Human Parainfluenza Virus Type 3 Infection Research Articles

Inefficient antiviral response in reconstituted small-airway epithelium from chronic obstructive pulmonary disease patients following human parainfluenza virus type 3 infection

Chronic obstructive pulmonary disease (COPD) affects over 250 million individuals globally and stands as the third leading cause of mortality. Respiratory viral infections serve as the primary drivers of acute exacerbations, hastening the decline in lung function and worsening the prognosis. Notably, Human Parainfluenza Virus type 3 (HPIV-3) is responsible for COPD exacerbations with a frequency comparable to that of Respiratory Syncytial Virus and Influenza viruses. However, the impact of HPIV-3 on respiratory epithelium within the context of COPD remains uncharacterized.In this study, we employed in vitro reconstitution of lower airway epithelia from lung tissues sourced from healthy donors (n = 4) and COPD patients (n = 5), maintained under air–liquid interface conditions. Through a next-generation sequencing-based transcriptome analysis, we compared the cellular response to HPIV-3 infection.Prior to infection, COPD respiratory epithelia exhibited a pro-inflammatory profile, notably enriched in canonical pathways linked to antiviral response, B cell signaling, IL-17 signaling, and epithelial-mesenchymal transition, in contrast to non-COPD epithelia. Intriguingly, post HPIV-3 infection, only non-COPD epithelia exhibited significant enrichment in interferon signaling, pattern recognition receptors of viruses and bacteria, and other pathways involved in antiviral responses. This deficiency could potentially hinder immune cell recruitment essential for controlling viral infections, thus fostering prolonged viral presence and persistent inflammation.

Epidemiological Characteristics of Parainfluenza Virus Type 3 and the Effects of Meteorological Factors in Hospitalized Children With Lower Respiratory Tract Infection.

ObjectiveTo investigate the relationship between meteorological factors and Human parainfluenza virus type 3 (HPIV-3) infection among hospitalized children.MethodsAll hospitalized children with acute lower respiratory tract infections were tested for viral pathogens and enrolled, at the second affiliated hospital of Wenzhou medical university, between 2008 and 2017. Meteorological data were directly obtained from Wenzhou Meteorology Bureau's nine weather stations and expressed as the mean exposure for each 10-day segment (average daily temperatures, average daily relative humidity, rainfall, rainfall days, and wind speed). The correlation between meteorological factors and the incidence of HPIV-3 was analyzed, with an autoregressive integrated moving average model (ARIMA), generalized additive model (GAM), and least absolute shrinkage and selection operator (LASSO).ResultsA total of 89,898 respiratory specimens were tested with rapid antigen tests, and HPIV-3 was detected in 3,619 children. HPIV-3 was detected year-round, but peak activities occurred most frequently from March to August. The GAM and LASSO-based model had revealed that HPIV-3 activity correlated positively with temperature and rainfall day, but negatively with wind speed. The ARIMA (1,0,0)(0,1,1) model well-matched the observed data, with a steady R2 reaching 0.708 (Ljung-Box Q = 21.178, P = 0.172).ConclusionOur study suggests that temperature, rainfall days, and wind speed have significant impacts on the activity of HPIV-3. GAM, ARIMA, and LASSO-based models can well predict the seasonality of HPIV-3 infection among hospitalized children. Further understanding of its mechanism would help facilitate the monitoring and early warning of HPIV-3 infection.

Human parainfluenza virus type 3 infections in a haemato-oncology unit: social distancing measures needed in outpatient clinics

