MOLECULAR DETECTION OF ASTROVIRUS USING REVERSE TRANSCRIPTASE QUANTITATIVE POLYMERASE CHAIN REACTION TECHNIQUE

This study was aimed to highlight the importance of the melt curve-quantitative reverse transcriptase PCR (RT-qPCR) analysis in the detection of astrovirus (AstV) from both negative and positive rotavirus and enterovirus (EVs) samples, and the effectiveness of the AstV infection on the vaccine immunogenicity in the vaccinated infected children. By RT-qPCR based Sybre green associatedmelting curve assay, stool samples of 49 enterovirus suspected patients and of 39 rotavirus suspected patients were tested for AstV. Results of EVs group showed a 29 (59.2%) positive AstV contributed to 26 (89.5%) co-infection with Evs and 3 (10.3%) as a single infection in negative samples for Evs. Furthermore, AstV co-infection percentage is higher than the single infection. Moreover, the percentage of the Astrovirus among the vaccinated AFP-suspected cases was 53%, while the percentage of these viruses among the unvaccinated was 100%. Thus, MamAstrovirus1 MK948878 is the first local isolate recorded in the Genbank. In conclusion, the RT-qPCR based on SYBR Green showed the rapid and efficient detection of AstV with few copies number. This allow to be used for the diagnosis of AstV along with other gastroenteritis viruses in a multiplex assay to reduce processing


INTRODUCTION
Worldwide, acute gastroenteritis is now one of the most common infections among humankinds (36), which is characterized by vomiting, abdominal pain, nausea, fever and diarrhea that represents the most common symptoms and responsible for many childhood morbidity and mortality, especially in developing countries (35). Although rotavirus, norovirus and adenovirus have been documented as causative agents for gastroenteritis, presently, viruses like astroviruses are also vital, has been linked to frequent cases of acute gastroenteritis in young children, old people and immunocompromised individuals and supposed to represent up to 20% of sporadic viral diarrhea cases and 0.5-15% of total outbreaks (24), but it has been the topic for limited studies (10). astroviruses, are non-enveloped single-stranded positive-sense RNA viruses, belong to the family astroviridae which were firstly discovered in 1975 by electron microscopy in stool samples from children with acute gastroenteritis (9). Its genome divided into three open reading frames (ORFs): ORF1a (non-structural protein), ORF1b (non-structural protein) and ORF2 (capsid protein), a 3´ UTR and a polyA tail (21). Astroviruses classified into two genera: mammalian viruses (Mamastroviruses (MAstVs), 19 species and avian viruses (Avastroviruses (AAstVs), three species recognized both by (ICTV) (15). However, astroviruse infect mostly the pediatric population (28). The age of affected children with classic HAstV is highly variable, fluctuating from neonates to above 5 years, however, astroviral infection is more mutual among those younger than two (12). The usual circulation of astrovirus is more frequent in temperate regions with a better stability in the cold weather period, however HAstVs endure to circulate all over the year, and infections do happen in summer too (1). Other viral gastroenteritis agents such as rotaviruses and noroviruses, but not adenoviruses, present a parallel seasonal outline (16). In Iraq, there are several previous papers have studied astroviruses and its association with gastroenteritis in children by two molecular (27, 33) and immunological methods (6,8,19,7). It's good to know, that there are no previous local studies isolated the virus from vaccinated children with oral Polio vaccine for the enterovirus study group and with Rota vaccine for the rotavirus study group. Moreover, this is the first study relied on real time reverse transcriptase polymerase chain (RT-qPCR) reaction associated with melt curve analysis in detection of astrovirus. As well, the original stool samples were taken from previously identified samples for rotavirus and enterovirus by ELISA and by tissue culture for enterovirus to increase the accuracy (i.e the specifity and sensitivity) in the diagnosis. Furthermore, there is the no previous Iraqi study typed the astrovirus serotypes by the nucleotide sequence analysis and stated its similarity with other world reference serotypes. So that, this study aimed to highlight the importance of the melt curvequantitative reverse transcriptase PCR (RT-qPCR) analysis in the detection of astrovirus (AstV) from both negative and positive rotavirus and enterovirus (EVs) samples, and the effectiveness of the AstV infection on the vaccine immunogenicity in the vaccinated infected children.

MATERIALS AND METHODS Study population
From the National Central Public Health Laboratory/ Iraq (CPHL) / National Polio Laboratory, during March to December, 2018. Eighty eight stool samples of patients suffered from gastroenteritis and/ or acute diarrhea were collected with aged 1 to 48 month (47 male and 41 female). Ninety three samples of them were selected from patients suspected with rotavirus and previously tested for this virus by the Enzyme Linked Immunosorbent (ELISA). The second study subjected group was selected from 49 samples from patients infected with Acute Flaccid Paralysis (AFP) and previously tested for Enterovirus (Sabinepolio and non-polio Enterovirus) by tissue culture method.

