Temporal Dynamics of the Ruminant Type I IFN-Induced Antiviral State against Homologous Parainfluenza Virus 3 Challenge In Vitro.

Viruses have evolved diverse strategies to evade the antiviral response of interferons (IFNs). Exogenous IFNs were applied to eliminate the counteracting effect and possess antiviral properties. Caprine parainfluenza virus 3 (CPIV3) and bovine parainfluenza virus type 3 (BPIV3) are important pathogens associated with respiratory diseases in goat and cattle, respectively. To explore the feasibility of type I IFNs for control of CPIV3 and BPIV3 infection, the activated effects of IFN-stimulated genes (ISGs) and the immunomodulation responses of goat IFN-α were detected by transcriptomic analysis. Then, the antiviral efficacy of goat IFN-α and IFN-τ against CPIV3 and BPIV3 infection in MDBK cells was evaluated using different treatment routes at different infection times. The results showed that CPIV3 infection inhibited the production of type I IFNs, whereas exogenous goat IFN-α induced various ISGs, the IFN-τ encoding gene, and a negligible inflammatory response. Consequently, goat IFN-α prophylaxis but not treatment was found to effectively modulate CPIV3 and BPIV3 infection; the protective effect lasted for 1 week, and the antiviral activity was maintained at a concentration of 0.1 µg/mL. Furthermore, the antiviral activity of goat IFN-τ in response to CPIV3 and BPIV3 infection is comparable to that of goat IFN-α. These results corroborate that goat IFN-α and IFN-τ exhibit prophylactic activities in response to ruminant respiratory viral infection in vitro, and should be further investigated for a potential use in vivo.

Curcumin Inhibits Replication of Human Parainfluenza Virus Type 3 by Affecting Viral Inclusion Body Formation.

Human Parainfluenza Virus Type 3 (HPIV3) is one of the main pathogens that cause acute lower respiratory tract infections in infants and young children. However, there are currently no effective antiviral drugs and vaccines. Herein, we found that a natural compound, curcumin, inhibits HPIV3 infection and has antiviral effects on entry and replication of the virus life cycle. Immunofluorescence and western blotting experiments revealed that curcumin disrupts F-actin and inhibits viral inclusion body (IB) formation, thus inhibiting virus replication. Curcumin can also downregulate cellular PI4KB and interrupt its colocalization in viral IBs. This study verified the antiviral ability of curcumin on HPIV3 infection and preliminarily elucidated its influence on viral replication, providing a theoretical basis for antiviral drug development of HPIV3 and other parainfluenza viruses.

The in vitro multilineage differentiation and maturation of lung and airway cells from human pluripotent stem cell-derived lung progenitors in 3D.

Lung and airway epithelial cells generated in vitro from human pluripotent stem cells (hPSCs) have applications in regenerative medicine, modeling of lung disease, drug screening and studies of human lung development. Here, we describe a strategy for directed differentiation of hPSCs into mature lung and airway epithelial cells obtained through maturation of NKX2.1+ hPSC-derived lung progenitors in a 3D matrix of collagen I in the absence of glycogen synthase kinase 3 inhibition. This protocol is an extension of our previously published protocol on the directed differentiation of lung and airway epithelium from hPSCs that modifies the technique and offers additional applications. This protocol is conducted in defined media conditions, has a duration of 50-80 d, does not require reporter lines and results in cultures containing mature alveolar type II and I cells as well as airway basal, ciliated, club and neuroendocrine cells. We also present a flow cytometry strategy to assess maturation in the cultures. Several of these populations, including mature NGFR+ basal cells, can be prospectively isolated by cell sorting and expanded for further investigation.

Modeling Infection and Tropism of Human Parainfluenza Virus Type 3 in Ferrets.

