydroxyapatite and the results of isoelectric focusing of the virion glycoproteins substantiate this interpretation. Both viral glycoproteins E1 and E2 from the variant were more negatively charged than those of the standard virus but we were unable to find changes in tryptic peptides of the variant. Differences were found in stability of the two virus strains to heat and proteolytic enzymes.With 3 Figures     

Characterization of an influenza a host range mutant

A mixed infection of primary chick kidney cells at 38° with A/Ann Arbor/6/60 cold adapted virus and A/Alaska/6/77 wt virus yielded a cold-reassortant virus, CR43-clone 3, which had a host range different from that of either parent. It does not produce detectable virus when grown in Madin-Darby canine kidney cells, while growing normally in primary chick kidney cells at 33°. Both parents, however, grow well in either cell type at 33°. Genotypic analysis of viral RNA electrophoresed in polyacrylamide gels has shown that CR43-clone 3 virus has an aberrant NS gene different from the NS gene of either parent virus. Reassortant viruses made between CR43-clone 3 virus and A/California/ 10/78 (H1N1) virus in primary chick kidney cells at 33° showed the same host range restriction only if the NS gene was derived from the CR43-clone 3 virus. A mixed infection with these same parents, but in Madin-Darby canine kidney cells at 33°, produced reassortants that always contained the A/California/10/78 NS gene instead of the CR43clone 3 NS gene. Ferrets inoculated intranasally with the CR43-clone 3 reassortant do not become sick or infected, based on the lack of symptoms: no rhinitis, coryza, or fever; and no detectable virus recovered from nasopharyngeal swabs, turbinate, or lung tissues at 48 hr after infection. Thus, CR43-clone 3 virus contains an aberrant NS gene and manifests a restricted host range phenotype in Madin-Darby canine kidney cells and ferrets.     

Characterization of the polypeptides synthesized in cells infected with a temperature-sensitive mutant derived from an HVJ (Sendai virus) carrier culture

Summary The intracellular synthesis of virus-specific polypeptides in cells infected with the wild-type virus of HVJ (HVJ-W) (haemagglutinating virus of Japan—the Sendai strain of parainfluenza 1 virus) and with a temperature-sensitive(ts) mutant (HVJ-pB) derived from an HVJ carrier culture has been analysed by polyacrylamide gel electrophoresis. At the permissive temperature (32° C), all of the known virus structural polypeptides were identified in cells infected with each strain of virus and in addition to the non-structural polypeptides B and C, another polypeptide at the region with a molecular weight of 26,000 to 27,000 (26 to 27K) could be detected in infected cells. At the non-permissive temperature (38° C), the synthesis of the polypeptide M was markedly restrained in cells infected with HVJ-pB, while other major virus polypeptides were present in approximately comparable amounts to cells infected with the wild-type virus. A non-structural polypeptide with a molecular weight of 105K was dominant ints mutant infected cells at higher temperatures and disappeared after temperature-shift from 38° to 32° C. The production of the non-structural polypeptides B and 27K was also temperature-sensitive. The molecular weights of the polypeptides B, M and 27K in HVJ-pB infected cells were larger than those of the corresponding polypeptides in HVJ-W infected cells. The synthesis of the M protein in HVJ-pB infected cells started just after lowering the incubation temperature and the newly made M protein was successfully incorporated into virus particles.With 4 Figures     

Protection of mice against virulent virus infection by a temperature-sensitive mutant derived from an HVJ (Sendai virus) carrier culture

Summary Experimental infection with HVJ (haemagglutinating virus of Japan—the Sendai strain of parainfluenza 1 virus) in mice was studied. Aerosol infection of newborn mice with the wild-type virus (HVJ-W) retarded the development of body weight and killed the animals within a few weeks. Large amounts of virus were isolated from both the lungs and the nasal turbinates of infected mice. In contrast, newborn mice exposed by inhalation to a temperature-sensitive(ts) mutant (HVJ-pB) derived from an HVJ carrier culture showed no clinical signs and grew equally well as mock-infected animals. No infectious virus could be recovered from the lungs although thets mutant grew to moderate titre in the nasal turbinates.The prior inoculation of newborn mice with thets mutant virus induced a state of significant resistance to subsequent challenge with the virulent wild-type virus.No replication of challenge virus in both lungs and nasal turbinates could be detected and the animals were protected a lethal infection. It is suggested that an avirulent temperature-sensitive mutant which has lost the capacity to replicate in the lower respiratory tract but is still capable of multiplying in the nasal turbinates may be a promising candidate for use in live vaccines especially against the infectious disease of the lower respiratory tract.With 2 Figures     

Homologous interference induced by a temperature-sensitive mutant derived from an HVJ (Sendai virus) carrier culture.

