Emergence of Recombinant Subclade D3/Y in Coxsackievirus A6 Strains in Hand-Foot-and-Mouth Disease (HFMD) Outbreak in India, 2022.

Coxsackievirus-A6 (CV-A6) is responsible for more severe dermatological manifestations compared to other enteroviruses such as CV-A10, CV-A16, and EV-A71, causing HFMD in children and adults. Between 2005 and 2007, the recombinant subclade D3/RF-A started to expand globally, and a CV-A6 pandemic started. The study aimed to conduct whole-genome sequencing (WGS) of an isolated CV-A6 strain from currently circulating HFMD cases from India in 2022. Gene-specific RT-PCR and sequencing were used to perform molecular characterization of the isolated virus. Confirmation of these isolates was also performed by transmission electron microscopy and WGS. Among eleven positive clinical enterovirus specimens, eight CV-A6 strains were successfully isolated in the RD cell line. Isolates confirmed the presence of the CV-A6 strain based on VP1 and VP2 gene-specific RT-PCR. Sequences of isolates were clustered and identified as the novel CV-A6 strain of the D3/Y sub-genotype in India. The studies revealed that the D3/Y sub-genotype is being introduced into Indian circulation. The predicted putative functional loops found in VP1 of CV-A6 showed that the nucleotide sequences of the amino acid were a remarkably conserved loop prediction compatible with neutralizing linear epitopes. Therefore, this strain represents a potential candidate for vaccine development and antiviral studies.

Whole-genome sequencing and phylogenetic analysis of coxsackievirus-A16 strains causing hand, foot and mouth disease (HFMD) in India.

Hand, foot and mouth disease (HFMD) is a common childhood infectious disease, caused by enteroviruses (EVs), which can present with typical or atypical lesions. The illness is self-limiting, but it can also have serious complications. Since 1997, HFMD infections have become endemic and have increased to epidemic proportions across the Asia Pacific region, including India. Coxsackievirus-A16 (CV-A16) outbreaks occurred in India from 2005 onwards, although the clinical symptoms were noticeably different during this period. Understanding the population dynamics of enteroviruses that cause HFMD is crucial in the post-polio era because one of the circulating strain may replace another as the dominant strain. The aim of this study is to describe the genetic features of the CV-A16 strains isolated from hand, foot and mouth disease (HFMD) patients in India. Reverse transcription PCR (RT-PCR) and cell-culture-based isolation of CV-A16 was done from the 55 clinical samples. The entire genome of the CV-A16 isolate was performed from the seven isolates. After the sequences were analysed, a phylogenetic tree was created using bioinformatics tools. The total genomic length obtained was 7411 base pairs (bp). Nucleotide similarity across various regions, including 5'UTR, P1, P2 and 3'UTR, ranged from 87.0-97.9 %, 77.0-95.4 %, 80.3-96.9 %, and 77.9-96.2 %, respectively. Correspondingly, similarities in the VP1 region's nucleotide and amino acid sequences were 91.4-96.4 % and 99.3-99.7 %, respectively. Phylogenetic analysis highlighted that CV-A16 strains identified in Pune, Maharashtra, were grouped within the same cluster. The analysed CV-A16 isolates in this study aligned with subgenotype B1c. These findings have far-reaching implications for the surveillance, prevention and management of HFMD and CV-A16. Monitoring the dynamics of CV-A16 strains, informed by the genetic characteristics identified here, will significantly impact strategies aimed at tackling HFMD and its associated public health challenges.

Hand, Foot, and Mouth Disease (HFMD) in India: A Review on Clinical Manifestations, Molecular Epidemiology, Pathogenesis, and Prevention.

HFMD is a childhood viral disease initiated by enteroviruses (EVs). Symptoms are initiated with mild-to-moderate fever of short duration followed by oral and skin lesions. Skin lesions are papulovesicular which appears on palms/soles of feet, hands, knees, and elbows. Oral lesions appear as vesicles producing multiple small superficial ulcers. Disease is usually mild illness but sometimes progresses in severe form as meningitis, encephalitis, and polio-like paralysis. Etiological agents of the disease belong to Picornaviridae family. The causative viral agents are from genus human enterovirus (HEV) such as enterovirus-A 71 (EV-A71), coxsackievirus -A6 (CV-A6), CV-A10, CV-A16. Coxsackievirus A-16 (CV-A16) and enterovirus A-71 (EV-A71) are the major etiological agents of this disease, among children reported globally. In India, studies conducted on HFMD cases revealed CV-A16 as a major EV type and under circulation over a period of time. Molecular studies of different CV-A16 isolates and the viral kinetic studies conducted on organ tissues of experimental mouse model with complete VP1 gene sequencing revealed presence of B1c sub genotype which is currently in circulation. Genetic changes observed at nucleotide and amino acid level in vital organs of experimental infected mice model might predict some targets and can act as markers of virulence. Mice infected with CV-A16 strains revealed progressive pathological changes in mice organs. Major affected organs were to be as brain, heart, intestine, and skeletal muscles. The present review focuses on HFMD caused by CV-A16 with epidemiological, molecular, pathogenesis and need of antivirals against the disease.

