Jeffrey S. Kahn

Human Bocavirus Infection in Young Children in the United States: Molecular Epidemiological Profile and Clinical Characteristics of a Newly Emerging Respiratory Virus

Abstract BackgroundHuman bocavirus (HBoV) is a newly identified human parvovirus that was originally identified in the respiratory secretions of children with respiratory tract disease. To further investigate the epidemiological profile and clinical characteristics of HBoV infection, we screened infants and children <2 years of age (hereafter referred to as “children”) for HBoV MethodsChildren for whom respiratory specimens submitted to a diagnostic laboratory tested negative for respiratory syncytial virus, parainfluenza viruses (types 1–3), influenza A and B viruses, and adenovirus, as well as asymptomatic children, underwent screening for HBoV by use of polymerase chain reaction (PCR). Respiratory specimens were obtained from the children from 1 January 2004 through 31 December 2004 ResultsTwenty-two (5.2%) of the 425 children who had a respiratory specimen submitted to the diagnostic laboratory and 0 of the 96 asymptomatic children were found to be positive for HBoV by PCR (P=.02). Fever, rhinorrhea, cough, and wheezing were observed in ⩾50% of the HBoV-positive children. Of the 17 children who had chest radiography performed, 12 (70.6%) had abnormal findings. HBoV appeared to have a seasonal distribution. Nucleotide polymorphisms were detected in the viral capsid protein (VP) 1/VP2 genes. Two distinct HBoV genotypes circulated during the study period ConclusionsHBoV is circulating in the United States and is associated with both upper and lower respiratory tract disease in infants and young children

Epidemiology of Human Metapneumovirus

SUMMARY Since the discovery of human metapneumovirus (hMPV) in 2001, the virus has been identified worldwide. hMPV is a common respiratory pathogen, particularly in infants and young children. The virus is associated with both upper and lower respiratory tract infections and may be a trigger for asthma. At least two major genotypes of hMPV circulate during community outbreaks. Whether these genotypes represent distinct serotypes remains controversial. The major challenges faced by the medical and scientific communities are the understanding of the pathogenesis of hMPV disease and the development of a safe and effective vaccine to protect against infection and disease caused by this newly recognized respiratory virus.

A 1-Year Experience with Human Metapneumovirus in Children Aged <5 Years

Abstract Human metapneumovirus (hMPV) is a recently discovered respiratory pathogen. We tested respiratory specimens for the presence of hMPV by reverse-transcription polymerase chain reaction. These specimens were obtained over a 1-year period from children aged <5 years and had negative results by the direct fluorescent antibody test for respiratory syncytial virus, influenza A and B, parainfluenza viruses 1–3, and adenovirus. Overall, 54 (8.1%) of 668 individuals tested positive for hMPV. During March and April of the study period, hMPV was detected in 17.6% and 25.0% of specimens tested, respectively. At least 2 distinct genotypes of hMPV circulated during the study period. Fever, tachypnea, cough, rhinorrhea, retractions of the chest wall, and wheezing were common findings. Of hMPV-positive children, 60.4% were aged <12 months. hMPV accounted for a small but significant proportion of respiratory-tract disease in infants and children.

Association between a Novel Human Coronavirus and Kawasaki Disease

Abstract Kawasaki disease is a systemic vasculitis of childhood; its etiology is unknown. We identified evidence of a novel human coronavirus, designated “New Haven coronavirus” (HCoVNH), in respiratory secretions from a 6-month-old infant with classic Kawasaki disease. To further investigate the possible association between HCoV-NH infection and Kawasaki disease, we conducted a case-control study. Specimens of respiratory secretions from 8 (72.7%) of 11 children with Kawasaki disease and from 1 (4.5%) of 22 control subjects (children without Kawasaki disease matched by age and the time the specimens were obtained) tested positive for HCoVNH by reverse-transcriptase polymerase chain reaction (Mantel-Haenszel matched odds ratio, 16.0 [95% confidence interval, 3.4–74.4]; P = .0015). These data suggest that HCoV-NH infection is associated with Kawasaki disease.

Evidence of a novel human coronavirus that is associated with respiratory tract disease in infants and young children.

