Outbreak of Acute Respiratory Infection among Infants in Lisbon,Portugal, Caused by Human Adenovirus Serotype 3 and a New 7/3 Recombinant Strain

Human adenoviruses (AdVs) typically cause mild illnesses in otherwise healthy hosts. We investigated a pediatric outbreak of acute respiratory infection with fatal outcomes that occurred in Lisbon, Portugal, in 2004. Biological specimens were collected from 83 children attending two nurseries, a kinesiotherapy clinic, and the household of a nanny. Adenovirus infection was confirmed in 48 children by PCR and virus isolation. Most (96%) isolates were classified as being of subspecies B1. Phylogenetic analysis of fiber and hexon gene sequences revealed that most infants were infected with AdV serotype 3 (AdV3) strains. Infants attending one nursery harbored a new recombinant strain containing an AdV serotype 7 hexon and serotype 3 fiber (AdV7/3). Both the AdV3 and the AdV7/3 strains caused fatal infections. Two different serotype 3 strains were circulating in Lisbon in 2004, and the new AdV7/3 recombinant type originated from only one of those strains. These results demonstrate that recombination leads to the emergence of new adenovirus strains with epidemic and lethal potential.Human adenoviruses (HAdVs) have been associated with a wide spectrum of clinical diseases with respiratory and gastrointestinal symptoms (23, 32). Severe illness can occur in newborns, elderly individuals, and patients with underlying medical conditions. In otherwise healthy adults, infections caused by human adenoviruses do not represent a life-threatening clinical condition. Adenoviruses are characterized by a linear double-stranded DNA genome of 2 to 45 kbp that encodes 30 to 40 proteins (6). HAdV comprises 51 serotypes (HAdV-1 to HAdV-51), on the basis of type-specific antiserum-mediated neutralization of infectivity (10). The serotypes can be divided into seven species, named HAdV-A to HAdV-G, on the basis of hemagglutination inhibition and biochemical criteria (13). HAdV-B is further classified into subspecies B1 and B2, which use different cellular receptors for viral entry (29). These variants can be segregated by different geographic areas, time periods, and clinical conditions.Serotype identification is critical for epidemiological surveillance, the detection of new strains, assessment of treatment efficacy, and understanding the pathogenesis of HAdV. For example, acute respiratory disease is primarily caused by HAdV-B1 serotypes 3, 7, 16, and 21; HAdV-B2 serotypes 11 and 14; and HAdV-E serotype 4 (8, 23, 25, 28, 35, 38, 41). Respiratory infections caused by HAdV-B1 serotypes 3 and 7 (16) and HAdV-B2 serotype 14 (17) are potentially fatal. Neutralization tests are the classical reference method used for the typing of adenovirus and require virus isolation from infected organs or tissues (20). The main type-specific neutralizing epitope, the ɛ determinant, consists of loop 1 (L1) and loop 2 (L2) on the hexon protein, the major capsid protein and the most abundant structural protein (26). Cases of the failure of neutralization with the available antisera require extensive cross-neutralization studies to define a new HAdV type. To circumvent the practical problems associated with traditional serum neutralization studies, molecular methods for the typing of adenovirus have been established. Examples are restriction fragment length polymorphism (RFLP) analysis of adenoviral DNA (16), PCR-based assays (1, 36), and microarray-based methods (36). However, these methods cannot discriminate between all serotypes and do not allow detailed studies of molecular epidemiology and viral evolution to be performed.More recently, analysis of the nucleotide and amino acid sequences from different genes has shown that adenovirus species form three distinct phylogenetic clusters: HAdV-C belongs to cluster 1; HAdV-A and HAdV-F belong to cluster 2; and HAdV-B, HAdV-D, and HAdV-E belong to cluster 3 (6, 22). In addition, phylogenetic analysis of selected gene fragments has increasingly been used to classify human adenoviruses at the serotype and species levels (7, 19, 40), to detect cases of coinfection with multiple adenoviral species (36, 38), and to identify new recombinant strains formed between similar species (18, 37, 41) or different species (19). Finally, phylogenetic analysis has become an important tool in the epidemiological investigation of many disease outbreaks caused by adenovirus (11, 17, 27, 40, 41). In the present study, we have used epidemiological, virological, and molecular phylogenetic methods to investigate the causes and origin of a recent outbreak of acute respiratory infection in Lisbon, Portugal, that resulted in the deaths of two children.