Serotypic and genotypic profile of dengue virus outbreaks in India between 2011 and 2017.

The epidemiology of dengue has been demonstrating significant changes in recent years, with rising incidence of infection in countries with known endemicity and occurrence of fresh outbreaks in previously unaffected territories. India, which has been a traditional hotspot dengue virus (DENV) transmission in the endemic south-east Asian region, has also been affected by the sweeping changes in dengue epidemiology. Two remarkable developments characterize the alterations witnessed by India 2011-2017. Firstly, all parts of the country have witnessed abrupt replacement of existing DENV lineages with emerging novel strains. Secondly, co-circulation of multiple serotypes of DENV have been reported from all across the country 2011-2017; thereby hinting at the transition of the country towards hyper-endemicity. Considering the potential clinical implications of such epidemiological transformation in terms increasing involvement of pediatric patients and growing predisposition to serious complications, the present review provides an update on the serotypic and genotypic profile of dengue outbreaks that have been witnessed by different zones of India between 2011 and 2017. Dividing the country into northern, southern, eastern, western, central and north-eastern zones, we describe discrete zone-specific distribution patterns of DENV serotypes and genotypes and observe simultaneous circulation of different DENV strains in different parts of the country. Random shifts in the genetic characteristics of the circulating strains and the widespread co- circulation of all four serotypes underscore the need for undertaking continuous and representative molecular surveillance of the circulating DENV strains across the country for prompt identification of emerging strains and novel mutants; gain insights into the formulation of Dengue vaccines and develop a clearer understanding of the molecular basis of immune evasion, disease epidemiology and pathogenesis.

Glioblastoma Multiforme miRNA based Comprehensive Study to Validate Phytochemicals for Effective Treatment against Deadly Tumour through In Silico Evaluation.

BACKGROUND: Glioblastoma Multiforme (GBM) is a prevalent and deadly type of primary astrocytoma, constituting over 60% of adult brain tumors, and has a poor prognosis, with a high relapse rate within 7 months of diagnosis. Despite surgical, radiotherapy, and chemotherapy treatments, GBM remains challenging due to resistance. MicroRNA (miRNAs) control gene expression at transcriptional and post-transcriptional levels by targeting their messenger RNA (mRNA), and also contribute to the development of various neoplasms, including GBM. METHODS: The present study focuses on exploring the miRNAs-based pathogenesis of GBM and evaluating most potential plant-based therapeutic agents with in silico analysis. Gene chips were retrieved from the Gene Expression Omnibus (GEO) database, followed by the Robust- RankAggereg algorithm to determine the Differentially Expressed miRNAs (DEMs). The predicted targets were intersected with the GBM-associated genes, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the overlapping genes was performed. At the same time, five phytochemicals were selected for the Connectivity map (CMap), and the most efficient ones were those that had undergone molecular docking analysis to obtain the potential therapeutic agents. RESULTS: The hsa-miR-10b, hsa-miR-21, and hsa-miR-15b were obtained, and eight genes were found to be associated with glioma pathways; VSIG4, PROCR, PLAT, and ITGB2 were upregulated while, CAMK2B, PDE1A, GABRA1, and KCNJ6 were downregulated. The drugs Resveratrol and Quercetin were identified as the most prominent drugs. CONCLUSION: These miRNAs-based drugs can be used as a curative agent for the treatment of GBM. However, in vivo, experimental data, and clinical trials are necessary to provide an alternative to conventional GBM cancer chemotherapy.

Evaluation of the diagnostic utility of MCAM-1 (CD146) in a group of common gynecological cancers: A case-control study.

