RSV Replication Research Articles

Probenecid Inhibits Extracellular Signal-Regulated Kinase and c-Jun N-Terminal Kinase Mitogen-Activated Protein Kinase Pathways in Regulating Respiratory Syncytial Virus Response.

We have previously shown that probenecid inhibits RSV, influenza virus, and SARS-CoV-2 replication in vitro in preclinical animal models and in humans. In a Phase two randomized, placebo-controlled, single-blind, dose range-finding study using probenecid to treat non-hospitalized patients with symptomatic, mild-to-moderate COVID-19, we previously showed that a 1000 mg twice daily treatment for 5 days reduced the median time to viral clearance from 11 to 7 days, and a 500 mg twice daily treatment for 5 days reduced the time to viral clearance from 11 to 9 days more than the placebo. In this study, we sought to determine the mechanism of action of the probenecid inhibition of RSV replication in human respiratory epithelial (A549) cells. We show that probenecid inhibits the RSV-induced phosphorylation of JNKs and ERKs and the downstream phosphorylation of c-jun, a component of the AP-1 transcription complex needed for virus replication. The inhibition of JNKs by probenecid reversed the repression of transcription factor HNF-4. The probenecid inhibition of JNK and ERK phosphorylation involves the MAPK pathway that precludes virus replication.

Infant-derived human nasal organoids exhibit relatively increased susceptibility, epithelial responses, and cytotoxicity during RSV infection

Respiratory syncytial virus (RSV) causes significant morbidity and mortality, especially in young children. Why RSV infection in children is more severe compared to healthy adults is not fully understood. We used ex-vivo human nasal organoid platforms from infants and adults to investigate the underlying mechanism of this disease disparity at the initial site of RSV replication, the nasal epithelium. Infant-derived human nasal organoid-air liquid interface (HNO-ALIs) lines were more susceptible to early RSV replication. Moreover, infant-derived HNO-ALIs elicited a statistically significant greater overall cytokine response, enhanced mucous production, and greater cellular damage compared to their adult counterparts. Furthermore, the adult cytokine response was associated with a superior regulatory cytokine response, which could explain less cellular damage than in infant lines. Our data highlights substantial differences in how infant and adult upper respiratory tract epithelium responds to RSV infection at the cellular level. These differences in epithelial cellular response can lead to impaired mucociliary clearance, a more dysregulated innate immune response predisposing infants to more severe RSV infection compared to adults.

Lung ILC2s are activated in BALB/c mice born to immunized mothers despite complete protection against respiratory syncytial virus.

Activated lung ILC2s produce large quantities of IL-5 and IL-13 that contribute to eosinophilic inflammation and mucus production following respiratory syncytial virus infection (RSV). The current understanding of ILC2 activation during RSV infection, is that ILC2s are activated by alarmins, including IL-33, released from airway epithelial cells in response to viral-mediated damage. Thus, high levels of RSV neutralizing maternal antibody generated from maternal immunization would be expected to reduce IL-33 production and mitigate ILC2 activation. Here we report that lung ILC2s from mice born to RSV-immunized dams become activated despite undetectable RSV replication. We also report, for the first time, expression of activating and inhibitory Fcgamma receptors on ILC2s that are differentially expressed in offspring born to immunized versus unimmunized dams. Alternatively, ex vivo IL-33-mediated activation of ILC2s was mitigated following the addition of antibody: antigen immune complexes. Further studies are needed to confirm the role of Fcgamma receptor ligation by immune complexes as an alternative mechanism of ILC2 regulation in RSV-associated eosinophilic lung inflammation.

Screening antivirals with a mCherry-expressing recombinant bovine respiratory syncytial virus: a proof of concept using cyclopamine

Bovine respiratory syncytial virus (BRSV) is a pathogenic pneumovirus and a major cause of acute respiratory infections in calves. Although different vaccines are available against BRSV, their efficiency remains limited, and no efficient and large-scale treatment exists. Here, we developed a new reverse genetics system for BRSV expressing the red fluorescent protein mCherry, based on a field strain isolated from a sick calf in Sweden. Although this recombinant fluorescent virus replicated slightly less efficiently compared to the wild type virus, both viruses were shown to be sensitive to the natural steroidal alkaloid cyclopamine, which was previously shown to inhibit human RSV replication. Our data thus point to the potential of this recombinant fluorescent BRSV as a powerful tool in preclinical drug discovery to enable high throughput compound screening.

