Molecular characterization & recombination analysis of complete enterovirus-88 isolated from acute flaccid paralysis cases in India.

Background & objectives: Focus on non-polio enteroviruses (NPEVs) causing acute flaccid paralysis (AFP) due to myelitis has increased with the containment of the poliovirus. Enterovirus-B88 (EV-B88) has been associated with the AFP cases in Bangladesh, Ghana, South Africa, Thailand and India. In India, EV-B88 infection was linked to AFP a decade ago; however, to date, no complete genome has been made available. In this study, the complete genome sequence of EV-B88 was identified and reported from two different States (Bihar and Uttar Pradesh) in India using the next-generation sequencing technique. Methods: Virus isolation was performed on the three AFP suspected cases as per the WHO-recommended protocol. Samples showing cytopathic effects in the human Rhabdocarcinoma were labelled as NPEVs. Next-generation sequencing was performed on these NPEVs to identify the aetiological agent. The contiguous sequences (contigs) generated were identified, and reference-based mapping was performed. Results: EV-B88 sequences retrieved in our study were found to be 83 per cent similar to the EV-B88 isolate from Bangladesh in 2001 (strain: BAN01-10398; Accession number: AY843306.1). Recombination analyses of these samples demonstrate recombination events with sequences from echovirus-18 and echovirus-30. Interpretation & conclusions: Recombination events in the EV-B serotypes are known, and this work reconfirms the same for EV-B88 isolates also. This study is a step in increasing the awareness about EV-B88 in India and emphasizes future studies to be conducted in the identification of other types of EV present in India.

Urinary immunoglobulins in viral diagnosis: An overview.

Antibody detection by serological methods gained a lot of interest in recent years and has become the backbone of virological diagnosis. Despite the detection of all five classes of immunoglobulins in urine, not much attention has been paid to the use of urine as a diagnostic sample to detect viral antibodies. Unlike venipuncture, this non-invasive mode of sample collection can help cover all age groups, especially paediatric and old age patients, where blood collection is difficult. Using urine as a sample is also economical and involves lesser risk in sample collection. The antibodies are found to be stable in urine at room temperature for a prolonged period, which makes the sample transport management easier as well. A few recent studies, have also shown that the detection limit of antibodies in urine is at par with serum or other clinical material. So, the ease in sample collection, availability of samples in large quantity and stability of immunoglobulins in urine for prolonged periods can make urine an ideal sample for viral diagnosis.

Detection of Nipah virus in Pteropus medius in 2019 outbreak from Ernakulam district, Kerala, India.

BACKGROUND: In June 2019, Nipah virus (NiV) infection was detected in a 21-year-old male (index case) of Ernakulum, Kerala, India. This study was undertaken to determine if NiV was in circulation in Pteropus species (spp) in those areas where the index case had visit history in 1 month. METHODS: Specialized techniques were used to trap the Pteropus medius bats (random sampling) in the vicinity of the index case area. Throat and rectal swabs samples of 141 bats along with visceral organs of 92 bats were collected to detect the presence of NiV by real-time reverse transcriptase-polymerase chain reaction (qRTPCR). Serum samples of 52 bats were tested for anti-NiV Immunoglobulin (Ig) G antibodies by Enzyme-Linked Immunosorbent Assay (ELISA). The complete genome of NiV was sequenced by next-generation sequencing (NGS) from the tissues and swab samples of bats. RESULTS: One rectal swab sample and three bats visceral organs were found positive for the NiV. Interestingly, 20.68% (12/58) of Pteropus were positive for anti-NiV IgG antibodies. NiV sequences of 18,172; 17,200 and 15,100 nucleotide bps could be retrieved from three Pteropus bats. CONCLUSION: A distinct cluster of NiV sequences, with significant net-evolutionary nucleotide divergence, was obtained, suggesting the circulation of new genotype (I-India) in South India. NiV Positivity in Pteropus spp. of bats revealed that NiV is circulating in many districts of Kerala state, and active surveillance of NiV should be immediately set up to know the hotspot area for NiV infection.

Experiences of Indian Council of Medical Research with tick-borne zoonotic infections: Kyasanur Forest disease & Crimean-Congo haemorrhagic fever in India with One Health focus.

Emergence and re-emergence of several pathogens have been witnessed by this century in the form of outbreaks, epidemics and pandemics. In India, the influencing factor that promotes dissemination of emerging and re-emerging viral infections is the biogeographical zones: a megadiverse country, characterized by varied geographical, climatic conditions and ever-changing socio-economical and geopolitical issues. These influence the movement of humans and animals and add layers of complexity for the identification and timely management of infectious diseases. This review focuses on two tick-borne infections: Crimean-Congo haemorrhagic fever (CCHF) and Kyasanur forest disease (KFD). In the last two decades, these viruses have emerged and caused outbreaks in different parts of India. KFD virus was initially identified in 1957 and was known to be endemic in Karnataka State while CCHF virus was first identified during 2010 in Gujarat State, India. These viruses have managed to emerge in new areas within the last decade. With changing epidemiology of these arboviruses, there is a probability of the emergence of these viruses from new areas in future. The investigations on these two diseases under the One Health focus involved early detection, quickly developing diagnostic tools, identifying stakeholders, capacity building by developing collaboration with major stakeholders to understand the epidemiology and geographical spread in domestic animal reservoirs and tick vectors in the affected areas, developing laboratory network, providing diagnostic reagents and biosafety and laboratory diagnosis training to the network laboratories to control these diseases.

