Respiratory Syncytial Virus Replication Research Articles

Age-dependent replication of respiratory syncytial virus in the cotton rat.

Despite the documented disease burden of respiratory syncytial virus (RSV) in the elderly, little is known about the underlying risk factors or pathogenesis of RSV in a geriatric population. This report describes an age-dependent change of RSV clearance in the lung and nose of the cotton rat. Six days postinfection with RSV, lung and nose viral titers were significantly higher in all older age groups as compared with 4- to 6-week old cotton rats (P < 0.05). When comparing the 4- to 6-week old animals to the 15- to 16-month old animals 6 days postinfection, there was over an 800- and 100-fold increase in lung and nose viral titers, respectively. The cotton rat may prove to be a useful model in eliciting mechanisms of severe RSV disease in the elderly.

Cleavage at the furin consensus sequence RAR/KR(109) and presence of the intervening peptide of the respiratory syncytial virus fusion protein are dispensable for virus replication in cell culture.

Proteolytic processing of the respiratory syncytial virus F (fusion) protein results in the generation of the disulfide-linked subunits F1 and F2 and in the release of pep27, a glycopeptide originally located between the two furin cleavage sites FCS-1 (RKRR(136)) and FCS-2 (RAR/KR(109)). We made use of reverse genetics to study the importance of FCS-2 and of pep27 for BRSV replication in cell culture. Replacement of FCS-2 in the F protein of recombinant viruses by either of the sequences NANR(109), RANN(109) or SANN(109), respectively, abolished proteolytic processing at this position, whereas the cleavage of FCS-1 was not affected. All mutants replicated in calf kidney and Vero cells in the absence of exogenous trypsin, although somewhat higher titers of BRSV containing the NANR(109) or the RANN(109) motif were achieved in the presence of trypsin. The virus mutants showed a reduced cytopathic effect which was lowest in the case of the SANN(109) mutant. These findings demonstrate that cleavage at FCS-2 is dispensable for replication of respiratory syncytial virus in cell culture. A deletion mutant containing FCS-1 but lacking FCS-2 and most of pep27 replicated in cell culture as efficiently as the parental virus, indicating that this domain of the F protein is not essential for virus maturation and infectivity.

Perflubron Reduces Lung Inflammation in Respiratory Syncytial Virus Infection by Inhibiting Chemokine Expression and Nuclear Factor–κB Activation

Airway mucosa inflammation plays a critical role in the pathogenesis of lower respiratory tract infections caused by respiratory syncytial virus (RSV), the major etiologic agent of bronchiolitis in infancy. Type and intensity of cellular infiltration are dictated by inflammatory chemokines, which are rapidly and abundantly induced in lung tissue by RSV. This process is, to a large extent, transcriptionally regulated by RSV-mediated activation of the nuclear factor-kappa B. The administration of a perfluorocarbon (PFC) liquid, such as perflubron, during partial liquid ventilation improves lung function and also reduces inflammation. In this study we demonstrate that treatment of BALB/c mice with perflubron intranasally 6 hours after RSV infection significantly inhibited lung cellular inflammation as well as the expression of the chemokines RANTES, MIP-1 alpha, MIP-1 beta, and MIP-2, compared with phosphate-buffered saline-treated control mice. However, perflubron treatment did not affect RSV replication. Strikingly, treatment with perflubron abrogated nuclear factor-kappa B activation in lung of RSV-infected mice. These results demonstrate a novel mechanism by which PFC may exert antiinflammatory activity and suggest that partial liquid ventilation with PFC may be considered in future clinical trials for infants with severe RSV infections requiring mechanical ventilation.

Plasmid DNA encoding the respiratory syncytial virus G protein protects against RSV-induced airway hyperresponsiveness

Respiratory syncytial virus (RSV) is an important cause of childhood respiratory disease as well as exacerbations of asthma. Although previous studies have demonstrated that a DNA vaccine encoding the RSV G protein can inhibit RSV replication in mouse models of RSV infection, studies have not been performed to determine whether a DNA vaccine encoding the RSV G protein can protect against RSV induced mucus expression and airway hyperresponsiveness which was the focus of this study. The DNA-G vaccine we constructed significantly inhibited RSV viral replication, mucus expression, and importantly was associated with inhibition of RSV induced airway responsiveness.

