N6-methyladenosine modification enables viral RNA to escape recognition by RNA sensor RIG-I.

Internal N6-methyladenosine (m6A) modification is one of the most common and abundant modifications of RNA. However, the biological roles of viral RNA m6A remain elusive. Here, using human metapneumovirus (HMPV) as a model, we demonstrate that m6A serves as a molecular marker for innate immune discrimination of self from non-self RNAs. We show that HMPV RNAs are m6A methylated and that viral m6A methylation promotes HMPV replication and gene expression. Inactivating m6A addition sites with synonymous mutations or demethylase resulted in m6A-deficient recombinant HMPVs and virion RNAs that induced increased expression of type I interferon, which was dependent on the cytoplasmic RNA sensor RIG-I, and not on melanoma differentiation-associated protein 5 (MDA5). Mechanistically, m6A-deficient virion RNA induces higher expression of RIG-I, binds more efficiently to RIG-I and facilitates the conformational change of RIG-I, leading to enhanced interferon expression. Furthermore, m6A-deficient recombinant HMPVs triggered increased interferon in vivo and were attenuated in cotton rats but retained high immunogenicity. Collectively, our results highlight that (1) viruses acquire m6A in their RNA as a means of mimicking cellular RNA to avoid detection by innate immunity and (2) viral RNA m6A can serve as a target to attenuate HMPV for vaccine purposes.

SPINT2 inhibits proteases involved in activation of both influenza viruses and metapneumoviruses.

Viruses possessing class I fusion proteins require proteolytic activation by host cell proteases to mediate fusion with the host cell membrane. The mammalian SPINT2 gene encodes a protease inhibitor that targets trypsin-like serine proteases. Here we show the protease inhibitor, SPINT2, restricts cleavage-activation efficiently for a range of influenza viruses and for human metapneumovirus (HMPV). SPINT2 treatment resulted in the cleavage and fusion inhibition of full-length influenza A/CA/04/09 (H1N1) HA, A/Aichi/68 (H3N2) HA, A/Shanghai/2/2013 (H7N9) HA and HMPV F when activated by trypsin, recombinant matriptase or KLK5. We also demonstrate that SPINT2 was able to reduce viral growth of influenza A/CA/04/09 H1N1 and A/X31 H3N2 in cell culture by inhibiting matriptase or TMPRSS2. Moreover, inhibition efficacy did not differ whether SPINT2 was added at the time of infection or 24 h post-infection. Our data suggest that the SPINT2 inhibitor has a strong potential to serve as a novel broad-spectrum antiviral.

RNAi Targeting of Human Metapneumovirus P and N Genes Inhibits Viral Growth.

BACKGROUND/AIMS: Human metapneumovirus (hMPV) is an important human respiratory pathogen and is implicated in an array of respiratory illnesses, ranging from asymptomatic infection to severe bronchiolitis. Currently, there is no reliable vaccine or specific antiviral therapy for hMPV infection and treatment is supportive. The use of ribonucleic acid interference has the potential to change that with the targeting of essential viral genes via small interfering RNAs (siRNAs) offering the ability to directly and rapidly treat viral infections. METHOD: The human lung carcinoma epithelial cell line, A549, was transfected with siRNAs targeting the N and P genes before infecting with hMPV A2 CAN97-83. Viral growth inhibition was then measured by the viral plaque assay and nucleoprotein (N) and phosphoprotein (P) gene knockdown was determined by real-time PCR. RESULTS: In vitro prophylactic use of siRNAs targeting the 3'-abundantly expressed N and P genes of hMPV resulted in potent, sequence-specific viral inhibition. The viral inhibition was specific and not mediated by an anti-viral interferon-ß response or cell death. CONCLUSION: The findings presented here confirmed the highly potent, sequence-specific antiviral effect of siRNAs targeting the N and P gene of hMPV. These results may facilitate the development of a novel therapeutic agent for hMPV control.

Human lung epithelial cells support human metapneumovirus persistence by overcoming apoptosis.

