Incidence,molecular epidemiology and clinical presentations of human metapneumovirus; assessment of its importance as a diagnostic screening target

BackgroundHuman metapneumovirus (HMPV) is a recently discovered human paramyxovirus associated with a spectrum of respiratory symptoms from the common cold to pneumonia and bronchiolitis.ObjectivesTo assess the clinical significance and epidemiology of HMPV, standardized comparison of frequencies of infection, age profiles and disease associations were made with other respiratory viruses in Scotland.Study design7091 respiratory samples collected in Scotland between 1 July 2006 and 30 June 2008 from 4282 individuals were screened by multiplex RT-PCR for respiratory syncytial virus (HRSV), adenovirus (AdV), parainfluenzaviruses 1–3 (PIV-1, -2 and -3), influenza A and B and by nested RT-PCR for HMPV.ResultsHMPV was the fifth most prevalent virus (2.0% of samples), found predominantly in young children in winter months. In the 2006–2007 respiratory season, 70% of HMPV isolates were genotype A, but a switch to predominantly type B infections occurred next winter. For samples with information on clinical presentations, 26% of HMPV infections were from subjects with lower respiratory tract presentations, lower than recorded for HRSV, but similar to adenovirus, parainfluenza viruses and influenza viruses A and B. Around 13% of HMPV infections were associated with upper respiratory tract symptoms or disease, comparable with other respiratory virus infections.ConclusionsNumerically and through its association with respiratory disease, HMPV represents a diagnostically significant target that should be included in PCR-based routine screening of respiratory samples. Understanding the biological basis of observed rapid turnover of HMPV variants, including the observed HMPV genotype change between respiratory seasons requires further longitudinal studies.     

Frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections

Viruses are the major cause of pediatric acute respiratory tract infection (ARTI) and yet many suspected cases of infection remain uncharacterized. We employed 17 PCR assays and retrospectively screened 315 specimens selected by season from a predominantly pediatric hospital-based population. Before the Brisbane respiratory virus research study commenced, one or more predominantly viral pathogens had been detected in 15.2% (n = 48) of all specimens. The Brisbane study made an additional 206 viral detections, resulting in the identification of a microbe in 67.0% of specimens. After our study, the majority of microbes detected were RNA viruses (89.9%). Overall, human rhinoviruses (HRVs) were the most frequently identified target (n = 140) followed by human adenoviruses (HAdVs; n = 25), human metapneumovirus (HMPV; n = 18), human bocavirus (HBoV; n = 15), human respiratory syncytial virus (HRSV; n = 12), human coronaviruses (HCoVs; n = 11), and human herpesvirus-6 (n = 11). HRVs were the sole microbe detected in 37.8% (n = 31) of patients with suspected lower respiratory tract infection (LRTI). Genotyping of the HRV VP4/VP2 region resulted in a proposed subdivision of HRV type A into sublineages A1 and A2. Most of the genotyped HAdV strains were found to be type C. This study describes the high microbial burden imposed by HRVs, HMPV, HRSV, HCoVs, and the newly identified virus, HBoV on a predominantly paediatric hospital population with suspected acute respiratory tract infections and proposes a new formulation of viral targets for future diagnostic research studies.     

Human parainfluenza virus types 1–4 in hospitalized children with acute lower respiratory infections in China

