
==== Front
Ann Med
Ann Med
Annals of Medicine
0785-3890
1365-2060
Taylor & Francis

39221771
10.1080/07853890.2024.2398719
2398719
Version of Record
Research Article
Infectious Diseases
Molecular detection and genotyping of HMPV in patients with severe acute respiratory infection in India
P. Pragathi et al.
Annals of Medicine
P. Pragathi #
https://orcid.org/0000-0002-7707-8186
Shetty Ujwal #
https://orcid.org/0009-0001-6144-185X
Parida Preetiparna
https://orcid.org/0000-0003-0384-1078
Varamballi Prasad
https://orcid.org/0000-0003-0402-1143
Mukhopadhyay Chiranjay
https://orcid.org/0000-0002-3540-2865
N Sudheesh
Manipal Institute of Virology, Manipal Academy of Higher Education, Manipal, India
# Contributed equally.

CONTACT Sudheesh N sudheesh.n@manipal.edu Manipal Institute of Virology, Manipal Academy of Higher Education, Manipal, India.
2 9 2024
2024
2 9 2024
56 1 239871913 10 2023
3 8 2024
13 8 2024
KnowledgeWorks Global Ltd.31 8 2024
published online in a building issue31 8 2024
© 2024 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
2024
The Author(s)
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

Abstract

Background

Human metapneumovirus (HMPV) is a common respiratory pathogen that causes respiratory tract infections. In India, HMPV has been identified as one of the leading causes of morbidity and mortality in infants and young children with respiratory tract infections. The most reported sublineages of HMPV in India are B1, B2, A2b and A2c.

Objective

A retrospective study was conducted to determine the circulating genotypes of HMPV among SARI cases from January 2016 to December 2018.

Materials and methods

Positive throat swab samples were confirmed with real-time RT–PCR. Subsequently, these samples were analysed using semi-nested conventional RT–PCR targeting the G gene, followed by sequencing and phylogenetic analysis. Clinical data analysis was also performed using SPSS 15.0 software.

Results

All 20 samples from the SARI cases were classified under the A2c sublineage of HMPV. Phylogenetic analysis indicated that these strains were genetically related to those circulating in Japan, China, and Croatia. Among the samples, ten showed 111-nucleotide duplications, while the other ten had 180-nucleotide duplications.

Conclusion

Clinical analysis showed that four cases had coinfections with other pathogens. Our extensive analysis of patient samples determined that HMPV, especially the A2c genotype, significantly contributed to SARI cases within our study population, which signifies the importance of considering HMPV as a probable aetiological agent when investigating SARI outbreaks.

Keywords

HMPV
SARI
RT–PCR
climate action
good health and well-being
Manipal Institute of Virology, Manipal Academy of Higher Education Manipal Institute of Virology, Manipal Academy of Higher Education provided financial support for this study.
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pmc1. Introduction

In 2001, researchers in the Netherlands isolated the first human metapneumovirus (HMPV) from nasopharyngeal aspirates of young children less than five years of age [1,2]. HMPV is classified as a negative sense single-stranded RNA virus, a member of the Metapneumovirus genus within the Pneumoviridae family [3]. It is transmitted through infectious air droplets; the incubation period is 3 to 5 days [4]. HMPV has been isolated in all continents and is seasonally distributed, with outbreaks typically occurring during the spring and winter [4]. HMPV is linked to various respiratory conditions, including croup, bronchitis, bronchiolitis, pneumonia, chronic obstructive pulmonary disease (COPD), reactive airway disease, and asthma exacerbation in clinical settings. Reports suggest that HMPV is responsible for 5–7% of acute respiratory infections in patients younger than five [5]. Retrospective serological studies in the Netherlands have shown that by the age of five, virtually all the children possess antibodies against HMPV [6], and 6–40% of acute respiratory infection (ARI) cases in hospitalised and outpatient children are attributed to HMPV [7,8].

Acute respiratory tract infections (ARTIs) are a major cause of symptomatic illness worldwide, regardless of age, gender, or geographical location. They are one of the principal causes of morbidity and mortality [9]. Globally, ARI (acute respiratory illness) is one of the leading causes of death among children [10–12]. HMPV is a casual respiratory pathogen that causes ARI primarily in children and causes substantial morbidity in elderly and immunocompromised patients [13]. The lack of an etiologic diagnosis accounts for more than 50% of the unnecessary or inappropriate prescriptions of antibiotics for ARI [14].