Abstract Background. The etiological agents responsible for a substantial proportion of respiratory tract diseases have not been identified. We sought to determine whether novel human coronaviruses (HCoVs) are circulating in New Haven, Connecticut, and, if so, whether they are associated with respiratory tract disease in infants and young children. Methods. We developed a polymerase chain reaction (PCR)-based approach for screening specimens from the respiratory tracts of symptomatic children. PCR probes that target regions of the replicase 1a gene that are conserved among genetically diverse animal CoVs and HCoVs were designed. Using these probes, we identified genomic sequences of a novel HCoV, designated “New Haven coronavirus” (HCoV-NH). Thereafter, we designed specific probes to screen respiratory specimens from children <5 years old for this novel HCoV. Clinical features associated with HCoV-NH infection were identified. Results. Seventy-nine (8.8%) of 895 children tested positive for HCoV-NH. Cough, rhinorrhea, tachypnea, fever, abnormal breath sounds, and hypoxia were the most common findings associated with HCoV-NH infection. Sequence analysis revealed that HCoV-NH is closely related to a novel HCoV recently reported in The Netherlands. Conclusions. The novel HCoVs identified in New Haven and The Netherlands are similar and likely represent the same species. This newly discovered virus may have worldwide distribution and may account for a significant proportion of respiratory tract disease in infants and young children.

Correlation between respiratory syncytial virus genotype and severity of illness.

Respiratory syncytial virus (RSV) causes seasonal outbreaks of respiratory tract infections, but the viral factors associated with virulence remain unknown. To determine whether RSV genotype correlated with severity of illness, isolates were characterized by phylogenetic analysis of the RSV G gene, and a composite score was used to quantify severity of illness. During the 1998-1999 and 1999-2000 winter seasons, 137 subgroup A and 84 subgroup B isolates were identified. The severity of illness caused by subgroup A isolates did not differ from that caused by subgroup B isolates (P=.086). However, the GA3 clade was associated with significantly greater severity of illness, compared with clades GA2 (P=.004) and GA4 (P=.016). In a subpopulation of patients < or =24 months old who had no known risk factors for severe RSV disease, clade GA3 was again associated with greater severity of illness, compared with clade GA2 (P=.018). Severity of RSV infection is associated with RSV genotype.

RNA Interference-Mediated Silencing of the Respiratory Syncytial Virus Nucleocapsid Defines a Potent Antiviral Strategy

ABSTRACT We describe the design and characterization of a potent human respiratory syncytial virus (RSV) nucleocapsid gene-specific small interfering RNA (siRNA), ALN-RSV01. In in vitro RSV plaque assays, ALN-RSV01 showed a 50% inhibitory concentration of 0.7 nM. Sequence analysis of primary isolates of RSV showed that the siRNA target site was absolutely conserved in 89/95 isolates, and ALN-RSV01 demonstrated activity against all isolates, including those with single-mismatch mutations. In vivo, intranasal dosing of ALN-RSV01 in a BALB/c mouse model resulted in potent antiviral efficacy, with 2.5- to 3.0-log-unit reductions in RSV lung concentrations being achieved when ALN-RSV01 was administered prophylactically or therapeutically in both single-dose and multidose regimens. The specificity of ALN-RSV01 was demonstrated in vivo by using mismatch controls; and the absence of an immune stimulatory mechanism was demonstrated by showing that nonspecific siRNAs that induce alpha interferon and tumor necrosis factor alpha lack antiviral efficacy, while a chemically modified form of ALN-RSV01 lacking measurable immunostimulatory capacity retained full activity in vivo. Furthermore, an RNA interference mechanism of action was demonstrated by the capture of the site-specific cleavage product of the RSV mRNA via rapid amplification of cDNA ends both in vitro and in vivo. These studies lay a solid foundation for the further investigation of ALN-RSV01 as a novel therapeutic antiviral agent for clinical use by humans.

