Detection and genetic diversity of human metapneumovirus in hospitalized children with acute respiratory infections in India

Abstract

Human metapneumovirus (hMPV) causes acute respiratory infections in children and adults. It is classified into two major genetic lineages and each lineage into two sublineages. The purpose of the study was to identify and characterize hMPV in children who presented to the All India Institute of Medical Sciences, New Delhi, India with acute respiratory infection from April 2005 to March 2007. By reverse-transcription polymerase chain reaction, hMPV was detected in 21 (3%) of the 662 nasopharyngeal samples from children with acute respiratory infection and in none of the 120 control children. Seven of the 21 (33%) children infected with hMPV required hospital admission for pneumonia or bronchiolitis. Most hMPV detections were during the winter and spring seasons. The majority (67%, 11/21) of children positive for hMPV were within 24 months of age. Phylogenetic analysis of partial F and N gene and the full G gene sequences showed three sub-lineages of hMPV circulated during the study period, B1, B2, and the novel sub-lineage A2b. The circulation pattern of hMPV genotypes varied by season. Comparison of the F and G genes of eight strains revealed incongruencies in lineage assignments, raising the possibility that recombination had occurred. Sequence analysis also revealed the F gene was relatively conserved whereas the G gene was more variable between the A and B lineages. This study demonstrates that hMPV is an important contributor to acute respiratory infection in children in India, resulting in both outpatient visits and hospitalizations.

Figure 1

Neighbor-joining phylogenetic tree using 359 bp (nt 607–965) of F gene sequences of Indian hMPV strains and reference sequences of lineage A and B. The prototype strains from the Netherlands and Canada of each of the sub-lineages are indicted by triangles. The Indian hMPV strains detected during 2005–2006 winter-spring season are indicated by circles and those detected during 2006–2007 winter-spring season are indicated by squares. Bootstrap values greater than 50% are shown at the branch nodes. Bootstrap values of 98% and 80% that supported the observed bi-partitioning of A2 sub-lineage are encircled. F gene sequences from GenBank of the following reference strains were used to construct the phylogenetic tree: A1 sub-lineage: NL/00/1, CAN99-81, JPS03-180; A2a sub-lineage:NL/17/00, CAN97-83; A2b sub-lineage: JPS03-176, JPS03-178, JPS03-187, JPY88-12, BJ1887, O0601; B1 sub-lineage:NL/1/99, CAN97-82, JPS02-76, JPS03-194; B2 sub-lineage: NL/1/94, CAN98-75, BJ1816, CAN00-13, CS113. Accession numbers of F gene sequences of 19 Indian strains: DQ861908 (IND/06-9/294OP), DQ861909 (IND/06-17/344OP), DQ861910 (IND/06-18/347OP), DQ861911(IND/06-19/351OP), DQ855629 (IND/06-12/313OP), DQ855630 (IND/06-11/311OP), DQ855631 (IND/06-8/268OP), DQ855632 (IND/06-21/365OP), DQ861912 (IND/06-16/W113), DQ855633 (IND/06-10/W102), DQ855634 (IND/06-13/W108), DQ855635 (IND/06-14/W110), DQ855636 (IND/06-15/W112), DQ855637 (IND/06-20/W119), EU259878 (IND/06-23/440OP), EU259879 (IND/06-24/452OP), EU259880 (IND/07-26/507OP), EU259882 (IND/07-27/514OP), EU259881 (IND/07-25/W140).

Figure 2

Neighbor-joining phylogenetic tree using 340 bp (69–408 nucleotides) N gene sequences of Indian hMPV strains and reference sequences of lineage A and B. The prototype strains from the Netherlands and Canada of each of the sub-lineages are indicated by triangles. Twelve Indian hMPV strains detected during 2005–2006 winter-spring season are indicated by circles. Bootstrap values greater than 50% are shown at the branch nodes. Bootstrap values of 98% and 89% that supported the observed bi-partitioning of A2 sub-lineage are encircled. N gene sequences from GenBank of the following reference strains were used to construct the phylogenetic tree. A1 sub-lineage: NL/00/1, CAN99-81, JPS03-180; A2a sub-lineage:NL/17/00, CAN97-83; A2b sub-lineage: JPS03-176, JPS03-178, JPS03-240, JPS03-187, BJ1887; B1 sub-lineage:NL/1/99, CAN97-82, JPS02-76, JPS03-194; B2 sub-lineage: NL/1/94, CAN98-75, BJ1816, CAN00-13, CS113. Accession numbers of N gene sequences of 12 Indian strains: DQ908917(IND/06-9/294OP), DQ908914(IND/06-17/344OP), DQ908915(IND/06-18/347OP), DQ908916(IND 06-19/351OP), DQ908913(IND 06-12/313OP), DQ908907(IND 06-11/311OP), DQ908906(IND 06-8/268OP), DQ908908(IND 06-21/365OP), DQ908909(IND 06-10/W102), DQ908910(IND 06-13/W108), DQ908911(IND 06-14/W110), DQ908912(IND 06-15/W112)

