Optimization of plasmid-only rescue of highly attenuated and temperature-sensitive respiratory syncytial virus (RSV) vaccine candidates for human trials

Respiratory syncytial virus (RSV) is the most common cause of severe bronchiolitis in infants and young children in the U.S. No licensed RSV vaccines are currently available. Established techniques for recovering RSV from cDNA utilize mammalian cells, such as HEp-2 or BSR T7/5, that are not currently suitable for vaccine manufacture. When using HEp-2 cells, co-infection with an attenuated vaccinia virus that expresses T7 RNA polymerase is also required. For human clinical trials, processes that do not require the use of helper viruses and minimize the use of animal derived materials must be developed to reduce the potential theoretical risk of transmitting adventitious agents such as BSE. RSV was generated by electroporating Vero cells from a well characterized cell bank with 6 plasmids expressing T7 RNA polymerase, the full-length anti-genomic RSV and RSV N, P, M2-1 and L. The process was optimized such that highly attenuated and temperature-sensitive RSV vaccine candidates could be recovered in a system completely free of animal derived components. Efficiencies of virus recovery ranged from 30% to 100%. Human metapneumovirus was also readily recovered, suggesting that this protocol is applicable for the production of clinical trial material of other non-segmented negative sense RNA viruses.     

Reproduction of Lassa virus in different cell cultures

Sierra Leone strain of Lassa virus was growing to high titres of 10(5)-10(6) plaque forming units (PFU) per ml in Vero, L and swine kidney cell lines as well as in diploid human cells and primary human embryo kidney cells. As many as 80% of the cells became infected as demonstrated by the immunofluorescence (IF) technique. In BHK-21, CV-1, HeLa, FL, HEp-2 and dog kidney cell lines, the virus reproduced to lower titres (10(4)-10(5) PFU per ml), whereas in primary chick embryo fibroblasts it did not multiply at all. The virus formed plaques under agar overlay only in CV-1 and Vero cells.     

Contribution of the respiratory syncytial virus G glycoprotein and its secreted and membrane-bound forms to virus replication in vitro and in vivo.

The surface glycoproteins of viruses can play important roles in viral attachment, entry, and morphogenesis. Here, we investigated the role of the attachment G glycoprotein of human respiratory syncytial virus (RSV) in viral infection. RSV G is produced both as a complete, transmembrane form and as an N-terminally truncated form that is secreted. Using reverse genetics, we created mutant recombinant RSVs (rRSV) that do not express G (DeltaG) or express either the secreted or the membrane-bound form of G only (sG and mG, respectively). In Vero cells, the DeltaG virus formed plaques and grew as efficiently as wild-type rRSV and mG. In contrast, DeltaG replicated less efficiently and did not form distinct plaques in HEp-2 cells. This defect was primarily at the level of the initiation of infection, with only a minor additional effect at the level of packaging. Replication of DeltaG in the respiratory tract of mice was very highly restricted, indicating that G is important in vivo. Although the G protein expressed by the sG virus was confirmed to be secreted, this virus grew at least as efficiently as wild-type in HEp-2 cells and was only moderately attenuated in vivo. Thus, the G protein was important for efficient replication in HEp-2 cells and in vivo, but this function could be supplied in large part by the secreted form and thus does not require the cytoplasmic and transmembrane domains. Amino acids 184-198 have been identified as the major heparin-binding domain of the G protein and were implicated in mediating binding to cells [S. A. Feldman et al., 1999, J. Virol. 73, 6610-6617]. Heparin-like glycosaminoglycans also appeared to be important for infection in vitro by direct clinical isolates of RSV. Deletion of amino acids 187-197 from rRSV did not reduce its sensitivity to neutralization in vitro by incubation with soluble heparin, did not reduce its efficiency of growth in vitro, and resulted in only a modest reduction in vivo. Thus, the putative heparin-binding domain is not the sole determinant of heparin sensitivity and is not a critical functional domain.     