Human parainfluenza virus type 3 (HPIV-3) is a significant cause of lower respiratory tract infections, with the most severe disease in young infants, immunocompromised individuals, and the elderly. HPIV-3 infections are currently untreatable with licensed therapeutics, and prophylactic and therapeutic options are needed for patients at risk. To complement existing human airway models of HPIV-3 infection and develop an animal model to assess novel intervention strategies, we evaluated infection and transmission of HPIV-3 in ferrets. A well-characterized human clinical isolate (CI) of HPIV-3 engineered to express enhanced green fluorescent protein (rHPIV-3 CI-1-EGFP) was passaged on primary human airway epithelial cells (HAE) or airway organoids (AO) to avoid tissue culture adaptations. rHPIV3 CI-1-EGFP infection was assessed in vitro in ferret AO and in ferrets in vivo. Undifferentiated and differentiated ferret AO cultures supported rHPIV-3 CI-1-EGFP replication, but the ferret primary airway cells from AO were less susceptible and permissive than HAE. In vivo rHPIV-3 CI-1-EGFP replicated in the upper and lower airways of ferrets and targeted respiratory epithelial cells, olfactory epithelial cells, type I pneumocytes, and type II pneumocytes. The infection efficiently induced specific antibody responses. Taken together, ferrets are naturally susceptible to HPIV-3 infection; however, limited replication was observed that led to neither overt clinical signs nor ferret-to-ferret transmission. However, in combination with ferret AO, the ferret model of HPIV-3 infection, tissue tropism, and neutralizing antibodies complements human ex vivo lung models and can be used as a platform for prevention and treatment studies for this important respiratory pathogen. IMPORTANCE HPIV-3 is an important cause of pediatric disease and significantly impacts the elderly. Increasing numbers of immunocompromised patients suffer from HPIV-3 infections, often related to problems with viral clearance. There is a need to model HPIV-3 infections in vitro and in vivo to evaluate novel prophylaxis and treatment options. Currently existing animal models lack the potential for studying animal-to-animal transmission or the effect of immunosuppressive therapy. Here, we describe the use of the ferret model in combination with authentic clinical viruses to further complement human ex vivo models, providing a platform to study approaches to prevent and treat HPIV-3 infection. Although we did not detect ferret-to-ferret transmission in our studies, these studies lay the groundwork for further refinement of the ferret model to immunocompromised ferrets, allowing for studies of severe HPIV-3-associated disease. Such models for preclinical evaluation of prophylaxis and antivirals can contribute to reducing the global health burden of HPIV-3.

A synthetic STING agonist inhibits the replication of human parainfluenza virus 3 and rhinovirus 16 through distinct mechanisms.

Stimulator of interferon genes (STING), as a signaling hub in innate immunity, plays a central role for the effective initiation of host defense mechanisms against microbial infections. Upon binding of its ligand cyclic dinucleotides (CDNs) produced by the cyclic GMP-AMP synthase (cGAS) or invading bacteria, STING is activated, leading to the induction of both type I interferon responses and autophagy, which are critical for the control of certain microbial infections. RNA viruses, such as Parainfluenza virus (PIV) and Rhinovirus (HRV), are among the leading causes of respiratory infections that affect human health without effective treatments. Activation of STING pathway may provide a new therapeutic approach fighting against these viruses. However, the role of STING in the control of RNA virus infection remains largely unexplored. In this study, using dimeric amidobenzimidazole (diABZI), a newly discovered synthetic small molecule STING receptor agonist with much higher potency than CDNs, we found that activation of STING elicits potent antiviral effects against parainfluenza virus type 3 (PIV3) and human rhinovirus 16 (HRV16), two representative respiratory viral pathogens. Notably, while anti-PIV3 activity was depend on the induction of type I interferon responses through TANK-binding kinase 1 (TBK1), anti-HRV16 activity required the induction of autophagy-related gene 5 (ATG5)-dependent autophagy, indicating that two distinct antiviral mechanisms are engaged upon STING activation. Antiviral activity and individual specific pathway was further confirmed in infected primary bronchial epithelial cells. Our findings thus demonstrate the distinct antiviral mechanisms triggered by STING agonist and uncover the potential of therapeutic effect against different viruses.

Bta-miR-98 Suppresses Replication of Caprine Parainfluenza Virus Type 3 Through Inhibiting Apoptosis by Targeting Caspase-3.

