In recent years, sequence independent PCR approaches have revolutionized identification of unknown and novel viruses either alone or in combination with conventional methods. In this study a simplified version of VIDISCA was used to detect human enteroviruses, a human parechovirus type 3, a human astrovirus, and a tetnovirus-1 in cell culture. Our data show that a wide range of distinct viruses that remained unrevealed through routine assays can be identified in a relatively short amount of time with an easy to use method. The VIDISCA method is based on cDNA-AFLP and one characteristic of this method is that it uses a double PCR strategy, with in the second round of amplification selective primers (selective PCR-round). These selective primers are extended at the 3′ site with one or two nucleotides, and in the selective PCR various combinations of primers are used in order to amplify everything which was amplified in the first PCR. This selective PCR step is a complex and laborious step in VIDISCA, but was needed previously to distinguish viral PCR fragments from ribosomal RNA amplicons. However, an improved purification and reverse transcription step which strongly diminishes ribosomal RNA amplification has recently been published, and therefore the laborious selective amplification step might not be needed . Here, we show that in the majority of virus cultures simplified-VIDISCA can quickly reveal the infecting agent. We previously published that VIDISCA in its original setting can identify a virus in picornavirus-cultures in >90% of the cultures . A sensitivity of 70% which we have here is largely comparable, but we must mention here that we did not compare the sensitivity of traditional VIDISCA and simplified VIDISCA directly in our study.
The possible failure of screening of study samples via routine assays may be due to increased genetic diversity at PCR priming annealing sites [14, 15]. Seven viruses (EV-B97, EV-B100, E-9 and E-21, HPeV-3, HAstV-3/5 and TNV-1) were identified which originate from stool samples of AFP patients. The clinical significance of these viruses has great impact on public health and constitutes a health risk for the community. All isolated enteroviruses belong to enterovirus B species containing the most frequently isolated serotypes that more commonly cause meningitis, myocarditis and neurological disorders [16–20]. Some enteroviruses cause severe and potentially life-threatening illness and there is currently no antiviral treatment available for enterovirus infection . Phylogenetic clustering of our study isolates reveals that the enteroviruses are related to the circulating strains which have been reported in neighboring countries: India and Bangladesh. Our analysis also showed that the nucleotide sequence of echovirus 21 isolate (PAK-NIH VS1661A) has low nucleotide identity (78.7%) with prototype Farina strain (AF081334) that fulfils the criterion for a genotype  and therefore we suggested that it is a separate genotype of E-21 circulating in the area. Importantly, the emergence of new genetic lineages of enteroviruses in the community is an alarming situation for Pakistan where there is no enterovirus surveillance system. Aside from poliovirus, which is the target pathogen of the polio eradication strategy, the non-polio enterovirus detection in the laboratory is only a “part-outcome” of AFP surveillance. In Pakistan, despite the significant number of isolated non-polio enteroviruses, limited information is available with regard to its incidence, diversity and circulation pattern. Consequently, it is the right time to prepare for future tasks and to give attention towards non polio viruses causing AFP which is an equal cause of concern while we are approaching towards the polio eradication era.
Similarly, HPeVs are known to cause a variety of clinical symptoms similar to enteroviruses like gastroenteritis and occasionally flaccid paralysis and encephalitis [22, 23] particularly in infants, and they are considered a major cause of infant mortality worldwide. HPeV types 1, 5, 6, 7 and 12 were isolated from stool samples of non polio AFP patients <3 years of age [24–26]. In this study we identified HPeV-3 in a seven months old paralytic infant having fever. Our findings was in agreement with previous studies in which HPeV-3 was isolated from children aged less than three years having transient paralysis and sepsis like illness [22, 27].
Astrovirus is one of the known causes of acute gastroenteritis in humans, mostly in young children. It has been isolated earlier from stool of an AFP patient . In this study, an astrovirus was isolated from a six months old child. A follow up examination after 60 days indicated the complete recovery of patient from paralysis. Phylogenetic analyses of PAK-NIH-VS908 based on ORF1a and ORF2 genes clustered this isolate in two different genotypes; HAstV-5 and HAstV-3. This genotype discrepancy among phylogenetic analysis of both genes most likely represents a possible recombination event in the evolution of HAstVs. Recombination is a normal phenomenon among these viruses [29–32].
PAK-NIH-VS926 is the most divergent strain of the type 1 tetnoviruses. Tetnovirus-1 is an RNA virus containing two large ORFs encoding structural and non-structural proteins. The non-structural protein of tetnovirus-1 contains an RNA dependent RNA polymerase (RdRp) and a cysteine-like protease domain. The genomic organization of TNV-1 is more closely related to the viruses classified in the family Tetraviridae. Tetnoviruses are closely related to nodaviruses within the RdRp protein. The host of nodaviruses is fish and arthropods, including insects. Tetnovirus-1 and −2 have been isolated earlier from stool samples of AFP children of Afghanistan . Future studies are needed to identify the origins of these viruses and to clarify their in vitro replication and pathogenic potential in AFP children.
Nowadays sequence independent methods followed by high throughput sequencing [34–36] are becoming more promising means for detection of novel pathogens. However, highly developed technical skills, involvement of bioinformatics’ support, adequate computing resources, advanced data interpretation and high costs are major barriers for their use in developing and resource-limited countries, which are exactly those regions where outbreaks of new pathogens are likely to occur [28, 37–39]. Therefore, we consider the adaptation of VIDISCA a most appropriate method for resource poor countries and it can be successfully completed in a limited time without technical difficulties. This assay is applicable independently in any laboratory having only PCR and sequencing facilities and can be reproduced easily from the literature.