Human metapneumovirus infections among children with acute respiratory infections seen in a large referral hospital in India

1. Introduction 

Acute respiratory infections (ARI) are the leading cause of morbidity and mortality among infants and children less than 5 years of age in developing countries (Denny and Loda, 1986). A newly described respiratory pathogen, human metapneumovirus (hMPV), has been identified as a significant cause of hospitalization for ARI in young children (van den Hoogen et al., 2001, Boivin et al., 2003). The virus has been detected in both developed (Peret et al., 2002, Nissen et al., 2002, Peiris et al., 2003) and developing countries (Rao et al., 2004, IJpma et al., 2004). This study describes the prevalence of hMPV in children who presented with ARI to a large referral hospital in Delhi, India, and the genotype of circulating strains on the basis of F gene nucleotide sequence analysis.

2. Methods 

Children <5 years of age with ARI seen in the outpatient department (OPD) or as inpatients at the All India Institute of Medical Sciences (AIIMS), New Delhi, were enrolled in the study from June 2004 to March 2005. Informed consent was obtained from parents of the children and the human ethics committee of AIIMS approved the study. In most children, nasopharyngeal aspirates (NPAs) were collected and in some children from the OPD, nasopharyngeal swabs (NPSs) were collected using sterile cotton tipped applicators (Hardwood Products Company LLC, USA). Samples were stored at −70°C until use.

RNA was isolated from clinical samples and reverse transcription using 500ng of random hexamer primer and AMV reverse transcriptase (Promega Inc., USA) was performed. Amplification was performed by semi-nested PCR using primers designed from a conserved region in the N gene of hMPV. Primer positions are provided relative to hMPV strain 00–1 (van den Hoogen et al., 2001). External PCR was done with forward primer N79 (AAG CAT GCT ATA TTA AAA GAG TCT CA, nucleotides 79–104) and reverse primer N518 (ATT ATG GGT GTG TCT GGT GCT GA, nucleotides 496–518). The semi-nested PCR was done with the forward primer N79 and the internal reverse primer N443 (ATT GTT TTT CTT GCT TCT TTG TCT AT, nucleotides 418–443). External PCR cycling conditions were 95°C for 3min followed by 35 cycles of 95°C for 1min, 52°C for 45s and 72°C for 1min, with a final extension step of 7min at 72°C. Semi-nested PCR cycling conditions were similar to the external but with an annealing temperature of 53°C. F gene RT-PCR was done on the N gene PCR positive samples to allow further characterization of the hMPV strains. Primer positions are given relative to hMPV strain NL/1/94 (van den Hoogen et al., 2004). Primers designed for the F gene amplification and sequencing were, forward primer F571 (GTC AGC TTC AGT CAA TTC AAC AGA, nucleotides 571–594) and reverse primer F977 (CAG TCT TTT TCA TTT GGG TAG TAA, nucleotides 954–977) which amplified a 407 base region of F gene. PCR cycling used 95°C for 3min followed by 35 cycles of 95°C for 1min, 55°C for 45s, 72°C for 1min and a final extension step of 72°C for 7min. The PCR products were visualized in 2% agarose gels, extracted and the nucleotide sequences of bases 607–965bp of the F gene were determined (Big Dye Terminator DNA sequencing kit v 3.1, ABI, USA). The GenBank accession numbers are in the figure legend. The phylogenetic tree was constructed using 751–965bp of the F gene.

3. Results 

Of the 97 children enrolled for this study, 14 had URI and 83 had ALRI as per WHO criteria (WHO, 2000). hMPV was detected in 12 children, 2 had URI and 10 had ALRI (12% of the ALRI). hMPV was detected in 3 (13%) of the 24 inpatients. The majority of hMPV detections were in the months of December–February (8 out of 12), one positive sample was detected each in June, August, November and March. Eight of the patients with hMPV were less than 1 year of age.

