Phylogenetic Analysis of Respiratory Syncytial Virus Isolated from Children with Respiratory Tract Infections in Baghdad City, Iraq

Respiratory syncytial virus (RSV) is the leading cause of hospitalization in infants worldwide, genotypes responsible of disease severity and host immune responses. This study aims to determine the infection rate of respiratory syncytial virus in children with respiratory tract infection and identify the genotyping among the study population. Cross sectional study which enrolled 150 infants with acute respiratory tract infection, males 81(54%) and females 69(46%) aged under five years old, who was admitted to Al-Imamin Al-Kadhimin Medical City and Pediatrics Protection Hospital in Baghdad during the period from December 2017 till April 2018. Nasopharyngeal swabs were collected from each participant and stored as frozen at -70 °C until to use for RNA extraction and convention polymerase chain reaction to detect of respiratory syncytial virus in the study population. According to result of this study out of all these samples, 26 samples were positive for RSV(17.33%). The infection rate of RSV is more common in males 17(65.39%), than females 9(34.61%) and in children ≤ one year (46.15%) also high frequency were noticed among patients live in an urban area (73.08%) and winter 20 (76.92%) than spring 6 (23.08%). According to different clinical feature, fever, cough, and wheezing were more common than other sign. The sequence conducted for all RSV- positive isolates, 11 respiratory syncytial virus positive isolates was in genotype B and 1 in genotype A. The sequence of RSV B the local isolates were closed to Argentina isolation and Tailwind isolate while in genotype A isolates were closed to isolates from different regions (Saudi Arabia, German, India isolation). The conclusion of this study revealed that respiratory syncytial virus B infections were more frequent than RSV A among children with acute respiratory tract infection.


Introduction
Human respiratory syncytial virus (RSV) is one of the most common viruses to infect children worldwide and increasingly is recognized as an important pathogen in adults, especially the elderly and immunocompromised patients [1]. Respiratory syncytial virus is responsible for more than three million yearly hospitalizations and up to 118 000 deaths in children under 5 years, is the leading pulmonary cause of death for this age group that lacks a licensed vaccine [2].
Respiratory syncytial virus particles are pleomorphic with both spherical and filamentous particles of different sizes; which comprise of a nucleocapsid bundled in a lipid envelope got from the host cell plasma membrane [3]. It is a cytoplasmic with linear, negative sense, ssRNA genome of approximately 15,000 nucleotides that is classified in the Pneumovirus genus of the Paramyxoviridae family. The viral genome encodes 11 proteins. Of these, the Gand F-proteins are the major surface antigens of RSV, which is involved in virus attachment to cell receptors and the mediation of cell membrane fusion, respectively. Both G-and Fproteins are accessible to neutralizing antibodies, however, only the G-protein is known to accumulate mutations in response to host immunological pressures [4].
Respiratory syncytial virus has been classified into groups A and B based on antigenic differences of the G, F, and N proteins [5]. Further genetic analysis of the nucleotide sequence of the second hypervariable region of the G gene allowed for the classification into  [6]. Many studies have reported the molecular epidemiology and genetic variability of RSV worldwide [7, 8 ,7]. Group A strains have been categorized into 14 genotypes (GA1 to GA7, SAA1, CB-A, NA1 to NA4 and ON1) [8]. While subgroup B is categorized into 27 genotypes (BA1 to BA12, BA-C, SAB1 to SAB4, GB1 to GB4, URU1 to URU2, CB-B, CB-1, BA-CCB and BA-CCA) [9].
Respiratory syncytial virus is transmitted through close contact with a person who has an active infection or direct contact with infectious secretions on environmental surfaces such as droplets, saliva, or large particle aerosols. Although fomites may also be a source of contamination [10].
Signs and symptoms of respiratory syncytial virus infection most commonly appear about four to six days after exposure to the virus. In children, RSV usually causes mild cold-like signs and symptoms. These include fever, congested or runny nose, sore throat, dry cough and mild headache [11]. And then progress down into the lower respiratory tract to cause bronchiolitis, pneumonia, and they implicated with allergy and asthma exacerbation [12].
The present study aims to determine the infection rate and genotyping of human respiratory syncytial virus in children with acute respiratory tract infection.