Human metapneumovirus (hMPV) has been identified as a major cause of lower respiratory tract infection in children. Epidemiological and molecular evidence has highlighted an association between severe childhood respiratory viral infection and chronic lung diseases, such as asthma and chronic obstructive pulmonary disease. Currently, animal models have demonstrated the ability of hMPV to persist in vivo suggesting a role of the virus in asthma development in children. However, mechanisms involved in hMPV persistence in the respiratory tract are not yet understood. In the present study we monitored hMPV infection in human alveolar epithelial A549 cells in order to understand if the virus is able to persist in these cells upon acute infection. Our data show that hMPV initially induces an apoptotic process in A549 cells through poly (ADP-ribose) polymerase 1 cleavage, caspase-3/7 activation and Wee1 activity. The hMPV-infected cells were then able to overcome the apoptotic pathway and cell cycle arrest in G2/M by expressing B-cell lymphoma 2 and to acquire a reservoir cell phenotype with constant production of infectious virus. These findings provide evidence of the ability of hMPV to persist in alveolar epithelial cells and help in understanding the mechanisms responsible for hMPV persistence in the human respiratory tract.

Efficient isolation of human metapneumovirus using MNT-1, a human malignant melanoma cell line with early and distinct cytopathic effects.

Isolation of human metapneumovirus (HMPV) from clinical specimens is currently inefficient because of the lack of a cell culture system in which a distinct cytopathic effect (CPE) occurs. The cell lines LLC-MK2, Vero and Vero E6 are used for isolation of HMPV; however, the CPE in these cell lines is subtle and usually requires a long observation period and sometimes blind passages. Thus, a cell line in which an early and distinct CPE occurs following HMPV inoculation is highly desired by clinical virology laboratories. In this study, it was demonstrated that, in the human malignant melanoma cell line MNT-1, obvious syncytium formation occurs shortly after inoculation with HMPV-positive clinical specimens. In addition, the growth and efficiency of isolation of HMPV were greater using MNT-1 than using any other conventional cell line. Addition of this cell line to our routine viral isolation system for clinical specimens markedly enhanced isolation frequency, allowing isolation-based surveillance. MNT-1 has the potential to facilitate clinical and epidemiological studies of HMPV.

Mutations in the fusion protein heptad repeat domains of human metapneumovirus impact on the formation of syncytia.

Human metapneumovirus (HMPV) is an important cause of respiratory tract infections. The mechanism by which its fusion (F) protein is responsible for variable cytopathic effects in vitro remains unknown. We aligned the F sequences of the poorly fusogenic B2/CAN98-75 strain and the hyperfusogenic A1/C-85473 strain and identified divergent residues located in the two functional heptad repeats domains (HRA and HRB). We generated recombinant viruses by inserting the mutations N135T-G139N-T143K-K166E-E167D in HRA and/or K479R-N482S in HRB, corresponding to swapped sequences from C-85473, into CAN98-75 background and investigated their impact on in vitro phenotype and fusogenicity. We demonstrated that the five HRA mutations enhanced the fusogenicity of the recombinant rCAN98-75 virus, almost restoring the phenotype of the wild-type rC-85473 strain, whereas HRB substitutions alone had no significant effect on cell-cell fusion. Altogether, our results support the importance of the HRA domain for an HMPV-triggered fusion mechanism and identify key residues that modulate syncytium formation.

Influenza and other respiratory viruses: standardizing disease severity in surveillance and clinical trials.

INTRODUCTION: Influenza-Like Illness is a leading cause of hospitalization in children. Disease burden due to influenza and other respiratory viral infections is reported on a population level, but clinical scores measuring individual changes in disease severity are urgently needed. Areas covered: We present a composite clinical score allowing individual patient data analyses of disease severity based on systematic literature review and WHO-criteria for uncomplicated and complicated disease. The 22-item ViVI Disease Severity Score showed a normal distribution in a pediatric cohort of 6073 children aged 0-18 years (mean age 3.13; S.D. 3.89; range: 0 to 18.79). Expert commentary: The ViVI Score was correlated with risk of antibiotic use as well as need for hospitalization and intensive care. The ViVI Score was used to track children with influenza, respiratory syncytial virus, human metapneumovirus, human rhinovirus, and adenovirus infections and is fully compliant with regulatory data standards. The ViVI Disease Severity Score mobile application allows physicians to measure disease severity at the point-of care thereby taking clinical trials to the next level.

TMPRSS12 Is an Activating Protease for Subtype B Avian Metapneumovirus.