Homologous interference between a temperature-sensitive small plaque mutant (HVJ-pB) derived from an HVJ (haemagglutinating virus of Japan - the Sendai strain of parainfluenza I virus) carrier culture of BHK cells and the original wild-type virus (HVJ-W) has been investigated. Prior infection of LLCMK2, HeLa, BHK or mouse L cells with HVJ-pB, both at permissive and non-permissive temperatures, for 24 h resulted in a reduced yield of superinfecting HVJ-W, reflecting a smaller number of cells capable of producing the superinfecting virus. However, HVJ-pB did not interfere with the replication of vesicular stomatitis virus, Sindbis virus or Newcastle disease virus. Interference in this system seems to be due to inhibition of the attachment of superinfecting HVJ-W as a result of intracellular mechanisms operating at a late stage in the replication of the interfering virus. There is also blocking or destruction of cellular receptors by extra-cellular particles of the interfering virus. Protein synthesis coded for by the complete virus genome is required to establish and maintain the interference, and treatment with actinomycin D has no effect on the interference phenomenon.     

Experimental infection of suckling mice with a host range mutant of Junin virus.

Experimental infection of three mouse strains with a non-pathogenic mutant of Junin virus named Cl67 was compared with respect to the parental XJCl3 strain. After intracerebral (ic) or intraperitoneal inoculation, XJCl3 was highly virulent for 2 day-old C3H/HeJ, OF1, and BALB/cJ mouse strains, whereas its derivative Cl67 was attenuated. Survival of the Cl67-infected mouse was associated with a restricted replication at the site of inoculation which would impair spread of virus. Thus, the reduced virulence of Cl67 for suckling mice is independent of the mouse strain and the route of viral entry. When Cl67 was preinoculated ic 10 days before the challenge inoculation with XJCl3 by the same route, mice were partially protected from lethal infection. Since neutralizing antibodies were first detected at 30 days post-infection, an interference mechanism is postulated as a mechanism of protection of the mice.     

Generation and characterization of a Cowpox virus mutant lacking host range factor CP77

Cowpox virus (CPXV) host range factor CP77 was identified to be required for virus replication in Chinese hamster ovary (CHO) cells, but the underlying molecular mechanism by which CP77 modulates host range has remained unclear. Therefore, a CPXVΔCP77 deletion mutant was constructed by applying bacterial artificial chromosome (BAC) technology. Integrity of BAC-derived viral DNA was confirmed by whole genome sequencing. In vitro growth characteristics of CPXV wild type (WT), BAC-derived vCPXV WT and vCPXVΔCP77 were virtually indistinguishable in HEK293T cells, whereas in CHO-K1 cells replication of virus lacking CP77 was unambiguously attenuated. This block of viral replication was confirmed by lack of late viral protein expression. The replication defect of various Orthopoxviruses lacking CP77 in CHO cells could be restored by recombinant expression of CP77. Thus, for the first time, the described CP77-dependent host range effect in CHO cells was shown in the background of CPXV as well as Camelpox virus. To further characterize the mutant virus, cells of several different species were comparably infected with vCPXV WT and vCPXVΔCP77, respectively. Interestingly, except for CHO-K1 cells, vCPXV WT and vCPXVΔCP77 showed no significant difference in terms of morphology of cytopathic effects, expression of a late transcribed virus-encoded green fluorescent protein and virus reproduction, even in other hamster-derived cells. Additionally, in ovo inoculation with either virus revealed the same red-pock phenotype on chicken egg chorioallantoic membranes. Since the data presented indicate a CP77-dependent host range effect only for CHO cells, we conclude that the protein might mediate additional functions not identified yet. The vCPXVΔCP77 deletion mutant generated can now be applied as a useful tool to investigate the function of the putative host range protein CP77.     