Even in a few developed countries, severe acute respiratory illness (SARI) has led to high morbidity and mortality among children, thus significantly burdening society. A wide range of potential pathogens with similar clinical symptoms complicates the diagnosis of ARI. HMPV has been reported in 5–10% of children admitted to hospitals with acute respiratory infections (ARI). However, in a study conducted in India, HMPV was detected in 12% of patients with acute lower respiratory infections (ALRI), including 13% of those hospitalized with ALRI [15].

HMPV is classified into two genotypes, A and B, each of which is further divided into two subgroups: A1 and A2, and B1 and B2. Subgroup A2 is additionally subdivided into A2a, A2b and A2c [1,2,5,16].

The main surface polymers of virally encoded proteins are G and F glycoproteins. These two proteins are antigenically significant and are responsible for evoking protective immunity. The HMPV G protein exhibits a high degree of nucleotide variation, which explains the genetic variance between genotypes. The G gene’s nucleotide identity varies between genotypes by approximately 45–53%, although its amino acid identity varies by approximately 22–27% [17].

Studies suggest using reverse transcription polymerase chain reaction (RT–PCR)-based molecular techniques to detect HMPV targeting the G gene [18–21]. However, in India, there exists limited data regarding the prevalence and genetic variability of human metapneumovirus (HMPV) strains [22–24]. Diagnostic HMPV assays need to be developed to detect all HMPV subtypes.

HMPV is identified as one of the important causes of lower respiratory tract infections in children, adults and immunocompromised individuals. Previously, few studies have focused on the correlation of HMPV with SARI cases. However, limited data on the molecular epidemiology of HMPV causing SARI are available in India. The present study focuses on clinical, haematological and biological data on HMPV.

2. Materials and methods

2.1. Samples

A total of 206 HMPV infection cases were identified using QuantStudio™ 5 Real-Time PCR System (Applied Biosystems) and FTD™ Respiratory Pathogens 21 Multiplex Real-Time PCR Kit as per the manufacturer’s instructions (Fast-track Diagnostics© Luxembourg S.à.r.l) from samples collected during acute febrile illness (AFI) surveillance study conducted at the Manipal Institute of Virology (MIV) from January 2016 to December 2018. Cases were recruited after obtaining written consent from all patients or their guardians for minors (assent form). Archived samples from this earlier study (AFI) were retrospectively selected using a purposive sampling method. Of these cases (n = 206), 36 met the World Health Organization (WHO) criteria for severe acute respiratory infection (SARI), and 20 cases with cycle threshold (Ct) values below 20 were selected for modified Sanger sequencing. The study was conducted following the principles of the 2013 Helsinki Declaration [25], and ethical approval was obtained from the Institutional Ethics Committee (IEC2:181/2022).

2.2. PCR

Viral RNA was extracted from 140 µl of throat swab sample using the FavorPrep™ Viral DNA/RNA Kit (FAVORGEN, Taiwan) according to the manufacturer’s instructions. Semi-nested RT–PCR was performed to amplify the G gene [23], resulting in a product size of 930 bp spanning the complete gene. The thermocycling protocol was followed as described by Preeti et al. [26]. Subsequently, the final PCR products were electrophoresed on a 1% gel (w/v) agarose gel and visualised using an ultraviolet transilluminator (G: BOX Chemi XRQ, Syngene, A Division of Synoptics Ltd). The band of interest was excised and purified using the GenElute™ gel purification kit (Sigma–Aldrich, Merck, USA).

2.3. Modified Sanger sequencing and analysis

Following purification, the PCR products were directly sequenced using specific primers [23] and the BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies, Thermo Fisher Scientific, USA) according to the manufacturer’s instructions in a 3500 XL Genetic Analyser (Applied Biosystems, USA). The obtained chromatograms were analysed using Sequencher 5.4.6 software. For multiple sequence alignment (MSA), Clustal W in Mega 11.0 (https://www.megasoftware.net/) was utilised for all 20 sequences. Subsequently, the nucleotide and deduced amino acid sequences of the G gene of the strains were compared with previously published G gene sequences in the NCBI nucleotide database. The maximum-likelihood method with reliability testing through 1000 bootstrap replications was employed to construct the phylogenetic tree. The best evolutionary model, determined by model selection in MEGA 11.0, was the General Time Reversible model.