Partial F gene sequences were available from seven patients. Nucleotide identity was high among the Delhi viruses (97%), two of the viruses had identical sequences. The Delhi strains clustered with A2 lineage strain NL/17/00 (nucleotide and amino acid identity both 97–99%). The Delhi strains differed from viruses in other lineages, i.e., nucleotide and amino acid identity with A1 strain (NL/1/00) was 91–93% and 96–97%, with B1 (NL/1/99) was 79–81% and 95–96% and with B2 strain (NL/1/94) was 80–82% and 95–96%, respectively. The phylogenetic tree demonstrates the relationship of the Indian isolates to those from other parts of the world (Fig. 1). All the viruses from Delhi were placed in one of two major branches of the A2 lineage.

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Fig. 1. Phylogenetic tree for Indian hMPV strains based on partial F gene nucleotide sequences. The nucleotide sequences for a 215 bases (position 751-965) region of the F gene were aligned with CLUSTAL X program. Phylogenetic and molecular evolutionary analyses were conducted using MEGA Version 3.0 (Kumar et al., 2004), Kimura-2 parameter and neighbour joining algorithm (bootstrap value of 1000 replications). Major lineages are shown. Delhi strains are encircled. Percent bootstrap support is indicated by the values at each node, values <50 are omitted. Comparisons were made to the following viruses (Genbank accession number). A1 lineage: NL/1/00 [AF371337], CAN′99-81 [AY145294], CAN′00-14 [AY145299], JPS03-180 [AY530092], FR02-7 [AY626969]; A2 lineage: FR01-1 [AY626963], JPOC04-125 [AB194959], JPY88-12 [AY622381], NI107 [AY789630], BJ1887 [AY830145], CAN′97 [AY145296], NL/17/00 [AY304360], CAN′00-12 [AY145297], CAN′00-16 [AY145301], NI143 [AY789627], NG03F27 [AB191396]; B1 lineage: NL/1/99 [AY304361]; B2 lineage: NL/1/94 [AY304362]. The Indian viruses from the present study belonging to A2 lineage have been annoted accession numbers: DQ083335 [IND/04-1], DQ083334 [IND/04-2], DQ083336 [IND/05-3], DQ083337 [IND/05-4], DQ083338 [IND/05-5], DQ083339 [IND/05-6], DQ083340 [IND/05-7].

4. Discussion 

This report establishes hMPV as an important cause of ARI in children in India. hMPV was found in 12% of patients with ALRI, including 13% of inpatients with ALRI. hMPV has been reported in 5–10% of children hospitalized with ARI. It has also been found in healthy and immunocompromised adults (Boivin et al., 2002, Prins and Wolthers, 2004).

Most infections with hMPV occur in children younger than 1 year of age, this was true in our study also. The seasonal distribution of hMPV infection varies, the majority of cases occur between December and April in temperate climates and in spring and early summer in the far-east (Boivin et al., 2003, Peiris et al., 2003, Prins and Wolthers, 2004). North India has a temperate climate with colder months from November through February. The majority of the hMPV detections in the present study were from December to February with a few in the summer, spring and fall. A preliminary report from Pune in central India analyzed 26 samples from children collected during July and August, 2003; hMPV was found in five patients (Rao et al., 2004). Thus, hMPV seasonality might vary in different parts of India.

To detect hMPV, we designed a RT-PCR assay based on the N gene as it is highly conserved and has been used in other studies (van den Hoogen et al., 2003, Prins and Wolthers, 2004). The F gene has been a target gene for phylogenetic studies as the F protein is a major antigenic determinant (Esper et al., 2003, Boivin et al., 2004). Group A viruses are identified more commonly than group B, and all of the viruses characterized here were in the group A lineage A2. The Delhi viruses clustered with viruses from Japan, China, France and Ireland (Fig. 1). There was very limited variability among the Delhi viruses based on F gene analysis. Because the F gene is highly conserved, further insights could be provided by analysis of more variable genes such as that of the G protein (Boivin et al., 2004). Additional studies including sampling for longer periods of time and more detailed genetic analyses will be required to better define hMPV molecular epidemiology and its contribution to ARIs in children in India.