Caprine parainfluenza virus type 3 (CPIV3) is an emerging respiratory pathogen that affects the sheep and goat industry in China and possibly other countries around the world. Accumulating evidence suggests that microRNAs play important roles in regulating virus-host interactions and can suppress or facilitate viral replication. In this study, we showed that CPIV3 infection induced apoptosis in Madin-Darby bovine kidney (MDBK) cells, as determined by morphological changes and flow cytometry. Caspase activity and the expression of pro-apoptotic genes further indicated that CPIV3 induced apoptosis by activating both the intrinsic and extrinsic pathways. We also demonstrated the involvement of bta-microRNA-98 (bta-miR-98) in regulating CPIV3-induced apoptosis. Bta-miR-98 was downregulated in MDBK cells infected with CPIV3. Overexpression of bta-miR-98 significantly decreased the activities of caspase-3, -8, and -9. Conversely, inhibition of bta-miR-98 had completely opposite effects. Furthermore, our data showed that bta-miR-98 markedly affected CPIV3 replication by regulating apoptosis. Importantly, we found that bta-miR-98 modulated CPIV3-induced apoptosis by targeting caspase-3, an effector of apoptosis. Collectively, our results may suggest that CPIV3 infection induced apoptosis and downregulated the levels of bta-miR-98, and this miRNA regulated viral replication through effected apoptosis. This study contributes to our understanding of the molecular mechanisms underlying CPIV3 pathogenesis.

Cholesterol-rich lipid rafts both in cellular and viral membrane are critical for caprine parainfluenza virus type3 entry and infection in host cells.

Cholesterol-rich lipid rafts have been shown to play important roles in the life cycle of various non-enveloped and enveloped viruses. Deletion of cholesterol from lipid rafts could influence different steps of viral replication cycle including entry, infection, assembly and release. Caprine parainfluenza virus type3 (CPIV3) is a newly identified member of Paramyxoviridae family. CPIV3 is highly prevalence and threatened the goat industry in China. The infection mechanism of CPIV3 is under exploring and still not fully understood, the roles of cholesterol and lipid rafts for CPIV3 infection remains unclear. In this study, we investigated the association of cholesterol and lipid rafts with CPIV3 during the different viral replication stages (binding, entry and infection) in two cells [MDBK and goat bronchial epithelial (GBE) cells]. Methyl-ß- cyclodextrin (MßCD) was used to deplete cholesterol from cell and viral membranes. The results showed that MßCD treatment significantly inhibited CPIV3 entry and infection in these two cells with a dose-dependent manner, but didn't impair the binding of CPIV3. Addition of exogenous cholesterol to the cells after MßCD treatment restored the viral infection. In addition, treatment of MßCD only before virus-entry showed inhibitory effect in MDBK cells. Depletion of cholesterol from virion envelop also decreased the entry and infection of CPIV3 in the two cells. Furthermore, lipid rafts isolation test indicated that viral proteins (HN and N) co-localized with lipid rafts during infection in MDBK and GBE cells. Viral N protein co-localized with caveolin-1 (the marker of lipid rafts) in these two cells both at the entry and infection steps, as detected by con-focal laser scanning microscopy test. In conclusion, the results presented here demonstrated that cholesterol rich lipid rafts play an important role in CPIV3 life cycle. The findings give new insights on understanding of the mechanism of CPIV3 infection and provide a new anti-CPIV3 strategy.

Exosomes promote caprine parainfluenza virus type 3 infection by inhibiting autophagy.

Caprine parainfluenza virus type 3 (CPIV3) is a novel important pathogen causing respiratory disease in goats, but the pathogenic mechanism is not clear yet. Evidence suggests that exosomes transfer biologically active molecules between cells. Viral infections can cause profound changes in exosome components, and exosomes have been involved in viral transmission and pathogenicity. In this study, we explored the characteristics and functions of exosomes purified from the supernatant of Madin-Darby bovine kidney (MDBK) cells inoculated with CPIV3. Infection of CPIV3 showed increased exosome secretion and the loading of viral proteins and RNA into exosomes. These exosomes were capable of transferring CPIV3 genetic materials to recipient cells to establish a productive infection and promote the viral replication. To explore the potential mechanism, small RNA deep sequencing revealed that CPIV3 exosomes contained a diverse range of RNA species, including miRNA and piRNA, in proportions different from exosomes isolated from mock-infected cells. Expression patterns of 11 differentially expressed miRNAs were subsequently validated by quantitative reverse transcriptase PCR (qRT-PCR). Targets of miRNAs were predicted and functional annotation analysis showed that the main pathways involved were autophagy signalling ways. Autophagy inhibited by the CPIV3-exosome was further verified, and miR-126-3 p_2 packaged in the vesicles was an important regulation factor in this process. Inhibition of autophagy may be one of the responsible reasons for promoting efficient replication of exosome-mediated CPIV3 infection. The study suggests that exosomes are key in pathogenesis or protection against CPIV3. Further understating of their role in CPIV3 infection may bring novel insight to the development of protection measures.

