Sharon J. Tollefson

Burden of Interpandemic Influenza in Children Younger than 5 Years: A 25-Year Prospective Study

Many respiratory viruses cause morbidity in young children, but a licensed vaccine and effective oral therapy are available only for influenzavirus. To determine the incidence of laboratory-confirmed influenza illness, we prospectively followed up 1665 healthy children aged <5 years who were enrolled in the Vanderbilt Vaccine Clinic at some point from 1974 through 1999. Viral cultures were obtained when the children presented with clinical illness. The isolation of influenzavirus was associated with an estimated 95 health care visits for children with symptoms of influenza, 46 episodes of acute otitis media, and 8 episodes of lower respiratory tract disease per 1000 children yearly. Rates of acute otitis media and lower respiratory tract disease were highest among children aged <2 years. Hospitalizations associated with culture-positive influenza occurred at an annual rate of 3-4 per 1000 children aged <2 years. Influenza is associated with substantial morbidity in otherwise healthy children aged <5 years.

The Role of Human Metapneumovirus in Upper Respiratory Tract Infections in Children: A 20-Year Experience

BACKGROUND The role that human metapneumovirus (hMPV) plays in the etiology of upper respiratory tract infections (URIs) in children over a period of many years has not been evaluated previously. METHODS By use of real-time reverse-transcriptase polymerase chain reaction, we retrospectively tested nasal wash (NW) specimens for hMPV that had been obtained from a cohort of 1532 infants and children with URIs who were prospectively followed for an average of 2.4 years during the period from 1982 to 2001. Virus genes were sequenced, and prospectively collected clinical data were analyzed. RESULTS There were 2710 visits for URIs for which routine cultures did not reveal a viral etiology. Archival NW specimens from 2384 of these visits were available. hMPV RNA was detected in 118 (5%) of 2384 specimens. The mean age of the children with hMPV infection was 20 months, and 78% of illnesses occurred from December through May. Acute otitis media (AOM) was detected in 50% of these children. hMPV circulated each year, but the numbers of isolates detected varied by year. Reinfections with both homologous and heterologous strains occurred. Four distinct genetic lineages were present over the 20 years of surveillance, with several different lineages circulating during some seasons. CONCLUSIONS hMPV was detected in a substantial number of children with URIs and concomitant AOM.

Epidemiology and Clinical Impact of Parainfluenza Virus Infections in Otherwise Healthy Infants and Young Children < 5 Years Old

Over a 20-year period in a population of otherwise healthy children, respiratory viruses have been cultured from nasal wash specimens from each child with a clinically significant respiratory illness. Since efforts are underway to develop vaccines for prevention of illness due to parainfluenza virus (PIV) type 3, the epidemiologic characteristics of PIVs were reviewed, and the population size necessary to demonstrate vaccine efficacy was estimated. A population of 1429 children was followed through early childhood. PIVs were isolated from 286 samples, 17.4% of positive viral cultures. PIV-3 was the most common: 10% of the children had at least one symptomatic, culture-proven PIV-3 infection. PIV-3 was endemic during the study period, while the other two PIVs, PIV-1 and -2, caused biennial flu epidemics. Only four PIV-related hospitalizations were seen. The efficacy of a PIV-3 vaccine could be demonstrated in a trial of 600 carefully monitored children vaccinated by 3 months and followed to 15 months of age.

The Cotton Rat (Sigmodon hispidus) Is a Permissive Small Animal Model of Human Metapneumovirus Infection, Pathogenesis, and Protective Immunity

ABSTRACT Human metapneumovirus (hMPV) is a newly described paramyxovirus that is an important cause of acute respiratory tract disease. We undertook to develop a small animal model of hMPV infection, pathogenesis, and protection. Hamsters, guinea pigs, cotton rats, and nine inbred strains of mice were inoculated intranasally with hMPV. The animals were sacrificed, and nasal and lung tissue virus yields were determined by plaque titration. None of the animals exhibited respiratory symptoms. The quantity of virus present in the nasal tissue ranged from 4.6 × 102 PFU/gram tissue (C3H mice) to greater than 105 PFU/gram (hamster). The amount of virus in the lungs was considerably less than in nasal tissue in each species tested, ranging from undetectable (<5 PFU/g; guinea pigs) to 1.8 × 105 PFU/gram (cotton rat). The peak virus titer in cotton rat lungs occurred on day 4 postinfection. hMPV-infected cotton rat lungs examined on day 4 postinfection exhibited histopathological changes consisting of peribronchial inflammatory infiltrates. Immunohistochemical staining detected virus only at the luminal surfaces of respiratory epithelial cells throughout the respiratory tract. hMPV-infected cotton rats mounted virus-neutralizing antibody responses and were partially protected against virus shedding and lung pathology on subsequent rechallenge with hMPV. Viral antigen was undetectable in the lungs on challenge of previously infected animals. This study demonstrates that the cotton rat is a permissive small animal model of hMPV infection that exhibits lung histopathology associated with infection and that primary infection protected animals against subsequent infection. This model will allow further in vivo studies of hMPV pathogenesis and evaluation of vaccine candidates.