Targeted therapy of respiratory syncytial virus in African green monkeys by intranasally administered 2-5A antisense.

Respiratory syncytial virus (RSV) is a leading cause of respiratory disease in infants, young children, immunocompromised patients, and the institutionalized elderly. Previous work had shown that RNase L, an antiviral enzyme of the interferon system, could be recruited to cleave RSV genomic RNA by attaching tetrameric 2prime prime or minute-5prime prime or minute-linked oligoadenylates (2-5A) to an oligonucleotide complementary to repetitive gene-start sequences within the RSV genome (2-5A antisense). A 2prime prime or minute-O-methyl RNA-modified analog of the lead 2-5A anti-RSV chimera is shown here to have enhanced antiviral activity in cell culture studies while also cleaving RSV genomic RNA in an RNase L- and sequence-specific manner. When administered intranasally to RSV-infected African green monkeys, this chimera reduced nasal RSV replication by up to four log(10) units in a dose- and time-dependent manner.

Granulocyte-macrophage colony-stimulating factor expressed by recombinant respiratory syncytial virus attenuates viral replication and increases the level of pulmonary antigen-presenting cells.

An obstacle to developing a vaccine against human respiratory syncytial virus (RSV) is that natural infection typically does not confer solid immunity to reinfection. To investigate methods to augment the immune response, recombinant RSV (rRSV) was constructed that expresses murine granulocyte-macrophage colony-stimulating factor (mGM-CSF) from a transcription cassette inserted into the G-F intergenic region. Replication of rRSV/mGM-CSF in the upper and lower respiratory tracts of BALB/c mice was reduced 23- to 74- and 5- to 588-fold, respectively, compared to that of the parental rRSV. Despite this strong attenuation of replication, the level of RSV-specific serum antibodies induced by rRSV/mGM-CSF was comparable to, or marginally higher than, that of the parental rRSV. The induction of RSV-specific CD8(+) cytotoxic T cells was moderately reduced during the initial infection, which might be a consequence of reduced antigen expression. Mice infected with rRSV/mGM-CSF had elevated levels of pulmonary mRNA for gamma interferon (IFN-gamma) and interleukin 12 (IL-12) p40 compared to animals infected by wild-type rRSV. Elevated synthesis of IFN-gamma could account for the restriction of RSV replication, as was observed previously with an IFN-gamma-expressing rRSV. The accumulation of total pulmonary mononuclear cells and total CD4(+) T lymphocytes was accelerated in animals infected with rRSV/mGM-CSF compared to that in animals infected with the control virus, and the level of IFN-gamma-positive or IL-4-positive pulmonary CD4(+) cells was elevated approximately twofold. The number of pulmonary lymphoid and myeloid dendritic cells and macrophages was increased up to fourfold in mice infected with rRSV/mGM-CSF compared to those infected with the parental rRSV, and the mean expression of major histocompatibility complex class II molecules, a marker of activation, was significantly increased in the two subsets of dendritic cells. Enhanced antigen presentation likely accounts for the maintenance of a strong antibody response despite reduced viral replication and would be a desirable property for a live attenuated rRSV vaccine.

Increased replication of respiratory syncytial virus (RSV) in pulmonary infiltrates is associated with enhanced histopathological disease in bonnet monkeys (Macaca radiata) pre-immunized with a formalin-inactivated RSV vaccine.