Objective: MCAM-1 (CD146) is an endothelial cell adhesion molecule belonging to the immunoglobulin superfamily. Recent studies have identified CD146 expression as a critical marker for tumor progression, migration, and metastasis in various malignancies. This study aimed to evaluate CD146 immunohistochemical expression in various gynecological cancers. Materials and Methods: This study was conducted in a tertiary medical center in central India. A total of 49 gynecological cancer cases and 16 site-matched controls were included. The cases comprised 27 cervical, 10 endometrial, 10 ovarian, and two miscellaneous cancers. CD146 immunohistochemistry was performed and assessed for immunoreactivity score (IRS), microvascular density (MVD), and microvascular caliber (MVC). An IRS of 5 or more was considered CD146 positive. Results: The p-values for CD146 positivity for cases vs. control were 0.0531, 0.0580, and 0.007 for cervical, endometrial, and ovarian sites, respectively. The mean MVD was found to be significantly higher in cases compared with benign tissues (p-value <0.00001), and the mean MVC of cases was found to be smaller when compared with the controls (p-value <0.0001). Conclusion: MVD by CD146 was found to be higher in gynecological malignancies, highlighting its role in cancer neo-angiogenesis and its potential therapeutic role. CD146 epithelial expression was also significantly higher in ovarian cancers. Further studies with a larger sample size are required to confirm that this protein may be a potential theognostic target in gynecological cancers.

Validation of the VirCapSeq-VERT system for differential diagnosis, detection, and surveillance of viral infections.

IMPORTANCE: Broad range assay for accurate and sensitive diagnostics.

Overexpression of XRCC1 is Associated with Poor Survival in Patients with Head and Neck Squamous Carcinoma and Has Potential to Be Used as Targeted Therapy by Synthetic Lethality.

Background: Head neck squamous cell carcinoma (HNSC) is globally prevalent cancer attributed to tobacco habit. Despite the significant advances in early diagnosis and treatment of HNSC chemo-radio resistance are routinely observed in patients. Aberrant DNA repair mechanisms mainly microhomology mediated DNA end joining (MMEJ) pathway causing deleterious mutations and is implicated in treatment resistance. X-ray cross complimenting group 1 (XRCC1) has recently been shown to play an essential role in MMEJ making XRCC1 a potential therapeutic target to render tumors chemo-radiosensitive. This study analyzes the correlation between the expression level of XRCC1 gene with survival, regulation by miRNA and synthetic lethality partners in HNSCC. Materials and Methods: XRCC1 gene expression was evaluated in 520 HNSC patients and 44 of normal tissues using the UALCAN (TCGA) database and its correlation with survival outcome of HNSC patients was analyzed by Kaplan-Meier plot. Infiltration of immune cells in tumors was analyzed by "Tumor-Infiltrating Immune Estimation Resource (TIMER) and promoter methylation status of XRCC1 in samples was analysed by UALCAN. STRING was used to find gene interacting partners of XRCC1.  Results: XRCC1 was significantly overexpressed in primary tumor of HNSCC and significantly increased with tumor stages and grade and associated with poor survival rate. High XRCC1 expression in HNSC was positively correlated with infiltration level of B cells naïsve, CD4+ and macrophages. Conclusion: These results indicate that XRCC1 is a prognostic marker for predicting survival in HNSC patients. Understanding how XRCC1 leads to treatment resistance and modulate immune response can lead to development of targeted therapy.

Polymorphism in Apolipoprotein C3 (APOC3) and Fatty Acid-Binding Proteins (FABP2) Genes in Nondiabetic Dyslipidemic Patients: A Tertiary Care Hospital-Based Pilot Study.

Context Dyslipidemia is a multifactorial disease in which lipoproteins play an important role as one of the early markers for coronary heart disease (CHD). Mixed dyslipidemia is common in people with diabetes mellitus, but nondiabetic dyslipidemics (NDD) remain unidentified for the risk of developing dyslipidemia and eventually CHD. Objectives This pilot study attempts to analyze the genetic basis of lipid metabolism alterations, emphasizing the association between fatty acid-binding protein-2 (FABP2-Ala54Thr) and apolipoprotein-C3 (APOC3-rs5128) genetic polymorphism, as a risk for developing dyslipidemia and CHD in NDD. Methods and Design Total 90 subjects-30 DD, 30 NDD, and 30 apparently healthy subjects representing Central India-were included. Biochemical analysis and DNA genotyping were done by polymerase chain reaction restriction fragment length polymorphism. Statistical Analysis The biochemical parameters were reported as means ± standard deviation. One-way analysis of variance test was used to compare biochemical parameters of three groups. Chi-squared test was done to compare genotype distributions. The strength of association was assessed by odds ratios (ORs) with 95% confidence intervals (CIs). All statistical analysis was done using SPSS-PC software and Graph Pad. Results In NDD, maximum polymorphism was observed followed by DD and least polymorphism was observed in controls. There was a significant association of APOC3 G allele with occurrence of hypertriglyceridemia ( p < 0.05); however, no such association was found for FABP2 A allele ( p > 0.05). Logistic regression analysis revealed APOC3 polymorphism to be significantly associated with dyslipidemia (OR = 2.6667, 95% CI = 1.0510-6.7663, p = 0.0341); no such association was found for FABP2 polymorphism (OR = 0.4643, 95% CI = 0.1641-1.3136, p = 0.1347). The triglyceride and cholesterol values in individuals with homozygous genotype indicate that genetic study is comparable to the biochemical findings in carriers of polymorphic allele than noncarriers, especially in NDD patients. Conclusions Pilot study indicates that the presence of APOC3 gene polymorphism is associated with pro-atherogenic dyslipidemia in nondiabetic patients and may raise risk of CHD. This information could be used for preventive strategies in NDD group that may otherwise go unnoticed.