Human parainfluenza virus type 3 (HPIV3) infections are associated with high mortality in immunocompromised settings, especially in bone marrow transplant recipients. Asymptomatic infection and lack of effective antiviral treatment makes HPIV3 prevention and treatment a real challenge. To retrospectively investigate the epidemiological characteristics, clinical characteristics and outcomes of 51 haematology patients with confirmed HPIV3 infections, detected between February and May 2019 in the haematology unit at King's College Hospital, London. Between February and May 2019, HPIV3 RNA was detected in combined nose and throat swab samples collected from 51 symptomatic haematology patients, 41 of whom attended the haematology outpatient unit. Clinical data were reviewed retrospectively and a timeline of patients' appointments drawn up to investigate transmission. Sequencing analysis was performed on 14 stored samples. Fifty-one patients were identified with HPIV3 infection. Mean age was 54 years (SD: 12; range: 19-72) and 60% (31/51) were male. There were 41 (80%) bone marrow transplant recipients, 24 had an allograft, and 17 an autograft. Thirty-day and 3-month mortality post HPIV3 was 6% and 14%, respectively. Lower respiratory tract infection and inpatient acquisition were associated with higher mortality (6/7 vs 1/7, P= 0.010; and 5/7 vs 2/7, P= 0.031). Onset of HPIV3 infection in patients within 6 days of attending the clinic was associated with the clusters identified in phylogenetic analysis (64% (9/14) vs 21% (8/37); odds ratio: 6.5 (confidence interval: 95% 1.7-25); P= 0.006). Timelines suggested community transmission, but also possible transmission patterns within the outpatients and subsequent nosocomial transmission within the same ward. Early recognition of HPIV3 infection and the use of polymerase chain reaction and sequence analysis is fundamental in identifying respiratory virus outbreaks and person-to-person transmission. Careful planning of outpatient clinic attendance is required to minimize contact and prevent respiratory virus transmission in immunosuppressed patients.

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.

Frequent lower respiratory tract disease in hematological patients with parainfluenza virus type 3 infection.

Human parainfluenza virus type 3 (HPIV-3) may cause lower respiratory tract infection disease (LRTI-D) after hematopoietic stem cell transplantation (HSCT). Most previous have studies focused on recipients of HSCT whereas data on characteristics and outcomes in patients with hematological malignancies (HMs) compared to non-hematological patients are limited. The prognostic value of viral load in respiratory specimens remains elusive. In a 2-year retrospective study, we determined the frequencies of LRTI-D in HM, HSCT, and in non-hematological patients, and HPIV-3 levels in respiratory tract secretions. Among 98 patients with HPIV-3 infection, including 31 HSCT and 40 HM, 36 had a diagnosis of LRTI-D. LRTI-D was significantly more frequent in patients with HM or HSCT (n = 32, 45.1%) than in non-hematological patients (n = 4, 14.8%) (p = 0.006). The median HPIV-3 loads were high in upper respiratory tract secretions regardless of the presence or absence of LRTI-D (8.3 log10 vs. 7.6 log10 TCID50 /106 cells). HPIV-3 loads in respiratory tract samples in HM were not significantly higher than those found in HSCT but significantly higher than in non-hematological patients (p = 0.007). In conclusion, LRTI-D was frequent in HM patients who were diagnosed with HPIV-3 infection.

Involvement of Actin-Regulating Factor Cofilin in the Inclusion Body Formation and RNA Synthesis of Human Parainfluenza Virus Type 3 via Interaction With the Nucleoprotein.

Human parainfluenza virus type 3 (HPIV3) is one of the primary pathogens that causing severe respiratory tract diseases in newborns and infants. It could induce inclusion bodies (IBs) in infected cells. Comprised of viral nucleoprotein (N) and phosphoprotein (P), as well as some cellular factors, HPIV3 IBs are unique platform for efficient viral synthesis. Although several studies have demonstrated the formation of IBs, little is known about cellular proteins involved in HPIV3 IBs formation. By quantitative real-time PCR assays after cytochalasin D treatment, we found actin microfilaments of the cytoskeleton were indispensible for HPIV3 RNA synthesis. Using co-immunoprecipitation and immunofluorescence assays, an actin-modulating protein, cofilin was found to involve in the IBs formation through interaction with the N protein in N–P induced IBs complex. Viral IBs formation reduced upon RNA interference knockdown of cellular cofilin, thus viral RNA synthesis and protein expression level were also suppressed. What’s more, the inactive form of cofilin, p-cofilin was increased after HPIV3 infection, and phosphorylation of cofilin was required for interacting with N–P complex and IBs formation. We further identified that the regions in cofilin interacting with N protein lies in the C-terminus. Our findings for the first time to state that cellular cofilin involves in HPIV3 IBs and interaction with N is critical for cofilin to aid IBs formation and enhancing viral RNA synthesis.