The pathology of respiratory syncytial virus (RSV) disease in bonnet monkeys parallels findings with human RSV disease. RSV-infected animals pre-immunized with a formalin-inactivated (FI) RSV vaccine develop inflammation in peribronchiolar, perivascular, interstitial and intra-alveolar sites with lung inflammation scores significantly higher than animals with a primary RSV infection and those pre-immunized with an FI-Vero cell control vaccine (P=0.05). Animals previously infected and re-exposed to RSV had significantly lower alveolar, interstitial and total lung inflammation scores than in primary infection (P=0.05). Immunization with two intra-muscular doses of 0.5 ml of the FI-RSV vaccine administered 21 days apart resulted in little serum-neutralizing and ELISA antibody, low levels of secretory IgA and a low lymphocyte proliferative response that was significantly lower than the response observed in animals that were previously infected with live RSV. Higher RSV virus titres were detected in the lungs and lung lavage fluid of monkeys immunized with the FI-RSV vaccine than in those with a primary infection (P=0.001). RSV was detected by in situ hybridization in pulmonary inflammatory infiltrates, where the single most abundant infiltrating cellular species was macrophages, so it may be these cells that support the enhanced virus replication that contributes to the enhanced pulmonary pathology of FI-RSV immunization.

Suppressive effect of locally produced interleukin-10 on respiratory syncytial virus infection.

Interleukin (IL)-10 is known to be a multifunctional cytokine. This study was designed to evaluate the role of IL-10 during respiratory syncytial virus (RSV) infection using a C57BL/6 transgenic (TG) mouse model in which the expression of murine IL-10 cDNA was regulated by a human salivary amylase promoter (IL-10 TG mice). These mice expressed a large amount of IL-10 in the nasal mucosa and in salivary glands. Viral replication in the respiratory tract after intranasal infection with RSV was suppressed significantly in IL-10 TG mice compared to non-transgenic controls. This suppression was IL-10 specific, because it was prevented by treating mice with neutralizing anti-IL-10 antibodies. We also found that IL-10-stimulated T cells displayed cytotoxic activity against infected murine nasal epithelial cells. Previous data indicated that IL-10 induces Fas ligand (L) expression on mouse T cells. Taken together, these data suggest that Fas/Fas L mediated cytotoxicity is involved in the suppression of RSV replication observed in IL-10 TG mice after intranasal infection.

Mechanism of selective inhibition of respiratory syncytial virus by a benzodithiin compound (RD3-0028).

RD3-0028, a compound with a benzodithiin structure, was found to be a potent inhibitor of respiratory syncytial virus (RSV) replication. Its action is specific; no activity is seen against influenza A virus, measles virus, herpes simplex virus type 1 or 2, or human cytomegalovirus. A time-dependent drug addition experiment indicated that the antiviral activity occurs in the late stage of the RSV replication cycle, since this compound completely inhibited syncytium formation even when added up to 16 hr after the infection of cell monolayers at an MOI of 3. RD3-0028 had no direct virucidal effect on RSV. Western blotting analysis showed that RD3-0028 significantly decreased the amount of RSV proteins released into the cell culture medium. Moreover, five independent isolates of the RSV long strain were selected for growth in RD3-0028 (5-20 microg/ml). These resistant viruses were more than 80-fold less sensitive to RD3-0028 than the long strain. The F gene segment of each of these viruses was sequenced and in each case the mutant RNA segment contained at least one sequence alteration, converting asparagine 276 to tyrosine (F1 protein). These results suggest that RD3-0028 inhibits RSV replication by interfering with intracellular processing of the RSV fusion protein, or a step immediately thereafter, leading to loss of infectivity.

Antiviral activity of lovastatin against respiratory syncytial virus in vivo and in vitro.

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 that the RSV F glycoprotein, which mediates viral fusion, binds to RhoA. One of the steps in RhoA activation involves isoprenylation at the carboxy terminus of the protein by geranylgeranyltransferase. This modification allows RhoA to be attached to phosphatidyl serine on the inner leaflet of the plasma membrane. Treatment of mice with lovastatin, a drug that inhibits prenylation pathways in the cell by directly inhibiting hydroxymethylglutaryl coenzyme A reductase, diminishes RSV but not vaccinia virus replication when administered up to 24 h after RSV infection and decreases virus-induced weight loss and illness in mice. The inhibition of replication is not likely due to the inhibition of cholesterol biosynthesis, since gemfibrozil, another cholesterol-lowering agent, did not affect virus replication and serum cholesterol levels were not significantly lowered by lovastatin within the time frame of the experiment. Lovastatin also reduces cell-to-cell fusion in cell culture and eliminates RSV replication in HEp-2 cells. These data indicate that lovastatin, more specific isoprenylation inhibitors, or other pharmacological approaches for preventing RhoA membrane localization should be considered for evaluation as a preventive antiviral therapy for selected groups of patients at high risk for severe RSV disease, such as the institutionalized elderly and bone marrow or lung transplant recipients.

