Frequency of Parechovirus Serotype 1 Among Young Infants With Sepsis

Background: Human parechovirus (HPeV) is recognized as a potentially severe viral infection such as gastrointestinal , respiratory and sepsis disease.In neonates and young infants. HPeV-1 is the most prevalent genotype and most commonly causes sepsis in young infants. The aim of study was to determine the prevalence of HPeV in hospitalized young infants with sepsis. Methods: The sera of 100 samples were collected from young infants [46 (46%) females and 54(54%) males < below 90 days] with clinical signs and symptoms of sepsis. The total RNA was extracted, cDNA was synthesized. The Nested PCR was carried out for detection of HPeV. The mean , chi square tests were used for distributions of HPeV genotypes among the gender, age group and season. Results: 5/100 (5%) of patients including 2/46(2%) females and 3/54(3%) males showed positive for HPeV (P=0.85). The results of sequencing and phylogenetic tree revealed that the isolated HPeV were genotype 1. Conclusion: Low prevalence of 5% HPeV were detected. HPeV was dominant in this region. The screening HPeV RNA in patients with sepsis may reduce the use of antibiotics and shorten the duration of hospitalization.

HPeVs have been classi ed in the genus Parechovirus, which is divided into two species: Parechovirus A and Parechovirus B. Parechovirus A is classi ed into 19 genotypes, HPeV-1 to -19, based on phylogenetic analysis of VP1 sequences, while Parechovirus B comprises Ljungan viruses 1 to 4 genotypes (3)(4). The prevalence of HPeVs -A genotypic varies globally. HPeVs -A1 genotype is dominant in Europe and USA followed by PeV-A3 and PeV-A4, while PeV-A2, and A7-A19 genotypes are rarely reported (5). In Asia, similar to Europe and the USA, PeV-A1, A3, and A4 genotypes are the most prevalent and a higher diversity of genotypes has been reported in India and Pakistan (6,7,8). In the African continent, PeV-A1, A2, and A3 are the most prevalent but nearly all HPeVs genotypes have been detected, indicating a much wider circulation of genotypes in this continent (9). The prevalence of HPeVs have been reported in young children with aseptic meningitis, sepsis-like illness and gastroenteritis in Iran (10)(11)(12) PeV-A1 has been detected in acute gastroenteritis, upper respiratory tract symptoms, fever, rash ,paralysis and encephalitis in children age 6 months to 5 years (13-16).
HPeV-4 has been identi ed in patients with nervous system infection (21). HPeV-6 has been reported from a 1-year old fatally child with Reye syndrome (22) and isolated from a stool sample of a 2.5-monthold girl with fever, dehydration, bilateral otitis media, and anemia (23).
HPeVs can be Transmitted via the fecal-oral route and the respiratory tract (24,25). At present There is no standard approach for diagnosis of viral sepsis but molecular approach such as PCR provides greater sensitivity and speci city than DFA and cell culture methods (26).
Serum samples are the most useful compared to CSF for detection of HPeV RNA from neonates and young infants with sepsis and encephalitis (27). Early recognition of HPeV infections is very important since it may reduce the use of antibiotics and shorten the duration of hospital admissions for neonates and young infants with sepsis (28). The patients ages were between 20-90 days with mean age of 54 ± 22 who were admitted to the intensive care unit with sepsis. The de nition of sepsis was based on the following criteria ; Fever > 38 °C, Poor feeding (76%), Lethargy(47%), irritability(97%),Tachypnea(78%),Tachycardia(91%),rash(67%),vomiting(27%),diarrhea(41%), Poor Perfusion(54%), apnea (12%). The analyses of proteins, CBC and serological tests such as C-reactive were carried out for all the participants.

Statistical analysis
Data analysis was performed using the Statistical Package for the Social Sciences 22.0 (SPSS Inc., Chicago, IL, USA). Chi-Square test was used to calculate. P < 0.05 was accepted as signi cant.

Extraction of RNA and cDNA synthesis
The total RNA was extracted from each serum sample using high pure viral nucleic acid( Roche company, Germany) according to instructor manufacture. The cDNA was prepared using (Thermo scienti c, USA) company, according to manufacture instructor.

Sequencing Of Isolated Hbv Dna
The results of two PCR products were sequenced using ABI 3730xl DNA sequencer (Applied Biosystems, USA). The partial sequences of parechovirus genome was sent to Gene Bank to obtain the accession number.