Figure 3

Alignment of the predicted amino acid sequences of partial F protein (202–321 aa) of Indian hMPV strains belonging to A2 sublineage (Figure 3A), B1 sub-lineage (Figure 3B) and B2 sub-lineage (Figure 3C) with the Canadian (CAN) and Dutch (NL) prototype strains. Only residues that differ from the consensus sequence are shown. Identical amino acids are represented by periods. The prototype strains of A1 sub-lineage: CAN 99-81, NL/00/1, A2 sub-lineage: CAN 97-83, NL-17-00, B1 sub-lineage: CAN 97-82, NL/1/99, B2 sub-lineage: CAN 98-75, NL/1/94. Amino acid residues unique to sub-lineage A are boxed. Amino acid residues unique to lineage B are shaded in black, the amino acid residue unique to sub-lineage B1 is shaded in dark grey and the amino acid residue unique to sub-lineage B2 is shaded in light gray.

Figure 4

Neighbor-joining phylogenetic tree using G gene sequences of Indian hMPV strains and reference sequences of lineage A and B. The prototype strains from the Netherlands and Canada of each of the sub-lineages are indicted by triangles. The Indian hMPV strains detected during 2005–2006 winter-spring season are indicated by circles and those detected during 2006–2007 winter-spring season are indicated by squares. Bootstrap values greater than 50% are shown at the branch nodes. Bootstrap values of 96% and 74% that supported the observed bipartitioning of A2 sub-lineage are encircled. The 18 Indian A2b strains group in two separate clusters; one comprising 13 strains (in dotted box) and other comprising 5 strains (in dotted oval). Bootstrap values of 83% and 99% indicated by arrows further confirm the true clustering of 18 Indian A2b strains. Eight A2b Indian strains (*) are the possible recombinants that grouped in B1 lineage in F gene phylogenetic analysis. G gene sequences from GenBank of the following reference strains were used to construct the phylogenetic tree. A1 sub-lineage: NL/00/1, CAN99-81, JPS03-180 A2a sub-lineage:NL/17/00, CAN97-83, CAN182-02, Arg/4/00 A2b sub-lineage: JPS03-240, BJ1887, CHN05-06, CAN197-02, O0601 B1 sub-lineage:NL/1/99, CAN97-82 B2 sub-lineage: NL/1/94, CAN98-75, UK/5/01, BJ5128, CS113, CHN01-06 Accession numbers of G gene sequences of 19 Indian strains: EU259863 (IND 06-9/294OP), EU259875 (IND 06-17/344OP), EU259871 (IND 06-18/347OP), EU259859 (IND 06-12/313OP), EU259874 (IND 06-11/311OP), EU259868 (IND 06-8/268OP), EU259860 (IND 06-21/365OP), EU259872 (IND 06-22/326OP), EU259869 (IND 06-16/W113), EU259870 (IND 06-10/W102), EU259865 (IND 06-13/W108), EU259867 (IND 06-14/W110), EU259861 (IND 06-15/W112), EU259862 (IND 06-20/W119), EU259876 (IND 06-23/440OP), EU259873 (IND 06-24/452OP), EU259866 (IND 07-25/W140), EU259864 (IND 07-26/507OP), EU259877 (IND 07-27/514OP).

Figure 5

Alignment of the predicted amino acid sequences of G protein of Indian A2b strains with the prototype strains of A2 sub-lineage of hMPV. Proposed intracellular, transmembrane and extracellular domains are indicated by the arrows above the alignment. Numbers indicate amino acid position. Conserved cysteine residue is marked with a dot. Potential N-glycosylation sites are in dotted boxes. The brackets, [ ], represent the potential O-glycosylation region.

Figure 6

Alignment of the predicted amino acid sequences of G protein of Indian B2 strains with the prototype strains and a Chinese strain, CS113 of B2 sub-lineage of hMPV. Proposed intracellular, transmembrane and extracellular domains are indicated by the arrows above the alignment. Numbers indicate amino acid position. Conserved cysteine residue is marked with a dot. Potential N-glycosylation sites are in dotted boxes. The brackets, [ ], represent the potential O-glycosylation region.