Role of human immunodeficiency virus (HIV) type 1 envelope in the anti-HIV activity of the betulinic acid derivative IC9564.

The betulinic acid derivative IC9564 is a potent anti-human immunodeficiency virus (anti-HIV) compound that can inhibit both HIV primary isolates and laboratory-adapted strains. However, this compound did not affect the replication of simian immunodeficiency virus and respiratory syncytial virus. Results from a syncytium formation assay indicated that IC9564 blocked HIV type 1 (HIV-1) envelope-mediated membrane fusion. Analysis of a chimeric virus derived from exchanging envelope regions between IC9564-sensitive and IC9564-resistant viruses indicated that regions within gp120 and the N-terminal 25 amino acids (fusion domain) of gp41 are key determinants for the drug sensitivity. By developing a drug-resistant mutant from the NL4-3 virus, two mutations were found within the gp120 region and one was found within the gp41 region. The mutations are G237R and R252K in gp120 and R533A in the fusion domain of gp41. The mutations were reintroduced into the NL4-3 envelope and analyzed for their role in IC9564 resistance. Both of the gp120 mutations contributed to the drug sensitivity. On the contrary, the gp41 mutation (R533A) did not appear to affect the IC9564 sensitivity. These results suggest that HIV-1 gp120 plays a key role in the anti-HIV-1 activity of IC9564.

Both immunisation with a formalin-inactivated respiratory syncytial virus (RSV) vaccine and a mock antigen vaccine induce severe lung pathology and a Th2 cytokine profile in RSV-challenged mice

Respiratory syncytial virus (RSV) is the most important cause of bronchiolitis and pneumonia in infants and young children. Immunopathology may play a role in RSV-induced disease and a severe RSV infection may also be associated with an increased risk of developing asthma. Vaccination with formalin-inactivated RSV (FI-RSV) prior to infection resulted both in human and in the mouse model in extensive lung pathology. In the mouse model, it has been shown that this aggravation of disease was associated with a shift in the balance between Th1 and Th2 cytokines towards a Th2-type response. The aim of the present study was to characterise the immunological and inflammatory responses in BALB/c mice upon RSV infection with or without prior vaccination with aluminium-adjuvanted FI-RSV or control antigens (FI-Mock). As previously reported by others, we also observed that a primary RSV infection in BALB/c mice resulted in a predominant Th1-type cytokine response, which was associated with slight bronchiolitis and alveolitis. FI-RSV vaccination prior to RSV challenge prevented virus replication and was associated with an aggravation of pulmonary histopathology and a shift towards a Th2-type response. Vaccination with FI-Mock did not prevent RSV replication in the lung but resulted in an even more pronounced Th2 response after infection while these mice were not sensitised to specific viral antigens. Thus, viral replication in a Th2 responding animal (induced by aluminium-adjuvanted mock vaccine) appears to boost the Th2 response upon RSV infection.