2.Patients and Methods
A cross sectional study was based on the processing of nasopharyngeal swab from 150 children with acute respiratory tract infections, aged under five years old, who was admitted to Al-Imamin Al-Kadhimin Medical City and Pediatrics Protection Hospital in Baghdad during the period from December 2017 till April 2018. Data were collected by interview with a parent or relevant of each participant, through structural questionnaire which include gender, age, residence, season, type of infection, cough, fever, wheezing, nasal discharge, history of asthma, diarrhea and nervous manifestations.
Swab specimens were preserved in viral transport media (VTM) without antibiotics, according to [15]. Then transported by a cool box to the Virology Unit at the National Central Public Health Laboratory. All samples were vortexes for 15 seconds to dislodge material on the swab into the transport medium. On completion of routine investigations of microbial causes of respiratory tract infection all residual nasopharyngeal aspirate samples were divided into aliquots, labelled and stored at -70°C until the time of analysis.

RNA extraction
The viral RNA to be used as a template for cDNA synthesis was extracted directly from nasopharyngeal swabs by using QIAamp Viral RNA Mini Kit (Cat. No. 52904, 52906, QIAGEN, Germany).

3-22, 5'-CAACTCCATTGTTATTTGCC-3') was used as reverse primer for both subgroups A and B[16].
In the a nested reverse transcription polymerase added 8μl of templet RNA to the component of One Step RT-PCR Kit (Cat.No.210210, 210212, and 210215, QIAGEN, Germany), consisting of 4 µl 5X buffer, 0.8 µl dNTPs, 0.8 µl Enzyme Mix, 1 µl of Mgcl2,1.2 µl of Forward and Reverse primers and 3 µl of RNase-free water. The total reaction volume was 20 µl. Protocol that used in a nested reverse transcription -polymerase chain reaction assay to amplify a fragment of the RSV G gene to detect of subgroup A and subgroup B respectively consisted of 30 min at 50°C of reverse transcribtion, Initial PCR activation at 15 min for 94°C, 35 cycle of 94°C for 30 sec, 50°C for 30 sec, and 72°C for 1 min and a final extension at 72°C for 7 min, two μl of external PCR product was used for hemi-nested PCR amplify a fragment of the RSV G gene was used to detect of subgroup A and subgroup B. Added to the component of Master Mix: Go Taq® green master mix (Cat No.K-2018 Promega -USA) consisting of 12.5µl Green master mix, 12.5 µl of Forward and Reverse primers and 8µl of Nuclease free-water. The total reaction volume was 25 µl.
A protocol that used in semi-nested PCR assay to amplify a fragment of the RSV G gene was used to detect of subgroup A and subgroup B consisted of 94°C for 30 min, 30 cycles of 94 for 30 Sec, 50 for 45 Sec and 72 for one min and a final extension at 72°C for five min.
The external and hemi-nested PCR amplicons were 550 and 500 bp, respectively. Both subgroups A and B.

Sequencing of PCR products and data analysis.
After successful amplification of the target regions of RSV by using primers, (25μl) of PCR product along with primers, were sent abroad to Macrogen company in South Korea for direct sequencing. Homology search was conducted using basic local alignment search tool (BLAST) program which is available at the National Center Biotechnology Information (NCBI) online at (http:// www.ncbi.nlm.nih.gov) to identify the resultant sequencing, then all local isolate were recorded in Gene Bunk

Results
The rate of respiratory syncytial virus infection among children under five years age was 17.33 % (26 out of 150) samples using conventional-PCR. Group A was 30.78% and group B was 69.23% as shown in (Figure 1and 2). Respiratory syncytial virus infection is predominant in males 17(65.38%) than female 9(34.62%), age group 1-12 month (46.15%) than another 13-24 months (46.15%), 25-36 months (19.23%) and 37-48 months (7.69%). The sequence conducted for all RSV-positive isolates, 11 respiratory syncytial virus positive isolates was 10 in subgroup B and 1 in subgroup A. The sequence of the local isolates RSV subgroup B were closed to Argentina isolation and Tailwind isolation while in RSV subgroup A isolates were closed to isolate from different regions (Saudi Arabia, German, India, China isolation. According to phylogenetic tree Figure (