Human parainfluenza viruses (HPIVs) are an important cause of acute lower respiratory tract infections (ALRTIs). HPIV‐4, a newly identified virus, has been associated with severe ALRTIs recently. A total of 771 nasopharyngeal aspirate samples were collected from hospitalized children between March 2010 and February 2011. HPIVs were detected by Nest‐PCR, and other known respiratory viruses were detected by RT‐PCR and PCR. All amplification products were sequenced. HPIVs were detected in 151 (19.58%) patients, of whom 28 (3.63%) were positive for HPIV‐4, 12(1.55%) for HPIV‐1, 4 (0.51%) for HPIV‐2, and 107 (13.87%) for HPIV‐3. Only three were found to be co‐infected with different types of HPIVs. All HPIV‐positive children were under 5 years of age, with the majority being less than 1 year. Only the detection rate of HPIV‐3 had a significant statistical difference (χ² = 29.648, P = 0.000) between ages. HPIV‐3 and HPIV‐4 were detected during the summer. Sixty (39.74%) were co‐infected with other respiratory viruses, and human rhinovirus (HRV) was the most common co‐infecting virus. The most frequent clinical diagnosis was bronchopneumonia, and all patients had cough; some patients who were infected with HPIV‐3 and HPIV‐4 had polypnea and cyanosis. No significant difference was found in clinical manifestations between those who were infected with HPIV‐4 and HPIV‐3. Two genotypes for HPIV‐4 were prevalent, although HPIV‐4a dominated. HPIV‐4 is an important virus for children hospitalized with ALRTIs in China. HRV was the most common co‐infecting virus. Two genotypes for HPIV‐4 are prevalent, HPIV‐4a dominated. J. Med. Virol. 88:2085–2091, 2016. © 2016 Wiley Periodicals, Inc.

     

Human parainfluenza virus 3 neuraminidase activity contributes to dendritic cell maturation

Mechanisms of dendritic cells (DCs) immunomodulation by parainfluenza viruses have not been characterized. We analyzed whether the human parainfluenza 3 (HPF3) virus hemagglutinin-neuraminidase glycoprotein (HN) might influence DC maturation. HN possesses a receptor binding function and a neuraminidase or desialidating activity. To assess whether the neuraminidase activity of HN affects DC maturation, human myeloid DCs were exposed to either live or UV-inactivated HPF3 viruses containing wild type or a mutated form of HN with decreased neuraminidase activity. Exposure of human DCs to either UV-inactivated or live virus induced up-regulation of CD83 and CD86 surface markers, morphological changes, and a cytokine expression pattern consistent with maturation. However, the level of maturation was found to be lower in DCs infected with the neuraminidase deficient variant as compared to the wild type. These results suggest that during the course of viral infection, HN's neuraminidase activity may play an important role contributing to maturation and activation of DCs.     

Infection of cultured human tracheal epithelial cells by human parainfluenza virus types 2 and 3.

Despite growing information of the effects of human respiratory virus infection on airway physiology, little information is available on the mechanisms of pathology and pathophysiology in these infections. The human respiratory pathogens, parainfluenza virus types 2 and 3 (hPIV2, hPIV3, respectively), clinically cause laryngotracheobronchitis (infection of the large proximal airways). In order to examine the pathobiology of these viruses in airway cells of human origin, we exposed primary cultures of human tracheal epithelial cells. Primary cultures of human tracheal epithelial cells were readily infected by these agents: cells exposed to hPIV2 and hPIV3 expressed viral antigens (demonstrated by indirect immunofluorescence assay), produced infectious virus, and demonstrated cytopathic effects (including early syncytium formation). Peak viral titers of 2 x 10(7) plaque-forming units per milliliter were obtained, similar to titers from permissive CV-1 cells. Trypan blue staining and direct cell counts demonstrated no difference in the viability of the control and infected cells until the infected cells began to detach from the culture substrate. However, infected cells release significantly more LDH than control cells by 48 h following infection at a multiplicity of infection of 1 virus/target cell. This system provides a model for studying the effects of infection of the human tracheal epithelium by human respiratory viral pathogens without confounding interactions with other cell and tissue types.     