Recombinant Respiratory Syncytial Viruses with Deletions in the NS1, NS2, SH, and M2-2 Genes Are Attenuated in Vitro and in Vivo

Respiratory syncytial virus (RSV) encodes several proteins that lack well-defined functions; these include NS1, NS2, SH, and M2-2. Previous work has demonstrated that NS2, SH, and M2-2 can each be deleted from RSV genome and thus are considered as accessory proteins. To determine whether RSV can replicate efficiently when two or more transcriptional units are deleted, we removed NS1, NS2, SH, and M2-2 genes individually and in different combinations from an infectious cDNA clone derived from human RSV A2 strain. The following six mutants with two or more genes deleted were obtained: ΔNS1NS2, ΔM2-2SH, ΔM2-2NS2, ΔSHNS1, ΔSHNS2, and ΔSHNS1NS2. Deletion of M2-2 together with NS1 was detrimental to RSV replication. It was not possible to obtain a recombinant RSV when all four genes were deleted. All of the double and triple deletion mutants exhibited reduced replication and small plaque morphology in vitro. Replication of these deletion mutants was more reduced in HEp-2 cells than in Vero cells. Among the 10 single and multiple gene deletion mutants obtained, ΔM2-2NS2 was most attenuated. ΔM2-2NS2 formed barely visible plaques in HEp-2 cells and had a reduction of titer of 3 log10 compared with the wild-type recombinant RSV in infected HEp-2 cells. When inoculated intranasally into cotton rats, all of the deletion mutants were attenuated in the respiratory tract. Our data indicated that the NS1, NS2, SH, and M2-2 proteins, although dispensable for virus replication in vitro, provide auxiliary functions for efficient RSV replication.

Liposome encapsulation of a soluble recombinant fragment of the respiratory syncytial virus (RSV) G protein enhances immune protection and reduces lung eosinophilia associated with virus challenge

Respiratory syncytial virus (RSV) is a leading cause of bronchiolitis and pneumonia in young children and infants. Previous animal studies have shown that immunizing intramuscularly or intraperitoneally with the RSV G protein has elicited protective as well as harmful immune responses upon RSV challenge. In an RSV immunization strategy designed to target the respiratory tract directly (the site of RSV replication), we immunized BALB/c mice intranasally with a liposome-encapsulated, prokaryotically expressed thioredoxin fusion protein consisting of amino acids 128–229 of the RSV G protein (Trx-G 128–229). Upon intranasal challenge with RSV, a 100 to 500-fold reduction in lung RSV replication was observed in mice immunized with liposome-encapsulated Trx-G 128–229 compared to a sham-immunized control group. Analysis of bronchoalveolar lavage fluids revealed an influx of eosinophils (18% of total cells) in mice immunized with Trx-G 128–229 alone. Such eosinophilic infiltration was diminished (to 4.5% of total cells), however, in mice immunized with liposome-encapsulated Trx-G 128–229. Histological analysis of lung tissue revealed an accumulation of cells around the bronchioles and vessels in mice immunized with Trx-G 128–229 alone followed by RSV challenge which was not increased further in mice immunized with liposome-encapsulated Trx-G 128–229. These results show that intranasal immunization of BALB/c mice with Trx-G 128–229, when encapsulated in liposomes, can reduce the level of RSV replication in the lung as well as specifically reduce the degree of eosinophilic infiltration compared to mice immunized with Trx-G 128–229 alone. This demonstrates the potential of liposomes and particular recombinant fragments of the RSV G protein as an effective combination in RSV vaccine studies.

Intranasal IFN-γ gene transfer protects BALB/c mice against respiratory syncytial virus infection

Respiratory syncytial virus (RSV) is a major respiratory pathogen in infants, young children and the elderly and causes severe bronchiolitis and asthma. In an effort to develop a preventive IFN-γ therapy against RSV infection, an intranasal gene transfer strategy was utilized. Intranasal administration of a plasmid expressing the IFN-γ cDNA (pIFN-γ) resulted in the expression of IFN-γ in murine lungs and decreased RSV replication. The mice administered with pIFN-γ and then infected with RSV exhibited a significant decrease in broncho-alveolar lavage lymphocyte and neutrophil counts. A significant reduction in epithelial cell damage, infiltration of mononuclear cells in the peribronchiolar and perivascular regions, and thickening of the septa was observed in the lungs of mice treated with pIFN-γ when compared to controls. These results suggest that intranasal IFN-γ gene transfer results in decreased RSV replication and pulmonary inflammation and may be useful against RSV infection.