Human parainfluenza virus type 3 expressing the respiratory syncytial virus pre-fusion F protein modified for virion packaging yields protective intranasal vaccine candidates.

Human respiratory syncytial virus (RSV) and parainfluenza virus type 3 (HPIV3) are among the most common viral causes of childhood bronchiolitis and pneumonia worldwide, and lack effective antiviral drugs or vaccines. Recombinant (r) HPIV3 was modified to express the RSV fusion (F) glycoprotein, the major RSV neutralization and protective antigen, providing a live intranasal bivalent HPIV3/RSV vaccine candidate. This extends previous studies using a chimeric bovine-human PIV3 vector (rB/HPIV3). One advantage is that rHPIV3 expresses all of the HPIV3 antigens compared to only two for rB/HPIV3. In addition, the use of rHPIV3 as vector should avoid excessive attenuation following addition of the modified RSV F gene, which may occur with rB/HPIV3. To enhance its immunogenicity, RSV F was modified (i) to increase the stability of the prefusion (pre-F) conformation and (ii) by replacement of its transmembrane (TM) and cytoplasmic tail (CT) domains with those of HPIV3 F (H3TMCT) to increase incorporation in the vector virion. RSV F (+/- H3TMCT) was expressed from the first (F/preN) or the second (F/N-P) gene position of rHPIV3. The H3TMCT modification dramatically increased packaging of RSV F into the vector virion and, in hamsters, resulted in significant increases in the titer of high-quality serum RSV-neutralizing antibodies, in addition to the increase conferred by pre-F stabilization. Only F-H3TMCT/preN replication was significantly attenuated in the nasal turbinates by the RSV F insert. F-H3TMCT/preN, F/N-P, and F-H3TMCT/N-P provided complete protection against wt RSV challenge. F-H3TMCT/N-P exhibited the most stable and highest expression of RSV F, providing impetus for its further development.

Hijacking the Fusion Complex of Human Parainfluenza Virus as an Antiviral Strategy.

The receptor binding protein of parainfluenza virus, hemagglutinin-neuraminidase (HN), is responsible for actively triggering the viral fusion protein (F) to undergo a conformational change leading to insertion into the target cell and fusion of the virus with the target cell membrane. For proper viral entry to occur, this process must occur when HN is engaged with host cell receptors at the cell surface. It is possible to interfere with this process through premature activation of the F protein, distant from the target cell receptor. Conformational changes in the F protein and adoption of the postfusion form of the protein prior to receptor engagement of HN at the host cell membrane inactivate the virus. We previously identified small molecules that interact with HN and induce it to activate F in an untimely fashion, validating a new antiviral strategy. To obtain highly active pretriggering candidate molecules we carried out a virtual modeling screen for molecules that interact with sialic acid binding site II on HN, which we propose to be the site responsible for activating F. To directly assess the mechanism of action of one such highly effective new premature activating compound, PAC-3066, we use cryo-electron tomography on authentic intact viral particles for the first time to examine the effects of PAC-3066 treatment on the conformation of the viral F protein. We present the first direct observation of the conformational rearrangement induced in the viral F protein.IMPORTANCE Paramyxoviruses, including human parainfluenza virus type 3, are internalized into host cells by fusion between viral and target cell membranes. The receptor binding protein, hemagglutinin-neuraminidase (HN), upon binding to its cell receptor, triggers conformational changes in the fusion protein (F). This action of HN activates F to reach its fusion-competent state. Using small molecules that interact with HN, we can induce the premature activation of F and inactivate the virus. To obtain highly active pretriggering compounds, we carried out a virtual modeling screen for molecules that interact with a sialic acid binding site on HN that we propose to be the site involved in activating F. We use cryo-electron tomography of authentic intact viral particles for the first time to directly assess the mechanism of action of this treatment on the conformation of the viral F protein and present the first direct observation of the induced conformational rearrangement in the viral F protein.