Experiential learnings from the Nipah virus outbreaks in Kerala towards containment of infectious public health emergencies in India.

Nipah virus (NiV) outbreak occurred in Kozhikode district, Kerala, India in 2018 with a case fatality rate of 91% (21/23). In 2019, a single case with full recovery occurred in Ernakulam district. We described the response and control measures by the Indian Council of Medical Research and Kerala State Government for the 2019 NiV outbreak. The establishment of Point of Care assays and monoclonal antibodies administration facility for early diagnosis, response and treatment, intensified contact tracing activities, bio-risk management and hospital infection control training of healthcare workers contributed to effective control and containment of NiV outbreak in Ernakulam.

Biorisk assessment for infrastructure & biosafety requirements for the laboratories providing coronavirus SARS-CoV-2/(COVID-19) diagnosis.

Novel coronavirus infection [coronavirus disease 2019 (COVID-19)] has spread to more than 203 countries of various regions including Africa, America, Europe, South East Asia and Western Pacific. The WHO had declared COVID-19 as the global public health emergency and subsequently as pandemic because of its worldwide spread. It is now one of the top-priority pathogens to be dealt with, because of high transmissibility, severe illness and associated mortality, wide geographical spread, lack of control measures with knowledge gaps in veterinary and human epidemiology, immunity and pathogenesis. The quick detection of cases and isolating them has become critical to contain it. To meet the increasing demand of the diagnostic services, it is necessary to enhance and expand laboratory capabilities since existing laboratories cannot meet the emerging demand. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a BSL-2 (Biosafety Level 2) agent and needs to be handled in biosafety cabinet using standard precautions. This review highlights minimum requirements for the diagnostic laboratories opting testing of material for the diagnosis of COVID-19 and associated biorisk to the individuals and to the community.

Proactive preparedness for Cat Que virus: An Orthobunyavirus existing in India.

Background & objectives: The presence of Cat Que virus (CQV) in Culex mosquitoes and pigs has been reported in China and Vietnam. Due to the spread of similar species of the Culex mosquitoes in India, there is a need to understand the replication kinetics of this virus in mosquito models. As a part of preparedness and to identify the presence of this CQV in humans and swine, this study was carried out to develop diagnostic tests. Methods: Serological and molecular diagnostic assays were developed for testing the mosquito population, human and swine serum samples. In this line, RNA-dependent RNA polymerase (L), glycoprotein (M) and nucleocapsid (S) genes-based reverse transcription-polymerase chain reaction (RT-PCR) assays were developed for CQV. Real-time RT-PCR was used for screening of retrospectively collected human serum samples (n=1020) with acute febrile illness during 2014-2017. Simultaneously, an in-house anti-CQV swine and human IgG ELISAs were also developed to detect anti-CQV IgG antibody. Human serum samples (n=883) with post-onset of disease (POD) >4 days and swine serum samples (n=459) were tested for the presence of anti-CQV IgG antibodies. CQV NIV 612,045 isolate was used for susceptibility and replication kinetics experiment using three different species of mosquitoes to understand its behaviour in Indian mosquitoes. Results: All human serum samples (n=1020) screened for the presence of CQV using real-time RT-PCR were found to be negative. Anti-CQV IgG antibody positivity was recorded in two of 883 human serum samples tested. Virus susceptibility experiments indicated that three species of mosquito, namely Aedes aegypti, Culex quinquefasciatus and Cx. tritaeniorhynchus supported multiplication of CQV by intrathoracic as well as artificial membrane/oral feeding routes. Interpretation & conclusions: Anti-CQV IgG antibody positivity in human serum samples tested and the replication capability of CQV in mosquitoes indicated a possible disease causing potential of CQV in Indian scenario. Screening of more human and swine serum samples using these assays is required as a proactive measure for understanding the prevalence of this neglected tropical virus.

Detection of coronaviruses in Pteropus & Rousettus species of bats from different States of India.