2-5A-DNA conjugate inhibition of respiratory syncytial virus replication: effects of oligonucleotide structure modifications and RNA target site selection

To define more fully the conditions for 2-5A-antisense inhibition of respiratory syncytial virus (RSV), relationships between 2-5A antisense oligonucleotide structure and the choice of RNA target sites to inhibition of RSV replication have been explored. The lead 2-5A-antisense chimera for this study was the previously reported NIH8281 that targets the RSV M2 RNA. We have confirmed and extended the earlier study by showing that NIH8281 inhibited RSV strain A2 replication in a variety of antiviral assays, including virus yield reduction assays performed in monkey (EC 90=0.02 μM) and human cells (EC 90=1 μM). This 2-5A-antisense chimera also inhibited other A strains, B strains and bovine RSV in cytopathic effect inhibition and Neutral Red Assays (EC 50 values=0.1–1.6 μM). The 2′- O-methylation modification of NIH8281 to increase affinity for the complementary RNA and provide nuclease resistance, the introduction of phosphothioate groups in the antisense backbone to enhance resistance to exo- and endonucleases, and the addition of cholesterol to the 3′-terminus of the antisense oligonucleotide to increase cellular uptake, all resulted in loss of activity. Of the antisense chimeras targeting other RSV mRNAs (NS1, NS2, P, M. G, F, and L), only those complementary to L mRNA were inhibitory. These results suggest that lower abundance mRNAs may be the best targets for 2-5A-antisense; moreover, the active 2-5A antisense chimeras in this study may serve as useful guides for the development of compounds with improved stability, uptake and anti-RSV activity.

Palivizumab.

The humanised monoclonal antibody palivizumab has been developed for prevention of serious lower respiratory tract disease caused by respiratory syncytial virus (RSV) in infants at high risk; RSV is the most common cause of lower respiratory tract infections in infants. Palivizumab specifically inhibits an epitope at the A antigenic site of the F protein of RSV subtypes A and B. RSV replication was inhibited in nasal and tracheal aspirates from infants receiving palivizumab 15 mg/kg. Mean 30-day trough serum concentrations of palivizumab were consistently about 70 mg/L in infants receiving repeated intramuscular or intravenous palivizumab 15 mg/kg. This is above the target serum concentration of 40 mg/L estimated to reduce pulmonary RSV replication by >99% in animal studies. In a large multicentre trial in 1502 infants at high risk of RSV infection, intramuscular palivizumab 15 mg/kg more than halved the incidence of RSV-attributable hospitalisation to 4.8% compared with 10.6% in placebo recipients. In the same group of high-risk infants, palivizumab significantly decreased total days in hospital attributable to RSV infection, days with increased supplemental oxygen requirement, days with moderate to severe lower respiratory tract infections and the incidence of admissions to intensive care. It had no effect on the incidence or total number of days of ventilation. Palivizumab was well tolerated during clinical trials in infants at risk of RSV infection. The incidence of adverse events was similar in placebo (10%) and palivizumab (11%) groups. Fever, irritability and injection site reaction were the most commonly reported adverse events.

Altered growth characteristics of recombinant respiratory syncytial viruses which do not produce NS2 protein.