Repurposing Probenecid to Inhibit SARS-CoV-2, Influenza Virus, and Respiratory Syncytial Virus (RSV) Replication.

Viral replication and transmissibility are the principal causes of endemic and pandemic disease threats. There remains a need for broad-spectrum antiviral agents. The most common respiratory viruses are endemic agents such as coronaviruses, respiratory syncytial viruses, and influenza viruses. Although vaccines are available for SARS-CoV-2 and some influenza viruses, there is a paucity of effective antiviral drugs, while for RSV there is no vaccine available, and therapeutic treatments are very limited. We have previously shown that probenecid is safe and effective in limiting influenza A virus replication and SARS-CoV-2 replication, along with strong evidence showing inhibition of RSV replication in vitro and in vivo. This review article will describe the antiviral activity profile of probenecid against these three viruses.

Evolution of protection after maternal immunization for respiratory syncytial virus in cotton rats.

Maternal anti-respiratory syncytial virus (RSV) antibodies acquired by the fetus through the placenta protect neonates from RSV disease through the first weeks of life. In the cotton rat model of RSV infections, we previously reported that immunization of dams during pregnancy with virus-like particles assembled with mutation stabilized pre-fusion F protein as well as the wild type G protein resulted in robust protection of their offspring from RSV challenge. Here we describe the durability of those protective responses in dams, the durability of protection in offspring, and the transfer of that protection to offspring of two consecutive pregnancies without a second boost immunization. We report that four weeks after birth, offspring of the first pregnancy were significantly protected from RSV replication in both lungs and nasal tissues after RSV challenge, but protection was reduced in pups at 6 weeks after birth. However, the overall protection of offspring of the second pregnancy was considerably reduced, even at four weeks of age. This drop in protection occurred even though the levels of total anti-pre-F IgG and neutralizing antibody titers in dams remained at similar, high levels before and after the second pregnancy. The results are consistent with an evolution of antibody properties in dams to populations less efficiently transferred to offspring or the less efficient transfer of antibodies in elderly dams.

Respiratory Syncytial Virus Phosphoprotein Residue S156 Plays a Role in Regulating Genome Transcription and Replication.

Respiratory syncytial virus (RSV) is a single-stranded, negative-sense RNA virus in the family Pneumoviridae and genus Orthopneumovirus that can cause severe disease in infants, immunocompromised adults, and the elderly. The RSV viral RNA-dependent RNA polymerase (vRdRp) complex is composed of the phosphoprotein (P) and the large polymerase protein (L). The P protein is constitutively phosphorylated by host kinases and has 41 serine (S) and threonine (T) residues as potential phosphorylation sites. To identify important phosphorylation residues in the P protein, we systematically and individually mutated all S and T residues to alanine (A) and analyzed their effects on genome transcription and replication by using a minigenome system. We found that the mutation of eight residues resulted in minigenome activity significantly lower than that of wild-type (WT) P. We then incorporated these mutations (T210A, S203A, T151A, S156A, T160A, S23A, T188A, and T105A) into full-length genome cDNA to rescue recombinant RSV. We were able to recover four recombinant viruses (with T151A, S156A, T160A, or S23A), suggesting that RSV-P residues T210, S203, T188, and T105 are essential for viral RNA replication. Among the four recombinant viruses rescued, rRSV-T160A caused a minor growth defect relative to its parental virus while rRSV-S156A had severely restricted replication due to decreased levels of genomic RNA. During infection, P-S156A phosphorylation was decreased, and when passaged, the S156A virus acquired a known compensatory mutation in L (L795I) that enhanced both WT-P and P-S156A minigenome activity and was able to partially rescue the S156A viral growth defect. This work demonstrates that residues T210, S203, T188, and T105 are critical for RSV replication and that S156 plays a critical role in viral RNA synthesis. IMPORTANCE RSV-P is a heavily phosphorylated protein that is required for RSV replication. In this study, we identified several residues, including P-S156, as phosphorylation sites that play critical roles in efficient viral growth and genome replication. Future studies to identify the specific kinase(s) that phosphorylates these residues can lead to kinase inhibitors and antiviral drugs for this important human pathogen.