2.4. Clinical data

The AFI database provided the cases’ sociodemographic, clinical and laboratory details. The statistical analysis was conducted using SPSS 15.0 for Windows (SPSS™ Inc., USA). Demographic data and baseline characteristics were summarised using numbers and percentages, while continuous data were presented as the mean and standard deviation.

3. Results

3.1. Clinical data

The 36 HMPV real-time PCR-positive cases with SARI were from different parts of India, such as Goa (n = 1, 2.8%), Odisha (n = 5,13.9%), Karnataka (n = 13, 36.1%), Kerala (n = 2, 5.6%), Jharkhand (n = 9, 25%), Maharashtra (n = 1, 2.8%), and Tamil Nadu (n = 5, 13.9%). A significant number of HMPV cases were observed during the spring and winter seasons. The mean age of the HMPV‑infected study participants was 24.6 years (standard deviation = 17.1). The most affected age group was 15–50 (55.6%), as shown in Table 1. The age-specific distribution of positive cases is shown in Table 1.

Table 1. Demographic characteristics of HMPV-positive SARI cases (n = 36).

Characteristics	HMPV Cases
(n = 36)
N (%)	
Age in years	 	
 Mean (SD)	24.6 (17.1)	
 Median (IQR)	22 (8.5 − 39.5)	
 Range	1 to 62	
Age in years (Male)	 	
 Mean (SD)	27 (20.6)	
 Median (IQR)	26 (8-45)	
 Range	1 to 62	
Age in years (Female)	 	
 Mean (SD)	23.1 (16.5)	
 Median (IQR)	19 (8.5-37)	
 Range	2 to 50	
Age Group	 	
 0 to 2	1 (2.8)	
 2 to 5	4 (11.1)	
 5 to 15	8 (22.2)	
 15 to 50	20 (55.6)	
 50 to 65	3 (8.3)	

Among the 36 HMPV‑positive cases, an equal number of males and females were infected with HMPV, as shown in Table 1. For males, the median age was 26 (8–45), and for females, it was 19 (8.5–37). Most of the HMPV cases had a middle socioeconomic status (61.1), and the remaining patients were students, agriculturists and working professionals. All the study participants presented with breathlessness, cough and fever. Other predominant symptoms were headache (n = 32, 88.9%), coryza (n = 31, 86.1%), myalgia (n = 29, 80.6%), chills (n = 26, 72.2%), sore throat (n = 24, 66.7%), night sweats (n = 20, 55.6%) and abdominal discomfort (n = 11, 30.6%) (Table 2).

Table 2. Clinical characteristics of HMPV-positive SARI cases (n = 36).

Clinical parameters	HMPV Cases
(n = 36)
N (%)	
Cough	36/36 (100)	
Breathlessness	36/36 (100)	
Headache	32/36 (88.9)	
General Weakness	32/36 (88.9)	
Coryza	31/36 (86.1)	
Myalgia	29/36 (80.6)	
Chills	26/36 (72.2)	
Sore throat	24/36 (66.7)	
Night sweats	20/36 (55.6)	
Joint Pain	17/36 (47.2)	
Nausea	16/36 (44.4)	
Retro-orbital pain	13/36 (36.1)	
Vomiting	12/36 (33.3)	
Chest pain	11/36 (30.6)	
Photophobia	11/36 (30.6)	
Abdominal pain	11/36 (30.6)	
Prostration	9/36 (25)	
Neck pain	8/36 (22.2)	
Pallor	6/36 (16.7)	
Lymphadenopathy	6/36 (16.7)	
Red eye	3/36 (8.3)	
Diarrhea	3/36 (8.3)	
Burning micturition	3/36 (8.3)	
Oral Ulcers	2/36 (5.6)	
Altered sensorium	1/36 (2.8)	
Seizures	1/36 (2.8)	

Coinfections among four patients were also observed in the study, where two of them had scrub typhus, one of the patients had influenza A (H3N2) infection and the other patient was infected with leptospirosis. Haematological parameters showed that five (14.7%) out of 36 patients had fluctuating total leukocyte counts, one (3.7%) participant had a total bilirubin level above 1.2 mg/dl, and three (8.8%) among them had a platelet count less than 140,000/mm3.