Structure-Guided Improvement of a Dual HPIV3/RSV Fusion Inhibitor.

Human parainfluenza virus 3 (HPIV3) and respiratory syncytial virus (RSV) are leading causes of lower respiratory tract infections. There are currently no vaccines or antiviral therapeutics to treat HPIV3 or RSV infections. We recently reported a peptide (VIQKI), derived from the C-terminal heptad repeat (HRC) domain of the HPIV3 fusion (F) glycoprotein that inhibits infection by both HPIV3 and RSV. The dual inhibitory activity of VIQKI is due to its unique ability to bind to the N-terminal heptad repeat (HRN) domains of both HPIV3 and RSV F, thereby preventing the native HRN-HRC interactions required for viral entry. Here we describe the structure-guided design of dual inhibitors of HPIV3 and RSV fusion with improved efficacy. We show that VIQKI derivatives possessing one (I456F) or two (I454F/I456F) phenylalanine substitutions near the N-terminus exhibit more stable assemblies with the RSV-HRN domain and enhanced antiviral efficacy against both HPIV3 and RSV infection. Cocrystal structures of the new Phe-substituted inhibitors coassembled with HPIV3 or RSV-HRN domains reveal that the I456F substitution makes intimate hydrophobic contact with the core trimers of both HPIV3 and RSV F.

Interferon-stimulated genes inhibit caprine parainfluenza virus type 3 replication in Madin-Darby bovine kidney cells.

Caprine parainfluenza virus type 3 (CPIV3) is the one of most common causative agents of caprine respiratory infection, resulting in significant economic losses in the goat and sheep industries. However, the molecular mechanisms and host genes involved in the pathogenesis of and immunity against CPIV3 infection remain poorly understood. In this study, we used RNA-Seq to understand the responses of madin-darby bovine kidney (MDBK) cells to CPIV3 infection. A total of 261 differentially-expressed genes (DEGs) were identified in CPIV3-infected compared with mock-infected MDBK cells at 24 h post-infection (hpi). The DEGs were mainly involved in immune system processes, metabolic processes, and signal transduction. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated that the most significantly enriched signaling pathways were MAPK, Wnt, PI3K-Akt, tumor necrosis factor, Toll-like receptor and ubiquitin-mediated proteolysis. STRING analysis revealed that seven interferon-stimulated genes (ISGs) were upregulated (IFI6, ISG15, OAS1Y, OAS1Z, MX1, MX2 and RSAD2) and may play a pivotal role during CPIV3 infection. Moreover, overexpression of these ISGs significantly reduced CPIV3 replication in vitro, while siRNA silencing markedly improved CPIV3 replication 24 and 48 hpi. Ours is the first study to profile the gene expression of CPIV3-infected MDBK cells. We identified seven ISGs that could be targeted in novel antiviral strategies against CPIV3.

Mutations in the HRB linker of human parainfluenza virus type 3 fusion protein reveal its importance for fusion activity.

Human parainfluenza virus type 3 (HPIV3) fuses the viral envelope with the host cell membrane through the concerted action of the fusion (F) protein and the hemagglutinin-neuraminidase (HN). Upon HN binding to sialic acid (SA), the F protein in a metastable prefusion form is activated to undergo a series of structural rearrangements into a stable postfusion form to actuate the fusion between membranes. Various domains of F protein of some other paramyxoviruses, including HPIV3, have been reported to be differently functional. However, it is not yet clear what roles HRB linker plays. To clarify the roles that HRB linker might play in the F-mediated membrane fusion process, here we examined the effects of mutations introduced into the HRB linker of HPIV3 F protein. Six Single amino acid mutants, three chimeric mutants, and one deletion mutant were obtained and analyzed for membrane fusion activity and cell surface expression. The results showed that the membrane fusion activity of mutants changed to varying degrees in comparison with wild-type (wt) F, and some mutants even forfeited fusogenicity absolutely. It is indicated that the HRB linker domain plays an important role in the F-mediated membrane fusion process.