A Safe and Effective Mucosal RSV Vaccine Consisting of RSV Phosphoprotein and Flagellin Variant

RSV is a major cause of serious acute lower respiratory tract infection in infants and the elderly. No licensed RSV vaccine available thus far calls for the development of vaccines with new target(s) and vaccination strategies. Here, we constructed a recombinant protein, designated P-KFD1, comprised of RSV phosphoprotein (P) and E. coli K12 strain-derived flagellin variant KFD1. Intranasal (i.n.) immunization with P-KFD1 inhibits RSV replication in both upper and lower respiratory tract, and protects mice against lung disease without vaccine-enhanced disease (VED). The P-specific CD4+ T cells provoked by P-KFD1 i.n. immunization, either reside in or migrate to respiratory tract, mediate protection against RSV infection. Sc-RNA seq and CFSE-labeled cell transfer further characterized the Th1 and Th17 responses induced by P-KFD1. Finally, we found the anti-viral protection depends on either IFN-γ or IL-17A. Collectively, P-KFD1 is promising as a safe and effective mucosal vaccine candidate to prevent RSV infection.

Wheezing in preschool children: New perspective.

In recent years the understanding about the mechanism of development of wheezing in children has improved and various management strategies were tried by different researchers. A search was made in PubMed by putting search term ‘Recurrent Wheezing in Children Diagnosis and Management’ and ‘Recurrent Wheezing in Children’. The respiratory syncytial virus induced bronchiolitis and rhinovirus infection in preschool children may lead to recurrent wheezing in preschool children. In infant immune system is immature and depends mainly on TLR ligation and maternal derived antibodies. Anti-inflammatory cytokines such as IL-10 and TGF beta are more common. RSV NS1 and NS2 proteins target RLR and TLR 3 dependent signaling and suppress the cellular response to RSV replication .This can lead to Th2 like response leading to asthma and allergy. CDHR3 acts as receptor in rhinovirus C infection. RV infection causes increase in IL 25 and IL 33 both induce Th2 type of immunity by increasing IL5 and IL 13 Daily Inhaled Corticosteroids (ICS) have been found useful in preventing exacerbations. Evidence is inconclusive about intermittent inhaled corticosteroids, intermittent montelukast and daily montelukast in recurrent wheezing. Azithromycin started early may decrease duration of wheezing episode. About intravenous magnesium sulfate and hypertonic saline evidence is inconclusive. Vitamin D supplementation in preterm babies for 6 months and avoidance of cow’s milk for first three days of life may be useful in prevention of recurrent wheezing in preschool children.

