Identification of new genotype of Echovirus 19 from children with Acute Flaccid Paralysis in Pakistan

Enteroviruses are known to cause childhood paralysis. The purpose of this study was to examine the genetic diversity and to determine the association of non-polio enteroviruses (NPEVs) with acute flaccid Paralysis (AFP). Stool samples (n = 1191) of children with AFP were collected from Khyber Pakhtunkhwa and Federally Administered Tribal Areas of Pakistan. Poliovirus was isolated in 205 (17.2%) samples and NPEVs were found in 215 (18.0%) samples. Out of 215 viruses, 124 (57.7%) were typed into 19 different types of enteroviruses while 91 (42.3%) remained untypeable on microneutralization assay that were reconfirmed as NPEVs by real time PCR. Echovirus 19 (20/35; 57.1%) was found the most prevalent type based on VP1 nucleotide sequencing with increased genetic diversity. Phylogenetic analysis revealed the circulation of a new genotype of E-19 in the country. The findings of this study are of great importance for future research and propose to establish the enterovirus surveillance system in the country to readily identify more enteroviruses and to monitor the emergence of new variants/genotypes especially at the moment when we are at the verge of polio eradication phase.

Most enteroviral infections are asymptomatic; however, sometimes they cause mild to severe illness such as aseptic meningitis, encephalitis conjunctivitis, myocarditis, poliomyelitis, neonatal enteroviral sepsis and acute flaccid paralysis 13,14 .
Echovirus 19 was first isolated from stool sample of a child suffering from diarrhea in 1955 15 and later from cerebrospinal fluid (CSF) of a man with aseptic meningitis in 1959 16 . However, epidemic of E-19 infection among infants and children has been observed in Europe during 1974 and 1975 17,18 . Similarly, association of this virus with other infections such as epidemic neuromyasthenia, uveitis, gastroenteritis and myalgia has been reported in previous years 19 and recently echovirus 19 has been isolated from a case of AFP child in Australia 20 .
In this study, we analyzed stool samples collected from acute flaccid paralytic children residing in Khyber Pakhtunkhwa (KP) and Federally Administered Tribal Areas (FATA) of Pakistan to investigate the association of NPEVs with AFP and to examine the genetic diversity among these viruses. The genetic data reveals that echovirus 19 is the most prevalent serotype and has some possible link in causation of paralysis. Furthermore, genome analysis confirms the circulation of a new indigenous genotype of E-19 with increased genetic diversity in the country.

Enterovirus Cell Culture and Microneutralization. A total of 1191 stool samples (887 from
Khyber Pakhtunkhwa and 304 from FATA; each represents a unique patient) collected during January to December 2013 from AFP patients were processed and inoculated on RD and L20B cell lines. Poliovirus was detected in 205 (17.2%) samples and non polio enteroviruses were found in 215 (18.0%) samples ( Table 1). Out of 215 samples (n = 45 from FATA and n = 170 from KP) 124 (57.7%) samples were successfully typed into 19 different enterovirus serotypes (E-1-7, 11-14, 20, 21, 25, 27, 29, 30, 33 and CV-A9) while 91 (42.3%) remained untypeable by microneutralization assay. Molecular Typing. All untypeable isolates (n = 91) were reconfirmed as NPEVs by real-time reverse transcriptase polymerase chain reaction targeting 5′ -UTR 21 and nucleotide sequencing of a partial VP1 specific fragment 22 placed 97.8% of these isolates into EV-B and 2.2% into EV-C species (data not shown). It has been found that majority ( Genetic Diversity among Pakistan Echovirus 19 isolates. Phylogenetic trees were constructed by aligning the partial VP1 sequences of E-19 study strains with their closely related sequences retrieved from GenBank to study their genetic diversity and possible relationships. Pair wise distance scores confirmed that all study isolates constitute a separate clade (bootstrap 85%) that further segregated into two distinct genetic groups (I and II) with 93.3% (89.7-94.8%) mean nucleotide identity with each other supported by high bootstrap value 90 and 86 respectively (Fig. 1a). Group