Microplaque immunoperoxidase detection of infectious respiratory syncytial virus in the lungs of infected mice

A rapid microplaque technique was developed for the detection of infectious respiratory syncytial virus (RSV) in the lungs of infected mice. Infected lung homogenates were titrated on microwell HEp-2 monolayers and incubated for 24 or 48 h. The microwells were then fixed with 4% formaldehyde in saline, or methanol containing 0.5% hydrogen peroxide. 24-h single cell infectious foci and 48-h microplaques were detected by an indirect immunoperoxidase (IIP) assay using monoclonal antibodies specific for RSV envelope glycoproteins as the first layer. This method can be used for the quantification of lung RSV in large numbers of samples much more rapidly and economically than conventional plaque assay techniques. In addition, the use of the IIP assay renders the system specific for RSV.     

Rapid detection of respiratory syncytial virus and influenza A virus in cell cultures by immunoperoxidase staining with monoclonal antibodies.

Peroxidase-labeled monoclonal antibodies against respiratory syncytial virus (RSV) and influenza A virus were used for immunoperoxidase staining (IPS) of cell cultures inoculated with nasopharyngeal aspirates. Cells were grown in 24-well plates, and specimens were inoculated by low-speed centrifugation. Cultures were incubated for 2 days at 37 degrees C and then fixed, stained, and observed by light microscopy. IPS was compared with standard virus isolation by using cultures of human diploid fibroblasts and Vero, HEp-2, and HeLa cell lines for RSV and Madin-Darby canine kidney cells for influenza A virus; these cultures were inoculated with specimens that were previously stored at -70 degrees C. Of 40 known RSV-positive specimens, 30 were found to be positive on reinoculation by both methods, and an additional 5 specimens were found to be positive by IPS only. Of 190 specimens tested for influenza A virus, 14 were positive by IPS and in tubes, and a further 8 specimens were positive by IPS only. IPS was also compared with direct detection of viral antigens in nasopharyngeal aspirates by a time-resolved fluoroimmunoassay (TR-FIA). Fresh nasopharyngeal aspirates were inoculated into human diploid fibroblasts and Madin-Darby canine kidney cells and tested for RSV and influenza A virus, respectively, by IPS. Of 110 specimens tested for RSV, 37 were positive in total, 32 were positive by IPS, and 33 were positive by TR-FIA. Of 150 specimens tested for influenza A virus, 39 were positive in total, 35 were positive by IPS, and 34 were positive by TR-FIA. IPS of cultures inoculated by centrifugation and incubated for 2 days is a sensitive method for the diagnosis of respiratory virus infections, and 24-well plates allow for the easy processing of a large number of specimens.     

Plaque formation by human-origin parainfluenza type 2 virus in established cell lines

Summary Two strains of parainfluenza type 2 virus formed well-defined plaques in cultures of Vero cells, an established line of African green monkey kidney cells. In the absence of trypsin, satisfactory plaques were formed by the Toshiba strain of virus. When trypsin was added in the overlay medium of Vero cell monolayers, another strain (62-M786) of virus produced plaques. The Toshiba strain was also able to make plaques in HeLa, BHK, and LLC-MK₂ (an established line of monkey kidney) cells without trypsin, but not in mouse L cells. The sensitivity of plaque assay was about equal to that of the hemadsorption method.With 3 Figures     

RhoA is activated during respiratory syncytial virus infection

Respiratory syncytial virus (RSV) is an important human pathogen that can cause severe and life-threatening respiratory infections in infants and immunocompromised adults. We have recently shown the RSV F glycoprotein, which mediates viral fusion and entry, interacts with the cellular protein RhoA in two-hybrid and in vitro binding assays. Whether this interaction occurs in living cells remains an open question. However, because RhoA signaling is associated with many cellular functions relevant to RSV pathogenesis such as actin cytoskeleton organization, expression of proinflammatory cytokines, and smooth muscle contraction, we asked whether RhoA activation occurred during RSV infection of HEp-2 cells. We found that the amount of isoprenylated and membrane-bound RhoA in RSV-infected cultures was increased. Further evidence of RhoA activation was demonstrated by downstream signaling activity mediated by RhoA. There was an increase in p130(cas) phosphorylation during RSV infection, which was prevented by Y-27632, a specific inhibitor of Rho kinase, or lovastatin, an HMG-CoA reductase inhibitor that reduces the synthesis of groups needed for isoprenylation. In addition, RSV infection of HEp-2 cells resulted in an increase in the formation of actin stress fibers. Pretreatment of HEp-2 cells with Clostridium botulinum C3 exotoxin, an enzyme that specifically ADP-ribosylates and inactivates RhoA, prevented RSV-induced stress fiber formation. These observations indicate that RhoA and subsequent downstream signaling events are activated during RSV infection, which has implications for RSV pathogenesis.     