Early Detection and Prediction of Infection using Infrared Thermography

Helicobacter pylori infection causes gastritis and gastric ulcers and associated gastric cancer. Approximately 50% of the world's population H pylori infection. Developing countries with high prevalence rates. In most individuals, low socio economic means and infections are acquired during childhood. Affected family members at higher risk for childhood acquisi tion. The definitive routes of transmission are not clear evidence oral-oral, gastro-oral and fecal-oral routes of transmission. If left untreated, H pylori infection can last a lifetime. The disease is usually clinical and often occurs decades after the initial infection. H pylori infection can cause gastritis in children. Lesions, mucus-associated lymphoid lymphoma, and rarely, intes tinal metaplasia with/without gastric dysplasia.Synovial fluid aspirates were analyzed total knee arthroplasty by polymerase chain reaction in 50 patients with symptoms. Its presence indicates a bacterial infection called deoxyribonucleic acid. Syn ovial fluid samples were processed using a rapid bacterial degradation and extracts are amplified using protocol polymerase chain reaction and products analyzed including universal bacterial primers polymerase chain reaction deoxyribonucleic acid compound. A total of 32 samples were positive for bacterial infection with pre-polymerase chain reaction aspirates. We ana lyzed antimicrobial use in 509 episodes with microbiology laboratory report implications. Antibiotic stewardship. Most the rapeutic interventions occurred during phlebotomy and results by telephone after reporting gram stain release antimicrobial susceptibility data were limited impact on microbial management.A significant cause of hospitalization is leading to infection North America the second leading cause of death children and under after 1 year of malaria worldwide. Because of its global and human health impact, relatively few treatments exist. Paradoxically, there is a huge amount of information available on RSV replication during purification, RSV-induced mechanisms and social spread. RSV adds to the high burden of infection with existing chronic respiratory diseases.A major concern in clinical practice is vancomycin-resistant enterococci (VRE). Increased prevalence and their potential other bacteria to develop resistance to vancomycin (including methicillin-resistant Staphylococcus aureus) subjected them to closer scrutiny and intensive investigation. Colonialism was generally acquired high colonization rate of VRE in patients surrounding susceptible hosts.

Luteolin inhibits respiratory syncytial virus replication by regulating the MiR-155/SOCS1/STAT1 signaling pathway

BackgroundRespiratory syncytial virus (RSV) is a major cause of acute lower respiratory tract infection in infants, children, immunocompromised adults, and elderly individuals. Currently, there are few therapeutic options available to prevent RSV infection. The present study aimed to investigate the effects of luteolin on RSV replication and the related mechanisms.Material and methodsWe pretreated cells and mice with luteolin before infection with RSV, the virus titer, expressions of RSV-F, interferon (IFN)-stimulated genes (ISGs), and production of IFN-α and IFN-β were determined by plaque assay, RT-qPCR, and ELISA, respectively. The activation of Janus kinase (JAK)-signal transducer and activator of transcription 1 (STAT1) signaling pathway was detected by Western blotting and luciferase assay. Proteins which negatively regulate STAT1 were determined by Western blotting. Then cells were transfected with suppressor of cytokine signaling 1 (SOCS1) plasmid and virus replication and ISGs expression were determined. Luciferase reporter assay and Western blotting were performed to detect the relationship between SOCS1 and miR-155.ResultsLuteolin inhibited RSV replication, as shown by the decreased viral titer and RSV-F mRNA expression both in vitro and in vivo. The antiviral activity of luteolin was attributed to the enhanced phosphorylation of STAT1, resulting in the increased production of ISGs. Further study showed that SOCS1 was downregulated by luteolin and SOCS1 is a direct target of microRNA-155 (miR-155). Inhibition of miR-155 rescued luteolin-mediated SOCS1 downregulation, whereas upregulation of miR-155 enhanced the inhibitory effect of luteolin.ConclusionLuteolin inhibits RSV replication by regulating the miR-155/SOCS1/STAT1 signaling pathway.

GBP5 Is an Interferon-Induced Inhibitor of Respiratory Syncytial Virus.