3.2. Real-time RT-PCR and sequencing

Out of 206 HMPV real-time RT-PCR-positive cases, 36 were SARI cases. The average Ct value of the 36 cases was 28.5. Twenty samples with Ct values less than 20 were selected for genotyping by modified Sanger sequencing and phylogenetic analysis. These sequences were submitted to the NCBI database (OR135588-OR135607).

Sequencing revealed that the predominant circulating lineage in India from 2016-2018 belonged to A2c (Figure 1). Of the 20 samples, 10 demonstrated 111-nucleotide duplication, and the remaining 10 displayed 180-nucleotide duplication.

Figure 1. The phylogenetic tree of HMPV strains, 2016-2018.

The reference strains were downloaded from the NCBI database. Only values >75% are shown on the major nodes. The 111-nucleotide duplication sequences are represented as red diamonds ◆ and 180-nucleotide duplication is shown as blue squares ■.

4. Discussion

Respiratory tract infections (RTIs) have emerged as a significant cause of hospitalisation, particularly affecting young children during winter. Previous research has identified HMPV as a prominent etiological agent responsible for lower respiratory tract infections (LRTIs) among children in China. However, the precise burden of HMPV among the general population is unknown due to limited available studies [27]. HMPV exhibits a global distribution, with sublineage A2b of genotype A2 being prevalent worldwide for several years [23, 26]. The prevalence of HMPV worldwide is estimated to be between 5 and 10% [15].

Usually, all SARI aetiologies are not usually known, and almost 50% go undiagnosed [28]. Through this study, we found that HMPV is also one of the causes of SARI. Hence, HMPV should be included in routine testing among SARI cases. Our study also found that patients with coinfections of scrub typhus, influenza and leptospirosis were more likely to have severe disease. Additionally, the impact of gene duplication on severity is not yet known in the case of HMPV. Hence, more genotyping and clinical studies on HMPV are needed.

Previous studies have suggested that HMPV likely causes severe disease in children, but in this study, the mean age of HMPV patients with SARI was 24.6 years. This suggests that HMPV can also cause severe disease in young and older adults. Additionally, some studies have shown that the A2c genotype is more likely to cause disease in younger adults than in children [29].

Duplications were also demonstrated by respiratory syncytial virus (RSV) in the G gene; however, the 180-nucleotide duplication observed in A2b strains is 2-3 times larger than that found in RSV [30]. In India, the studies conducted by Banerjee et al. and Choudhary et al. indicate that the average prevalence of HMPV varies from 4-12%. The circulating lineages identified in India include B1, B2, A2b and A2c [23,26,31]. Additional investigations were carried out by Singh et al. and Broor et al. who assessed pediatric patients in tertiary hospitals to determine the presence of other respiratory viruses. HMPV A and B contributed to nearly 1.1% of the cases [32]. In a Kolkata study conducted by Agarwal et al. a higher prevalence of the A2 (77%) subgroup compared to B1 (23%) was reported for 2005-2007, with a similar trend observed in eastern India. In contrast, Banerjee et al. in 2007, reported A2 (47.3%) and B1 (47.3%) predominance in 2006 and B2 (5.2%) prevalence in Delhi, northern India.

Phylogenetic analysis of the G gene of HMPV in this study showed the circulation of A2c sublineage with nucleotide duplication, among SARI cases in India. Our findings are consistent with Parida et al. [26], who previously reported the circulation of the A2c sublineage with nucleotide duplication between 2016 and 2018 using archived samples from the same AFI study. None of the samples belonged to the B1 or B2 lineages. Ten samples clustered with the Chinese strain (MK087726) and the Yokohama strain (LC270124) and showed 111-nucleotide duplications in the G gene. The remaining ten samples clustered together and showed 180-nucleotide duplications in the G gene respectively. Similar strains were reported from Malaysia between 2012 and 2014 and Croatia between 2015 and 2016 [33]. The 111-nucleotide duplication was previously reported in Yokohama in 2017, and the 180-nucleotide duplication was reported earlier in Spain and Yokohama, Japan, in 2011 and 2013 [30].