Replication-Competent or Attenuated, Nonpropagating Vesicular Stomatitis Viruses Expressing Respiratory Syncytial Virus (RSV) Antigens Protect Mice against RSV Challenge

ABSTRACT Foreign glycoproteins expressed in recombinant vesicular stomatitis virus (VSV) can elicit specific and protective immunity in the mouse model. We have previously demonstrated the expression of respiratory syncytial virus (RSV) G (attachment) and F (fusion) glycoprotein genes in recombinant VSV. In this study, we demonstrate the expression of RSV F and G glycoproteins in attenuated, nonpropagating VSVs which lack the VSV G gene (VSVΔG) and the incorporation of these RSV proteins into recombinant virions. We also show that intranasal vaccination of mice with nondefective VSV recombinants expressing RSV G (VSV-RSV G) or RSV F (VSV-RSV F) elicited RSV-specific antibodies in serum (by enzyme-linked immunosorbent assay [ELISA]) as well as neutralizing antibodies to RSV and afford complete protection against RSV challenge. In contrast, VSVΔG-RSV F induced detectable serum antibodies to RSV by ELISA, but no detectable neutralizing antibodies, yet it still protected from RSV challenge. VSVΔG-RSV G failed to induce any detectable serum (by ELISA) or neutralizing antibodies and failed to protect from RSV challenge. The attenuated, nonpropagating VSVΔG-RSV F is a particularly attractive candidate for a live attenuated recombinant RSV vaccine.

Seroepidemiology of Human Bocavirus Defined Using Recombinant Virus-Like Particles

BACKGROUND Human bocavirus (HBoV) is a newly identified human parvovirus for which seroepidemiology and antigenic properties remain undefined. METHODS The HBoV VP2 gene, expressed from a baculovirus vector, produced virus-like particles (VLPs), which were used to raise rabbit anti-HBoV antisera and to develop an enzyme-linked immunosorbent assay (ELISA). The VLP-based ELISA was used to screen for HBoV-specific immunoglobulin G antibodies in a convenience sample of 270 serum specimens, mostly from children, obtained at Yale-New Haven Hospital; 208 specimens were also screened for erythrovirus B19-specific antibodies by a B19 VLP-based ELISA. RESULTS Immunofluorescence and ELISA showed that human parvoviruses HBoV and B19 are antigenically distinct. By the HBoV VLP-based ELISA, 91.8% and 63.6% of serum specimens from infants in the first and second months of life, respectively, were found to be seropositive, as were 45.4% from 3-month-old infants and 25.0% from 4-month-old infants. The percentages of HBoV-seropositive children increased to 40.7%-60.0% for children 5-47 months of age and to >85% for individuals >or=48 months old. However, the overall percentage of B19-seropositive individuals was <40.5% for all age groups screened. CONCLUSIONS HBoV infection is common during childhood, but a minority of children and young adults screened have evidence of B19 infection.

Seroepidemiology of human metapneumovirus (hMPV) on the basis of a novel enzyme-linked immunosorbent assay utilizing hMPV fusion protein expressed in recombinant vesicular stomatitis virus.

ABSTRACT Human metapneumovirus (hMPV) is a newly identified human respiratory virus now recognized as a major respiratory pathogen of infants and children. To define the seroepidemiology of hMPV, we developed a novel enzyme-linked immunosorbent assay (ELISA) based on expression of the fusion protein of hMPV (hMPV F) in recombinant vesicular stomatitis virus (VSV). Western blot analysis using an hMPV F-specific antiserum confirmed the expression of hMPV in recombinant VSV. The ELISA is specific for hMPV F; antibody specific for the most closely related human paramyxovirus, respiratory syncytial virus, does not bind to hMPV F. Overall, 216 serum specimens were tested. The percentages of seropositive individuals were 89.1% in children ≤5 months old, 55.0% in children 6 to 11 months old, 36.0% in children 12 to 23 months old, 45.0% in children 24 to 47 months old, 77.3% in children 48 to 59 months old, 91.3% in children 5 to 10 years old, and 95.5% for individuals 11 to 20 years old. This is the first seroepidemiological survey of hMPV in the United States and the first analysis to determine the prevalence of antibody to a specific hMPV protein. The data suggest that exposure to hMPV is common in childhood and that hMPV F is an antigenic determinant of hMPV.