Infection of human dendritic cells with recombinant vaccinia virus MVA reveals general persistence of viral early transcription but distinct maturation-dependent cytopathogenicity

Vector-infected dendritic cells (DC) are evaluated for antigen delivery in experimental therapy of cancer and infectious diseases. Here, we investigated infections of immature or mature, monocyte-derived human DC with recombinant vaccinia virus MVA producing human Her-2/neu, a candidate tumor-associated antigen. Assessment of the molecular virus life cycle in infected DC revealed a general arrest at the level of viral early gene expression. When monitoring the phenotype of MVA-infected DC, including expression of cell surface markers, we found immature cells readily undergoing apoptosis. Nevertheless, we detected significant populations of viable DC being characterized by high level Her-2/neu expression and unimpaired display of costimulatory molecules. While infected viable immature DC failed to undergo maturation despite cytokine treatment, both DC populations efficiently presented MVA-produced target antigen. These findings allow to better define the requirements for MVA-mediated antigen delivery to DC and help to derive optimized vectors for this advanced therapy option.     

Reduced expression of surface glycoproteins in mouse fibroblasts persistently infected with human respiratory syncytial virus (HRSV)

Summary.  BCH4 cells, persistently infected with Human Respiratory Syncytial Virus (HRSV), were obtained by Fernie et al. [12] after infection of a BALB/c mouse embryo cell line with the Long strain of HRSV. To understand the basis of HRSV persistence, the expression of HRSV RNAs and proteins was evaluated in BCH4 cells and infected parental BALB/c and fully permissive HEp-2 cells. Production of viral mRNAs was severely impaired in BCH4 cells. In addition, the expression level of the surface glycoproteins F and G was markedly reduced relative to internal viral proteins. However, virus recovered from BCH4 cells could lytically infect HEp-2 cells and expressed normal levels of surface glycoproteins. No evidence of defective genomes or interfering particles was found in BCH4 cells. Taken together, these data indicate that reduction of both viral mRNA accumulation and surface glycoprotein biosynthesis are at the basis of HRSV persistence in BCH4 cells. Accepted November 3, 2000     

Detection of human respiratory syncytial virus genotype specific antibody responses in infants

Infection and reinfection of infants with human respiratory syncytial virus (HRSV) occur despite the presence of serum anti-viral glycoprotein antibodies similar to those, which afford protection in animal models of infection. Antigenic variation of the viral glycoproteins between different genotypes of the virus which co-circulate in the population may contribute to the ability of the virus to escape from antibody-mediated protection. In this study, we have investigated whether human infants infected with HRSV produced antibody responses recognising the antigenic differences between different contemporary genotypes of virus. Acute and convalescent sera from 26 infants were analysed for antibody responses to the glycoproteins of the virus isolated from their respiratory tract and to representative viruses of homologous and heterologous genotypes. All infants developed antibodies with similar reactivity for viruses of all contemporary isolates and genotypes when measured in an immunofluorescence assay against unfixed virus infected cells. However, when antibody responses to the individual glycoproteins were measured in a surace plasmon resonance (SPR) assay, although all infants developed genotype cross-reactive antibodies to the F glycoprotein, anti-G antibodies were detectable in only half of the infants and in all cases these were genotype specific. Possession of no or only genotype specific antibodies to the G glycoprotein may contribute to the susceptibility of infants to reinfection. In both assays, reactivity of anti-glycoprotein antibodies with the sub-group A archetypal strain, A2, was markedly lower than with any contemporary virus tested indicating that this strain alone is unsuitable for accurate assessment of infant antibody responses. .     

Phosphorylation of human respiratory syncytial virus P protein at serine 54 regulates viral uncoating

The human respiratory syncytial virus (HRSV) structural P protein, phosphorylated at serine (S) and threonine (T) residues, is a co-factor of viral RNA polymerase. The phosphorylation of S54 is controlled by the coordinated action of two cellular enzymes: a lithium-sensitive kinase, probably glycogen synthetase kinase (GSK-3) β and protein phosphatase 2A (PP2A).Inhibition of lithium-sensitive kinase, soon after infection, blocks the viral growth cycle by inhibiting synthesis and/or accumulation of viral RNAs, proteins and extracellular particles. P protein phosphorylation at S54 is required to liberate viral ribonucleoproteins (RNPs) from M protein, during the uncoating process. Kinase inhibition, late in infection, produces a decrease in genomic RNA and infectious viral particles.LiCl, intranasally applied to mice infected with HRSV A2 strain, reduces the number of mice with virus in their lungs and the virus titre. Administration of LiCl to humans via aerosol should prevent HRSV infection, without secondary effects.     