The 2022 outbreak and the pathobiology of the coxsackie virus [hand foot and mouth disease] in India.

Outbreaks of HFMD in children aged <5 years have been reported worldwide and the major causative agents are Coxsackievirus (CV) A16, enterovirus (EV)-A71 and recently CVA6. In India, HFMD is a disease that is not commonly reported. The purpose of the study was to identify the enterovirus type(s) associated with large outbreak of Hand, foot, and mouth disease during COVID-19 pandemic in 2022. Four hundred and twenty five clinical samples from 196-suspected cases were collected from different parts of the country. This finding indicated the emergence of CVA6 in HFMD along with CVA16, soon after the gradual easing of non-pharmaceutical interventions during-pandemic COVID-19 and the relevance of continued surveillance of circulating enterovirus types in the post-COVID pandemic era.

Clinical profile and molecular typing of viral etiological agents associated with Hand, Foot and Mouth Disease (HFMD): A study from Udhampur, Northern India.

PURPOSE: Hand, Foot and Mouth disease (HFMD) is a contagious pediatric viral disease caused due to enteroviruses (EV) of the family Picornaviridae. Cases of HFMD were reported from a tertiary care health centre, Udhampur, (Jammu and Kashmir), Northern India. The present study highlights the clinical and molecular virological aspects of HFMD cases. MATERIAL AND METHODS: Cases reported during August 2016-September 2017, and clinically diagnosed as HFMD of all age groups were included. Clinical, Biochemical and molecular virology aspects were compared. Clinical samples (n â€‹= â€‹50) such as vesicle swab, buccal and throat swabs were collected for enterovirus detection. EV-RNA was detected by 5'NCR based RT-PCR and genotyping by VP1 gene amplification and cycle sequencing. RESULTS: Of the cases of HFMD enrolled (n â€‹= â€‹50), highest (84%) were of children aged <5 years, presented either or both anathemas and exanthemas with prodromal symptoms (fever, irritability). Clinical presentations involved mainly oral ulcers on lips and tongue (48%). Oral erosions were either single or multiple in numbers. Exanthemas were seen on hand and palm, widely spread up to buttocks, legs, arms and trunk. Of these, six patients were found anemic. Complete blood count (CBC) indicated lymphocytosis and C-reactive protein (n â€‹= â€‹10) in children aged <5 years. EV-RNA was detected in 78% (39/50) of the clinical samples. VP1 gene based typing indicated the presence of CV-A16, CVA6 and EV-A71 types. CONCLUSIONS: The study highlights association of EVs in HFMD cases in the reported region. CV-A16, CV-A6 and EV-A71 types were reported for the first time from Udhampur (J&K), Northern India. No differences were observed in the clinical profile of EV strains detected. Circulation of the strains warrant and alarm outbreaks. More focused studies on HFMD and monitoring of viral strains is mandatory.

Prolonged Shedding of SARS-CoV-2 in Feces of COVID-19 Positive Patients: Trends in Genomic Variation in First and Second Wave.

The main route of the transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are through respiratory pathways and close contact of human-to-human. While information about other modes of transmission is comparatively less, some published literature supporting the likelihood of a fecal-oral mode of transmission has been accumulating. The diagnosis of SARS-COV-2 infected cases is based on the real-time reverse transcription-PCR (RT-PCR). The fecal excretion of SARS-COV-2 has been reported frequently, however, the role of fecal viral load with the severity of disease is not yet clear. Our study focused on the investigation of SARS-CoV-2 shedding in the fecal samples of patients with coronavirus disease 2019 (COVID-19). A total of 280 RT-PCR-positive patients were enrolled, among them 15.4% had gastrointestinal (GI) symptoms. It was shown that 62% of the patients were positive for SARS-CoV-2 RNA in fecal specimens. This positivity was not related to the presence of GI symptoms and the severity of disease. The next generation sequencing [NGS] of SARS-CoV-2 from fecal samples of patients was performed to analyze mutational variations. Findings from this study not only emphasized the potential presence of SARS-CoV-2 in feces, but also its continuing mutational changes and its possible role in fecal-oral transmission.

A real-time polymerase chain reaction-based approach for qualitative estimation of viral RNA in organ tissues of coxsackievirus A-16-infected neonatal mice.

Hand, foot and mouth disease (HFMD) is a paediatric disease associated with enteroviruses (EVs). Among EVs, coxsackievirus A-16 (CVA-16) strain is currently in circulation and causing outbreaks in India. Neonatal mice (Institute of Cancer Research) strains were infected with CVA-16 strain isolated from HFMD patients to conduct pathological and molecular studies. Infected organs were harvested as per time points. A real-time polymerase chain reaction was used for qualitative estimation of viral RNA in organ tissues of infected mice. Skeletal muscle, brain tissue and cardiac tissues were the major target sites of CVA-16 tropism. The first-ever study was conducted on CVA-16 strains using the current approach in India.

Pathological and molecular studies on Coxsackie virus A-16 isolated from hand, foot, and mouth disease cases in India: Approach using neonatal mouse model.