Human metapneumovirus infection in children hospitalized for wheezing

To the Editor: Recently, the prevalence of viral respiratory tract pathogens associated with acute wheezing exacerbations was reported in a study of infants and children admitted to the Children’s Hospital at the University of Virginia.1 Subsequently, we analyzed the nasal washes from these children for human metapneumovirus (hMPV), a paramyxovirus closely related to respiratory syncytial virus (RSV). hMPV has also been shown to be a significant cause of respiratory tract illnesses in young children.2–4 The study we previously reported from the University of Virginia was an observational, case-control study of 133 children (age 2 months to 18 years) hospitalized for wheezing between April 1, 2000, and March 31, 2001. This represented 93% of all admissions for acute wheezing during the study period. Children who had evidence of cardiopulmonary disease or immunosuppression were excluded. The control subjects included 133 age-matched and gender-matched hospitalized children without wheezing. They were enrolled during the same season of each matched wheezing subject. Demographic information was obtained from parent-administered questionnaires and hospital charts. The Human Investigation Committee at the University of Virginia approved the study. Informed consent was obtained from parents, and assent was obtained from older children. Nasal washes were obtained from patients to test for viruses by culture and for RSV and influenza by antigen testing. In addition, nucleic acids extracted from the samples had been tested previously by RT-PCR for rhinovirus, enterovirus, coronavirus, influenza virus, parainfluenza virus, and RSV, and by PCR for adenovirus.1 Metapneumovirus testing was performed by a real-time RT-PCR assay on a Smart Cycler (Cepheid, Sunnyvale, Calif) using primers and probe for the N gene5 with the Quantitect RT-PCR kit (Qiagen, Valencia, Calif). The probe was altered slightly, with BHQ-3 (Invitrogen, Carlsbad, Calif) substituted for 5-carboxytetramethyl-rhodamine as the 3′ fluorescent quencher. These primers and probe have been shown to detect all 4 genetic lineages of hMPV, and the limit of detection in our assay was 50 copies of viral genome per reaction. Patient demographic data and the frequencies for positive tests for virus among wheezing and control patients were analyzed by nonparametric exact methods. Exact 2-sided 95% CIs for the difference of proportion were constructed as described by Agresti and Min.6 Multivariate analyses related to predicting wheezing as a function of the patient’s atopic status and evidence for viral infection were performed by multiple logistic regression. Tests of association were evaluated on the basis of the generalized Wald χ2 statistic, and 95% CI construction for the adjusted odds ratio was based on the Wald approximation. Total serum IgE data were analyzed on the logarithmic scale by way of 1-way ANOVA. Demographic characteristics of the study population were previously described in detail.1 Thirteen children in this study tested positive for hMPV (10 children hospitalized for wheezing and 3 controls; Table I). Seven of the 10 wheezing children were <3 years old, and half were males. The mean age of the children <3 years old who were infected with hMPV was 7 months (range, 2–13 months). The majority of subjects with positive tests for hMPV (85%; 11/13) were hospitalized from January through April. TABLE I Characteristics of subjects infected with hMPV The prevalence of positive tests for hMPV among children <3 years old was 8.9% (7/79) in wheezing children compared with 1.3% (1/77) of controls (P = .035). Overall, the children in this age group who tested positive for any virus (including hMPV) were more likely to be hospitalized for wheezing than the children who tested negative for virus (odds ratio, 6.48; 95% CI, 2.83–14.81; P < .001). More wheezing children than controls who were younger than 3 years had positive tests for 1 or more viruses (32% compared with 16%; P = .02). Among those who tested positive for hMPV, 3 of the wheezing subjects as well as the control tested positive for other viral pathogens (Table I). Only 1 of these 4 children (all admitted during the midwinter) tested positive for RSV, and none tested positive for influenza. Mean serum total IgE levels were not different between hMPV-infected children with wheezing and hMPV-infected controls. Among the children 3 to 18 years old, the children who tested positive for any virus were more likely to be hospitalized for wheezing than the children who tested negative for virus (odds ratio, 6.00; 95% CI, 2.62–13.73; P < .001). As previously reported,1 rhinovirus was the dominant pathogen, whereas hMPV was not significantly associated with wheezing in this age group. Among children who were 3 through 9 years of age, hMPV was detected in 8.8% (3/34) of the wheezing subjects compared with 5.7% (2/35) of the controls (P = .71). None of the children who tested positive for hMPV in this age group were coinfected with another virus. No subject older than 9 years of age had a positive test for hMPV. In this analysis, we found a significant association between hMPV infection and wheezing among children younger than 3 years, especially during the midwinter months. This is consistent with a highly significant association between hMPV and wheezing exacerbations observed in a 25-year prospective study of lower respiratory tract illness in otherwise healthy outpatient children (in Tennessee) who were younger than 5 years.3 In contrast, hMPV was not significantly associated with wheezing requiring hospitalization among children 3 years of age and older in our study. Instead, rhinovirus was the dominant pathogen associated with severe exacerbations, which has also been observed in other studies of children hospitalized for wheezing.7 In addition, the large majority (at least 80%) of the wheezing children age 3 years and older who were hospitalized in Virginia had striking atopic characteristics.1 Combined with test results for other viral pathogens reported previously in the same patients,1 the detection of hMPV in our current analysis increased the overall prevalence of viral infections among the wheezing subjects younger than 3 years to almost 90%. These results confirm and strengthen the observation that viral respiratory tract pathogens are the dominant risk factor for wheezing exacerbations in early childhood. Similar to other viruses that are significantly associated with wheezing during early childhood, more information is needed about the infants who are infected with hMPV and their long-term prognosis with respect to persistent wheezing and their risk for developing asthma.8