RNA extraction
All the 88 original stool samples have been processed for RNA extraction. RNA was taken out according to the producer's directions from all the specimens using the QIAamp Viral RNA Mini kit (Qiagen GmbH, Germany) (3).

Quqntitative
Reverse Transcriptase Polymerase Chain Reaction (RT-qPCR): By GoTaq® one-Step RT-qPCR system protocol, using a nucleic acid stain (typically SYBR green by adding to PCR tube 5 µl of the GoTaq qpcr master mix; 2x, 0.8 µl of each primer and 0.5 µl of enzyme mix) associated with melting curve analysis (2), with mean = 33.45 for cycle number (Ct) and mean= 73.51 for Tm in astrovirus positive samples. The RT-qPCR, has been done for the detection 88 samples of Astrovirus, by amplifying the open reading frame-1b by the ORF1b primer (23) as a forward primer (5ʹ-CCGAGTAGGATCGAGGGT-ʹ3) and a reverse primer (5ʹ-GCTTCTGATTAAATCAATTTTAA-ʹ3) with a product size 93 nt in this study.

Reverse transcriptase Polymerase Chain Reaction (RT-PCR).
For further molecular typing to the Astrovirus-positive samples by the nucleotide sequence analysis, the open reading frame 1b of the samples extracted RNA has been amplified by the RT-PCR assay by using ORF1b primers, a forward primer (5ʹ-GGACTGCAAAGCAGCTTCGTG-ʹ3) and reverse primer (5ʹ-GTGAGCCACCAGCCATCCCT-ʹ3) with a product size 719 base pairs (37). The amplicons were run on agarose gel 1% and visualized with Transilluminator, using the ladder ranged 100-1000 bp (5). Sequencing for RT-PCR product For ORF1b gene sequencing, the RT-PCR product has been sent to Macrogen Company, USA by using forward primer ORF1b on a genetic analyzer (Applied Biosystems). The homology search was performed using the Basic Local Alignment Search Tool (BLAST) program online using blastn and blastx algorithm which is available at the NCBI online at (http://www.ncbi.nlm.nih.gov). To classify the positive local Astrovirus strains, the phylogenetic tree was performed applying the neighbor-joining method and the genetic distances were calculated according to the maximum composite likelihood model with 100-bootstrap using the MEGA version 6.0 software. A bootstrap value of 70 was used as an indicator of the significance of the clusters. Statistical analysis The statistical analysis system was analyzed by IBM SPSS Statistics version 25. All values were calculated according to the positive results as percentages (%). Differences between study groups and assays were analyzed by cross-tab and Pearson chi-square (X 2 ) test, while the Kruskal-Wallis test applied to check the signs between age groups. A value of P<0.05 was considered statistically significant.

RESULTS AND DISCUSSION Detection of astrovirus by RT-qPCR assay in the enterovirus and rotavirus suspected groups
Using RT-qPCR based Sybre green associated-melting curve assay (Fig.1  It's good to be mentioned that the co-infection model of the astrovirus with enteroviruses was higher than with the rotaviruses while the astovirus-single infection showed the contrary (figure-1). Moreover, the percentage of the astrovirus among the vaccinated AFPsuspected cases was 53%, while the percentage of these viruses among the unvaccinated was 100%.

Figure 1. Frequency of astrovirus single and mixed infection tested by Sybre green assay and accompanied with the enteroviruss and rotavirus groups. Asterisk pointed out significance appearance with enteroviruses
To our knowledge, there are no Astrovirus specific probes have been used with RT-qPCR in this study, otherwise, the one step Sybr green RT-qPCR (30) followed by melting curve analysis have introduced a good alternative way in detection of these viruses and this agreed with (29) study about the molecular ways of Astrovirus diagnosis. Sybr green assay has ability to detect sensitive and rapid a few copies numbers (low viral load) by melting curve than Taqman assay more clearly diagnosed with Ct below 38 (31,17).