Discussion
According to the results of the current study, the infection rate of RSV was 17.33% using conventional PCR. This result is comparable with several studies conducted in different area such as study done by Al-Charrakh et al., (2016) recorded (18.75%) in asthmatic patients used real time polymerase chain reaction in Wasit city [17], and with Hassan et al., (2018) revealed a seroconversion rate of RSV was (20.4%) among young children in the Kurdistan region [18].
The infection rate of RSV in this study is relatively low compared with data reported in Baghdad by Odisho et al., (2009) the percentage is reached to 79% among the children who have respiratory tract infection [19]. In neighboring countries the infection of the RSV was (36.8%) by Khadadah et al., (2010) in hospitalized children with respiratory tract infection in Kuwait [20] and to study done by Parsania et al., (2016) shows that 31.1% of RSV infections among Iranian children < 5 years of age [21]. In this study the infection rate seems to be higher with than other studies such as study done by Atyah et al., 2017 who recorded (6.6%) in children under 15 years old in Baghdad [22]. These variations in incidence among studies might reflect different epidemiological patterns of RSV infection in different countries, which in turn might be related to environmental factors, geographical factors, differences in host genetic susceptibility, immune status, sampling size, detection techniques, and different viral strains circulating in different geographical areas [23].
According to gender, it has been found that RSV infection in males more than females seems to be similar with those who participated in other studies such as Rodriguez-Fernandez et al., (2017) in Texas [24], Zahran et al., (2017) in Egypt [25], Hassan et al., (2018) in Iraq [18]. And Jepsen et al., (2018) Infant boys were at higher risk of severe RSV infection as compared to infant girls in Denmark [26]. While the current study is inconsistent with a study conducted by Reina et al., (2008) which revealed that the gender, females (53.2%) was higher than males [27].
Regarding the age, it has been found that RSV infections were higher among age group <1 year, 12(46.15%) use conventional-PCR technique, when compared to older children. This result was comparable to that Chen et al.,(2010) in china [28], Khalil et al., (2015) in Egypt [29], Al-Mossawi et al., (2016) in Iraq; the incidence of severe RSV infections was highest among infants and young toddlers and peaked in the 1 to 2 month-old infants [30].
Result of phylogenic analysis, which demonstrated that all local isolated did not had any similarity with references isolate at gene bank, this could be related with limited studies done around Iraq as well as this is the first study done in Iraq to determine the genotyping according to our knowledge. This study found a remarkably higher rate of RSV subgroup B (69.23%) than RSV subgroup A (30.78%), this result is in agreement with a study done by an Al-Mossawi et al., (2016) who recorded 8% and 14% of respiratory syncytial virus type A and B respectively, among children suffering from respiratory tract in Al-Amarah city [30]. And with study done by a Kenmoe et al., (2018) found RSV group A and group B co-circulated in this population at 17.4 and 82.6%, respectively [31]. On the other hand, many studies report the same results conducted in different areas such as (Hirsh et al., 2014;Gimferrer et al., 2015;Liu et al., 2014) [32,33,34]. While this result was disagreement with other studies such as Fieldhouse et al., (2018) revealed a high frequency of RSV-A(30.2% overall) among patients with pneumonia admitted to the two hospitals in Sarawak, Malaysia [35]. Faghihloo et al., (2011) found 66.6% RSV-positive belonged to subgroup A while others to B (33.4%) [16]. Also, this result disagreed with other global studies such as [36,24]. On the other hand large sample size study consists of 1589 hospitalized children with bronchiolitis from 2007 through 2010 in the U.S.A found no difference in disease severity between infants with RSV-A and RSV-B [37]. Another study found two types of RSV responsible for all seasonal outbreaks [38]. Regarding severity Some studies have reported that RSV-A demonstrated a higher (more severe) clinical score index in RSV-A infection [39], on the basis of respiratory rate, wheezing, heart rate, difficulty in feeding and oxygen saturation associated with more severe clinical disease [40]. While in few other studies group B infections have been reported to cause more severe disease [41]. This difference could be related to sample size and seasons. Infectivity of the virus, the development of immunological resistance in the community, and viral genetic drift due to spontaneous mutation may be important in the patterns of seasonal circulation and genetic evaluation of RSV strains [42].
The sequence conducted for all RSV-positive isolates, 10 respiratory syncytial virus positive isolates was in genotype B and 1 in genotype A. The sequence of RSV B the local isolates were closed to Argentina isolation and Tailwind isolation while in genotype A isolates were closed to isolate from different regions (Saudi Arabia, German, India isolation). This could be related to increase travel in a different area for tourism, treatment, study as well as increase in the religious travel. This contradiction could be attributed to difference in study design and population, definition of disease severity, the distribution of RSV subtypes [32]. And the interplay between host and virus factors, including RSV load [43]. Also a possible explanation for these alterations is the development of specific immunity against a specific RSV type that is prevalent in the country in the preceding year. For that, subgroups shift of RSV from year to year that may affect the immunity acquired against the previously circulating viruses [44].

Conclusions
The RSV subgroup B was more than subtype A by conventional PCR. All local RSV subgroup B strains were closed to Argentina isolation and Tailwind isolation while in genotype A isolates were closed to isolate from different regions (Saudi Arabia, Germany, India, China isolation). Further studies with large sample size to clarify this issue and identify new local isolates.