Viral acute lower respiratory infections impair CD8+ T cells through PD-1

Viruses are leading causes of severe acute lower respiratory infections (LRIs). These infections evoke incomplete immunity, as individuals can be repeatedly reinfected throughout life. We report that acute viral LRI causes rapid pulmonary CD8+ cytotoxic T lymphocyte (TCD8) functional impairment via programmed death-1/programmed death ligand-1 (PD-1/PD-L1) signaling, a pathway previously associated with prolonged antigenic stimulation during chronic infections and cancer. PD-1-mediated TCD8 impairment occurred acutely in mice following infection with human metapneumovirus or influenza virus. Viral antigen was sufficient for PD-1 upregulation, but induction of PD-L1 was required for impairment. During secondary viral infection or epitope-only challenge, memory TCD8 rapidly reexpressed PD-1 and exhibited severe functional impairment. Inhibition of PD-1 signaling using monoclonal antibody blockade prevented TCD8 impairment, reduced viral titers during primary infection, and enhanced protection of immunized mice against challenge infection. Additionally, PD-1 and PD-L1 were upregulated in the lungs of patients with 2009 H1N1 influenza virus, respiratory syncytial virus, or parainfluenza virus infection. These results indicate that PD-1 mediates TCD8 functional impairment during acute viral infection and may contribute to recurrent viral LRIs. Therefore, the PD-1/PD-L1 pathway may represent a therapeutic target in the treatment of respiratory viruses.

Integrin αvβ1 promotes infection by human metapneumovirus

Human metapneumovirus (hMPV) is a recently described paramyxovirus that causes lower respiratory infections in children and adults worldwide. The hMPV fusion (F) protein is a membrane-anchored glycoprotein and major protective antigen. All hMPV F protein sequences determined to date contain an Arg-Gly-Asp (RGD) sequence, suggesting that F engages RGD-binding integrins to mediate cell entry. The divalent cation chelator EDTA, which disrupts heterodimeric integrin interactions, inhibits infectivity of hMPV but not the closely related respiratory syncytial virus (RSV), which lacks an RGD motif. Function-blocking antibodies specific for αvβ1 integrin inhibit infectivity of hMPV but not RSV. Transfection of nonpermissive cells with αv or β1 cDNAs confers hMPV infectivity, whereas reduction of αv and β1 integrin expression by siRNA inhibits hMPV infection. Recombinant hMPV F protein binds to cells, whereas Arg-Gly-Glu (RGE)-mutant F protein does not. These data suggest that αvβ1 integrin is a functional receptor for hMPV.