Clinical characteristics and viral load of respiratory syncytial virus and human metapneumovirus in children hospitaled for acute lower respiratory tract infection

Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are two common viral pathogens in acute lower respiratory tract infections (ALRTI). However, the association of viral load with clinical characteristics is not well‐defined in ALRTI. To explore the correlation between viral load and clinical characteristics of RSV and HMPV in children hospitalized for ALRTI in Lanzhou, China. Three hundred and eighty‐seven children hospitalized for ALRTI were enrolled. Nasopharyngeal aspirates (NPAs) were sampled from each children. Real‐time PCR was used to screen RSV, HMPV, and twelve additional respiratory viruses. Bronchiolitis was the leading diagnoses both in RSV and HMPV positive patients. A significantly greater frequency of wheezing (52% vs. 33.52%, P = 0.000) was noted in RSV positive and negative patients. The RSV viral load was significant higher in children aged <1 year (P = 0.003), children without fever and wheezing (P = 0.015 and P = 0.000), days of illness <14 days (P = 0.002), children with bronchiolitis (P = 0.012) and children with RSV single infections (P = 0.000). No difference was found in the clinical features of HMPV positive and negative patients. The HMPV viral load had no correlation with any clinical characteristics. The incidences of severe disease were similar between single infection and coinfection for the two viruses (RSV, P = 0.221; HMPV, P = 0.764) and there has no statistical significance between severity and viral load (P = 0.166 and P = 0.721). Bronchiolitis is the most common disease caused by RSV and HMPV. High viral load or co‐infection may be associated with some symptoms but neither has a significant impact on disease severity for the two viruses. J. Med. Virol. 89:589–597, 2017 . © 2016 Wiley Periodicals, Inc.

     

Prevalence of human respiratory viruses in adults with acute respiratory tract infections in Beijing, 2005–2007

To determine the aetiological role and epidemiological profile of common respiratory viruses in adults with acute respiratory tract infections (ARTIs), a 2-year study was conducted in Beijing, China, from May 2005 to July 2007. Nose and throat swab samples from 5808 ARTI patients were analysed by PCR methods for common respiratory viruses, including influenza viruses (IFVs) A, B, and C, parainfluenza viruses (PIVs) 1–4, enteroviruses (EVs), human rhinoviruses (HRVs), respiratory syncytial virus (RSV), human metapneumovirus (HMPV), human coronaviruses (HCoVs) OC43, 229E, NL63, and HKU1, and adenoviruses (ADVs). Viral pathogens were detected in 34.6% of patient samples, and 1.6% of the patients tested positive for more than one virus. IFVs (19.3%) were the dominant agents detected, followed by HRVs (6.5%), PIVs (4.3%), EVs (3.2%), and HCoVs (1.1%). ADVs, RSV and HMPV were also detected (<1%). The viral detection rates differed significantly between infections of the lower and upper respiratory tracts in the sample population: PIVs, the second most commonly detected viral agents in lower acute respiratory tract infections (LRTIs), were more prevalent than in upper acute respiratory tract infections, indicating that the pathogenic role of PIVs in LRTIs should be investigated. Currently, this study is the largest-scale investigation of respiratory virus infections in China with multiple agent detection, providing baseline data for further studies of respiratory virus infections in adults with ARTIs.     