Neutralization activity of some Coxsackie B3 antibodies is compromized by protein factor(s) secreted by Vero cells

Summary When various anti-Coxsackie B3 virus antibodies were examined for the neutralizing activity in cultures under liquid medium, some antibodies including monoclonal antibodies gave abnormally low titers in the neutralization test in Vero cells, in comparison with the other cells such as HeLa, FL, HEp-2, or primary monkey kidney cells. The neutralization titer of these antibodies was, however, similar in all these cells by plaque reduction assays under agar overlay, i.e., the above phenomenon was restricted to cultures under liquid medium. The reduced neutralization titer in Vero cells under liquid medium was found to be brought about by protease-sensitive factor(s) released by Vero cells, because (1) the addition of Vero cell culture fluid resulted in a marked reduction of neutralizing titer in primary monkey kidney cells, and (2) the activity of the Vero cell factor was destroyed by trypsin (20 µg/ml for 1 h). As three-day incubation of virus-antibody complex in Vero cell culture fluid resulted in a partial restoration of virus infectivity, the binding of antibody to virions appears to be competed by the Vero cell factor.     

Sensitive plaque neutralization assay for parainfluenza virus types 1, 2, and 3 and respiratory syncytial virus.

A sensitive plaque neutralization assay for parainfluenza virus types 1, 2, and 3 and respiratory syncytial virus was developed in Vero and MA 104 cell cultures. The tests were performed in semimicrotoiter trays containing 24 wells, 16 mm in diameter. Parainfluenza virus type 1 formed plaques in Vero and MA 104 cells only when trypsin was added to the overlay medium. Plaquing of parainfluenza virus type 1 was more sensitive and technically reproducible in MA 104 cells than in Vero cells. Parainfluenza virus types 2 and 3 and respiratory syncytial virus readily formed plaques in Vero cells. Plaques with all viruses were necrotic in character, except for plaques produced by parainfluenza virus type 3, which appeared red due to an increased uptake of neutral red by infected cells. Different conditions for plaquing of the four viruses had to be used to obtain plaques of suitable size. Antibody titers of commercially prepared guinea pig typing sera were 5- to 50-fold higher by the plaque neutralization test than by complement fixation. The addition of guinea pig immunoglobulin G antiglobulin to the serum-virus mixtures enhanced the conventional neutralization test 5- to 10-fold. The sensitivity and specificity of the plaque neutralization test was also determined with sera of marmosets experimentally infected with parainfluenza virus types 1 and 3. The generally low postinfection titers could be enhanced, on the average, 40-fold by using human immunoglobulin G antiglobulin in the neutralization test. A low degree of cross-reactivity was shown between parainfluenza virus types 1 and 3 both in the conventional neutralization test and in the anti-immunoglobulin enhanced neutralization test.     

Rapid detection of respiratory viruses by centrifugation enhanced cultures from children with acute lower respiratory tract infections.

BACKGROUND: Acute respiratory tract infection (ARI) is the major cause of morbidity and mortality in young children in developing countries. Information on viral aetiology in ARI in India is very limited. OBJECTIVE: The aim of the study was to define the role of viruses in acute lower respiratory tract infections (ALRTI) in children in India using centrifugation enhanced cultures followed by indirect immunofluorescence (IIF). STUDY DESIGN: Nasopharyngeal aspirates (NPAs) were collected from children from September 1995 to April 1997, attending paediatric clinic of All India Institute of Medical Sciences (AIIMS) with symptoms of ALRTI. Virus isolation was done by centrifugation enhanced cultures using HEp-2, LLC-MK2 and MDCK cells. The viruses were identified at 24-48 h post inoculation by IIF staining using monoclonal antibodies to respiratory syncytial virus (RSV), parainfluenza virus (PIV), influenza virus and adenovirus. RESULTS: Of 200 NPA samples, 89 (44.5%) were positive for one or more viral pathogens. RSV was detected in 34 (17%) of all ALRTI cases followed by influenza viruses in 29 (14.5%), PIVs in 23 (11.5%) and adenoviruses in three (1.5%). In 79 children with bronchiolitis, RSV was most frequently isolated (25%) pathogen, while in bronchopneumonia cases (101) the most common viral pathogen was influenza virus (17%). In eight cases (4%) of ALRTI dual infections were detected. In 100 NPA specimens IIF staining on direct cell smears was carried out and viruses were detected in only 17%. RSV and influenza virus infection peaked from September to December, where as PIV infections were more frequent from January to April. CONCLUSION: Respiratory viruses accounted for 44.5% of cases of ALRTI in India and the results of viral aetiology could be given in 24-48 h using centrifugation enhanced cultures. RSV was the most common viral agent associated with ALRTI in children under 5 years of age with greater association with bronchiolitis.     