Human Metapneumovirus Fusion Protein Vaccines That Are Immunogenic and Protective in Cotton Rats

ABSTRACT Human metapneumovirus (hMPV) is a recently described paramyxovirus that is a major cause of upper and lower respiratory infection in children and adults worldwide. A safe and effective vaccine could decrease the burden of disease associated with this novel pathogen. We previously reported the development of the cotton rat model of hMPV infection and pathogenesis (J. V. Williams et al., J. Virol. 79:10944-10951, 2005). We report here the immunogenicity of an hMPV fusion (F) protein in this model. We constructed DNA plasmids that exhibited high levels of expression of hMPV F in mammalian cells (DNA-F). These constructs were used to develop a novel strategy to produce highly pure, soluble hMPV F protein lacking the transmembrane domain (FΔTM). We then immunized cotton rats at 0 and 14 days with either control vector, DNA-F alone, DNA-F followed by FΔTM protein, or FΔTM alone. All groups were challenged intranasally at 28 days with live hMPV. All three groups that received some form of hMPV F immunization mounted neutralizing antibody responses and exhibited partial protection against virus shedding in the lungs compared to controls. The FΔTM-immunized animals showed the greatest degree of protection (>1,500-fold reduction in lung virus titer). All three immunized groups showed a modest reduction of nasal virus shedding. Neither evidence of a Th2-type response nor increased lung pathology were present in the immunized animals. We conclude that sequence-optimized hMPV F protein protects against hMPV infection when delivered as either a DNA or a protein vaccine in cotton rats.

Evaluation of Combined Live, Attenuated Respiratory Syncytial Virus and Parainfluenza 3 Virus Vaccines in Infants and Young Children

We evaluated a combination respiratory syncytial virus (RSV) and parainfluenza 3 virus (PIV3) live, attenuated intranasal vaccine for safety, viral replication, and immunogenicity in doubly seronegative children 6-18 months old. RSV cpts-248/404 and PIV3-cp45 vaccines were combined in a dose of 10(5) plaque-forming units of each per 0.5-mL dose and compared with monovalent vaccines or placebo. The virus shedding pattern of RSV was not different between monovalent RSV cpts-248/404 vaccine and combination vaccine. Modest reductions in the shedding of PIV3-cp45 vaccine virus were found after the administration of RSV cpts-248/404 and PIV3-cp45 vaccine, relative to monovalent PIV3 vaccine; 16 (76%) of 21 children given combination vaccine shed PIV3-cp45 versus 11 (92%) of 12 of those given monovalent PIV3 vaccine. Both vaccines were immunogenic, and antibody responses were similar between the monovalent groups and the combination group. Combined RSV/PV3 vaccine is feasible for simultaneous administration, and further studies are warranted.

Genetic diversity and evolution of human metapneumovirus fusion protein over twenty years.

BackgroundHuman metapneumovirus (HMPV) is an important cause of acute respiratory illness in children. We examined the diversity and molecular evolution of HMPV using 85 full-length F (fusion) gene sequences collected over a 20-year period.ResultsThe F gene sequences fell into two major groups, each with two subgroups, which exhibited a mean of 96% identity by predicted amino acid sequences. Amino acid identity within and between subgroups was higher than nucleotide identity, suggesting structural or functional constraints on F protein diversity. There was minimal progressive drift over time, and the genetic lineages were stable over the 20-year period. Several canonical amino acid differences discriminated between major subgroups, and polymorphic variations tended to cluster in discrete regions. The estimated rate of mutation was 7.12 × 10-4 substitutions/site/year and the estimated time to most recent common HMPV ancestor was 97 years (95% likelihood range 66-194 years). Analysis suggested that HMPV diverged from avian metapneumovirus type C (AMPV-C) 269 years ago (95% likelihood range 106-382 years).ConclusionHMPV F protein remains conserved over decades. HMPV appears to have diverged from AMPV-C fairly recently.