Guanylate binding protein 5 (GBP5) belongs to the GTPase subfamily, which is mainly induced by interferon gamma (IFN-γ) and is involved in many important cellular processes, including inflammasome activation and innate immunity against a wide variety of microbial pathogens. However, it is unknown whether GBP5 inhibits respiratory syncytial virus (RSV) infection. In this study, we identified GBP5 as an effector of the anti-RSV activity of IFN-γ and found that in children, the weaker immune response, especially the weaker IFN-γ response and the decreased GBP5 expression, leads to RSV susceptibility. Furthermore, we revealed that GBP5 reduced the cell-associated levels of the RSV small hydrophobic (SH) protein, which was identified as a viroporin. In contrast, overexpression of the SH protein rescued RSV replication in the presence of GBP5. The GBP5-induced decrease in intracellular SH protein levels is because GBP5 promotes the release of the SH protein into the cell culture. Moreover, the GBP5 C583A mutants with changes at the C terminus or the GBP5 ΔC mutant lacking the C-terminal region, which impairs GBP5 localization in the Golgi, could not inhibit RSV infection, whereas the GTPase-defective GBP5 maintained RSV inhibition, suggesting that Golgi localization but not the GTPase activity of GBP5 is required for RSV inhibition. Interestingly, we found that RSV infection or RSV G protein downregulates GBP5 expression by upregulating DZIP3, an E3 ligase, which induces GBP5 degradation through the K48 ubiquitination and proteasomal pathways. Thus, this study reveals a complicated interplay between host restrictive factor GBP5 and RSV infection and provides important information for understanding the pathogenesis of RSV.IMPORTANCE RSV is a highly contagious virus that causes multiple infections in infants within their first year of life. It can also easily cause infection in elderly or immunocompromised individuals, suggesting that individual differences in immunity play an important role in RSV infection. Therefore, exploring the pathogenic mechanisms of RSV and identifying essential genes which inhibit RSV infection are necessary to develop an effective strategy to control RSV infection. Here, we report that the IFN-inducible gene GBP5 potently inhibits RSV replication by reducing the cell-associated levels of the RSV small hydrophobic (SH) protein, which is a viroporin. In contrast, the RSV G protein was shown to upregulate the expression of the DZIP3 protein, an E3 ligase that degrades GBP5 through the proteasomal pathway. Our study provides important information for the understanding of the pathogenic mechanisms of RSV and host immunity as well as the complicated interplay between the virus and host.

Respiratory Syncytial Virus Induces a Functional tRNA-derived Fragment to Promote Infection by Targeting SYNE2

Abstract RSV is the most common cause of lower respiratory tract infection, such as pneumonia and bronchiolitis, in infants. RSV also leads to significant outpatient visits. However the host-virus interaction is still poorly understood. The tRNA-derived RNA fragments (tRFs) is a recently discovered family of small non-coding RNAs. They are significantly induced by RSV and some have been demonstrated to play a role in mediating virus replication and host responses. However, the mechanisms used by most tRFs for the regulation are not clear. Previously, we have shown that an RSV-induced tRF derived from the 5′-end of mature tRNAs decoding GlyCCC (named as tRF5-GlyCCC) promotes RSV replication and has a regulatory function in gene expression. To identify target(s) of tRF5-GlyGCC, we first investigated tRF5-GlyCC-associated mRNAs by HIgh-Throughput Sequencing of RNAs Associated with Biotinylated tRF (HITS-ABt). The targets were prioritized by identifying common mRNAs whose expression is suppressed by RSV and predicted free energy for the interaction of tRF5-GlyCCC and targets. SYNE-2 is one of the most likely target genes for tRF5-GlyCCC. Our experiments confirmed that SYNE-2 was downregulated by RSV infection. In addition, we found that antisense oligonucleotides of tRF5-GlyCCC reversed the SYNE-2 downregulation by RSV. We also found that SYNE2 is not involved in the replication of RSV, but important in RSV-regulated innate cellular response. Two immune mediators MIP-1β and IP-10 are selectively regulated by tRF5-GlyGCC, possibly through controlling the activation of transcription factor p50. In summary, this study provides accumulative evidence that tRFs induced by RSV are not degraded byproducts, but functional molecules.

Respiratory syncytial virus exhibits differential tropism for distinct human placental cell types with Hofbauer cells acting as a permissive reservoir for infection.