Virion-Associated Cholesterol Regulates the Infection of Human Parainfluenza Virus Type 3.

The matrix (M) proteins of paramyxoviruses bind to the nucleocapsids and cytoplasmic tails of glycoproteins, thus mediating the assembly and budding of virions. We first determined the budding characterization of the HPIV3 Fusion (F) protein to investigate the assembly mechanism of human parainfluenza virus type 3 (HPIV3). Our results show that expression of the HPIV3 F protein alone is sufficient to initiate the release of virus-like particles (VLPs), and the F protein can regulate the VLP-forming ability of the M protein. Furthermore, HPIV3F-Flag, which is a recombinant HPIV3 with a Flag tag at the C-terminus of the F protein, was constructed and recovered. We found that the M, F, and hemagglutinin-neuraminidase (HN) proteins and the viral genome can accumulate in lipid rafts in HPIV3F-Flag-infected cells, and the F protein mainly exists in the form of F1 in VLPs, lipid rafts, and purified virions. Furthermore, the function of cholesterol in the viral envelope and cell membrane was assessed via the elimination of cholesterol by methyl-ß-cyclodextrin (MßCD). Our results suggest that the infectivity of HPIV3 was markedly reduced, due to defective internalization ability in the absence of cholesterol. These results reveal that HPIV3 might assemble in the lipid rafts to acquire cholesterol for the envelope of HPIV3, which suggests the that disruption of the cholesterol composition of HPIV3 virions might be a useful method for the design of anti-HPIV3 therapy.

Inhibiting Human Parainfluenza Virus Infection by Preactivating the Cell Entry Mechanism.

Paramyxoviruses, specifically, the childhood pathogen human parainfluenza virus type 3, are internalized into host cells following fusion between the viral and target cell membranes. The receptor binding protein, hemagglutinin (HA)-neuraminidase (HN), and the fusion protein (F) facilitate viral fusion and entry into the cell through a coordinated process involving HN activation by receptor binding, which triggers conformational changes in the F protein to activate it to reach its fusion-competent state. Interfering with this process through premature activation of the F protein has been shown to be an effective antiviral strategy in vitro. Conformational changes in the F protein leading to adoption of the postfusion form of the protein-prior to receptor engagement of HN at the host cell membrane-render the virus noninfectious. We previously identified a small compound (CSC11) that implements this antiviral strategy through an interaction with HN, causing HN to activate F in an untimely process. To assess the functionality of such compounds, it is necessary to verify that the postfusion state of F has been achieved. As demonstrated by Melero and colleagues, soluble forms of the recombinant postfusion pneumovirus F proteins and of their six helix bundle (6HB) motifs can be used to generate postfusion-specific antibodies. We produced novel anti-HPIV3 F conformation-specific antibodies that can be used to assess the functionality of compounds designed to induce F activation. In this study, using systematic chemical modifications of CSC11, we synthesized a more potent derivative of this compound, CM9. Much like CSC11, CM9 causes premature triggering of the F protein through an interaction with HN prior to receptor engagement, thereby preventing fusion and subsequent infection. In addition to validating the potency of CM9 using plaque reduction, fusion inhibition, and binding avidity assays, we confirmed the transition to a postfusion conformation of F in the presence of CM9 using our novel anti-HPIV3 conformation-specific antibodies. We present both CM9 and these newly characterized postfusion antibodies as novel tools to explore and develop antiviral approaches. In turn, these advances in both our molecular toolset and our understanding of HN-F interaction will support development of more-effective antivirals. Combining the findings described here with our recently described physiologically relevant ex vivo system, we have the potential to inform the development of therapeutics to block viral infection.IMPORTANCE Paramyxoviruses, including human parainfluenza virus type 3, are internalized into host cells by fusion between viral and target cell membranes. The receptor binding protein, hemagglutinin-neuraminidase (HN), and the fusion protein (F) facilitate viral fusion and entry into cells through a process involving HN activation by receptor binding, which triggers conformational changes in F to activate it to reach its fusion-competent state. Interfering with this process through premature activation of the F protein may be an effective antiviral strategy in vitro We identified and optimized small compounds that implement this antiviral strategy through an interaction with HN, causing HN to activate F in an untimely fashion. To address that mechanism, we produced novel anti-HPIV3 F conformation-specific antibodies that can be used to assess the functionality of compounds designed to induce F activation. Both the novel antiviral compounds that we present and these newly characterized postfusion antibodies are novel tools for the exploration and development of antiviral approaches.