A molecular epidemiological study of human parainfluenza virus type 3 at a tertiary university hospital during 2013–2015 in Catalonia, Spain

Human parainfluenza virus type 3 (HPIV-3) is one of the most common respiratory viruses particularly among young children and immunocompromised patients. The seasonality, prevalence and genetic diversity of HPIV-3 at a Spanish tertiary-hospital from 2013 to 2015 are reported. HPIV-3 infection was laboratory-confirmed in 102 patients (76%, under 5 years of age). Among <5 years-old patients, 9 (11.5%) were under any degree of immunosuppression, whereas this percentage was significantly higher (19; 79.2%) among patients older than 5 years. HPIV-3 was detected at varying levels, but mainly during spring and summer. All characterized HN/F sequences fell within C1b, C5 and in other two closely C3a-related groups. Furthermore, a new genetic lineage (C1c) was described. Genetic similarity and epidemiological data confirmed some nosocomial infections, highlighting the importance of the HPIV-3 surveillance, particularly in high-risk patients. This study provides valuable information on HPIV-3 diversity due to the scarce information in Europe.

Human parainfluenza virus type 3 (HPIV3) induces production of IFNγ and RANTES in human nasal epithelial cells (HNECs).

BackgroundHuman parainfluenza virus type 3 (HPIV3), while infecting lower airway epithelial cells induces pneumonia and bronchiolitis in infants and children, and may lead to asthma exacerbations in children and adults. Respiratory viruses invading the airway epithelium activate innate immune response and induce inflammatory cytokine release contributing to the pathophysiology of upper and lower airway disorders. However, the effects of HPIV3 infection on nasal epithelial cells have not been well defined.The aim of this study was to evaluate the effect of the HPIV3 infection on cultured human nasal epithelial cells (HNECs) and the release of interferon gamma and other cytokines.MethodsRPMI 2650, a human nasal epithelial cell line was cultured into confluence and was infected with HPIV3 (MOI of 0.1, 0.01 and 0.001). The protein release into supernatants and mRNA expression of selected cytokines were assessed 24, 48 and 72 h after infection. Cytokine concentrations in supernatants were measured by ELISA and expression of cytokine mRNA in RPMI 2650 cells confirmed by real time RT-PCR analysis.ResultsHNECs infection with HPIV3 did not induce cytotoxicity for at least 48 hours, but significantly increased IFN-γ protein concentration in the cell supernatants at 24 h and 48 h post infection (by 387% and 485% respectively as compared to mock infected cells). At 24 h a significant increase in expression of mRNA for IFNγ was observed. RANTES protein concentration and mRNA expression were significantly increased at 72 h after infection (mean protein concentration: 3.5 ± 1.4 pg/mL for 0.001 MOI, 10.8 ± 4.6 pg/mL for 0.01 MOI and 61.5 ± 18.4 pg/mL for 0.1 MOI as compared to 2.4 ± 1.3 pg/mL for uninfected cells). No measurable concentrations of TNF-α, IL-10, TSLP, IL-8, GM-CSF or eotaxin, were detected in virus infected cells supernatants.ConclusionsHPIV3 effectively infects upper airway epithelial cells and the infection is associated with induction of IFN-γ and generation of RANTES.

Interaction between the Hemagglutinin-Neuraminidase and Fusion Glycoproteins of Human Parainfluenza Virus Type III Regulates Viral Growth In Vivo

ABSTRACT Paramyxoviruses, enveloped RNA viruses that include human parainfluenza virus type 3 (HPIV3), cause the majority of childhood viral pneumonia. HPIV3 infection starts when the viral receptor-binding protein engages sialic acid receptors in the lung and the viral envelope fuses with the target cell membrane. Fusion/entry requires interaction between two viral surface glycoproteins: tetrameric hemagglutinin-neuraminidase (HN) and fusion protein (F). In this report, we define structural correlates of the HN features that permit infection in vivo. We have shown that viruses with an HN-F that promotes growth in cultured immortalized cells are impaired in differentiated human airway epithelial cell cultures (HAE) and in vivo and evolve in HAE into viable viruses with less fusogenic HN-F. In this report, we identify specific structural features of the HN dimer interface that modulate HN-F interaction and fusion triggering and directly impact infection. Crystal structures of HN, which promotes viral growth in vivo, show a diminished interface in the HN dimer compared to the reference strain’s HN, consistent with biochemical and biological data indicating decreased dimerization and decreased interaction with F protein. The crystallographic data suggest a structural explanation for the HN’s altered ability to activate F and reveal properties that are critical for infection in vivo.IMPORTANCE Human parainfluenza viruses cause the majority of childhood cases of croup, bronchiolitis, and pneumonia worldwide. Enveloped viruses must fuse their membranes with the target cell membranes in order to initiate infection. Parainfluenza fusion proceeds via a multistep reaction orchestrated by the two glycoproteins that make up its fusion machine. In vivo, viruses adapt for survival by evolving to acquire a set of fusion machinery features that provide key clues about requirements for infection in human beings. Infection of the lung by parainfluenzavirus is determined by specific interactions between the receptor binding molecule (hemagglutinin-neuraminidase [HN]) and the fusion protein (F). Here we identify specific structural features of the HN dimer interface that modulate HN-F interaction and fusion and directly impact infection. The crystallographic and biochemical data point to a structural explanation for the HN’s altered ability to activate F for fusion and reveal properties that are critical for infection by this important lung virus in vivo.