Isolation of homologous arbovirus cultures from heterologous mixtures using limit dilution and virus-specific enzyme immunoassays

Viral cultures were identified recently that contained both Kunjin virus and the closely related flavivirus West Nile. The observation that the KUN virus population grew more efficiently in a mosquito cell line (C6/36) while the WN population replicated more effectively in mammalian cells (Vero) allowed enrichment for either virus by culturing the mixture in the appropriate cell line. Limit dilution of the enriched virus preparations was then performed by infecting microtitre cultures with serial ten fold dilutions. Culture wells that contained a pure population of virus were then identified by immunostaining fixed cell monolayers with virus-specific monoclonal antibodies. Subsequent passage of the 'cloned' viruses in either C6/36 or Vero cells and analysis of the infected cultures by specific monoclonal antibody staining, PCR and nucleotide sequencing confirmed the identity of the virus and that in each case an homogeneous virus population had been obtained. This procedure is particularly useful for isolating virus populations from heterogeneous mixtures that fail to develop discrete plaques in infected cell monolayers.     

Plaque assay for avian metapneumovirus using a Japanese quail fibrosarcoma cell line (QT-35)

A plaque assay for detecting, isolating and titrating avian pneumoviruses using a Japanese quail fibrosarcoma cell line (QT-35) is described. Plaques are produced after application of either an agarose or carboxymethyl-cellulose (CMC) overlay onto cell monolayers infected with representative avian metapneumoviruses which belong to subgroup A, B or C. Virus plaques can be easily visualized by light microscopy or after staining. The parameters affecting plaque appearance include: cell seeding concentration, virus strain, overlay composition and incubation time following infection. Optimal conditions for plaquing involve seeding QT-35 cells at 40,000 cells per cm(2) when using a 1.5% CMC overlay or 100,000 cells per cm(2) when using a 1.0 or 0.8% agarose overlay. In both cases, cell monolayers are infected with virus 24 h post-seeding and clearly visible plaques develop in 6 days. Due to the robust nature of these cells, the incubation time can be extended to a maximum of 13 days after infection in order to produce larger plaques.     

Rapid detection of respiratory viruses using mixtures of monoclonal antibodies on shell vial cultures.

Eleven hundred and thirty-three clinical specimens submitted to the laboratory for diagnosis of respiratory virus infections were tested by direct immunofluorescence (DIF) for respiratory syncytial virus (RSV), by shell vial culture, and by conventional cell culture. The shell vial cultures were stained with 8 different monoclonal antibodies both 1 day and 3-7 days after inoculation. In order to limit the cost and the workload, mixtures of monoclonal antibodies were used. Coverslips with HEp-2 cells were incubated with a mixture of FITC-labeled monoclonal antibody to RSV and nonlabeled monoclonal antibody to adenovirus. When no RSV positive IF staining was observed after the first incubation step, the same coverslip was incubated once more with FITC-labeled anti-mouse antibody. A positive reaction at this stage indicated the presence of adenovirus. Similarly, cultures of tertiary monkey kidney cells were investigated with a mixture of two FITC-labeled monoclonals to the influenza viruses A and B and three nonlabeled monoclonals to the parainfluenza viruses 1, 2 and 3. If influenza virus or parainfluenza virus was detected, the exact type was determined by staining different parts of a duplicate coverslip. Shell vial cultures for cytomegalovirus (CMV) were always performed separately on human embryonic lung fibroblasts. Using this approach, we detected RSV (n = 248), CMV (n = 42), parainfluenza virus (n = 31), influenza virus (n = 28), and adenovirus (n = 6), in most cases after only one day of culture. For RSV, the sensitivity of the shell vial method was too low (74%) to allow omission of DIF (sensitivity 95%).(ABSTRACT TRUNCATED AT 250 WORDS)     