The present study highlights pathogenesis and molecular aspects of Coxsackie virus A-16 (CVA-16) strains isolated from hand, foot, and mouth disease (HFMD) cases from India using a neonatal mice model. ICR mice were intraperitoneally inoculated with CVA-16/311 strain isolated from HFMD cases. Mice developed hind and forelimb paralysis on day 3 of post infection. Histopathological observations of hind limb muscles showed necrosis, dissolution of muscle fiber cells, infiltration of inflammatory cells, marked dilated ventricle, hemorrhages, and neuronal degeneration in the brain. Immunohistochemical studies revealed high expression of CVA-16/311-specific viral antigen in limb muscles, brain, heart from day 3 till day 7 of post-infection. VP1 gene-based reverse transcription polymerase chain reaction conducted in RNA samples of different tissue organs of infected mice followed by sequencing of the positive amplimers revealed presence of CVA-16/311-specific viral sequences. Phylogenetic analysis based on the VP1 gene showed the presence of B1c sub genotype of CVA-16/311 strain in targeted tissue organs. Sequence analysis revealed major genetic changes in heart, skeletal muscle tissues at the nucleotide and amino acid levels. Genetic changes occurred in organs of mice might predict some potential targets and might act as markers of virulence for neuronal tropism. Pathogenesis and molecular studies of CVA-16 strains isolated from HFMD cases using neonatal mice model was conducted for the first time from India.

Chandipura virus encephalitis outbreak among children in Nagpur division, Maharashtra, 2007.

BACKGROUND & OBJECTIVES: An outbreak of acute encephalitis syndrome (AES) among children from Nagpur division, Maharashtra was investigated to confirm the aetiology and to describe clinico-epidemiological features. METHODS: AES cases among children<15 yr, from Nagpur division, hospitalized between June-September 2007, were investigated. Serum and cerebrospinal fluid (CSF) were tested for IgM antibodies against Chandipura virus (CHPV) and Japanese encephalitis virus (JEV) and for CHPV RNA by RT-PCR. Partial N gene sequences were used for phylogenetic analysis. Virus isolations were attempted in rhabdomyosarcoma (RD) cell line. Sandflies were collected, pooled and tested for CHPV RNA by RT-PCR. RESULTS: A total of 78 AES cases were recorded in children<15 yr of age. Case fatality ratio was 43.6 per cent. Male to female ratio was 1:1.2. Chandipura (CHP) was confirmed in 39 cases. CHPV RNA was detected in both CSF and serum specimens of 2 cases and in serum of 22 cases. Phylogenetic analysis showed 99.98-100 per cent nucleotide identity in the sequences studied. Anti-CHPV IgM antibodies were detected in CSF of 2 cases and in serum of 8 cases. Seroconversion to anti-CHPV IgM antibodies was observed in 5 cases. Clinical manifestations of CHP cases (n=38) were fever (100%), convulsion (76.3%), altered sensorium (34.2%), headache (23.7%), vomiting (44.7%) and diarrhoea (23.7%). CHPV RNA was detected in one of two pools of sandflies from affected locality. INTERPRETATION & CONCLUSIONS: Chandipura virus was confirmed as the aetiological agent of this acute encephalitis outbreak with high case-fatality among children.

Chandipura virus: a major cause of acute encephalitis in children in North Telangana, Andhra Pradesh, India.

A hospital-based surveillance was undertaken between May 2005 and April 2006 to elucidate the contribution of Chandipura virus (CHPV) to acute viral encephalitis cases in children, seroconversion in recovered cases and to compare the seroprevalences of anti-CHPV IgM and N antibodies in areas reporting cases with those without any case of acute viral encephalitis. During this period, 90 cases of acute encephalitis were hospitalized in the pediatric wards of Mahatma Gandhi Memorial (MGM) Hospital, Warangal. There were 49 deaths (Case Fatality Rate, i.e., CFR of 54.4%). Clinical samples and records were obtained from 52 suspected cases. The cases were below 15 years, majority in 0-4 years (35/52, 67.3%). Computerized tomography (CT) scans and cerebro-spinal fluid (CSF) picture favored viral etiology. No neurological sequelae were observed. CHPV etiology was detected in 25 cases (48.1%, n = 52; RNA in 20, IgM in 3 and N antibody seroconversion in 2). JEV etiology was detected in 5 cases (IgM in 4 cases and seroconversion in 1 case). Anti-CHPV IgM seroprevalence in contacts (26/167, 15.6%) was significantly higher (P < 0.05) than in non-contacts (11/430, 2.6%); which was also observed in children <15 years (19/90, 21.1% vs. 3/109, 2.7%). Anti-CHPV N antibody seroprevalence in <15 years contacts (66/90, 73.3%) and non-contacts (77/109, 70.6%) was significantly lower (P < 0.05) than in contacts (75/77, 97.4%) and non-contacts (302/321, 94.1%) more than 15 years respectively. CHPV appears to be the major cause of acute viral encephalitis in children in endemic areas during early monsoon months.