Detection of human metapneumovirus in respiratory secretions by reverse-transcriptase polymerase chain reaction, indirect immunofluorescence, and virus isolation in human bronchial epithelial cells

Over two winters in Newcastle upon Tyne, respiratory secretions, negative by immunofluorescence staining for other respiratory viruses, were tested for the presence of human metapneumovirus (HMPV) by RT-PCR. In the second winter, specimens were also tested by immunofluorescence staining with an anti-HMPV polyclonal rabbit antiserum and immunofluorescence positive specimens were inoculated into a line of human bronchiolar cells, 16HBE140. Overall, 55 of 549 (10%) specimens tested were positive for HMPV by RT-PCR. Of 162 specimens tested by both RT/PCR and immunofluorescence staining, 23 were positive by both techniques. Of five specimens positive by RT-PCR alone, only one was confirmed with a second set of primers. Of three specimens positive by immunofluorescence alone, only one was confirmed by virus culture. All four previously recognized sub-genotypes of the virus were identified by both RT-PCR and immunofluorescence staining. Sub-genotype A1 was prevalent in the first winter and B1 prevalent in the second. HMPV replication and virus isolation rates were higher in 16HBE140 cells than in monkey kidney cells and did not require exogenous trypsin. Low passage isolates of both sub-genotypes A2 and B1 replicated slowly reaching peak titers only 12 days after inoculation. In summary, single round RT/PCR and immunofluorescence staining with a polyclonal rabbit antiserum proved of equal sensitivity in the diagnosis of HMPV infection in respiratory secretions both detecting 96% of confirmed positive specimens. 16HBE40 cells provided a significant improvement on monkey kidney cells for the isolation and propagation of the virus.     

Dengue virus inoculation to human skin explants: an effective approach to assess in situ the early infection and the effects on cutaneous dendritic cells

Although dengue virus (DV) enters through skin while mosquitoes feed, early contacts remain unexplored regarding the cutaneous viral fate and in situ immune responses. We addressed this by exposing healthy, non-cadaveric, freshly obtained human skin explants to a human DV2 isolate. We demonstrated negative-strand DV-RNA and non-structural protein-1, both suggestive of viral replication in skin. Although control, mock-infected and DV-infected explants showed less (MHC-CII(+)/CD1a(+)/Langerin+) Langerhans cells, deranged morphology and decreased frequency were more apparent in DV-infected explants. Whereas DV+ cells were infrequent in epidermis and completely absent in dermis, some areas of basal epidermis were clearly DV+, presumably keratinocytes, cells where TUNEL positivity revealed apoptosis. Unlike fresh, control and mock-infected skin, DV-infected explants expressed CD80 and CD83, indicative of dendritic cell (DC) activation and maturation, respectively. However, sequential sections indicated that these cells were not DV+, suggesting that activated/mature DCs capable of priming T cells, probably, were not infected. Alternatively, the occasionally infected epidermal DC might not have reached maturation. Interestingly, skin DV infection apparently uncouples the DC activation/maturation process from another crucial DC function, the subsequent migration into dermis. This was suggested, because upon cutaneous DV infection, the few emerging CD83+ (mature) DCs remained within the outer epidermis, while no dermal CD83+ DCs were observed. These paradoxical effects might represent unknown DV subversion strategies. This approach is relatively easy, quick (results in 48 h), economical for developing countries where dengue is re-emerging and advantageous to evaluate in situ viral biology, immunity and immunopathology and potential antiviral strategies.     

High prevalence of human metapneumovirus infection in young children and genetic heterogeneity of the viral isolates

RNA of the newly identified human metapneumovirus (HMPV) was detected in nasopharyngeal aspirates of 11 of 63 (17.5%) young children with respiratory tract disease. Markers of infection caused by another member of the Pneumovirinae subfamily of the family Paramyxoviridae, respiratory syncytial virus (RSV), were identified in 15 of these patients (23.8%). Three patients were simultaneously infected with HMPV and RSV. Studies of the clinical characteristics of HMPV-infected children did not reveal any difference between HMPV-infected patients and a control population of RSV-infected patients with regard to disease severity, but the duration of symptoms was significantly shorter for HMPV-infected patients. Phylogenetic analysis of the amplified viral genome fragments confirmed the existence and simultaneous circulation within one epidemic season of HMPV isolates belonging to two genetic lineages.     