Phylogenetic Analyses of Pakistan Echovirus 19
Isolates. The molecular evolution of PAK E-19 strains was also investigated by phylogenetic analysis together with closely related E-19 VP1 sequences from the GenBank database (Fig. 1a). The phylogenetic tree revealed that study strains of E-19 represent a clade which is completely different from those detected earlier from China, Australia and Central African Republic. All PAK study isolates had distinct genetic relationship with other E-19 strains and constitute a separate genetic clade representing a new genotype of E-19.
In addition, the partial VP1 gene sequences were also analyzed using a Bayesian method to estimate the phylogenetic tree topology, to investigate the genetic relationship of study isolates sequences and to classify them consistently among the current echovirus 19 strains. The Bayesian phylogram topology ( Fig. 1b) was congruent with the topology previously observed in Maximum Likelihood tree (Fig. 1a). Overall, all the sequences of E-19 serotype sampled from 1955 to 2013 were distributed into two major partitions identified by ancestral nodes 1 and 2 (Fig. 1b). First partition contained only three isolates reported from Central African Republic. The second major tree partition was characterized by three different clades (1-3) with posterior probability 1. In this partition, a distinct clade 2 corresponding to Pakistan isolates clearly indicates a strong temporal clustering of sequences with two different groups supported by posterior probability 1 (Fig. 1b). Additionally, the study isolates displayed a close relatedness with an earlier identified E-19 PAK isolate (PAK-RRL-15-2009; Accession No. GU355715).
The most recent common ancestor of all PAK isolates dated back to about seven years ago 2006.3 (95% HPD: 2003.9-2008.4 years). Later on, the study isolates has diverged into two groups through node 6 (pp = 1) whose time was estimated to be 2008.7 (95% HPD: 2006.7-2010.5) years. Furthermore, group I was originated first as compared to Group II and the time origin of isolates into group I was estimated to be 2009.9 (95% HPD: 2008.2-2011.3) years while for Group II was 2010.5 (95% HPD: 2009.11-2011.7) years. The evolutionary timescale analysis of E-19 VP1 sequences revealed that the study strains diverged 58 years ago (1955) from their first isolated prototype (Burke). The tree root was estimated to the year 1949 with a credibility interval ranging between the years 1936.1 and 1955 (Fig. 1b).      (Fig. 2). In addition, three substitutions were found in BC loop region (82 to 92 amino acid residues) between E-19 study strains and Burke prototype strains. Among these substitutions, one was glutamine (Q) to lysine (K) at amino acid 82 except in two isolates (PAK-MEV3422B and PAK-MEV4343A) where glutamine was replaced by Asparagine (N) and Arginine (R) respectively. The second substitution was serine (S) to histidine (H) at amino acid 84 and third was aspartic acid (D) to glutamic acid (E) at amino acid 85. No genotype specific amino acid signatures were observed in the sequence.