Limited studies have investigated the epidemiology and molecular characterisation of HMPV among patients with SARI in India. HMPV was identified only in 2001, yet evidence suggests it has been endemic in the human population for at least two centuries and likely far longer [6,34]. In healthy adults, it typically causes mild to moderate ARIs. However, severe ARIs are more common in older adults and patients with underlying conditions such as cardiopulmonary disease and diabetes. In this study, we did not find a significant difference in severity between older and younger adults, nor did we find a significant difference in severity between people with higher and lower viral loads. However, the sample size was too small to draw firm conclusions about the relationship between severity and viral load. More extensive studies with a more representative sample population are needed to determine whether there is a correlation between these two factors. Nevertheless, among the 206 available samples that tested positive for HMPV, our findings underscored a heightened susceptibility to coinfections among individuals affected with SARI instead of those without SARI symptoms. Further studies are required to gain a more comprehensive understanding of the influence of SARI on cases involving HMPV and coinfections.

5. Conclusion

Our study underscores the significance of HMPV, specifically the A2c genotype, in the context of SARI, highlighting its etiological importance and genetic characteristics. The information on HMPV and their circulating genotypes is limited, with very few studies conducted on HMPV despite it being one of the leading causes of SARI. These findings provide a deeper perception of respiratory infections and can inform public health strategies for preventing and managing SARI cases associated with HMPV. Moreover, the findings of this study will contribute to researchers for future studies in better understanding the fundamental molecular mechanisms of disease in the population. It is imperative to consistently monitor HMPV infections to identify the epidemiologic emergence and spread of these relevant strains and their role in SARI. Additionally, further studies are needed to ascertain whether these novel variants have any impact on evading the immune system. There is also a need to increase the application of whole genome sequencing studies to characterise sub-group and strain variation better. This approach is being actively implemented for RSV, and similar advancements are anticipated for HMPV in the near future.

Acknowledgements

All authors express their gratitude to the scientific, laboratory, and office staff of Manipal Institute of Virology, Manipal Academy of Higher Education for their invaluable support and assistance.

Ethical conduct

The study obtained clearance from the Institutional Ethical Committee (IEC), Kasturba Hospital, Manipal, a constituent unit of Manipal Academy of Higher Education (IEC2:181/2022).

Authors contributions

PP performed the experiment, analysed data and drafted the manuscript. US performed data analysis, and interpretation and critically reviewed and edited the manuscript. PPP analysed and interpreted the data. PV contributed to clinical data analysis and interpretation. CM provided valuable insights into revising the paper for intellectual content and critically reviewed the manuscript. SN conceived and designed the study, provided critical feedback, and final approval of the version to be published. All authors agree to be accountable for all aspects of the work.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data and materials supporting the findings and analyses presented in this paper are available upon request, except in cases where ethical, privacy, or security considerations prevent disclosure.
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References