The fusion and hemagglutinin-neuraminidase glycoproteins of human parainfluenza virus 3 are both required for fusion.

Recombinant vaccinia viruses, VF and VHN, expressing the fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins of human parainfluenza virus 3 (HPIV3) were constructed. Infection of HeLa T4 cells with VF and VHN led to the synthesis of glycoproteins, with the correct apparent molecular weights, that were recognized by monoclonal antibodies specific for HPIV3F and HN. The HN glycoprotein was present on the surface of cells infected with VHN and these cells demonstrated both hemadsorbing and neuraminidase activities. The F glycoprotein was present in cleaved and uncleaved forms and was also expressed on the surface of VF-infected cells. Fusion activity, however, as evidenced by syncytium formation and lysis of human erythrocytes, could only be demonstrated when HeLa T4 cells were coinfected with VF and VHN. Fusion events that are mediated by HPIV3, therefore, require both the F and HN glycoproteins.     

Development and evaluation of NucliSens basic kit NASBA for diagnosis of parainfluenza virus infection with 'end-point' and 'real-time' detection

New methods for the detection of human parainfluenza viruses (HPIVs) were developed. These were based on nucleic acid sequence-based amplification (NASBA) and utilised the NucliSens Basic Kit. Primers and probes were selected from the haemagglutinin neuraminidase (HN) gene of HPIV1, HPIV2 and HPIV3, and from the phosphoprotein (P) of HPIV4a and -4b. Synthetic RNA, titrated control virus stocks and respiratory specimens (n=44) were utilised to evaluate performance of the assays. Detection of NASBA products was by probe hybridisation and electrochemiluminescence (ECL) ('end-point' detection) or using molecular beacons ('real-time' detection). The assays using ECL detection proved to be both sensitive and specific. Typically, less than or equal to 100 RNA copies or one TCID(50) input was detectable with no cross-reaction between the specific HPIV assays and other respiratory viruses. Results for clinical samples were concordant with those obtained by 'conventional' procedures by classical viral diagnostic methods. 'Real-time' detection utilised probes specific for either HPIV1 or HPIV3 with similar performance characteristics to the assays with 'end-point' detection. The feasibility of multiplexing targets together was confirmed using a combined HPIV1 and HPIV3 assay with good results for ECL and molecular beacon detection on control material and clinical samples.     

Concurrent detection of other respiratory viruses in children shedding viable human respiratory syncytial virus

Human respiratory syncytial virus (HRSV) is an important cause of respiratory disease. The majority of studies addressing the importance of virus co‐infections to the HRSV‐disease have been based on the detection of HRSV by RT‐PCR, which may not distinguish current replication from prolonged shedding of remnant RNA from previous HRSV infections. To assess whether co‐detections of other common respiratory viruses are associated with increased severity of HRSV illnesses from patients who were shedding viable‐HRSV, nasopharyngeal aspirates from children younger than 5 years who sought medical care for respiratory infections in Ribeirão Preto (Brazil) were tested for HRSV by immunofluorescence, RT‐PCR and virus isolation in cell culture. All samples with viable‐HRSV were tested further by PCR for other respiratory viruses. HRSV‐disease severity was assessed by a clinical score scale. A total of 266 samples from 247 children were collected and 111 (42%) were HRSV‐positive. HRSV was isolated from 70 (63%), and 52 (74%) of them were positive for at least one additional virus. HRSV‐positive diseases were more severe than HRSV‐negative ones, but there was no difference in disease severity between patients with viable‐HRSV and those HRSV‐positives by RT‐PCR. Co‐detection of other viruses did not correlate with increased disease severity. HRSV isolation in cell culture does not seem to be superior to RT‐PCR to distinguish infections associated with HRSV replication in studies of clinical impact of HRSV. A high rate of co‐detection of other respiratory viruses was found in samples with viable‐HRSV, but this was not associated with more severe HRSV infection. J Med. Virol. 85:1852–1859, 2013. © 2013 Wiley Periodicals, Inc.