Intestinal viral infections of nSARS-CoV2 in the Indian community: Risk of virus spread in India.

In December 2019, novel severe acute respiratory syndrome coronavirus 2 (nSARS-CoV-2) virus outbreaks emerged from Wuhan, China, and spread all over the world, including India. Molecular diagnosis of Coronavirus Disease 2019 (COVID) 19 for densely and highly populated countries like India is time-consuming. A few reports have described the successful diagnosis of nSARS-CoV-2 virus from sewage and wastewater samples contaminated with fecal matter, suggesting the diagnosis of COVID 19 from the same to raise an alarm about the community transmission of virus for implementation of evacuation and lockdown strategies. So far, the association between the detection of virus and its concentration in stool samples with severity of the disease and the presence or absence of gastrointestinal symptoms have been rarely reported. We led the search utilizing multiple databases, specifically PubMed (Medline), EMBASE, and Google Scholar. We conducted a literature survey on gastrointestinal infection and the spread of this virus through fecal-oral transmission. Reports suggested that the existence and persistence of nSARS-CoV-2 in anal/rectal swabs and stool specimens for a longer period of time than in nasopharyngeal swabs provides a strong tenable outcome of gastrointestinal contamination and dissemination of this infection via potential fecal-oral transmission. This review may be helpful to conduct further studies to address the enteric involvement and excretion of nSARS-CoV-2 RNA in feces and control the community spread in both COVID-19 patients ahead of the onset of symptoms and in asymptomatic individuals through wastewater and sewage surveillance as an early indication of infection. The existence of the viral genome and active viral particle actively participate in genomic variations. Hence, we comprehended the enteric spread of different viruses amongst communities with special reference to nSARS-CoV-2.

In silico analysis and molecular characterization of Influenza A (H1N1) pdm09 virus circulating and causing major outbreaks in central India, 2009-2019.

BACKGROUND AND OBJECTIVES: Influenza A/H1N1pdm09 causes respiratory illness and remains a concern for public health. Since its first emergence in 2009, the virus has been continuously circulating in the form of its genetic variants. Influenza A/H1N1pdm09 surveillance is essential for uncovering emerging variants of epidemiologic and vaccine efficacy. The present study attempts in silico analysis and molecular characterization of Influenza A (H1N1) pdm09 virus circulating and causing major outbreaks in central India during 2009-2019. MATERIALS AND METHODS: We have investigated the antigenic drift analysis of 96 isolates' hemagglutinin (HA) gene sequences (59 central Indian and 37 local Indian and 28 global reference HA gene sequences) of Influenza A/H1N1pdm09 viruses from 2009 to 2019. The study includes mutational (Multiple sequence Alignment), phylogenetic (Maximum Likelihood Method), and statistical analysis (Covariance and correlation) of HA sequences submitted in NCBI, IRD and GISAID from central India. RESULTS: Phylogenetic analysis indicated maximum clustering of central Indian HA gene sequences in genogroup 6B. Analysis of amino acid sequence alignment revealed changes in receptor binding site (RBS). The frequency of S220T amino acid substitution was found to be high followed by S202T, K300E A273T, K180Q. The Karl Pearson correlation coefficient (r) and covariance between the number of mutations and the death toll was found +0.246 and +100.3 respectively. CONCLUSION: The study identifies the continuous genetic variations in the HA gene sequences of circulating Influenza A/H1N1pdm09 in central India from the year 2009 to 2019. Further suggesting importance of monitoring the gradual evolution of the virus with regards to an increase in virulence, pathogenicity and vaccine efficacy timely.