The entry of avian metapneumovirus (aMPV) into host cells initially requires the fusion of viral and cell membranes, which is exclusively mediated by fusion (F) protein. Proteolysis of aMPV F protein by endogenous proteases of host cells allows F protein to induce membrane fusion; however, these proteases have not been identified. Here, we provide the first evidence that the transmembrane serine protease TMPRSS12 facilitates the cleavage of subtype B aMPV (aMPV/B) F protein. We found that overexpression of TMPRSS12 enhanced aMPV/B F protein cleavage, F protein fusogenicity, and viral replication. Subsequently, knockdown of TMPRSS12 with specific small interfering RNAs (siRNAs) reduced aMPV/B F protein cleavage, F protein fusogenicity, and viral replication. We also found a cleavage motif in the aMPV/B F protein (amino acids 100 and 101) that was recognized by TMPRSS12. The histidine, aspartic acid, and serine residue (HDS) triad of TMPRSS12 was shown to be essential for the proteolysis of aMPV/B F protein via mutation analysis. Notably, we observed TMPRSS12 mRNA expression in target organs of aMPV/B in chickens. Overall, our results indicate that TMPRSS12 is crucial for aMPV/B F protein proteolysis and aMPV/B infectivity and that TMPRSS12 may serve as a target for novel therapeutics and prophylactics for aMPV. IMPORTANCE: Proteolysis of the aMPV F protein is a prerequisite for F protein-mediated membrane fusion of virus and cell and for aMPV infection; however, the proteases used in vitro and vivo are not clear. A combination of analyses, including overexpression, knockdown, and mutation methods, demonstrated that the transmembrane serine protease TMPRSS12 facilitated cleavage of subtype B aMPV (aMPV/B) F protein. Importantly, we located the motif in the aMPV/B F protein recognized by TMPRSS12 and the catalytic triad in TMPRSS12 that facilitated proteolysis of the aMPV/B F protein. This is the first report on TMPRSS12 as a protease for proteolysis of viral envelope glycoproteins. Our study will shed light on the mechanism of proteolysis of aMPV F protein and pathogenesis of aMPV.

Replication and pathogenicity of attenuated human metapneumovirus F mutants in severe combined immunodeficiency mice.

This study was to evaluate the replication and pathogenicity of attenuated human metapneumovirus (HMPV) mutants in severe combined immunodeficiency (SCID) mice. SCID mice were intranasally infected with either wild type GFP-rHMPV (WT), or mutant viruses (M1, M2 and M4) with the N-linked glycosylation(s) of the F protein removed. The organs were collected for viral isolation, titration, pulmonary histopathology and mRNA detection by PCR at different time points. WT or mutant viruses were successfully isolated from the lungs of infected mice after inoculation. The titers of WT and M1 peaked on 5th day and remained detectable until 14th day post-inoculation. M2 reached approximately 4 logs lower titer on 5th and 9th day post-inoculation as compared to WT and M1. M4 showed similar growth kinetics to M2. Viral signal was never detected from the heart, liver, spleen, kidney and brain on 5th day post-inoculation. The pulmonary pathology score in the M1 infected mice was similar to WT infected mice but higher than in M2 or M4 infected mice. WT and HMPV mutants can thus only replicate in the lungs of SCID mice. Attenuated M2 and M4 may be considered as candidates for the preparation of vaccine against HMPV.

Generation and biological assessment of recombinant avian metapneumovirus subgroup C (aMPV-C) viruses containing different length of the G gene.

Genetic variation in length of the G gene among different avian metapneumovirus subgroup C (aMPV-C) isolates has been reported. However, its biological significance in virus replication, pathogenicity and immunity is unknown. In this study, we developed a reverse genetics system for aMPV-C and generated two Colorado (CO) strain-based recombinant viruses containing either the full-length G gene derived from a Canadian goose isolate or a C-terminally truncated G gene of the CO strain. The truncated short G (sG) gene encoded 252 amino acids (aa), which is 333 aa shorter than the full-length G (585 aa). The biological properties of these two recombinant G variants were assessed in Vero cells and in specific-pathogen-free (SPF) turkeys. In Vero cells, the short G variant displayed a similar level of growth dynamics and virus titers as the parental aMPV-CO strain, whereas the full-length G variant replicated less efficiently than the sG variant during the first 72 h post-infection. Both of the G variants induced typical cytopathic effects (CPE) that were indistinguishable from those seen with the parental aMPV-CO infection. In SPF turkeys, both of the G variants were attenuated and caused little or no disease signs, but the full-length G variant appeared to grow more readily in tracheal tissue than the sG variant during the first 5 days post-infection. Both G variants were immunogenic and induced a slightly different level of antibody response. These results demonstrated that the large portion (333 aa) of the extracellular domain of the viral attachment protein is not essential for virus viability in vitro and in vivo, but may play a role in enhancing virus attachment specificity and immunity in a natural host.