Cellular microRNA bta-miR-222 suppresses caprine parainfluenza virus type 3 replication via downregulation of interferon regulatory factor 2.

Caprine parainfluenza virus type 3 (CPIV3) is an important respiratory pathogen in the goat industry and was detected in China in 2014. Numerous studies have shown that microRNAs (miRNAs) play critical roles in viral infections, but the involvement of miRNAs during CPIV3 infection is poorly understood. In this study, we showed by deep sequencing that a panel of miRNAs, including bta-miR-222, were significantly downregulated in Madin-Darby bovine kidney (MDBK) cells infected with CPIV3 strain JS2013 compared with uninfected MDBK cells. Overexpression of bta-miR-222 significantly reduced CPIV3 replication in vitro, while inhibition of endogenous bta-miR-222 enhanced CPIV3 replication. Bta-miR-222 enhanced type I interferon expression and suppressed CPIV3 replication in MDBK cells. Moreover, we showed using luciferase reporter assays that bta-miR-222 directly targeted the 3'-untranslated region of interferon regulatory factor 2 (IRF2). Transfection of cells with bta-miR-222 mimics resulted in decreased IRF2 mRNA and protein levels, with a consequent increase in type I interferon levels and potentiation of antiviral responses. Together, these data demonstrate the important role of bta-miR-222 in restricting CPIV3 replication and suggest potential antiviral strategies against CPIV3.

The Sialic Acid Binding Activity of Human Parainfluenza Virus 3 and Mumps Virus Glycoproteins Enhances the Adherence of Group B Streptococci to HEp-2 Cells.

In the complex microenvironment of the human respiratory tract, different kinds of microorganisms may synergistically interact with each other resulting in viral-bacterial co-infections that are often associated with more severe diseases than the respective mono-infections. Human respiratory paramyxoviruses, for example parainfluenza virus type 3 (HPIV3), are common causes of respiratory diseases both in infants and a subset of adults. HPIV3 recognizes sialic acid (SA)-containing receptors on host cells. In contrast to human influenza viruses which have a preference for α2,6-linked sialic acid, HPIV3 preferentially recognize α2,3-linked sialic acids. Group B streptococci (GBS) are colonizers in the human respiratory tract. They contain a capsular polysaccharide with terminal sialic acid residues in an α2,3-linkage. In the present study, we report that HPIV3 can recognize the α2,3-linked sialic acids present on GBS. The interaction was evident not only by the binding of virions to GBS in a co-sedimentation assay, but also in the GBS binding to HPIV3-infected cells. While co-infection by GBS and HPIV3 had a delaying effect on the virus replication, it enhanced GBS adherence to virus-infected cells. To show that other human paramyxoviruses are also able to recognize the capsular sialic acid of GBS we demonstrate that GBS attaches in a sialic acid-dependent way to transfected BHK cells expressing the HN protein of mumps virus (MuV) on their surface. Overall, our results reveal a new type of synergism in the co-infection by respiratory pathogens, which is based on the recognition of α2,3-linked sialic acids. This interaction between human paramyxoviruses and GBS enhances the bacterial adherence to airway cells and thus may result in more severe disease.

Viral Entry Properties Required for Fitness in Humans Are Lost through Rapid Genomic Change during Viral Isolation.