Human Parainfluenza Virus Type 3 in Wild Nonhuman Primates, Zambia

Human parainfluenza virus type 3 (HPIV3) genome was detected in 4 baboons in Zambia. Antibody for HPIV3 was detected in 13 baboons and 6 vervet monkeys in 2 distinct areas in Zambia. Our findings suggest that wild nonhuman primates are susceptible to HPIV3 infection.

Multiplex real-time PCR for prompt diagnosis of an outbreak of human parainfluenza 3 virus in children with acute leukemia

IntroductionHuman parainfluenza virus type 3 (HPIV-3) causes significant morbimortality in immunocompromised patients. Outbreaks of severe pneumonitis have been previously described in this setting.Materials and methodsRetrospective observational study in children diagnosed with acute leukemia and a documented HPIV-3 infection in the context of a nosocomial outbreak occurred in a single center.ResultDuring summer 2012, an HPIV-3 infection was detected in six hospitalized children with acute leukemia. All the patients had respiratory symptoms and one of them suffered from parotitis.ConclusionEarly diagnoses using multiplex real-time polymerase chain reaction (PCR) let us control this outbreak. A phylogenetic analysis confirmed person-to-person transmission of a single HPIV-3 variant.

Retinoic acid inducible gene I Activates innate antiviral response against human parainfluenza virus type 3

Human parainfluenza virus type 3 (HPIV3) is a respiratory paramyxovirus that infects lung epithelial cells to cause high morbidity among infants and children. To date, no effective vaccine or antiviral therapy exists for HPIV3 and therefore, it is important to study innate immune antiviral response induced by this virus in infected cells. Type-I interferons (IFN, interferon-α/β) and tumor necrosis factor-α (TNFα activated by NFκB) are potent antiviral cytokines that play an important role during innate immune antiviral response. A wide-spectrum of viruses utilizes pattern recognition receptors (PRRs) like toll-like receptors (TLRs) and RLH (RIG like helicases) receptors such as RIGI (retinoic acid inducible gene -I) and Mda5 to induce innate antiviral response. Previously it was shown that both TNFα and IFNβ are produced from HPIV3 infected cells. However, the mechanism by which infected cells activated innate response following HPIV3 infection was not known. In the current study, we demonstrated that RIGI serves as a PRR in HPIV3 infected cells to induce innate antiviral response by expressing IFNβ (via activation of interferon regulatory factor-3 or IRF3) and TNFα (via activation of NF-κB).

Inhibition of human parainfluenza virus type 3 infection by novel small molecules

Human parainfluenza virus type 3 (HPIV3) is an important respiratory tract pathogen of infants and children. There are no vaccines or antivirals currently approved for prevention or treatment of HPIV3 infection. Towards developing an antiviral therapy to combat HPIV3 infection, we have established a green fluorescent protein (GFP)-tagged HPIV3 infected-cell assay and used it for screening of a small molecule library obtained from ChemBridge Diver. Two novel small molecules (C5 and C7) which shared structural similarities were identified and their inhibitory effects on HPIV3 were confirmed in CV-1 and human lung epithelium A549 cells by plaque assay, Western blot and Northern blot analyses. C5 and C7 effectively prevented the cytopathic effect in cells infected with HPIV3, achieving IC(50) values of 2.36 microM and 0.08 microM, respectively, for infectious virus production. The inhibition appears to be at the primary transcriptional level of HPIV3 life cycle based on sequential time course test, binding and internalization assays, and finally by a minigenome transcription assay in cells as well as measuring viral transcripts in cells in the presence of anisomycin. Interestingly, vesicular stomatitis virus (VSV), another member of mononegavirales order, was also inhibited by these compounds, whereas poliovirus-a picornavirus was not. Use of these inhibitors has a strong potential to develop novel antiviral agents against this important human pathogen.