[A comparative study of direct immunofluorescence, enzyme immunoassay, and culture for diagnosing metapneumovirus infection]. / Estudio comparativo de inmunofluorescencia directa, enzimoinmunoanálisis y cultivo para el diagnóstico de las infecciones por metapneumovirus humano.

INTRODUCTION: Human metapneumovirus (hMPV) is an important cause of lower respiratory tract infections in children, accounting for 14% to 24% of all viral respiratory infections with an etiological diagnosis. This study compares a direct fluorescent antibody (DFA) test, enzyme immunoassay (EIA), and shell-vial culture for diagnosing acute bronchiolitis in infants. METHODS: A total of 124 nasopharyngeal aspirates from 108 infants with lower respiratory tract infection were analyzed. Incoming samples were processed for DFA using a commercial anti-hMPV antibody (Diagnostic Hybrids Inc.); 76 were inoculated in an LLC-MK2 cell line, and after an incubation period of 48 h, were stained and fixed with the aforementioned serum. The remaining sample was processed according to the routine diagnostic procedure and aliquots were frozen for EIA analysis (Biotrin). RESULTS: Twenty (16.12%) samples were positive for hMPV by DFA, 27 (21.77%) by EIA, and 15 (19.73%) by culture. DFA and EIA results were consistent in 92.73% of the 124 samples. Considering the 3 techniques, the same results were obtained in 90.78% of the 76 specimens. Considering only the first specimen from each patient (acute phase), the sensitivity, predictive values, and Kappa index for DFA improved and were very close to the EIA values. CONCLUSION: DFA and EIA are useful for antigen detection in the diagnosis of acute hMPV infection, particularly in pediatric hospitals that do not have amplification techniques for this virus, and when a rapid diagnosis is required. It should be kept in mind that DFA analysis is a suitable test for this purpose only in the acute phase of the infection.

Efficient multiplication of human metapneumovirus in Vero cells expressing the transmembrane serine protease TMPRSS2.

Human metapneumovirus (HMPV) is a major causative agent of severe bronchiolitis and pneumonia. Its fusion (F) protein must be cleaved by host proteases to cause membrane fusion, a critical step for virus infection. By generating Vero cells constitutively expressing the transmembrane serine protease TMPRSS2 and green fluorescent protein-expressing recombinant HMPV, we show that TMPRSS2, which is expressed in the human lung epithelium, cleaves the HMPV F protein efficiently and supports HMPV multiplication. The results indicate that TMPRSS2 is a possible candidate protease involved in the development of lower respiratory tract illness in HMPV-infected patients.

Identification of human metapneumovirus-induced gene networks in airway epithelial cells by microarray analysis.

Human metapneumovirus (hMPV) is a major cause of lower respiratory tract infections in infants, elderly and immunocompromised patients. Little is known about the response to hMPV infection of airway epithelial cells, which play a pivotal role in initiating and shaping innate and adaptive immune responses. In this study, we analyzed the transcriptional profiles of airway epithelial cells infected with hMPV using high-density oligonucleotide microarrays. Of the 47,400 transcripts and variants represented on the Affimetrix GeneChip Human Genome HG-U133 plus 2 array, 1601 genes were significantly altered following hMPV infection. Altered genes were then assigned to functional categories and mapped to signaling pathways. Many up-regulated genes are involved in the initiation of pro-inflammatory and antiviral immune responses, including chemokines, cytokines, type I interferon and interferon-inducible proteins. Other important functional classes up-regulated by hMPV infection include cellular signaling, gene transcription and apoptosis. Notably, genes associated with antioxidant and membrane transport activity, several metabolic pathways and cell proliferation were down-regulated in response to hMPV infection. Real-time PCR and Western blot assays were used to confirm the expression of genes related to several of these functional groups. The overall result of this study provides novel information on host gene expression upon infection with hMPV and also serves as a foundation for future investigations of genes and pathways involved in the pathogenesis of this important viral infection. Furthermore, it can facilitate a comparative analysis of other paramyxoviral infections to determine the transcriptional changes that are conserved versus the one that are specific to individual pathogens.

Airway epithelial cell response to human metapneumovirus infection.