Human parainfluenza viruses cause a large burden of human respiratory illness. While much research relies upon viruses grown in cultured immortalized cells, human parainfluenza virus 3 (HPIV-3) evolves in culture. Cultured viruses differ in their properties compared to clinical strains. We present a genome-wide survey of HPIV-3 adaptations to culture using metagenomic next-generation sequencing of matched pairs of clinical samples and primary culture isolates (zero passage virus). Nonsynonymous changes arose during primary viral isolation, almost entirely in the genes encoding the two surface glycoproteins-the receptor binding protein hemagglutinin-neuraminidase (HN) or the fusion protein (F). We recovered genomes from 95 HPIV-3 primary culture isolates and 23 HPIV-3 strains directly from clinical samples. HN mutations arising during primary viral isolation resulted in substitutions at HN's dimerization/F-interaction site, a site critical for activation of viral fusion. Alterations in HN dimer interface residues known to favor infection in culture occurred within 4 days (H552 and N556). A novel cluster of residues at a different face of the HN dimer interface emerged (P241 and R242) and imply a role in HPIV-3-mediated fusion. Functional characterization of these culture-associated HN mutations in a clinical isolate background revealed acquisition of the fusogenic phenotype associated with cultured HPIV-3; the HN-F complex showed enhanced fusion and decreased receptor-cleaving activity. These results utilize a method for identifying genome-wide changes associated with brief adaptation to culture to highlight the notion that even brief exposure to immortalized cells may affect key viral properties and underscore the balance of features of the HN-F complex required for fitness by circulating viruses.IMPORTANCE Human parainfluenza virus 3 is an important cause of morbidity and mortality among infants, the immunocompromised, and the elderly. Using deep genomic sequencing of HPIV-3-positive clinical material and its subsequent viral isolate, we discover a number of known and novel coding mutations in the main HPIV-3 attachment protein HN during brief exposure to immortalized cells. These mutations significantly alter function of the fusion complex, increasing fusion promotion by HN as well as generally decreasing neuraminidase activity and increasing HN-receptor engagement. These results show that viruses may evolve rapidly in culture even during primary isolation of the virus and before the first passage and reveal features of fitness for humans that are obscured by rapid adaptation to laboratory conditions.

Human Parainfluenza Virus Type 3 Matrix Protein Reduces Viral RNA Synthesis of HPIV3 by Regulating Inclusion Body Formation.

Human parainfluenza virus type 3 is one of the main causes of lower respiratory illness in newborns and infants. The role of the matrix protein (M) in viral budding is extensively studied, but the effect of M on viral replication remains to be determined. Using an HPIV3 minigenome assay, we found that M reduced HPIV3 mingenome-encoded reporter activity even though it had an unspecific effect on the expression of cellular genes. Furthermore, the inhibition effect of M on viral RNA synthesis was proven to be independent of its virus-like particles (VLPs)' release ability. A VLP's defective mutant (ML302A) decreased the expression of minigenome reporter as wild type M did. Using an immunofluorescence assay, we found that M weakened the formation of inclusion bodies (IBs), although it did not co-localize with the IBs. Moreover, using another mutant, ML305A , which is defective in M-nucleoprotein (N) interaction, we found that ML305A had no effect on reporter activity and IB formation as the wild type of M did. Taken together, we conclude that M reduces the replication of HPIV3 and IB formation by M-N interaction.

Inclusion bodies of human parainfluenza virus type 3 inhibit antiviral stress granule formation by shielding viral RNAs.

Viral invasion triggers the activation of the host antiviral response. Besides the innate immune response, stress granules (SGs) also act as an additional defense response to combat viral replication. However, many viruses have evolved various strategies to suppress SG formation to facilitate their own replication. Here, we show that viral mRNAs derived from human parainfluenza virus type 3 (HPIV3) infection induce SG formation in an eIF2α phosphorylation- and PKR-dependent manner in which viral mRNAs are sequestered and viral replication is inhibited independent of the interferon signaling pathway. Furthermore, we found that inclusion body (IB) formation by the interaction of the nucleoprotein (N) and phosphoprotein (P) of HPIV3 correlated with SG suppression. In addition, co-expression of P with NL478A (a point mutant of N, which is unable to form IBs with P) or with NΔN10 (lacking N-terminal 10 amino acids of N, which could form IBs with P but was unable to synthesize or shield viral RNAs) failed to inhibit SG formation, suggesting that inhibition of SG formation also correlates with the capacity of IBs to synthesize and shield viral RNAs. Therefore, we provide a model whereby viral IBs escape the antiviral effect of SGs by concealing their own newly synthesized viral RNAs and offer new insights into the emerging role of IBs in viral replication.