Phylogenetic tree
The phylogenetic tree was constructed by Neighbor joining method for partial VP1 region of two isolated

Results of Sequencing
The sequences of two isolated HPeVes were deposited in GenBank with accession numbers, MN845069 and MN845070. Both isolated HPeVes showed 97% nucleotide identity with HPeV genotype1( MK792785.1) isolated from Tehran, Iran.

Phylogenetic Tree
The analysis of phylogenetic tree revealed that both the isolated HPeV (MN845069 and MN845070) are cluster with HPeVes serotype 1 isolated from different regions of the world ( Figure A).

Discussion
Sepsis due to HPeV may result in notable morbidity in neonates and young children. Although, most of HPeV infections are self-limited, but long-term neurological de cits such as learning disability, paralysis, epilepsy and encephalitis have been reported in patients with HPeV infections (16)(17)(18)(19)(20)(21)(22)30). It has been repoted the prevalence of HPeV3 is much more associated with sepsis disease than HPeV1 (17-20).
In the present survey, the prevalence of HPeV1 was 5/100 (5%) [2(2%) Females and 3(3%) males] in young infants with sepsis (P = 0.85). No HPeV3 was detected in the patients. The studies have shown HPeV1 was dominant in young children with aseptic meningitis and sepsis-like illness and gastroenteritis In Iran (10-12). The results of sequencing of two VP1 region of isolated HPeV1 (MN845069 and MN845070) showed 97% nucleotide identity with HPeV genotype 1 isolated from Tehran, Iran ( MK792785.1). The results of phylogenic tree of two isolated HPeV1 (MN845069 and MN845070) were cluster with HPeV1 isolated from different regions of the world. In this study, the incidence of HPeV1 was found in 2(2%)cases in autumn and 3(3%) cases winter season. No HPeV1 detection was detected in summer and spring seasons.
Rahimi et al, (2013) have detected HPeV 1 infections throughout the years with no signi cant difference in infection rate between seasons. However, the most HPeV 1 detections were found during summer and the lowest HPeV detection rate was in spring in Tehran ,Iran (10).
In a multinational epidemiological study have been described the etiology of one-third children with severe sepsis, were viral infections (31). The most frequent sites of infection were the respiratory tract (40%) with rhinovirus, Respiratory syncytial virus, and adenovirus most commonly isolated in children with sepsis (31). In Australia and New Zealand the etiology of 50% of patients with sepsis were RSV, HVR, CMV, EBV, HSV, VZV and in uenza (32). The previous study have reported that 15% of neonates with sepsis, were bacterial etiology (33).
In the present study the role of other viruses such as human rhinovirus, human Respiratory syncytial virus, human Enterovirus (EV), HAvd, CMV, EBV, HSV, VZV and in uenza have not been investigated in patients with sepsis, but it requires comprehensive investigation.
To date, no antiviral drug has been shown to be effective against HPeV, and no vaccines are currently available to protect against infection. The IVIGs have been used for the treatment of severe disease. An infant with severe, dilated cardiomyopathy caused by HPeV-1 was fully recovered after treatment with Intravenous immunoglobulin) IVIG (34).
Although low detection of 5% HPeV were found among young infant but the screening of HPeV RNA should be implemented in young infants with sepsis. The implementation of molecular detection of HPeV is very pivotal as it reduce the use of antibiotics and shorten the duration of hospitalization. the role of other viruses such as HRV, RSV, and HAdV, HCMV, EBV, HSV, VZV and in uenza have not been investigated in patients with sepsis, but it requires comprehensive investigation.

Conclusion
The main goal of the current study was to determine Frequency of Parechovirus serotype 1 among young infants with sepsis. This study has identi ed low prevalence of 5% HPeV were detected. HPeV was dominant in this region. A phylogenic tree was constructed with Neighbor joining method for partial VP1 region of two isolated HPeV from two patients with sepsis in Ahvaz city. The two isolated HPeV RNA with accession numbers (MN845069, MN845070) were compared with the different HPeVes genotypes retrieved from GenBank.
The results of phylogenetic revealed that the Iranian isolates with black circle color are cluster with HPeV genotype 1 isolatedfrom different regions of the world . The accuracy was assessed by 1000 bootstrap replicates. Scale bar=0.005