key: cord-0751430-4e1q0di3
authors: Palani, Nandhini; Sistla, Sujatha
title: Epidemiology and phylogenetic analysis of respiratory viruses from 2012 to 2015 – A sentinel surveillance report from union territory of Puducherry, India
date: 2020-04-28
journal: Clin Epidemiol Glob Health
DOI: 10.1016/j.cegh.2020.04.019
sha: e0021fca0e1b1795ab1e6fe61fc16d185d24137d
doc_id: 751430
cord_uid: 4e1q0di3

Abstract Background Acute respiratory infections (ARI) are the most common illnesses affecting people of all ages worldwide. Viruses contribute to 30–70% of acute respiratory infections. Identification of these respiratory viruses is not given high priority except influenza; however, the knowledge about prevalence of non-influenza viruses, their seasonal pattern and genetic evolution have significant epidemiological value. Methods As a part of National Influenza-like illness surveillance programme, respiratory specimens were collected children and adults with symptoms of ILI or ARI, between January 2012 and March 2015 (including SARI cases). Real-time PCR was done to identify 13 respiratory viruses. Sequencing was done for representative isolates of each virus using ABI 3730 Genetic Analyzer. Results During the study period between January 2012 and March 2015, a total of 648 patients with symptoms of ARI were included in this study. The mean age of the patients was 20.2 years (SD = 19.13, median = 18); 292 (45.1%) were children (≤13 years) and 356 (54.9%) were adults. Respiratory viruses were identified in 44% (287/648) of all patients. Influenza accounted for the maximum number of cases- 179/648 (27.6%). Among the non-influenza viruses, RSV predominated with 34 cases (5.2%), followed by HMPV 24 (3.7%) and PIV-3 20 (3%). Four patients died due to INF A/H1N1 as a result of acute respiratory distress syndrome (ARDS) (CFR 3.7%). Among the non-influenza viruses, no particular seasonality pattern was observed over the different months of the study period. Conclusion Antibiotic usage in treating acute respiratory infections empirically is not justified as nearly half of ARI are due to viruses; nearly 28% of them were due to influenza viruses. Among the non-influenza viruses, RSV predominated, followed by HMPV. This study is based on an active influenza surveillance initiated after 2009 pandemic influenza outbreak, in the Union territory of Puducherry which has contributed significantly to the knowledge of the burden of influenza and non-influenza viruses among children and adults. Such surveillance network has paved the way for better diagnosis and timely therapeutic interventions.

Acute respiratory infections (ARI) are the most common illnesses affecting people of all ages worldwide. Viruses contribute to 30-70% of acute respiratory infections (1, 2) .

Influenza viruses are one of the leading causes of respiratory infections, with an annual global attack rate of about 5-10% in adults and 20-30% in children (3) and it is known to cause severe respiratory illness and death in high-risk individuals. Although anti-influenza drugs and vaccines are available, due to the continuous antigenic variations (in the form of drifts and shifts), this virus can lead to yearly epidemics and occasional pandemics; hence influenza remains a global threat. The next common virus associated with significant hospitalization and respiratory distress, particularly in children, is the respiratory syncytial virus (RSV). Nearly 70% of children are infected with RSV by the age of 1 year (4). Other common viruses are parainfluenza viruses, coronaviruses, human metapneumovirus, measles, adenoviruses and bocavirus. Identification of these respiratory viruses is not given high priority as they mostly cause self-limiting disease and no specific antiviral drugs are available (except for influenza and RSV). However, the knowledge about prevalence of non-influenza viruses, their seasonal pattern and genetic evolution have significant epidemiological value.

As a part of National Influenza surveillance programme, influenza surveillance was initiated in the Union territory of Puducherry/Pondicherry in 2008, under National center for disease control (NCDC) and Integrated Disease Surveillance Programme (IDSP). In 2009, Regional Influenza Laboratory was set up in JIPMER hospital and till date surveillance activity is continued for identifying influenza viruses in patients with ARI. This study discusses the burden of respiratory viruses in patients with ARI from Union territory of Puducherry as a part of a hospital-based surveillance program between 2012 and 2015, the seasonal pattern of respiratory viruses over these three years and the phylogenetic analysis of representative viral strains.

The Union Territory of Puducherry/Pondicherry is in Southern India and lies at a latitude of 11°46' to 12°30' North and a longitude of 79°36' to 79°52' East. The territory lies on the Coromandel Coast of Bay of Bengal extending over 479 Sq. km. The total population is 12, 44, 464 (2011 census 

Samples were collected using flocked nylon swabs in Hi-viral transport medium (HiMedia, Mumbai, India) and immediately transported to the laboratory on ice. Samples were stored at −80°C until further use. QIAamp viral RNA/DNA extraction method (Qiagen, Germany) was used as per manufacturer's instructions and the viral nucleic acid was stored at -80°C.