Validation of leaf and microbial pectinases: commercial launching of a new platform technology.

Almost all current genetically modified plant commercial products are derived from seeds. The first protein product made in leaves for commercial use is reported here. Leaf pectinases are validated here with eight liquid commercial microbial enzyme products for textile or juice industry applications. Leaf pectinases are functional in broad pH/temperature ranges as crude leaf extracts, while most commercial enzyme products showed significant loss at alkaline pH or higher temperature, essential for various textile applications. In contrast to commercial liquid enzymes requiring cold storage/transportation, leaf pectinase powder was stored up to 16 months at ambient temperature without loss of enzyme activity. Commercial pectinase products showed much higher enzyme protein PAGE than crude leaf extracts with comparable enzyme activity without protease inhibitors. Natural cotton fibre does not absorb water due to hydrophobic nature of waxes and pectins. After bioscouring with pectinase, measurement of contact-angle water droplet absorption by the FAMAS videos showed 33 or 63 (leaf pectinase), 61 or 64 (commercial pectinase) milliseconds, well below the 10-second industry requirements. First marker-free lettuce plants expressing pectinases were also created by removal of the antibiotic resistance aadA gene. Leaf pectinase powder efficiently clarified orange juice pulp similar to several microbial enzyme products. Commercial pilot scale biomass production of tobacco leaves expressing different pectinases showed that hydroponic growth at Fraunhofer yielded 10 times lower leaf biomass per plant than soil-grown plants in the greenhouse. Pectinase enzyme yield from the greenhouse plants was double that of Fraunhofer. Thus, this leaf-production platform offers a novel, low-cost approach for enzyme production by elimination of fermentation, purification, concentration, formulation and cold chain.

Co-circulation of dengue virus serotypes in Central India: Evidence of prolonged viremia in DENV-2.

In view of paucity of information on serotype distribution of Dengue virus (DENV) in Central India, we undertook a cross-sectional study to identify clinical and virological characteristics of DENV serotypes that circulated in this region during the 2016 outbreak. Suspected cases were screened by ELISA for NS1 antigen and anti-DENV IgM antibodies. Serologically confirmed cases were subjected to RT-PCR based detection and serotyping. The RT-PCR results were confirmed by nucleotide sequencing. Genome-wide association was undertaken with DENV sequences from ViPR database and the immune evasion potential of infecting serotypes was ascertained by computing antigenic variability in B cell and Cytotoxic T cell (CTL) epitopes of all DENV proteins. The immunological basis of more prolonged viremia in DENV2-infected patients was also addressed through sequencing of NS2a gene and comparing the CTL activity in NS2a sequences identified among patients with ≤5 days and >5 days of illness. Among 166 serologically confirmed Dengue patients, 75 were positive for DENV RNA. Serotyping revealed predominance of DENV-1 and DENV-2, followed by DENV-3. Co-infection with multiple serotypes was observed in 15.5% of cases. In ~40% cases, DENV RNA was detectable beyond 5 days, among whom majority were DENV-2 infected (p = .044). Highest prevalence of antigenic variability was observed in B cell and CTL epitopes of DENV-2. The potential association between prolonged viremia and higher ability for immune evasion in DENV-2 patients was further corroborated with the observation of poorer HLA-I binding affinity in CTL epitopes observed in NS2a sequences retrieved from patients with >5 days of illness, compared to those with ≤5 days. This is the first report from central India revealing circulation of all DENV serotypes and high prevalence of co-infection with multiple serotypes. We also observed prolonged viremia upon DENV-2 infection, which could be potentially associated with its superior immune evasion potential.

Identification and characterization of nonpolio enterovirus associated with nonpolio-acute flaccid paralysis in polio endemic state of Uttar Pradesh, Northern India.