Detection of Astrovirus in the Rotavirus and Enterovirus suspected group using RT-PCR:
Among the 88 isolates, few isolates have high viral load from co-infection sample with Rotavirus appeared 17 Ct (cycle number) of amplification curve and 76.1 Tm of melting curve in the RT-qPCR (Fig.2) as a positive result for Astrovirus, and 719 base pair when the RT-PCR assay performed. This isolate was further analyzed by the nucleotide sequence analysis in order to type it and examine its evolutionary relationships with other international strains. The remaining astrovirus positive samples were low viral load with Ct more than 32 in RT-qPCR and less clear in observing the specific band with RT-PCR assay (Fig.3).  (8,19). The highest incidence rate in this study has matched only with Mitab, 2013 study (6), this may be explained the high accuracy and sensitivity of the RT-qPCR assay in the detection of these viruses and this matched with what mentioned in other previous studies (14,25). In comparison to other global molecular based studies from Italy (11), Germany (20), lebanon (38), Egypt (4) and Iran (26). The prevalence of HAstV in this study is the highest, telling that this virus exists, flowing and contributing expressively to gastroenteritis in different Iraqi provinces. The high incidence of Astrovirus relies on the age of patients, analytical approaches used and the season of isolates assembly in which HAstV infectious rate is more common among those younger than 2 and in temperate regions in the cold weather period beside of using RT-qPCR assays that provide high speed, sensitivity and reproducibility and reduction of contamination risk in the diagnostic of HAstVs (30, 26). As a result, our findings have corresponded to the previous ideal criteria (age <1 year, spring to winter seasons of collection, PCR assay) for Astrovirus distribution and occurrence in Iraq community. In the subsequent step of detection, out of the RT-qPCR positive samples, conventional PCR method introduced isolate with high viral load (2.5%) of 39 patients for astrovirus with 719 bp., which was recorded with the lowest cut value (17 Ct) and the Tm (76.1) in RT-qPCR. That's as may belong to the high sensitivity of RT-qPCR other than the RT-qPCR as what mentioned with Royuela et al., 2006, study (30) in which RT-qPCR identified IUs down to a 10 −6 dilution, whereas the RT-qPCR identified down to IUs 10 −2 dilution. However the percentage of RT-qPCR astrovirus positive results agreed with Thewiny, et al 2014 study (33) that detected 2.6% astrovirus from 200 infected patients. Notably, in this study the gastroenteritis infection showed a higher frequency in isolates with co-infection models (52.9% with EVs and 30.7 % with Rotavirus) other than with the single infection models (6.1% with EVs and 20.5% with Rotavirus). These results have agreed with local study (6) and many global studies (11,22,34) which revealed that the gastroenteritis may not only come from single viral infection, but also from co-viral infection with some enteric viruses, such as enterovirus, adenovirus, Rotavirus, norovirus and Astrovirus. Moreover, the high frequency of the co-infection model for the astrovirus with rotavirus and enterovirus have proved the efficacy of the real time PCR as an efficient alternative method in the detection of astrovirus from these dual gastroenteritis infections. In this study, there is a significant incidence of astrovirus in males other than females, in the enterovirus group, the result reversed in the rotavirus group but with no significant difference. However, these outcomes matched with several studies that the infected males' percentage was higher than females (6,19,22). The percentage of coinfection model to the Astrovirus with Enterovirus and Rotavirus was higher in the vaccinated children with OPV and RV for poliovirus and Rotavirus respectively, other than the single infection. That's explained by Taniuchi et al., 2016 study (32) in which the previous infection with enteric viruses decrease the immunization of the immune system of the vaccinated children against the OPV and RV vaccines which make the child would easily infect with rotavirus and Sabinepolio virus Marker (ladder) ranged 100-1000 bp Astrovirus serotypes sequencing Overall, the nucleotide sequence analysis of the ORFb1 region of the Astrovirus isolate was successfully done by amplifying the RT-PCR product (719 bp). The Iraqi\local Astrovirus isolate in this study was analyzed and compared with the reference strains available in the Genbank database at NCBI (Fig. 4)   Generally speaking, AstV-1 is the most frequently isolated type worldwide (13), in this study the sequenced strain has typed as AstV-1 serotype in a patient with a diarrheal infection aged 7 months old and this matched with Guix et al., 2002 study (18) which demonstrated that most infections with AstV-1 and AstV-3 occurred in children younger than 2 years old. As a result of its international distribution, a phylogenetic tree was established for the local strain to display its evolutionary relationship with other global stains. The tree mirrored this distribution in which the local strain presented a high relatedness with 98-99% identical to the reference strains that isolated from different continents, as Europe (Russia, Italy, Hungary), Asia (China, South Korea, Thailand, Japan) and North America (USA). The most related strain was from Russia that grouped with the Iraqi strain under the same cluster with 99% identity. Method of RT-qPCR based on SYBR Green showed the rapid and efficient detection of astrovirus with few copies number. This allows to be used for the diagnosis of AstV along with other gastroenteritis viruses in a multiplex assay to reduce processing time. Subsequent applied nested PCR to overcome low viral load.