The Real-time PCR reactions were done in Step one real-time system (Applied Biosystems, USA) as one-step reverse transcriptase PCR for RNA or DNA PCR using AgPath-IDTM One

Step RT-PCR kit. The primers and probes for influenza A(H1N1)pdm09 were designed The quality of specimen collection was checked by testing all the samples for internal control, RNase P and positive, negative controls were included for all the tests. 

Data was represented as proportions or percentages. Statistical analysis was performed using GraphPad InStat software. All the categorical variables were tested using Fisher's exact t test or Chi-square test. P-value of <0.05 was considered statistically significant.

During the study period between January 2012 and March 2015, a total of 648 patients with symptoms of ARI were included in this study ( (Table. 3).

Thirteen patients had co-infections with more than one virus; there were two cases with RSV-HMPV co-infections. A single case each of the following co-infections were also found: 

Respiratory virus infections were common among adults (171/356, 48%) compared to children (129/292, 44%), but the proportion difference was not statistically significant (P= 0.34) (Fig.1) . INF A/H1N1, INF B, HMPV and PIV-3 were more common in adults, while RSV was common in children, but the proportional differences were only significant for INF A/H1N1 and RSV (P=0.0001). Children were more commonly infected with OTHER viruses (PIV-1&2, adenovirus, measles, corona virus or bocavirus) compared to adults (8.2% vs.

5.3%). Maximum positivity of INF A/H1N1 was found in age group of 21-50 years (Fig.2) .

Of the 109 patients with INF A/H1N1 infection, 76% (83/109) had their residence in Puducherry town and 23% (26/109) in Karaikal (Fig.3 ).

Of the 648 cases, 211 patients were diagnosed with lower respiratory tract infections (LRI)

while the remaining 437 patients had upper respiratory tract infections (URI). Clinical symptoms were not distinct in influenza and non-influenza infections, however, in influenza ARI cases, fever, myalgia and respiratory distress were found to be significantly higher than non-influenza viral ARI cases whereas runny nose and nasal congestion was more often associated with non-influenza cases ( 

Of the total 648 samples collected from patients with ARI, 299 (46%), 314 (48%), 35 (5%) were collected during the first 3 days, 4-7 days and >7 days of symptoms onset respectively.

About 52% (157) an isolate reported from Assam (Fig.8) .

All the RSV strains sequenced belonged to type A and they formed a separate subgroup along with strains isolated from other parts of India like Chennai and Maharashtra (Fig.9) . Two HMPV strains isolated in the same year, 2011 were entirely distinct from each other, forming two separate subgroups (Fig.10) . One of the HPIV-3 strains, KR704265, isolated in 2013 was closely related to the strain from the neighboring region Chennai which was isolated in the same year, while the other strain KR902754 had a molecular composition similar to isolates from Lithuania, Australia and Seattle (Fig.11) . No Indian study has analyzed HCoV-229E virus and the strains isolated from this study were genetically similar to strains reported from Germany, Malaysia, Japan and Netherlands (Fig.12) . Similarly, no study from India has analyzed the genetic sequence HCoV-OC43 and single isolate sequenced in our study showed a close relationship with a strain from the United Kingdom and the United States (Fig.13) . The measles strains were of genotype D8 and closed circulation of measles strains between Pondicherry and other parts of the neighboring state, Tamil Nadu was observed.

These strains were distinct from measles strains of Spain, Moscow, South Africa and it formed a separate subgroup along with other Indian strains (Fig.14) . Human bocavirus strains of this study were not closely related to other Indian strains, particularly one isolate, KR376140, isolated in 2012 showed a distinct lineage (Fig.15 ).

In Union territory of Puducherry, summer is from March through July and winter from December through February. September to December is the rainy months in this area.

During this study, two outbreaks of influenza A(H1N1)pdm09 occurred; first one in 2012 (58 cases) and the next in 2015 (50 cases). Seasonal influenza A(H3N2) and influenza B cases were found in all years, with a slight increase in 2013 and 2015 respectively. The detailed seasonality of influenza viruses in this region has been described in our earlier study on "Epidemiology of influenza viruses from 2009-2013" (11) . Among the non-influenza viruses, no particular seasonality pattern was observed over the different months of the study period;

however, more number of RSV cases was observed during the rainy and winter season between August and January, while HMPV and PIV-3 were found more during two summer months; April and May (Fig.16 ).