Human metapneumovirus (hMPV) is a major cause of lower respiratory tract infections (LRTIs) in infants, elderly and immunocompromised patients. In this study, we show that hMPV can infect in a similar manner epithelial cells representative of different tracts of the airways. hMPV-induced expression of chemokines IL-8 and RANTES in primary small alveolar epithelial cells (SAE) and in a human alveolar type II-like epithelial cell line (A549) was similar, suggesting that A549 cells can be used as a model to study lower airway epithelial cell responses to hMPV infection. A549 secreted a variety of CXC and CC chemokines, cytokines and type I interferons, following hMPV infection. hMPV was also a strong inducer of transcription factors belonging to nuclear factor (NF)-kappaB, interferon regulatory factors (IRFs) and signal transducers and activators of transcription (STATs) families, which are known to orchestrate the expression of inflammatory and immunomodulatory mediators.

Inhibition of avian metapneumovirus (AMPV) replication by RNA interference targeting nucleoprotein gene (N) in cultured cells.

Avian metapneumovirus (AMPV) is the primary causative agent of severe rhinotracheitis in turkeys. It is associated with swollen head syndrome in chickens and is the source of significant economic losses to animal food production. In this study, we designed specific short interfering RNA (siRNA) targeting the AMPV nucleoprotein (N) and fusion (F) genes. Three days post-virus infection, virus titration, real time RT-PCR, and RT-PCR assays were performed to verify the effect of siRNA in AMPV replication. A marked decrease in virus titers from transfected CER cells treated with siRNA/N was observed. Also, the production of N, F, and G mRNAs in AMPV was decreased. Results indicate that N-specific siRNA can inhibit virus replication. In future studies, a combination of siRNAs targeting the RNA polymerase complex may be used as a tool to study AMPV replication and/or antiviral therapy.

Comparison of avian cell substrates for propagating subtype C avian metapneumovirus.

Avian metapneumovirus (AMPV) is a respiratory viral pathogen that causes turkey rhinotracheitis (TRT) or swollen head syndrome (SHS) in chickens. AMPV was first isolated in South Africa during the early 1970s and has subsequently spread worldwide during the 1980s to include Europe, Asia, and South America. In 1996, a genetically distinct AMPV subgroup C was isolated in the US following an outbreak of TRT. Vero cells are currently the best available substrate for AMPV propagation but are of non-avian origin. A number of different avian cell substrates have been compared to determine which is the most suitable for the propagation of AMPV to sufficiently high titers. Of the cell substrates tested, primary turkey turbinate and kidney and chicken kidney cells produced titers equal to or greater than Vero cells. Turkey turbinate and kidney epithelial cells that were life-span extended by the ectopic expression of human telomerase catalytic subunit (HTERT) initially displayed AMPV titers comparable to Vero cell controls, but declined in virus production with increased passage in culture. Interestingly, plaques emanating from Vero propagated virus were relatively small and dispersed, when analyzed by immunofluorescent assays (IFA), while both turkey turbinate and kidney cell propagated AMPV produced larger plaques. Even with these differences, there were no changes in the predicted amino acid sequences of the nucleocapsid (N) and phosphoprotein (P) genes of AMPV propagated in either turkey turbinate or Vero host cells. However, the fusion (F) gene showed 11 amino acid differences (98.7% identity) between the two host cell types. These results suggest that AMPV propagated in homologous avian cellular substrates may produce more infectious virus with possibly more effective fusion activity, compared to Vero cell propagation.

An S101P substitution in the putative cleavage motif of the human metapneumovirus fusion protein is a major determinant for trypsin-independent growth in vero cells and does not alter tissue tropism in hamsters.

Human metapneumovirus (hMPV), a recently described paramyxovirus, is a major etiological agent for lower respiratory tract disease in young children that can manifest with severe cough, bronchiolitis, and pneumonia. The hMPV fusion glycoprotein (F) shares conserved functional domains with other paramyxovirus F proteins that are important for virus entry and spread. For other paramyxovirus F proteins, cleavage of a precursor protein (F0) into F1 and F2 exposes a fusion peptide at the N terminus of the F1 fragment, a likely prerequisite for fusion activity. Many hMPV strains have been reported to require trypsin for growth in tissue culture. The majority of these strains contain RQSR at the putative cleavage site. However, strains hMPV/NL/1/00 and hMPV/NL/1/99 expanded in our laboratory contain the sequence RQPR and do not require trypsin for growth in Vero cells. The contribution of this single amino acid change was verified directly by generating recombinant virus (rhMPV/NL/1/00) with either proline or serine at position 101 in F. These results suggested that cleavage of F protein in Vero cells could be achieved by trypsin or S101P amino acid substitution in the putative cleavage site motif. Moreover, trypsin-independent cleavage of hMPV F containing 101P was enhanced by the amino acid substitution E93K. In hamsters, rhMPV/93K/101S and rhMPV/93K/101P grew to equivalent titers in the respiratory tract and replication was restricted to respiratory tissues. The ability of these hMPV strains to replicate efficiently in the absence of trypsin should greatly facilitate the generation, preclinical testing, and manufacturing of attenuated hMPV vaccine candidates.