Discussion
Acute flaccid paralysis (AFP) is a heterogeneous neurologic condition defined by the sudden onset of weakness and floppiness in any part of the body in a child < 15 years of age or paralysis in a person of any age in whom poliomyelitis is suspected 28 .The predominant cause of AFP is infection with poliovirus but a number of non polio enteroviruses including coxsackieviruses, echoviruses and enterovirus 68, 70, 71 have also been reported to cause many neurological disorders such as encephalitis, meningitis, meningoencephalitis and AFP 29,[30][31][32][33] . Similarly, in Pakistan many non polio enteroviruses have been isolated from AFP cases but unfortunately, these cases were never being explored further either by the polio eradication programme or the country health care system. As a result, no clear and detailed information about the epidemiology of NPEVs in the country is available except few reports [34][35][36][37] . Therefore, present study was designed to genetically characterize the NPEVs isolated from paralytic cases, as well as to understand the role of echovirus 19 in acute flaccid paralysis owing to the fact that Pakistan is at the verge to achieve the goal of polio eradication. Echoviruses frequently cause aseptic meningitis 38 but they may involve in paralysis, encephalitis, neonatal sepsis, exanthema, respiratory disease, myalgia, myocarditis, uveitis and hand-foot and mouth disease 19,39 . It was observed that proportion of echoviruses isolated in this study was high (73.1%) as compared to the numbered enteroviruses (21.7%) and coxsackieviruses (4.6%). Similarly, among echoviruses, the echovirus 19 was found the most prevalent human enterovirus (12.6%). Although echovirus 19 may cause mild to severe neurological disorders as reported from Malaysia, China, United States of America and Australia 20,40-43 but unfortunately, no clinical information, molecular epidemiology and disease correlation of this serotype with AFP have been reported except two case reports. In first case, E-19 was isolated from CSF sample of a 39 year old immunosuppressed transplant recipient who admitted to the hospital with acute onset of flaccid paralysis of the left leg 43 and in second case a three years old Australian child admitted to hospital with a 2-day history of fever to 38.8 °C and a 24-h history of painless inability to move her left upper arm and shoulder. A detailed medical history showed weakness restricted to the left upper limb, with power at the shoulder and elbow. The muscle tone was flaccid, but there was some degree of wrist flexion and extension and preservation of finger movements, with an absence of biceps, triceps and brachioradialis reflexes. Her stool sample was processed for NPEVs and confirmed it as E-19 20 . On discharge from hospital she had physiotherapy and rehabilitation but had residual weakness of her upper limb after 6 months. This case report supports our current study finding that suggests E-19 may have contribution in paralysis. Although, NPEVs are rarely associated with sporadic cases of AFP but we have isolated twenty E-19 strains from paralytic patients reside in a small geographic area of Pakistan. Clinically, all these patients presented fever, asymmetrical paralysis and rapid progression of paralysis at the time of onset of the disease. Additionally, residual weakness was seen in a marked proportion of AFP cases on 60 days follow up investigation. Consequently, all these findings suggest E-19 as the possible causative agent of paralysis and to the best of our knowledge; this is first report about the epidemiological linkage of E-19 with AFP published from Pakistan so far.
Furthermore, all viruses of this study, isolated from AFP cases grouped together in E-19 cluster and form a distinct genotype having 26.3% nucleotide divergence from their prototype and fulfill the criterion for assessment of new genotype as described previously 5,[23][24][25][26][27]36,44,45 . Moreover, it was perceived by Bayesian evolutionary analysis (Fig. 1b), that the most recent common ancestors for both groups 1 and 2 viruses were dated to 2008.7, indicating that the two groups of new E-19 genotype were circulated for ~4.5 years in the community and were not simply the result of any current transmission. The maximum clade credibility (MCC) tree was also notable for the presence of separate clade containing the study isolates that were shown to have diverged into two groups and been introduced from China in year 2006.2 in the country. Similarly, the frequent presence of long branches in the tree point out towards increased genetic diversity that indicates a gap, probably created by a small number of samples collected and analyzed during the study period.
The BC loop in the VP1 region is, in particular, one of the regions associated with viral antigenicity, and substitution mutations may result in changes in this region, can play a role in host adaptation for enteroviruses 46 . All PAK E-19 isolates had three substitutions within the BC loop on comparison with prototype. Therefore, the effect of these mutations may need further investigations to rule out any changes in antigenic and biochemical properties.
In conclusion, we presented a genetic overview of E-19 with increased genomic diversity and explored its possible association with acute flaccid paralysis. Although Pakistan is a polio endemic country where more efforts are focused on poliovirus eradication but there is a dire need to assess the impact of the disease burden caused by this group of viruses as polio vaccine offers no protection against NPEVs infection. Moreover, we speculate that NPEVs may predominate in its toll to cause paralysis after post-eradication. Therefore, enterovirus surveillance is needed to strengthen in the country in order to get more epidemiological and clinical information about NPEVs. Nucleotide Sequencing and Phylogenetic analysis. The purified RT-PCR products were sequenced with overlapping primer set 224-222 22 using a PRISM Big Dye Terminator cycle sequencing reaction kit version 3.1 (Perkin Elmer Applied Biosystem). Sequence data was generated by an automated ABI 3130 Genetic Analyzer and sequences were assembled and edited by using Sequencher 4.9 software (GeneCodes) (http://www.genecodes.com).

Methods
Genetic differences among E-19 isolates were analyzed by MEGA 5.0 software using partial (399 bp) VP1 region 50 . Sequence of E-19 prototype and all the representative strains retrieved from GenBank (http://www.ncbi.nlm.nih. gov/BLAST 51 and aligned using ClustalW 52 . Evolutionary distances have been computed by using Maximum Composite Likelihood model and Jones-Thornton-Taylor (JTT) model for nucleotide and deduced amino acid sequences respectively 53 . Phylogenetic tree was constructed by maximum-likelihood method 54 with 1000 bootstrap replicates 55,56 .