Respiratory viral infections are under-diagnosed due to non-specific clinical symptoms, self-limiting nature of the disease and expensive cost of testing. Literature indicates that clinicians prescribe antibiotics for ARI 40-50% of the time. It is a well-known fact that inappropriate use and abuse of antibiotics for the treatment of ARI has played a significant role in worsening the global problem of antimicrobial resistance (12) . Through this study, we reiterate the fact that nearly half the ARI cases (44%) could be viral, as reported in many previous studies (1, (13) (14) (15) (16) (17) and all the cases do not require antibiotic therapy.

The most common virus detected in this study was influenza A(H1N1)pdm09, with a prevalence of 17%. Globally, influenza A(H1N1)pdm09 virus has been reported to cause 10-30% of ARI (13, (17) (18) (19) . In India, over 20,000 people were affected by the pandemic and over 1700 people died by the end of 2010 influenza A(H1N1)pdm09 outbreak (20) .

Although WHO announced the end of the pandemic in August 2010, influenza viruses cause significant morbidity during the annual outbreaks. During this prospective study, we observed two influenza A(H1N1)pdm09 outbreaks, one each in 2012 and 2015 with laboratory-confirmed infection in more than100 patients altogether. During this study, all the 109 cases with influenza A(H1N1)pdm09 infection, were treated with oseltamivir, despite this, four patients died due to ARDS (Acute respiratory disease syndrome).

The case fatality rate of influenza A(H1N1)pdm09 (3.7%) reported in this study (2011) (2012) (2013) (2014) (2015) was much lower when compared to 2009-2010 (11) . Generally, the impact of pandemic influenza in this region was relatively lower when compared to many Indian and foreign states (21) (22) (23) (24) . This could be due to the continuing influenza surveillance activities in this region from 2008, ensuring rapid detection of influenza, timely admission and prompt initiation of prophylaxis in the suspected cases. Patients affected by influenza A(H1N1)pdm09 were mostly of age group 20-40 years; hence this study yet again corroborates this well-known observation made worldwide. This might be due to their active lifestyle environment, where they are at a higher risk of getting infected with this novel influenza A(H1N1) virus, to which they have never been exposed previously (25) .

The second most common virus identified in this study population was Influenza B virus (6.5%); however, the proportion was much lower compared to a recent survey from East India, where Influenza B was reported as the leading cause of ARI with 12% prevalence (13) . A recent report from India attributed 40% hospitalizations in children to influenza B infections (19) , however, in our study population, influenza B infection was predominantly found in patients aged 30-50 years and was mild. Seasonal influenza A(H3N2) virus also caused mild respiratory infection in <5% of the our study population; mostly in children aged 0-5 years due to their non-exposure to this virus previously.

In children, viruses are the most common cause of respiratory infections and they are considered as a significant health threat in this group. In the present study, of the 292 children with ARI, 44% (129) had one or more viruses and RSV was the most common cause of infection particularly in 0-5 year-old children. Worldwide, RSV predominance in children has been reported (26) and the same scenario exists in India. RSV is known to co-infect with HMPV and influenza viruses; we observed 3 mixed RSV infections, but no clinical correlation could be made due to the low frequency of the same. After the first Indian report of HMPV appeared in 2004 (27), many studies from Lucknow, Vellore and New Delhi have identified it as the second most common cause of ILI in children, next only to RSV (1, 28, 29) . In this study, the prevalence of human metapneumovirus (HMPV) was 3.7%; the predominant group affected was age 14-30 years. This adult preponderance to HMPV is no more a rare phenomenon as recent reports indicate the severity of HMPV in adults', particularly in elderly leading to hospitalization (30, 31) . (36) .

Clinical features of both influenza and non-influenza viruses were similar and nonspecific except for a few symptoms like fever, myalgia and respiratory distress, which were more often associated with influenza ARI; however, this may not be useful for clinical diagnosis as >25% respiratory infections are associated with febrile illness (37, 38) . Viral ARI is initiated with mild respiratory symptoms which are likely to be neglected in the first three to five days; during when the maximum virus shedding occurs. This has been well demonstrated in the pathogenesis of influenza and RSV infection (39, 40) and it is emphasized to collect samples in the first 3 days of symptoms onset. This study finding also suggests the importance of sample collection within 3 days of symptoms onset to achieve the maximum viral detection rate. Apart from the timing, the other important aspect of sample collection is the type of sample collected. In this study, we used nasopharyngeal sample collected using flocked nylon swabs, which have been shown to have excellent sensitivity when compared to rayon swabs, and is considered as an alternative to nasopharyngeal aspirate sampling (41, 42) .