1 Jartti T, van den Hoogen B, Garofalo RP, et al. Metapneumovirus and acute wheezing in children. Lancet. 2002;360 (9343 ):1393–1394. doi: 10.1016/S0140-6736(02)11391-2.12423987
2 Boivin G, Abed Y, Pelletier G, et al. Virological features and clinical manifestations associated with human metapneumovirus: a new paramyxovirus responsible for acute respiratory-tract infections in all age groups. J Infect Dis. 2002;186 (9 ):1330–1334. doi: 10.1086/344319.12402203
3 Nao N, Saikusa M, Sato K, et al. Recent molecular evolution of human metapneumovirus (HMPV): Subdivision of HMPV A2b strains. Microorganisms. 2020;8 (9 ):1280. doi: 10.3390/microorganisms8091280.32839394
4 Panda S, Mohakud NK, Pena L, et al. Human metapneumovirus: review of an important respiratory pathogen. Int J Infect Dis. 2014;25 :45–52. doi: 10.1016/j.ijid.2014.03.1394.24841931
5 Van Den Hoogen BG, Bestebroer TM, Osterhaus ADME, et al. Analysis of the genomic sequence of a human metapneumovirus. Virology. 2002;295 (1 ):119–132. doi: 10.1006/viro.2001.1355.12033771
6 van den Hoogen BG, de Jong JC, Groen J, et al. A newly discovered human pneumovirus isolated from young children with respiratory tract disease. Nat Med. 2001;7 (6 ):719–724. doi: 10.1038/89098.11385510
7 Huang J, Chopra P, Liu L, et al. Structure, immunogenicity, and conformation-dependent receptor binding of the postfusion human metapneumovirus F protein. J Virol. 2021;95 (18 ):e00593-21. doi: 10.1128/JVI.00593-21.34160259
8 Yi L, Zou L, Peng J, et al. Epidemiology, evolution and transmission of human metapneumovirus in Guangzhou China, 2013–2017. Sci Rep. 2019;9 (1 ):14022. doi: 10.1038/s41598-019-50340-8.31575919
9 Divarathna MVM, Rafeek RAM, Noordeen F. A review on epidemiology and impact of human metapneumovirus infections in children using TIAB search strategy on PubMed and PubMed Central articles. Rev Med Virol. 2020;30 (1 ):e2090. doi: 10.1002/rmv.2090.31788915
10 Fujitsuka A, Tsukagoshi H, Arakawa M, et al. A molecular epidemiological study of respiratory viruses detected in Japanese children with acute wheezing illness. BMC Infect Dis. 2011;11 (1 ):168. doi: 10.1186/1471-2334-11-168.21663657
11 Kim CK, Choi J, Callaway Z, et al. Clinical and epidemiological comparison of human metapneumovirus and respiratory syncytial virus in Seoul, Korea, 2003-2008. J Korean Med Sci. 2010;25 (3 ):342–347. doi: 10.3346/jkms.2010.25.3.342.20191030
12 Tregoning JS, Schwarze J. Respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology. Clin Microbiol Rev. 2010;23 (1 ):74–98. doi: 10.1128/CMR.00032-09.20065326
13 Jallow MM, Fall A, Kiori D, et al. Epidemiological, clinical and genotypic features of human Metapneumovirus in patients with influenza-like illness in Senegal, 2012 to 2016. BMC Infect Dis. 2019;19 (1 ):457. doi: 10.1186/s12879-019-4096-y.31117983
14 Zhang C, Zhu N, Xie Z, et al. Viral etiology and clinical profiles of children with severe acute respiratory infections in China. PLoS One. 2013;8 (8 ):e72606. doi: 10.1371/journal.pone.0072606.23991128
15 Banerjee S, Bharaj P, Sullender W, et al. Human metapneumovirus infections among children with acute respiratory infections seen in a large referral hospital in India. J Clin Virol. 2007;38 (1 ):70–72. doi: 10.1016/j.jcv.2006.07.003.17085070
16 Xie Z, Xu J, Ren Y, et al. Emerging human metapneumovirus gene duplication variants in patients with severe acute respiratory infection, China, 2017–2019. Emerg Infect Dis. 2021;27 (1 ):275–277. doi: 10.3201/eid2701.201043.33350918
17 Ludewick HP, Abed Y, van Niekerk N, et al. Human metapneumovirus genetic variability, South Africa. Emerg Infect Dis. 2005;11 (7 ):1074–1078. doi: 10.3201/eid1107.050050.16022783
18 Bouscambert-Duchamp M, Lina B, Trompette A, et al. Detection of human metapneumovirus RNA sequences in nasopharyngeal aspirates of young French children with acute bronchiolitis by real-time reverse transcriptase PCR and phylogenetic analysis. J Clin Microbiol. 2005;43 (3 ):1411–1414. doi: 10.1128/JCM.43.3.1411-1414.2005.15750120