The second gene in the 3'-to-5' gene order in respiratory syncytial virus (RSV) encodes the nonstructural protein NS2, for which there is no assigned function. To study the function of NS2, we have used a recently developed reverse genetics system to ablate expression of NS2 in recombinant RSV. A full-length cDNA copy of the antigenome of RSV A2 strain under the control of a T7 promoter was modified by introduction of tandem termination codons within the NS2 open reading frame (NS2stop) or by deletion of the entire NS2 gene (DeltaNS2). The NS2 knockout antigenomic cDNAs were cotransfected with plasmids encoding the N, P, L, and M2-1 proteins of RSV, each controlled by the T7 promoter, into cells infected with a vaccinia virus recombinant expressing T7 RNA polymerase. Recombinant NS2stop and DeltaNS2 RSVs were recovered and characterized. Both types of NS2 knockout virus displayed pinpoint plaque morphology and grew more slowly than wild-type RSV. The expression of monocistronic mRNAs for the five genes examined (NS1, NS2, N, F, and L) was unchanged in cells infected with either type of NS2 knockout virus, except that no NS2 mRNA was detected with the DeltaNS2 virus. Synthesis of readthrough mRNAs was affected only for the DeltaNS2 virus, where the NS1-NS2, NS2-N, and NS1-NS2-N mRNAs were replaced with the predicted novel NS1-N mRNA. Upon passage, the NS2stop virus stock rapidly developed revertants which expressed NS2 protein and grew with similar plaque morphology and kinetics wild-type RSV. Sequence analysis confirmed that the termination codons had reverted to sense, albeit not the wild-type assignments, and provided evidence consistent with biased hypermutation. No revertants were recovered from recombinant DeltaNS2 RSV. These results show that the NS2 protein is not essential for RSV replication, although its presence greatly improves virus growth in cell culture. The attenuated phenotype of these mutant viruses, coupled with the expected genetic stability associated with gene deletions, suggests that the DeltaNS2 RSV is a candidate for vaccine development.

Permissiveness of Guinea Pig Alveolar Macrophage Subpopulations to Acute Respiratory Syncytial Virus Infection In Vitro

Alveolar macrophages (AMs) are targets for respiratory syncytial virus (RSV) infection in vivo and in vitro. However, only a minority of AMs are permissive to acute RSV infection in vitro, and it is unknown whether this permissiveness may be related to the degree of cellular maturation that is achieved in vivo. By using density gradient centrifugation, in which the degree of AM maturation is inversely related to buoyant density, we prepared three subpopulations of guinea pig AMs (designated as hypodense, intermediate-density, and high-density AMs). Twenty-four hours after exposure to RSV in vitro, the percentage of RSV-positive cells in each subpopulation was determined by immunocytochemistry; intracellular virus was released from cells by sonication and quantified by plaque assay, and intracellular localization of RSV proteins was evaluated by immunogold electron microscopy. High-density AMs had a significantly higher proportion of RSV-positive cells than hypodense AMs (p < 0.001), with intermediate-density AMs having intermediate values. The amounts of intracellular virus significantly increased from hypodense to intermediate density to high-density AMs (p < 0.001). Hypodense cells showed immunogold labeling principally within phagolysosomes, whereas intermediate-density and high-density cells showed immunolabeling of free cytoplasmic viral proteins and nucleocapsids. The permissiveness of guinea pig AMs to acute RSV infection in vitro is inversely related to their degree of maturation achieved in vivo. In addition, these results suggest that immature, high-density AMs support RSV replication whereas more mature, hypodense AMs may restrict viral replication.

Novel Anti-respiratory Syncytial(RS) Viral Compounds: Benzodithiin Derivatives

Benzodithiin derivatives are highly potent and specific inhibitors of respiratory syncytial virus (RSV) replicationin vitro.The most potent and selective congener of a benzodithiin derivative is 1,4-dihydro-2,3-benzodithiin(RD3-0028). According to the modified 3-(4,5-dimethylthiazole-2-yl) 2, 5-diphenyl tetrazolium bromide (MTT) assay developed in our laboratories, this compound has a 50% effective concentration of 4.5 μM and a 50% cytotoxic concentration of 271.0 μM, which is superior to that of ribavirin. This compound also inhibits RSV strain subgroups A and B and clinical isolates. RD3-0028, however, does not inhibit the replication of influenza A virus, measles virus, herpes simplex virus types 1 and 2, or human cytomegalovirus. Two other benzodithiin derivatives [1,4-dihydro-6-methyl-2,3-benzodithiin (RD3-0270) and 1,4-dihydro-5-methyl-2,3-benzodithiin (RD3-0284)] also inhibit RSV replication at a selectivity index greater than a factor of 20. These results suggest that the benzodithiin skeleton is an important structure for inhibitory activity against RSV replication.