Despite polio eradication, nonpolio enterovirus (NPEV) detection amid polio surveillance, which is considered to have implications in paralysis, requires attention. The attributes of NPEV infections in nonpolio-AFP (NPAFP) cases from Uttar Pradesh (UP), India, remain undetermined and are thus investigated. A total of 1839 stool samples collected from patients with acute flaccid paralysis (AFP) from UP, India, between January 2010 and October 2011 were analyzed as per the WHO algorithm. A total of 359 NPAFP cases yielded NPEVs, which were subjected to microneutralization assay, partial VP1 gene-based molecular serotyping and phylogenetic analysis. Demographic and clinical-epidemiological features were also ascertained. Echoviruses (29%) and Coxsackievirus (CV)-B (17%) were the most common viruses identified by the microneutralization assay. The molecular genotyping characterized the NPEVs into 34 different serotypes, corresponding to Enterovirus (EV)-A (1.6%), EV-B (94%) and EV-C (5.3%) species. The rarely described EV serotypes, such as EV-C95, CV-A20, EV-C105, EV-B75, EV-B101, and EV-B107, were also identified. NPEV-associated AFP was more prevalent in younger male children, peaked in the monsoon months and was predominantly found in the central part of the state. The NPEV strains isolated in the study exhibited genetic diversity from those isolated in other countries. These form part of a different cluster or subcluster existing in cocirculation, limited to India only. This study augments the understanding of epidemiological features and demonstrates the extensive diversity exhibited by the NPEV strains in NPAFP cases from the polio-endemic region. It also underscores the need or effective long-term strategies to monitor NPEV circulation and its associated health risks in the post-polio eradication era.

Analysis of Arbovirus Isolates from Australia Identifies Novel Bunyaviruses Including a Mapputta Group Virus from Western Australia That Links Gan Gan and Maprik Viruses.

The Mapputta group comprises antigenically related viruses indigenous to Australia and Papua New Guinea that are included in the family Bunyaviridae but not currently assigned to a specific genus. We determined and analyzed the genome sequences of five Australian viruses isolated from mosquitoes collected during routine arbovirus surveillance in Western Australia (K10441, SW27571, K13190, and K42904) and New South Wales (12005). Based on matching sequences of all three genome segments to prototype MRM3630 of Trubanaman virus (TRUV), NB6057 of Gan Gan virus (GGV), and MK7532 of Maprik virus (MPKV), isolates K13190 and SW27571 were identified as TRUV, 12005 as GGV, and K42904 as a Mapputta group virus from Western Australia linking GGV and MPKV. The results confirmed serum neutralization data that had linked SW27571 to TRUV. The fifth virus, K10441 from Willare, was most closely related to Batai orthobunyavirus, presumably representing an Australian variant of the virus. Phylogenetic analysis also confirmed the close relationship of our TRUV and GGV isolates to two other recently described Australian viruses, Murrumbidgee virus and Salt Ash virus, respectively. Our findings indicate that TRUV has a wide circulation throughout the Australian continent, demonstrating for the first time its presence in Western Australia. Similarly, the presence of a virus related to GGV, which had been linked to human disease and previously known only from the Australian southeast, was demonstrated in Western Australia. Finally, a Batai virus isolate was identified in Western Australia. The expanding availability of genomic sequence for novel Australian bunyavirus variants supports the identification of suitably conserved or diverse primer-binding target regions to establish group-wide as well as virus-specific nucleic acid tests in support of specific diagnostic and surveillance efforts throughout Australasia.

Upolu virus and Aransas Bay virus, two presumptive bunyaviruses, are novel members of the family Orthomyxoviridae.

UNLABELLED: Emerging and zoonotic pathogens pose continuing threats to human health and ongoing challenges to diagnostics. As nucleic acid tests are playing increasingly prominent roles in diagnostics, the genetic characterization of molecularly uncharacterized agents is expected to significantly enhance detection and surveillance capabilities. We report the identification of two previously unrecognized members of the family Orthomyxoviridae, which includes the influenza viruses and the tick-transmitted Thogoto and Dhori viruses. We provide morphological, serologic, and genetic evidence that Upolu virus (UPOV) from Australia and Aransas Bay virus (ABV) from North America, both previously considered potential bunyaviruses based on electron microscopy and physicochemical features, are orthomyxoviruses instead. Their genomes show up to 68% nucleotide sequence identity to Thogoto virus (segment 2; ∼74% at the amino acid level) and a more distant relationship to Dhori virus, the two prototype viruses of the recognized species of the genus Thogotovirus. Despite sequence similarity, the coding potentials of UPOV and ABV differed from that of Thogoto virus, instead being like that of Dhori virus. Our findings suggest that the tick-transmitted viruses UPOV and ABV represent geographically distinct viruses in the genus Thogotovirus of the family Orthomyxoviridae that do not fit in the two currently recognized species of this genus. IMPORTANCE: Upolu virus (UPOV) and Aransas Bay virus (ABV) are shown to be orthomyxoviruses instead of bunyaviruses, as previously thought. Genetic characterization and adequate classification of agents are paramount in this molecular age to devise appropriate surveillance and diagnostics. Although more closely related to Thogoto virus by sequence, UPOV and ABV differ in their coding potentials by lacking a proposed pathogenicity factor. In this respect, they are similar to Dhori virus, which, despite the lack of a pathogenicity factor, can cause disease. These findings enable further studies into the evolution and pathogenicity of orthomyxoviruses.