19 Côté S, Abed Y, Boivin G. Comparative evaluation of real-time PCR assays for detection of the human metapneumovirus. J Clin Microbiol. 2003;41 (8 ):3631–3635. doi: 10.1128/JCM.41.8.3631-3635.2003.12904367
20 Mackay IM, Jacob KC, Woolhouse D, et al. Molecular assays for detection of human metapneumovirus. J Clin Microbiol. 2003;41 (1 ):100–105. doi: 10.1128/JCM.41.1.100-105.2003.12517833
21 Maertzdorf J, Wang CK, Brown JB, et al. Real-time reverse transcriptase PCR assay for detection of human metapneumoviruses from all known genetic lineages. J Clin Microbiol. 2004;42 (3 ):981–986. doi: 10.1128/JCM.42.3.981-986.2004.15004041
22 Agrawal AS, Roy T, Ghosh S, et al. Genetic variability of attachment (G) and Fusion (F) protein genes of human metapneumovirus strains circulating during 2006-2009 in Kolkata, Eastern India. Virol J. 2011;8 (1 ):67. doi: 10.1186/1743-422X-8-67.21314961
23 Banerjee S, Sullender WM, Choudekar A, et al. Detection and genetic diversity of human metapneumovirus in hospitalized children with acute respiratory infections in India. J Med Virol. 2011;83 (10 ):1799–1810. doi: 10.1002/jmv.22176.21837798
24 Piyaratna R, Tollefson SJ, Williams JV. Genomic analysis of four human metapneumovirus prototypes. Virus Res. 2011;160 (1–2 ):200–205. doi: 10.1016/j.virusres.2011.06.014.21740936
25 World Medical Association. World Medical Association Declaration of Helsinki: ethical principles for medical research involving human subjects. JAMA. 2013;310 (20 ):2191–2194. doi: 10.1001/jama.2013.281053.24141714
26 Parida P, Sudheesh N, Sanjay ER, et al. The emergence of human metapneumovirus G gene duplication in hospitalized patients with respiratory tract infection, India, 2016–2018. Mol Biol Rep. 2023;50 (2 ):1109–1116. doi: 10.1007/s11033-022-08092-8.36399244
27 Zhang C, Du L-N, Zhang Z-Y, et al. Detection and genetic diversity of human metapneumovirus in hospitalized children with acute respiratory infections in Southwest China. J Clin Microbiol. 2012;50 (8 ):2714–2719. doi: 10.1128/JCM.00809-12.22692746
28 Aman AT, Wibawa T, Kosasih H, et al. Etiologies of severe acute respiratory infection (SARI) and misdiagnosis of influenza in Indonesia, 2013‐2016. Influenza Other Respir Viruses. 2021;15 (1 ):34–44. doi: 10.1111/irv.12781.32666619
29 Piñana M, Vila J, Maldonado C, et al. Insights into immune evasion of human metapneumovirus: novel 180- and 111-nucleotide duplications within viral G gene throughout 2014-2017 seasons in Barcelona, Spain. J Clin Virol. 2020;132 :104590. doi: 10.1016/j.jcv.2020.104590.32957052
30 Saikusa M, Kawakami C, Nao N, et al. 180-nucleotide duplication in the G gene of human metapneumovirus A2b subgroup strains circulating in Yokohama City, Japan, since 2014. Front Microbiol. 2017;8 :402. doi: 10.3389/fmicb.2017.00402.28352258
31 Choudhary ML, Anand SP, Sonawane NS, et al. Development of real-time RT-PCR for detection of human metapneumovirus and genetic analysis of circulating strains (2009-2011) in Pune, India. Arch Virol. 2014;159 (2 ):217–225. doi: 10.1007/s00705-013-1812-6.23929232
32 Singh AK, Jain A, Jain B, et al. Viral aetiology of acute lower respiratory tract illness in hospitalised paediatric patients of a tertiary hospital: one year prospective study. Indian J Med Microbiol. 2014;32 (1 ):13–18. doi: 10.4103/0255-0857.124288.24399381
33 Saikusa M, Nao N, Kawakami C, et al. A novel 111-nucleotide duplication in the G gene of human metapneumovirus. Microbiol Immunol. 2017;61 (11 ):507–512. doi: 10.1111/1348-0421.12543.28960538
34 de Graaf M, Osterhaus ADME, Fouchier RAM, et al. Evolutionary dynamics of human and avian metapneumoviruses. J Gen Virol. 2008;89 (Pt 12 ):2933–2942. doi: 10.1099/vir.0.2008/006957-0.19008378