BackgroundRespiratory syncytial virus (RSV) is capable of transient viremia and extrapulmonary dissemination. Recently, this virus has been identified in fetal cord blood, suggesting the possibility of in utero acquisition in humans. Here, we assess permissivity and kinetics of RSV replication in primary human placental cells, examine their potential to transfer this infection to neighboring cells, and measure the inflammatory response evoked by the virus.Methods and findingsHuman placental villus tissue was collected immediately upon delivery and processed for isolation of placental cytotrophoblast, fibroblast, and macrophage (Hofbauer) cells. Isolated cells were infected with a recombinant RSV-A2 strain (rrRSV) expressing red fluorescent protein (RFP) and analyzed by fluorescence microscopy, Western blot, and quantitative PCR (qPCR). Based on RFP expression, rrRSV exhibited differential tropism for the three major placental cell types. Placental fibroblasts and Hofbauer cells were permissive and supported productive rrRSV replication. While infected cytotrophoblast cells expressed viral glycoprotein (G protein), only limited RSV replication was detected. Importantly, qPCR and fluorescence-focused unit assay revealed that the viral progeny remains trapped within infected Hofbauer cells for up to 30 days, with no release into surrounding media. Yet, Hofbauer cells passed the infection onto overlaid naïve 16HBE cells, suggesting contact-dependent trans-infection. Lastly, a significant increase in proinflammatory cytokines, particularly IL-6, TNF-alpha, and IFN-gamma was measured in the supernatant of infected Hofbauer cells by multiplex cytokine assay and conventional ELISA.ConclusionsThis study demonstrates that RSV can replicate in human placenta, exhibits differential tropism for distinct placental cell types, can be stored and transferred to neighboring cells by Hofbauer cells, and elicits an inflammatory response. It also supports the hypothesis that this respiratory virus can be vertically transferred to the fetus and potentially affect its development and the outcome of pregnancies.

Anti-Viral Vaccine Activity of Zinc(Ⅱ) for Viral Prevention, Entry, Replication, and Spreading During Pathogenesis Process

Anti-viral vaccine activity of Zn2+ ions for viral prevention, pathogenesis processes, and ROS generation causing to oxidative stress have been investigated. AZP is efficient for viral prevention by inhibitions of BSCTV and DNA virus replications. The AZP phenotypes show strongly resistant to virus infection and viral DNA replication could be applied to the prevention of virus infections in humans. ZnOTs exhibit the ability to neutralize HSV-2 virions and blocking HSV-2 attachment activity. Zinc salts can mediate antiviral activity on RSV by altering the ability of the cell to support RSV replication. The effect of zinc sulfate on seroconversion after a simple method vaccination had been identified that accelerated HB vaccination can shorten duration of immunization of this clinical trial for showing its effectiveness. The inhibition of zinc binding activity of hMPV M2-1 protein can lead to the development of novel, live attenuated vaccines as well as antiviral drugs for pneumoviruses. The CCHH zinc finger motif provides potential vaccine candidates for the development of live species-specific attenuated influenza virus vaccines. Chelates zinc ions inhibit HIV-1 replication. The LAIVs are attracting attention as several advantages over inactivated vaccines. Zinc finger reactive compounds also inactivate retroviruses. ZOTEN promoted the presentation of bound HSV-2 virions. The regulated ZFNs in the presence of HIV-1 Tat may provide a safer and novel genome-editing technology for eradicating HIV-1 proviral DNA from infected host cells. Zinc ions inhibit vaccine virus growth. The ZAP inhibits HIV-1 infection by viral mRNAs degradation. ZAP also inhibits the spreading against XMRV. The treatment of viral infections with viral spreading will be achieved and improved the therapeutic effects by using zinc oxide nanoparticles. ROS and RNS within viruses of viral entry, viral replication, and viral spread are generated in all situations. ROS in virus pathogenesis play an important role in cell signaling and regulate hormone action, growth factors, cytokines, transcription, apoptosis, immunomodulation, and neuromodulation, leading to chronic oxidative stress. Oxidative stress has been occurred in various viral infections. The antioxidant components lead to an excess storage of H2O2, which further increases the hydroxyl radicals and lipid peroxide that signal the cell to undergo a programmed cell death. Thus, zinc-associated vaccine activity mechanism against viruses is indicated that the anti-viral vaccine activity of released Zn2+ ions from zinc solutions and ZnO NPs, may be enhanced by Zn2+ ion-induced Zn2+ ions-coordinated adapted immunity, viral growth regulation, and viral apoptosis and death.