Successful Topical Respiratory Tract Immunization of Primates against Ebola Virus

Ebola virus causes outbreaks of severe viral hemorrhagic fever with high mortality in humans. The virus is highly contagious and can be transmitted by contact and by the aerosol route. These features make Ebola virus a potential weapon for bioterrorism and biological warfare. Therefore, a vaccine that induces both systemic and local immune responses in the respiratory tract would be highly beneficial. We evaluated a common pediatric respiratory pathogen, human parainfluenza virus type 3 (HPIV3), as a vaccine vector against Ebola virus. HPIV3 recombinants expressing the Ebola virus (Zaire species) surface glycoprotein (GP) alone or in combination with the nucleocapsid protein NP or with the cytokine adjuvant granulocyte-macrophage colony-stimulating factor were administered by the respiratory route to rhesus monkeys--in which HPIV3 infection is mild and asymptomatic--and were evaluated for immunogenicity and protective efficacy against a highly lethal intraperitoneal challenge with Ebola virus. A single immunization with any construct expressing GP was moderately immunogenic against Ebola virus and protected 88% of the animals against severe hemorrhagic fever and death caused by Ebola virus. Two doses were highly immunogenic, and all of the animals survived challenge and were free of signs of disease and of detectable Ebola virus challenge virus. These data illustrate the feasibility of immunization via the respiratory tract against the hemorrhagic fever caused by Ebola virus. To our knowledge, this is the first study in which topical immunization through respiratory tract achieved prevention of a viral hemorrhagic fever infection in a primate model.

Recombinant Newcastle Disease Virus Expressing a Foreign Viral Antigen Is Attenuated and Highly Immunogenic in Primates

Paramyxoviruses such as human parainfluenza viruses that bear inserts encoding protective antigens of heterologous viruses can induce an effective immunity against the heterologous viruses in experimental animals. However, vectors based on common human pathogens would be expected to be restricted in replication in the adult human population due to high seroprevalence, an effect that would reduce vector immunogenicity. To address this issue, we evaluated Newcastle disease virus (NDV), an avian paramyxovirus that is serotypically distinct from common human pathogens, as a vaccine vector. Two strains were evaluated: the attenuated vaccine strain LaSota (NDV-LS) that replicates mostly in the chicken respiratory tract and the Beaudette C (NDV-BC) strain of intermediate virulence that produces mild systemic infection in chickens. A recombinant version of each virus was modified by the insertion, between the P and M genes, of a gene cassette encoding the human parainfluenza virus type 3 (HPIV3) hemagglutinin-neuraminidase (HN) protein, a test antigen with considerable historic data. The recombinant viruses were administered to African green monkeys (NDV-BC and NDV-LS) and rhesus monkeys (NDV-BC only) by combined intranasal and intratracheal routes at a dose of 10(6.5) PFU per site, with a second equivalent dose administered 28 days later. Little or no virus shedding was detected in nose-throat swabs or tracheal lavages following immunization with either strain. In a separate experiment, direct examination of lung tissue confirmed a highly attenuated, restricted pattern of replication by parental NDV-BC. The serum antibody response to the foreign HN protein induced by the first immunization with either NDV vector was somewhat less than that observed following a wild-type HPIV3 infection; however, the titer following the second dose exceeded that observed with HPIV3 infection, even though HPIV3 replicates much more efficiently than NDV in these animals. NDV appears to be a promising vector for the development of vaccines for humans; one application would be in controlling localized outbreaks of emerging pathogens.

MHC Class II Gene Expression Is Not Induced in HPIV3-Infected Respiratory Epithelial Cells

The major histocompatibility complex (MHC) class II transactivator (CIITA) typically is required for both constitutive and inducible expression of MHC class II genes. However, transcription of class II MHC genes has been observed in specific cell types (e.g., thymic epithelial cells) in CIITA-deficient mice as well as in specific situations (e.g., following viral infections or in natural killer [NK]/target cell interaction). These observations have been interpreted by some to indicate that a CIITA-independent pathway of class II gene expression might be germane to processes such as the acquisition of tolerance during thymic selection or in the evasion of immune surveillance by a subset of viruses. One of the most striking examples of CIITA-independent, inducible class II gene expression has involved the de novo expression of class II MHC molecules on respiratory epithelial cells following infection by human parainfluenza virus type 3 (HPIV3). We report here that despite careful analysis using multiple techniques, we have been unable to detect HPIV3-dependent, CIITA-independent (or CIITA-dependent) induction of class II MHC genes. Thus, whereas there may still be an intriguing role for CIITA-independent gene expression in facets of the immune response, this is unlikely to manifest in the analysis of HPIV3 infection of respiratory epithelial cells.