Tryptic peptide analysis of the structural proteins of a temperature-sensitive mutant derived from an HVJ (Sendai virus) carrier culture

Summary The individual structural polypeptides of HVJ (haemagglutinating virus of Japan—the Sendai strain of parainfluenza 1 virus) were examined by tryptic peptide analysis.[³H]-methionine-labelled structural proteins of the wild-type virus of HVJ (HVJ-W) and [³⁵S]-methionine-labelled corresponding constituent proteins of a temperature-sensitive(ts) mutant (HVJ-pB) derived from an HVJ carrier culture were compared by ion-exchange chromatography on columns of P-type chromobeads. The tryptic peptides of the individual structural proteins showed characteristic elution profiles. In all the structural proteins tested, the chromatographic elution profiles of both strains generally showed a close resemblance. However, certain minor peaks which were present in one strain but absent in the other strain were detected in the preparations of the P, HN, and F polypeptides. Further, analysis of the NP polypeptide showed that a major peak of one strain appeared at a position in the pH gradient different from a seemingly corresponding major peak of the other strain. In the M protein some possibly homologous minor peaks were found to differ between the two strains.With 2 Figures     

The properties of recombinant Sendai virus having the P gene of Sendai virus pi strain derived from BHK cells persistently infected with Sendai virus

We prepared the chimeric recombinant Sendai virus [rSeV(Ppi)] by replacing the P gene of the Z strain with that of pi strain for analyzing the function of Ppi, Vpi and Cpi proteins. Intriguingly, HA production by rSeV(Ppi) is significantly lower at 38°C than at 32°C, showing that virus growth of rSeV(Ppi) is slightly suppressed at 38°C. However, the main phenotypes of SeVpi, a marked temperature sensitivity as viral replication and an ability of establishing persistent infection, are not explained by the Ppi, Vpi and Cpi proteins. The V and C proteins form inclusion bodies in L929 cells infected with rSeV(Ppi) and incubated at 38°C. L929 cells infected with rSeV(Ppi) and L929 cells stably expressing the Cpi protein show resistance to interferon-β at 32 and 38°C, indicating that the Cpi protein per se is not temperature-sensitive to inhibition of IFN signaling. The complete genome sequences of Sendai virus (SeV) pi and parent Nagoya strains were determined. Fifty nucleic acid substitutions are found in the genome sequence of SeV pi strain in comparison with Nagoya strain. There are three nucleic acid substitutions in the leader sequence, while the trailer, intergenic, gene-end and gene-start sequences of both strains are completely identical. Deletions and insertions of nucleotide are not found. There are 32 amino acid substitutions in Sendai virus pi strain. The specific amino acid substitutions unique to the SeVpi are 18. Information about the complete genome sequences of SeVpi strain is important to totally understand the persistent infection and lower pathogenicity of SeV.Machiko Nishio and Ai Nagata have contributed equally.     

Eriophyid mite transmission and host range of a Brome streak mosaic virus isolate derived from a full-length cDNA clone

Summary Biolistic inoculation of Hordeum vulgare and Phalaris paradoxa with a brome streak mosaic virus (BrSMV) full-length cDNA clone (pBrSMV_fl) led to typical leaf streak symptoms in both plant species. Infected H. vulgare plants showed a more stunted growth 8 weeks after symptom appearance compared to BrSMV wild type (BrSMV_wt)-infected plants. Moreover, a slightly higher virus titer was observed in BrSMV_fl-inoculated H. vulgare, A. sativa and P. paradoxa plants. The biological activity of BrSMV_fl and BrSMV_wt was verified in vector transmission assays, providing the first experimental evidence that Aceria tosichella can act as a natural vector of BrSMV. Correspondence: Prof. Edgar Maiss, Institute of Plant Disease and Plant Protection, Leibniz University of Hannover, Herrenhaeuser Str. 2, 30419 Hannover, Germany     

Assembly of viral structural proteins in cells infected with a temperature-sensitive mutant derived from an HVJ (Sendai virus) carrier culture

Summary The processing of virus polypeptides synthesized in cells infected with HVJ (haemagglutinating virus of Japan—the Sendai strain of parainfluenza 1 virus) was studied. Maturation of a temperature-sensitive(ts) mutant (HVJ-pB) derived from an HVJ carrier culture was inhibited at 38° C incubation. A considerable amount of viral components were made at the restrictive temperature. They were, with the exception of the polypeptide HN, well preserved without a great loss of their function and successfully incorporated into virus particles released after lowering the incubation temperature. The membrane (M) protein seems to be essential for virus morphogenesis.With 1 Figure     

Plaque formation by a host range mutant of vaccinia virus in non-permissive cells co-infected with Yaba virus.