Genetic diversity of cosaviruses in nonpolio acute flaccid paralysis cases of undefined etiology, Northern India, 2010-2011.

BACKGROUND: No cases of wild poliovirus have been reported for more than one and a half years from India. Cases of acute flaccid paralysis (AFP) of undefined etiology continue to occur in the region. Despite the recent discovery of the human Cosavirus (HCoSV) in the feces of children from developing countries, there have been no studies of cosavirus infection in India. OBJECTIVES: To detect and characterize HCoSVs in stool specimens of nonpolio AFP cases by RT-PCR followed by sequencing. STUDY DESIGN: A total of 387 fecal samples collected from AFP cases in Uttar Pradesh, India, between May 2010 and April 2011, tested negative on cell culture according to WHO algorithm, were subjected to 5'-UTR region specific RT-PCR followed by sequencing to detect HCoSV. Molecular characterization of HCoSV strains was done by sequencing followed by phylogenetic analysis. RESULT: 123 (32%) samples tested positive for cosaviruses and 87 (70.7%) were identified for genetic variants by sequencing a 316-nucleotide interval in the partial 5'-UTR region. Cosavirus strains were characterized as putative species HCoSV-A (n=70; 82%), HCoSV-B (n=7; 8%), HCoSV-C (n=1; 1.1) and HCoSV-D (n=4; 4.5%) while 5 (5%) strains remain uncharacterized. The cosavirus infection appeared highest (63.5%) in younger children, and showed a distinct seasonality, with a late summer peak and winter low. CONCLUSION: This study demonstrates a diversity of cosavirus strains in circulation, and reports the first investigation of HCoSV infection in children with nonpolio acute flaccid paralysis in India. Currently, this study provides baseline data for further studies of HCoSV infections in children with common enteric infections in India.

Genetic variants of echovirus 13, northern India, 2010.

Nonpolio acute flaccid paralysis is increasing in India. To determine viral causes, we conducted cell culture and molecular analysis identification of nonpolio human enteroviruses associated with acute flaccid paralysis during March-August 2010 in northern India. The predominant nonpolio enterovirus found was echovirus 13, a serotype rarely isolated in India.

Genetic characterization of the Wyeomyia group of orthobunyaviruses and their phylogenetic relationships.

Phylogenetic analyses can give new insights into the evolutionary history of viruses, especially of viruses with segmented genomes. However, sequence information for many viral families or genera is still limited and phylogenies based on single or short genome fragments can be misleading. We report the first genetic analysis of all three genome segments of Wyeomyia group viruses Wyeomyia, Taiassui, Macaua, Sororoca, Anhembi and Cachoeira Porteira (BeAr328208) in the genus Orthobunyavirus of the family Bunyaviridae. In addition, Tucunduba and Iaco viruses were identified as members of the Wyeomyia group. Features of Wyeomyia group members that distinguish them from other viruses in the Bunyamwera serogroup and from other orthobunyaviruses, including truncated NSs sequences that may not counteract the host's interferon response, were characterized. Our findings also suggest genome reassortment within the Wyeomyia group, identifying Macaua and Tucunduba viruses as M-segment reassortants that, in the case of Tucunduba virus, may have altered pathogenicity, stressing the need for whole-genome sequence information to facilitate characterization of orthobunyaviruses and their phylogenetic relationships.

Non-polio enteroviruses in acute flaccid paralysis children of India: vital assessment before polio eradication.