Human parainfluenza virus type 3 HN-receptor interaction: effect of 4-guanidino-Neu5Ac2en on a neuraminidase-deficient variant.

The envelope of human parainfluenza virus type 3 (HPF3) contains two viral glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion protein (F). HN, which is responsible for receptor attachment and for promoting F-mediated fusion, also possesses neuraminidase (receptor-destroying) activity. We reported previously that 4-guanidino-neu5Ac2en (4-GU-DANA) and related sialic acid-based inhibitors of HPF3 neuraminidase activity also inhibit HN-mediated receptor binding and fusion processes not involving neuraminidase activity. We have now examined this mechanism, as well as neuraminidase's role in the viral life cycle, using a neuraminidase-deficient HPF3 variant (C28a) and stable cell lines expressing C28a or wild-type (wt) HN. C28a, which has a wt F sequence and two point mutations in the HN gene corresponding to two amino acid changes in the HN protein, is the first HPF3 variant with insignificant neuraminidase activity. Cells expressing C28a HN did not bind erythrocytes at 4 degrees C unless pretreated with neuraminidase, but no such pretreatment was required for hemadsorption activity (HAD) at 22 or 37 degrees C. HAD was blocked by 4-GU-DANA, attesting to the ability of this compound to inhibit HN's receptor-binding activity. C28a or wt plaque enlargement, a process that involves cell-cell fusion and does not depend on virion release, is diminished by the presence of 4-GU-DANA, confirming the inhibitory effect of 4-GU-DANA on the fusogenic function of C28a HN. In C28a-infected cell monolayers, virion release and thus multicycle replication are severely restricted. This defect was corrected by supplementation of exogenous neuraminidase and also by the addition of 4-GU-DANA; neuraminidase destroys the receptors whereby newly formed C28a virions would remain attached to the cell surface, whereas 4-GU-DANA prevents the attachment itself, obviating the need for receptor cleavage. In accord with the ability of 4-GU-DANA to prevent attachment, the neuraminidase inhibitory effect of 4-GU-DANA on wt HPF3 did not diminish virion release into the medium. Thus, it is by inhibition of viral entry and syncytium formation that sialic acid analogs like 4-GU-DANA may counteract wt HPF3 infection.

Human parainfluenza virus type 3 upregulates ICAM-1 (CD54) expression in a cytokine-independent manner.

Human parainfluenza virus type 3 (HPIV3) causes bronchiolitis, pneumonia, and croup in newborns and infants. Several studies have implicated intercellular adhesion molecule-1 (ICAM-1) in inflammation during infection by viruses. In this study, we investigated the potential for HPIV3 to induce ICAM-1 in HT1080 cells. FACS analysis showed that HPIV3 strongly induced ICAM-1 expression in these cells. The ICAM-1 induction was significantly reduced when the virions were UV inactivated prior to infection, indicating that ICAM-1 induction was mostly viral replication dependent. Culture supernatant of HPIV3-infected cells induced ICAM-1 at an extremely low level, indicating that virus-induced cytokines played only a minor role in the induction process. Consistent with this, potent inducers of ICAM-1 such as IFN-gamma, TGF-beta, and TNF-alpha were absent in the culture supernatant, but a significant amount of IFN type 1 was present. By using U2A cells, which are defective in IFN type I signaling, we confirmed that ICAM-1 induction by HPIV3 occurred in a JAK/STAT signaling-independent manner. These data strongly indicate that HPIV3 induces ICAM-1 directly by viral antigens in a cytokine-independent manner; this induction may play a role in the inflammation during HPIV3 infection.

Mechanism of interference mediated by human parainfluenza virus type 3 infection.