Design, Synthesis, Antiviral Evaluation, and SAR Studies of New 1-(Phenylsulfonyl)-1H-Pyrazol-4-yl-Methylaniline Derivatives.

A series of N-((3-phenyl-1-(phenylsulfonyl)-1H-pyrazol-4-yl)methyl)anilines 7a-p and 8a-l, structurally related to previously synthesized and tested (N-(1,3-diphenyl-1H-pyrazol-4-yl)methyl)anilines (1a-v), were designed and synthesized. The new derivatives were evaluated in cell-based assays for their cytotoxicity and antiviral activity against a large panel of RNA and DNA viruses of public health significance. Generally, the tested compounds did not display cytotoxicity toward the cell lines used. The majority of derivatives 7a-p were able to interfered with YFV and RSV replication in the micromolar range showing a marked improvement in potency and selectivity with respect to the reference inhibitors 6-azauridine and ribavirin, respectively. The introduction of a p-methoxy substituent on the phenylsulfonyl group (compounds 8a-l) completely abolished the anti-RSV activity and reduced or eliminated the potency against YFV. On the contrary, several p-methoxy analogs were able to interfere with BVDV replication with a comparable (8b, 8c, 8g, and 8k) or better (8a and 8f) potency than the reference inhibitor, ribavirin. Compound 7e, selected for time of addition experiments on BHK-21 cell cultures infected with YFV, achieved the highest reduction of virus titer when added 2 h post infection and maintained up to 4 h post infection.

Identification of novel factors associated with severe respiratory syncytial virus disease in infants

Background Almost all infants are infected with RSV by 2 years. 1–3 % of RSV-infected infants are hospitalised with severe disease. Reasons for susceptibility to severe disease remain obscure. We aimed to identify factors that might explain such susceptibility. Methods We generated well-differentiated primary nasal epithelial cell (WD-PNEC) cultures from infants with histories of severe or mild RSV disease. Following infection with RSV BT2a (clinical isolate), virus growth kinetics, cytopathogenesis, chemokines, and IL-29/IFNλ1 responses and differential gene expression were determined. In an exciting development, 1 differentially expressed gene, ptn, encodes pleiotrophin (PTN), which interacts with nucleolin (NCL), an RSV entry co-factor. The PTN antiviral activity was determined in BEAS-2B cells and WD-PBECs (where B=bronchial). Results Viral growth kinetics, cell tropism, IP-10/CXCL10, TRAIL and RANTES/CCL5 responses did not differ significantly between cohorts. However, apical cell sloughing and IL-29/IFNλ1 were diminished in severe WD-PNECs following infection, while expression of isg15, ifi6, irf9, duox2 and tap1 was also reduced. Interestingly, diminished ptn expression was evident in severe WD-PNECs, irrespective of RSV infection. Furthermore, pre-treatment with PTN blocked RSV infection in BEAS-2B cells and WD-PBECs, while neutralisation of PTN with either antibodies or siRNAs resulted in increased RSV replication. Conclusions Diminished apical cell sloughing and expression/secretion of IL-29/IFNλ1 and specific interferon stimulated genes in WD-PNECs were associated with severe RSV. Importantly, PTN was identified as a novel endogenously expressed RSV antiviral protein in human airway epithelium. Lower expression of PTN in paediatric airway epithelium may explain, in part, increased susceptibility to severe disease.