小发卡RNA抗呼吸道合胞病毒效应的研究

目的 为从基因水平抑制呼吸道合胞病毒（respiratory syncytial virus，RSV）的复制，针对RSV的M2-2基因构建小发卡结构状RNA（short hairpin RNA，shRNA）重组质粒，在细胞水平观察shRNA对RSV复制的影响。方法 将成功构建的针对RSV M2-2基因的重组质粒pshRNA8260转染HEp-2细胞，利用光镜观察pshRNA8260对RSV致HEp-2细胞病变效应（cytopathogenic effect，CPE）的影响并计算CPE抑制率，空斑形成实验检测RSV滴度变化。结果 成功构建了针对人RSV M2-2基因mRNA的pshRNA8260重组质粒，研究发现pshRNA8260能明显改善RSV所致的病变效应，降低RSV在细胞内复制的病毒滴度。结论 针对RSV M2-2基因的pshRNA82（g）重组质粒具有明显的特异性抗RSV效应的作用。     

Epitope analysis of mumps virus proteins in a persistent cell culture infection: changes in the hemagglutinin-neuraminidase polypeptide

Monoclonal antibodies (MABs) against major structural mumps virus proteins were used for epitope analysis in primarily and persistently infected HEp-2 cells by means of immunofluorescence and radioimmunoprecipitation techniques. Qualitatively, no differences were found in MAB binding between corresponding proteins of the original and persistent viruses, whereas quantitative differences observed might be explained in terms of weakened viral protein synthesis in persistent infection. Limited proteolysis of MAB-bound antigen has revealed alterations in certain epitopes on persistent virus HN polypeptide. Despite the inability of HEp-2 infected cells for hemadsorption, HN protein was expressed on the surface of these cells to the same extent as in the hemadsorbing system of mumps virus-infected Vero cells.     

Inhibition of respiratory syncytial virus infection with the CC chemokine RANTES (CCL5)

Respiratory syncytial virus (RSV) is a major cause of respiratory tract disease in infants, aged adults, and immunosuppressed patients. The only approved medicines for RSV disease are administration of prophylatic antibodies or treatment with a synthetic nucleoside. Both approaches are expensive and the latter is not without risk and of controversial benefit. The present investigation studied whether pharmaceutical or biologic compounds based upon chemokines might be useful in preventing RSV disease. Of interest was RANTES/CCL5, which inhibits infection by HIV strains that use chemokine receptor (CCR)-5 as co-receptor. Herein, we report that prior or simultaneous treatment of HEp-2 cells with recombinant human CCL5 provides dose-dependent inhibition of infection with RSV. Other recombinant chemokines (MIP-1alpha/CCL3, MIP-1beta/CCL4, MCP-2/CCL8, eotaxin/CCL11, MIP-1delta/CCL15, stromal cell derived factor (SDF)-1alpha/CXCL12) were not inhibitory. The data suggested that CCL5 might inhibit infection by blocking fusion (F) protein-epithelial cell interactions. Infections by mutant RSV strains deleted of small hydrophobic and/or attachment proteins and only expressing F protein in the envelope were inhibited by prior treatment with CCL5 or a biologically inactive N-terminally modified met-CCL5. Inhibition was also observed when virus adsorption and treatment with CCL5 were performed at 4 degrees C. Flow cytometry further revealed that epithelial cells were positive for CCR3, but not CCR1 or CCR5. Thus, novel mimetics of CCL5 may be useful prophylatic agents to prevent respiratory tract disease caused by RSV.     