Viral interference is characterized by the resistance of infected cells to infection by a challenge virus. Mechanisms of viral interference have not been characterized for human parainfluenza virus type 3 (HPF3), and the possible role of the neuraminidase (receptor-destroying) enzyme of the hemagglutinin-neuraminidase (HN) glycoprotein has not been assessed. To determine whether continual HN expression results in depletion of the viral receptors and thus prevents entry and cell fusion, we tested whether cells expressing wild-type HPF3 HN are resistant to viral infection. Stable expression of wild-type HN-green fluorescent protein (GFP) on cell membranes in different amounts allowed us to establish a correlation between the level of HN expression, the level of neuraminidase activity, and the level of protection from HPF3 infection. Cells with the highest levels of HN expression and neuraminidase activity on the cell surface were most resistant to infection by HPF3. To determine whether this resistance is attributable to the viral neuraminidase, we used a cloned variant HPF3 HN that has two amino acid alterations in HN leading to the loss of detectable neuraminidase activity. Cells expressing the neuraminidase-deficient variant HN-GFP were not protected from infection, despite expressing HN on their surface at levels even higher than the wild-type cell clones. Our results demonstrate that the HPF3 HN-mediated interference effect can be attributed to the presence of an active neuraminidase enzyme activity and provide the first definitive evidence that the mechanism for attachment interference by a paramyxovirus is attributable to the viral neuraminidase.

The Use of a Quantitative Fusion Assay to Evaluate HN–Receptor Interaction for Human Parainfluenza Virus Type 3

Sialic acid is the receptor determinant for the human parainfluenza virus type 3 (HPF3) hemagglutinin–neuraminidase (HN) glycoprotein, the molecule responsible for binding of the virus to cell surfaces. In order for the fusion protein (F) of HPF3 to promote membrane fusion, HN must interact with its receptor. In addition to its role in receptor binding and fusion promotion, the HPF3 HN molecule contains receptor-destroying (sialidase) activity. The putative active sites are in the extracellular domain of this type II integral membrane protein. However, HN is not available in crystalline form; the exact locations of these sites, and the structural requirements for binding to the cellular receptor, which has not yet been isolated, are unknown. Nor have small molecular synthetic inhibitors of attachment or fusion that would provide insight into these processes been identified. The strategy in the present study was to develop an assay system that would provide a measure of a specific step in the viral cycle—functional interaction between viral glycoproteins and the cell during attachment and fusion—and serve to screen a variety of substances for inhibitory potential. The assay is based on our previous finding that CV-1 cells persistently infected (p.i.) with HPF3 do not fuse with one another but that the addition of uninfected CV-1 cells, supplying the critical sialic acid containing receptor molecules that bind HN, results in rapid fusion. In the present assay two HeLa cell types were used: we persistently infected HeLa-LTR-βgal cells, assessed their fusion with uninfected HeLa-tat cells, and then quantitated the β-galactosidase (βgal) produced as a result of this fusion. The analog α-2-S-methyl-5-N-thioacetylneuraminic acid (α-Neu5thioAc2SMe) interfered with fusion, decreasing βgal production by 84% at 50 mM and by 24% at 25 mM. In beginning to extend our studies to different types of molecules, we tested an unsaturated derivative of sialic acid, 2,3-dehydro-2-deoxy-n-acetyl neuraminic acid (DANA), which is known to inhibit influenza neuraminidase by virtue of being a transition-state analog. We found that 10 mM DANA inhibited neuraminidase activity in HPF3 viral preparations. More significantly, this compound was active in our assay of HN–receptor interaction; 10 mM DANA completely blocked fusion and βgal production, and hemadsorption inhibition by DANA suggested that DANA blocks attachment. In plaque reduction assays performed with the compounds, the active analog α-Neu5thioAc2SMe reduced plaque formation by 50% at a 50 mM concentration; DANA caused a 90% inhibition in the plaque reduction assay at a concentration of 25 mM. Our results indicate that specific sialic acid analogs that mimic the cellular receptor determinant of HPF3 can block virus cell interaction and that an unsaturated n-acetyl-neuraminic acid derivative with affinity to the HN site responsible for neuraminidase activity also interferes with HN–receptor binding. Strategies suggested by these findings are now being pursued to obtain information regarding the relative locations of the active sites of HN and to further elucidate the relationship between the receptor-binding and receptor-destroying activities of HN during the viral life cycle. The quantitative assay that we describe is of immediate applicability to large-scale screening for potential inhibitors of HPF3 infection in vivo.