Development of a reverse-genetics system for Avian pneumovirus demonstrates that the small hydrophobic (SH) and attachment (G) genes are not essential for virus viability.

Avian pneumovirus (APV) is a member of the genus Metapneumovirus of the subfamily Pneumovirinae. This study describes the development of a reverse-genetics system for APV. A minigenome system was used to optimize the expression of the nucleoprotein, phosphoprotein, M2 and large polymerase proteins when transfected into Vero cells under the control of the bacteriophage T7 promoter. Subsequently, cDNA was transcribed from the virion RNA to make a full-length antigenome, which was also cloned under the control of the T7 promoter. Transfection of the full-length genome plasmid, together with the plasmids expressing the functional proteins in the transcription and replication complex, generated APV in the transfected cells. The recombinant virus was passaged and was identified by cytopathic effect (CPE) that was typical of APV, the presence of a unique restriction-endonuclease site in the cDNA copy of the genome and immunofluorescence staining with anti-APV antibodies. Replacement of the full-length wild-type antigenome with one lacking the small hydrophobic (SH) protein and the attachment (G) genes generated a virus that grew more slowly and produced atypical CPE with syncytia much larger than those seen with wild-type virus.

Human metapneumovirus infection in hospital referred South African children.

Human metapneumovirus (hMPV) was first described in Dutch children with acute respiratory symptoms. A prospective analysis of the epidemiology, clinical manifestation, and seroprevalence of hMPV and other respiratory viruses in South African children referred to hospital for upper or lower respiratory tract infection were carried out during a single winter season, by using RT-PCR, viral culture, and enzyme-linked immunosorbent assays. In nasopharyngeal aspirates from 137 children, hMPV was detected by RT-PCR in 8 (5.8%) children (2-43 months of age) as a sole viral pathogen, respiratory syncytial virus (RSV) in 21 (15%), influenza A virus in 18 (13%) and influenza B virus in 20 (15%). Pneumonia was diagnosed in seven children and upper respiratory tract infection in one of the hMPV-infected children. One hMPV-infected child was admitted to the intensive care unit in need of mechanical ventilation and one child was infected with human immunodeficiency virus (HIV). No statistically significant differences were found between hMPV, RSV, and influenza virus infected groups with regard to clinical signs and symptoms and chest radiograph findings. The seropositive rate of hMPV specific IgG antibodies was 92% in children aged 24-36 months, the oldest seronegative child in our study was 7 years and 6 months of age. In conclusion, hMPV contributes to upper and lower respiratory tract morbidity in South African children.

Substituted benzimidazoles with nanomolar activity against respiratory syncytial virus.

A cell-based assay was used to discover compounds inhibiting respiratory syncytial virus (RSV)-induced fusion in HeLa/M cells. A lead compound was identified and subsequent synthesis of >300 analogues led to the identification of JNJ 2408068 (R170591), a low molecular weight (MW 395) benzimidazole derivative with an EC(50) (0.16 nM) against some lab strains almost 100,000 times better than that of ribavirin (15 microM). Antiviral activity was confirmed for subgroup A and B clinical isolates of human RSV and for a bovine RSV isolate. The compound did not inhibit the growth of representative viruses from other Paramyxovirus genera, i.e. HPIV2 and Mumps Virus (genus Rubulavirus), HPIV3 (genus Respirovirus), Measles virus (genus Morbillivirus) and hMPV. Efficacy in cytopathic effect inhibition assays correlated well with efficacy in virus yield reduction assays. A concentration of 10nM reduced RSV production 1000-fold in multi-cycle experiments, irrespective of the multiplicity of infection. Time of addition studies pointed to a dual mode of action: inhibition of virus-cell fusion early in the infection cycle and inhibition of cell-cell fusion at the end of the replication cycle. Two resistant mutants were raised and shown to have single point mutations in the F-gene (S398L and D486N). JNJ 2408068 was also shown to inhibit the release of proinflammatory cytokines IL-6, IL-8 and Rantes from RSV-infected A549 cells.