Exchange Proteins Directly Activated by cAMP and Their Roles in Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) is the leading cause of respiratory infection in young children and high-risk adults. However, a specific treatment for this viral infection is not currently available. In this study, we discovered that an exchange protein directly activated by cyclic AMP (EPAC) can serve as a potential therapeutic target for RSV. In both lower and upper epithelial cells, treatment with EPAC inhibitor (ESI-09), but not protein kinase A inhibitor (H89), significantly inhibits RSV replication and proinflammatory cytokine/chemokine induction. In addition, RSV-activated transcriptional factors belonging to the NF-κB and IRF families are also suppressed by ESI-09. Through isoform-specific gene knockdown, we found that EPAC2, but not EPAC1, plays a dominant role in controlling RSV replication and virus-induced host responses. Experiments using both EPAC2 knockout and EPAC2-specific inhibitor support such roles of EPAC2. Therefore, EPAC2 is a promising therapeutic target to regulate RSV replication and associated inflammation.IMPORTANCE RSV is a serious public health problem, as it is associated with bronchiolitis, pneumonia, and asthma exacerbations. Currently no effective treatment or vaccine is available, and many molecular mechanisms regarding RSV-induced lung disease are still significantly unknown. This project aims to elucidate an important and novel function of a protein, called EPAC2, in RSV replication and innate inflammatory responses. Our results should provide an important insight into the development of new pharmacologic strategies against RSV infection, thereby reducing RSV-associated morbidity and mortality.

Unrecognized prolonged viral replication in the pathogenesis of human RSV infection

BackgroundRespiratory symptoms in RSV persist long after the virus is no longer detected by culture. Current concepts of RSV pathogenesis explain this by RSV inducing a long-lasting pathogenic immune cascade. We alternatively hypothesized that prolonged unrecognized RSV replication may be responsible and studied this possibility directly in a human wild-type RSV experimental infection model. ObjectiveThe objective of the current report was to define the duration of true human RSV replication by studying it directly in immunocompetent adults experimentally infected with a clinical strain of RSV utilizing this previously established safe and reproducible model. Study Design35 healthy adult volunteers were inoculated with RSV-A (Memphis-37, a low11 passage clinical strain virus, manufactured from a hospitalized bronchiolitic infant) and evaluated over 12 days. Viral load by culture, parallel quantitative PCR (genomic, message) and RSV-specific IgA, were measured twice daily from serially collected nasal washes. ResultsAfter inoculation, 77% (27/35) of volunteers became RSV infected. As expected, culture-detectable RSV ceased abruptly by the 5–6 t h 15 infection day. However, infected volunteers demonstrated prolonged RSV presence by both genomic and message PCR. RSV-specific IgA rose within respiratory secretions of infected volunteers during same time frame. ConclusionsRSV replication appears to continue in humans far longer than previously thought. The rise in nasal RSV-specific IgA shortly after infection likely neutralizes culture detectable virus producing misleadingly short durations of infection. Prolonged viral replication helps explain RSV’s extended disease manifestations and increases the potential utility of antivirals.

Exchange Proteins Directly Activated by cAMP and the Role of these Proteins in Respiratory Syncytial Virus Infection

Abstract Respiratory syncytial virus (RSV) is a single-stranded, negative-strand RNA virus belonging to Paramyxoviridae family, and is in the genus Pneumovirus. It was discovered in chimpanzees in 1955 and subsequently confirmed to be a human pathogen shortly after that. RSV is one of the most common viruses to infect young children and high-risk adults worldwide, and the leading cause of inflammation of the lungs (pneumonia) or the lungs’ airways (bronchiolitis) in infants. Due in part to the lack of long-term immunity after infection, making reinfection frequent. There is a single antiviral treatment for RSV currently approved, but its use is limited by questionable efficacy, side effects, and cost, and it is recommended that it be used only for patients at-risk for severe disease, on a case-by-case basis. In this study, we discovered an exchange protein directly activated by cyclic AMP (EPAC) that can serve as a potential therapeutic target for RSV. In both lower and upper epithelial cells, the treatment of EPAC inhibitor (ESI-09), but not PKA inhibitor (H89), significantly inhibits the RSV replication and pro-inflammatory cytokine/chemokine induction. In addition, RSV-activated transcriptional factors belonging to the NF-κB and IRF families are also suppressed by ESI-09. Through isoform-specific gene knock down, we found that EPAC2, but not EPAC1, plays a dominant role in controlling RSV replication and viral-induced host responses. Experiments using both EPAC2 knock out and EPAC2 specific inhibitor support such roles of EPAC2. Therefore, EPAC2 could be a promising therapeutic target to regulate RSV replication and associated inflammation.