A host-dependent conditional lethal mutant of vaccinia virus strain DIs was rescued to form plaques in a non-permissive cell line JINET co-infected with Yaba virus, a poxvirus serologically distant from the rescued virus. The efficiency of plaque formation under optimal conditions in this system was comparable to that in Vero cells which were permissive for the mutant. The plaque number of the mutant in JINET cells was influenced greatly by the multiplicity of Yaba virus, the interval between inoculations with DIs and Yaba virus and the maintenance medium for pre-infection of cells with Yaba virus. The plaque formation by DIs in JINET cells was suppressed or inhibited when the duration of pre-infection with high multiplicities of Yaba virus was prolonged. Thus, Yaba virus possessed both rescuing and inhibitory activities on DIs and the plaque number of DIs in doubly infected cells was thought to be determined by the balance between the two activities. Ultraviolet light-inactivated Yaba virus retained rescuing activity on DIs in JINET cells.     

Characteristics of the progeny derived from multiplication of Sendai virus in a measles virus carrier cell line

Summary After infection of a carrier cell line of measles virus in Lu 106 cells with Sendai virus a heterogeneous product was obtained. Some particles apparently contained only one or the other of the two antigens, but a considerable fraction of particles seemed to carry both antigens. The occurrence of the latter was suggested from analyses of hemagglutinating activity and immunological characteristics of particles adsorbed and eluted on rooster erythrocytes. Tween-ether treatment of such particles caused a breakdown into fragments seemingly carrying either one or both antigens.Dedicated to the Honor of the 60th birthday of ProfessorSven Gard.Supported by grants from the Swedish Medical Research Council (Project no. Y 575).     

Recovery of a Sendai virus variant with temperature-sensitive hemolytic activity from persistently infected cells from mouse brain

Summary A persistently infected cell line designated MB/Sen_AS was established by cultivation of mouse brain cells from four-day-old C3H mice infected intracerebrally at birth with 10⁶ PFU of Sendai virus, strain 52. After 5 passages, 0.16 per cent of Sendai₅₂ antiserum (containing two 50 per cent plaque reducing doses/ml of serum) was introduced into the culture medium. The addition of antiserum was accompanied by a rise in cell-associated viral antigen from a level of 5 per cent antigen positive cells to 100 per cent demonstrable by both intracellular and membrane immunofluorescence.A variant of Sendai₅₂ virus, designated Sendai_AS, was recovered from MB/Sen_AS by inoculation of supernatant medium into chick embryos. Infection of chick embryos at 37° C was abortive. Fifty per cent or less of chick embryos infected at dilutions 10^–1 to 10^–9 yielded detectable virus. Hemagglutination (HA) was weak but could be improved by trypsinization of allantoic fluids. Neuraminidase (NA) activity was barely detectable. Hemolysis (HE) was absent. Propagation of Sendai_AS virus at 33° C showed no change from weak HA and NA activities but HE activity was now apparent which was temperature sensitive. Mortality of infected chick embryos increased to 100 per cent. HE activity and lethality for chick embryos was thermolabile at 45° C.With 3 Figures     

Vaccinia virus host range genes

A gene encoding an 18-kDa polypeptide (ORF C7L) located in the vaccinia virus HindIII C fragment was shown to be functionally equivalent to previously described host range gene (ORF K1L) spanning the HindIII K/M fragment junction. Either C7L or K1L host range gene is necessary and sufficient by itself to allow replication of vaccinia virus on human cells. Deletion of both C7L and K1L genes from the wild-type vaccinia genome is required to derive a virus deficient for replication on human cells. Further, an ORF encoding a 77-kDa polypeptide derived from cowpox (CP77kDa) and previously shown to allow vaccinia to overcome the restriction for replication on Chinese hamster ovary cells could substitute for the vaccinia host range genes C7L and K1L in permitting replication of the virus on human cells. Additionally, the three unique host range genes C7L, K1L, and CP77kDa were functionally equivalent for vaccinia replication on pig kidney cells, but not on rabbit kidney cells.     

The role of host responses in the recovery of mice from Sendai virus infection

The antiviral responses in mice to intranasal inoculation with Sendai virus are described. To investigate the relative importance of the humoral, cell-mediated and interferon responses, the pathogenesis of this infection was studied in animals which were immunocompetent, T cell-deprived or immunosuppressed with cyclophosphamide. Treatment with cyclophosphamide converted the mild, self-limiting infection observed in immunocompetent mice into a severe and frequently lethal pneumonic disease. This was associated with an enhanced interferon response but no detectable antibody or cell-mediated immune response. T cell-deprived mice suffer an infection of intermediate severity associated with an increased interferon response, a normal humoral immune response and no cell-mediated immune response. The implications of these results in relation to the role of the antiviral responses in recovery from Sendai virus infection are discussed.