A specific pattern in the seasonality of respiratory viruses (other than influenza) was not appreciated in this study as their prevalence was low in this population. In tropical countries, the seasonal pattern of respiratory viruses is reported to be less evident due to the local variations in precipitation, temperature and humidity. This can be demonstrated from the varied seasonality data reported from different parts of India by Chadha et al (43) . In our study from the southern part of India, the maximum ARI cases were recorded during the months between August and December, except in 2012 and 2015. In both these years, a surge of suspected ARI cases was observed during the early months of these years, due to the outbreak of influenza A(H1N1)pdm09. As described in our earlier studies, both these outbreaks occurred during high mean maximum temperature period of these years and we found no correlation with rainfall (11).

This is the first Indian study to submit gene sequences of HCoV OC43 and 229E in the Genbank database, and hence, no intra-national evolutionary comparisons could be made.

A few studies have reported human bocavirus (HBoV), but the strains isolated from Union territory of Puducherry, were not closely associated with other Indian strains. However, conclusive information could not be made with single gene sequencing. Co-circulation of RSV-A and RSV-B has been observed in many countries including India (44) (45) (46) (47) but RSV-B

is considered as the predominant type in Western part of India (48) . However, in this study, all the RSV isolates sequenced, belonged to type A. Similar observation of exclusive RSV-A circulation have been reported from North East India (49) .

In our study, both Yamagata and Victoria lineage strains of influenza B were found in this region, similar to other reports of co-circulation of both the lineages from India, China and Singapore (50) (51) (52) . Globally, Victoria lineage of influenza B viruses is considered the predominant type of influenza B viruses (53) and the current influenza vaccine contains only the Victoria-like lineage virus (B/Brisbane/60/2008). However, previous studies conducted in humans have proved that this vaccine could not provide immunity against the Yamagata lineage viruses (54) and hence recently, CDC has approved the usage of Quadrivalent vaccine with both the types of influenza B viruses (55) .

To conclude, we reconfirm that, antibiotic usage in treating acute respiratory infections empirically is not justified as nearly half of ARI are due to viruses; nearly 28% of them were due to influenza viruses. Among the non-influenza viruses, RSV predominated, followed by HMPV. This study is based on an active influenza surveillance initiated after 2009 pandemic influenza outbreak, in the Union territory of Puducherry which has contributed significantly to the knowledge of the burden of influenza and non-influenza viruses among children and adults. Such surveillance network has paved the way for better diagnosis and therapeutic interventions. 

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EU100604.1| Inf B/Florida/4/2006/Yamagata lineage EU100732.1|

KP456225.1| Inf A(H3N2)/India/Latur/2010

CY110806.1| Inf A(H3N2)/India/Kerala/2011

KP335865.1| Inf A(H3N2)/Thailand/2011 KX446984.1| Inf A(H3N2)/India/Puducherry/JOP

KU198899.1| Inf A(H3N2)/Moscow/2015

CY194001.1|Inf A(H3N2)/Japan/2015

CY194037.1|Inf A(H3N2)/South

KU182656.1| Inf A(H3N2)/Iran/2014 KX446985.1| Inf A(H3N2)/India/Puducherry

KT369731.1| Inf A(H3N2)/India/Assam/2013

KX765886.1|RSV/NZL/2015-genotype A KX510138.1|RSV/KILIFI/2010-genotype A KC731482

KU950487.1|RSV/USA/2012-genotype A

KF826820.1|RSV/ITA/2009-genotype B

KF826829.1|RSV/MEX/2005-genotype B KJ627254.1|RSV/PER/2011-genotype B KX765888.1|RSV/NZL/2014-genotype B

gb|KX268229.1|hCoV 229E/ India/Puducherry/POM

AY391777.1|HCoV OC43/Belgium/2005

Phylogenetic tree of human coronavirus

Phylogenetic tree of human coronavirus

KR347116.1|HBoV/India/Pondicherry

We acknowledge National Center for Disease Control (NCDC), New Delhi for technical support and Integrated Disease Surveillance Programme (IDSP), New Delhi for financial support.

• First data on the epidemiology of respiratory viruses from this region after 1974 • First study to report corona virus, HCoV OC43 from India, and only the second Indian study to document corona virus, HCoV229E. This study is the first study to analyse the genetic sequence of HCoV-229E and OC-43 • Influenza accounted for the maximum number of cases in the study population, 27%; four patients died of Acute respiratory distress syndrome (ARDS) due to influenza A/H1N1; CFR-3.7%• Among the non-influenza viruses, RSV pre-dominated followed HMPV and PIV-3. No deaths were reported due to non-influenza viral ARI. RSV was detected almost equally in adults and children • Distinct pattern was observed in seasonality of influenza viruses but not for non-influenza viruses