AIM: This study is an overview of non-polio enterovirus (NPEV) circulating in North India studied from the perspective of poliomyelitis eradication. Wild polio cases declined because of intensive oral polio vaccine immunization. As we approach global eradication of poliovirus (PV), NPEV causing acute flaccid paralysis (AFP) are equal cause of concern. METHODS: A total of 46 653 AFP samples (World Health Organization) and apparently 1000 healthy contacts living in the same geographical area were studied (2004-2007). Serological identification of NPEV was done using RIVM-specific pools (The Netherlands). Untyped (UT)-NPEVs were sequenced directly from reverse transcription-polymerase chain reaction using pan-enterovirus (Pan-EV) primer (CDC, Atlanta, GA) targeting highly conserved 5'un-translated regions of the enterovirus. RESULTS: In this study, 12 513 NPEVs were isolated from the collected stool samples. Seroneutralization had identified 67% of NPEV isolates, whereas 32.6% remained as UT- NPEV. Of the typed NPEVs, Coxsackie-B accounted for 32.3%; followed by echoviruses-11, 12, 13, 7 between 8 and 28%. In sequencing few UT-NPEVs, some were identified also as echovirus-30, 11 and 18 which were probably present in mixtures as they remained UT-NPEV in ENT. Newly classified human enterovirus virus-86 (HEV) (EU079026), HEV-97(EU071767) and HEV-B isolate (EU071768) were isolated in AFP samples. CONCLUSIONS: This study provided definitive information about circulation, prevalence and new emerging NPEV in the polio-endemic region of India, hence they should be considered in AFP surveillance. This would help in adopting and planning new strategies in post-PV eradication era in the country. This is the right time to prepare for the future tasks while we head towards a polio-free region.

Interrupting wild poliovirus transmission using oral poliovirus vaccine: environmental surveillance in high-risks area of India.

Global eradication of poliomyelitis has reached critical stage. Sabin Oral Poliovirus Vaccine (OPV) has been successful in three major regions of the world. In India eradication of poliomyelitis from states of Uttar Pradesh (UP) and Bihar has been difficult due to high population and low-socioeconomic standards of living. Acute flaccid paralysis (AFP) surveillance and intensive OPV rounds continues with the World Health Organization (WHO) operational strategies. Yet apparent lack of progress in reducing the number of wild cases has resulted in occasional impatience and frustration, even leading to questions about ultimate feasibility of global eradication using OPV. Lucknow in UP is in geographical area endemic for poliomyelitis and is surrounded by high-risk areas yet maintains a polio-free status since 2002. Environmental surveillance study was conducted (2004-2006) to authenticate the decline in the wild poliovirus (PV) cases in Lucknow. Sewage sample analyses were compared with stools of AFP patients and healthy children from same geographical area. Study reveals useful information on OPV circulation and proves important epidemiological tool to trust WHO's OPV immunization program. Genetic sequencing had detected silent wild PV-1 circulation of RCP1PGI (EU049849), RCP2PGI (EU049850), RCP3PGI (EU049851), and RCP4PGI (EU049852) in sewage waters. Properties of isolates from sewage reflected those of viruses excreted from human. This study provides valuable information and encouragement to AFP surveillance to maintain high levels of OPV immunization campaigns in the most difficult endemic region of India to interrupt the wild PV transmission.

Rapid detection of sewage sample polioviruses by integrated cell culture polymerase chain reaction.

The authors' objective in this study was to introduce and evaluate integrated cell culture polymerase chain reaction (ICC-PCR) as a technique for the rapid screening of poliovirus in sewage samples. Researchers are in the last stage of poliomyelitis eradication; however, in a densely populated country such as India, time is the prime factor in the identification of poliovirus circulation and transmission because this virus follows the fecal-oral route for transmission and is excreted in nature. The authors used ICC-PCR to detect poliovirus in sewage samples, and they compared this nonconventional method with conventional cell culture methods to determine sensitivity, accuracy, and the time from sample collection to final results. The ICC-PCR method provided results within 4-5 days of sewage-sample collection; in contrast, the conventional method takes more than 18 days to provide such results. The ICC-PCR method proved to be sensitive, reproducible, and accurate, as well as rapid in its screening of sewage samples for poliovirus. This diagnostic tool may indeed prove quite useful in polio eradication.