Respiratory syncytial virus G glycoprotein expressed using the Semliki Forest virus replicon is biologically active

Summary.  The respiratory syncytial virus (RSV) G glycoprotein mediates attachment of RSV to cells via an unknown receptor. To study G glycoprotein function we have cloned two variants of the RSV G gene into a Semliki Forest virus (SFV) expression vector, a full length (rG) and soluble (srG) G glycoprotein variant. By immunofluorescence microscopy, rG was found to be predominantly membrane associated, while srG was mostly cytoplasmic. The rG (80–85 kDa) and srG (75–80 kDa) constructs produced heavily glycosylated proteins, however they were slightly smaller than the G glycoprotein expressed in RSV infected HEp-2 cells (85–90 kDa). The biological activity of purified srG was tested by its ability to bind to RSV permissive cells. Purified srG bound to HEp-2 cells and the amount bound increased linearly with the quantity added. Binding was not saturable with the small quantities of protein available. Binding of srG to HEp-2 cells was inhibited (67–68%) by MAb 30 and neutralising anti-G MAb 29. Nonpermissive SF9 insect cells bound 20–50 times less srG than HEp-2 cells. SFV expressed recombinant RSV G glycoprotein should be useful for studying interactions between the RSV G glycoprotein and cells. Accepted December April 10, 1998 Received July 29, 1998     

Attenuation of a reactivated cardiovirulent coxsackievirus B3: The 5′-nontranslated region does not contain major attenuation determinants

To investigate the molecular basis of pathogenicity of Coxsackieviruses, a virus was reactivated by transfection from a full-length cDNA clone derived from cardiovirulent Coxsackievirus B3 (CVB3). The reactivated virus, rCVB3, was passaged serially in human dermatofibroblasts (HDF). No cytopathic effect was observed up to 12 days after inoculation with rCVB3 or earlypassage virus, although disintegration of the monolayers was observed with late-passage virus (10th to 14th passages). Approximately 10% of HDF inoculated with rCVB3 were positive for viral antigens by immunofluorescence using enterovirus- or CVB3-specific monoclonal antibodies. These observations, together with the low infectivity titre of rCVB3 in HDF, suggest that HDF initially support only carrier state infection. After the 14th passage, the cardiovirulence of passaged virus (p14V) in mice was attenuated by a factor of >10⁴. Phenotypic changes of plaque size were also noticed in p14V: An attenuated variant (p14V-1) that produced larger plaques than rCVB3 in Vero cells has been plaque purified. The 5′-terminus of the genome of attenuant p14V-1 was amplified by polymerase chain reaction (PCR) and its sequence determined. Only one point mutation was found within the 5′-nontranslated region (5′NTR) at position 690 (A to U) compared to the viral RNA sequence obtained for rCVB3. An intertypic chimeric virus was reactivated from a cDNA clone after replacing the 5′-terminal 891 nucleotides of the wild-type genome with the corresponding region of the attenuant p14V-1. This chimeric virus, CB3/p14V1/1, produced wild-type plaques in Vero cells and showed cardiovirulence similar to that of rCVB3 in mice. It is concluded that, unlike polioviruses, the 5′NTR of p14V-1 does not contain major determinants of attenuation.     

TCID50 determination by an immuno dot blot assay as exemplified in a study of storage conditions of infectious pancreatic necrosis virus.

Some cell lines are difficult to grow in confluent monolayers and, therefore, the plaque assay cannot be applied since plaques may be hard to distinguish from other blank areas of the cell monolayers. To avoid this problem a rapid and sensitive immuno dot blot TCID50 method was developed using antibodies against virus antigens to detect infection and virus production. An alternative statistical method was developed to treat the scoring data and thereby obtained a coefficient of variation of 10%. To speed up the total procedure and to increase the proliferation rate of cells grown in 96-well cell culture plates, the plates were pretreated for 4 h at 4 degrees C with growth medium obtained from cell culturing flasks containing confluent cell monolayers. This immuno dot blot TCID50 method was applied for a study of the infectivity maintenance upon storage of infectious pancreatic necrosis virus (IPNV). Storage of IPNV at -70 degrees C with a cryoprotective agent (10% glycerol) preserved the TCID50 level even after as many as ten cycles of freezings and thawings, whereas the infectivity decreased by four orders of magnitude after storage at 4-8 degrees C for 2 months in the salt buffer used commonly.