Background & objectives: Bats are considered to be the natural reservoir for many viruses, of which some are potential human pathogens. In India, an association of Pteropus medius bats with the Nipah virus was reported in the past. It is suspected that the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also has its association with bats. To assess the presence of CoVs in bats, we performed identification and characterization of bat CoV (BtCoV) in P. medius and Rousettus species from representative States in India, collected during 2018 and 2019. Methods: Representative rectal swab (RS) and throat swab specimens of Pteropus and Rousettus spp. bats were screened for CoVs using a pan-CoV reverse transcription-polymerase chain reaction (RT-PCR) targeting the RNA-dependent RNA polymerase (RdRp) gene. A single-step RT-PCR was performed on the RNA extracted from the bat specimens. Next-generation sequencing (NGS) was performed on a few representative bat specimens that were tested positive. Phylogenetic analysis was carried out on the partial sequences of RdRp gene sequences retrieved from both the bat species and complete viral genomes recovered from Rousettus spp. Results: Bat samples from the seven States were screened, and the RS specimens of eight Rousettus spp. and 21 Pteropus spp. were found positive for CoV RdRp gene. Among these, by Sanger sequencing, partial RdRp sequences could be retrieved from three Rousettus and eight Pteropus bat specimens. Phylogenetic analysis of the partial RdRp region demonstrated distinct subclustering of the BtCoV sequences retrieved from these Rousettus and Pteropus spp. bats. NGS led to the recovery of four sequences covering approximately 94.3 per cent of the whole genome of the BtCoVs from Rousettus bats. Three BtCoV sequences had 93.69 per cent identity to CoV BtRt-BetaCoV/GX2018. The fourth BtCoV sequence was 96.8 per cent identical to BtCoV HKU9-1. Interpretation & conclusions: This study was a step towards understanding the CoV circulation in Indian bats. Detection of potentially pathogenic CoVs in Indian bats stresses the need for enhanced screening for novel viruses in them. One Health approach with collaborative activities by the animal health and human health sectors in these surveillance activities shall be of use to public health. This would help in the development of diagnostic assays for novel viruses with outbreak potential and be useful in disease interventions. Proactive surveillance remains crucial for identifying the emerging novel viruses with epidemic potential and measures for risk mitigation.

Outbreak Investigation of Nipah Virus Disease in Kerala, India, 2018.

BACKGROUND: Nipah Virus (NiV) is a highly fatal emerging zoonotic virus and a potential threat to global health security. Here we describe the characteristics of the NiV outbreak that occurred in Kerala, India, during May-June 2018. METHODS: We used real-time reverse transcription polymerase chain reaction analysis of throat swab, blood, urine, and cerebrospinal fluid specimens to detect NiV. Further, the viral genome was sequenced and subjected to phylogenetic analysis. We conducted an epidemiologic investigation to describe the outbreak and elucidate the dynamics of NiV transmission. RESULTS: During 2-29 May 2018, 23 cases were identified, including the index case; 18 were laboratory confirmed. The lineage of the NiV responsible for this outbreak was closer to the Bangladesh lineage. The median age of cases was 45 years; the sex of 15 (65%) was male. The median incubation period was 9.5 days (range, 6-14 days). Of the 23 cases, 20 (87%) had respiratory symptoms. The case-fatality rate was 91%; 2 cases survived. Risk factors for infection included close proximity (ie, touching, feeding, or nursing a NiV-infected person), enabling exposure to droplet infection. The public health response included isolation of cases, contact tracing, and enforcement of hospital infection control practices. CONCLUSION: This is the first recorded NiV outbreak in South India. Early laboratory confirmation and an immediate public health response contained the outbreak.

Nipah Virus Sequences from Humans and Bats during Nipah Outbreak, Kerala, India, 2018.

We retrieved Nipah virus (NiV) sequences from 4 human and 3 fruit bat (Pteropus medius) samples from a 2018 outbreak in Kerala, India. Phylogenetic analysis demonstrated that NiV from humans was 96.15% similar to a Bangladesh strain but 99.7%-100% similar to virus from Pteropus spp. bats, indicating bats were the source of the outbreak.

Canine Distemper Virus in Asiatic Lions of Gujarat State, India.

In September 2018, an epizootic infection caused by canine distemper virus emerged in an Asiatic lion population in India. We detected the virus in samples from 68 lions and 6 leopards by reverse transcription PCR. Whole-genome sequencing analysis demonstrated the virus strain is similar to the proposed India-1/Asia-5 strain.

Infections among Contacts of Patients with Nipah Virus, India.

We conducted a serosurvey of 155 healthcare workers and 124 household and community members who had close contact with 18 patients who had laboratory-confirmed Nipah virus infections in Kerala, India. We detected 3 subclinical infections; 2 persons had IgM and IgG and 1 only IgM against Nipah virus.

Crimean Congo hemorrhagic fever serosurvey in humans for identifying high-risk populations and high-risk areas in the endemic state of Gujarat, India.

BACKGROUND: Crimean Congo Hemorrhagic Fever (CCHF) is a highly infectious zoonotic disease of humans transmitted by Hyalomma ticks. Earlier studies have shown CCHF seroprevalence in livestock throughout India, yet sporadic outbreaks have been recorded mostly from the Gujarat state of India since 2011. Occupational vulnerability to CCHF for animal handlers, veterinarians, abattoir workers, and healthcare workers has been documented. The current study was planned to determine the seroprevalence of CCHF with an intention to identify the high -risk population and high -risk areas from Gujarat state, India. METHODS: Based on the socio-clinical data, the human population of Gujarat was divided into eight categories viz. A: CCHF affected person/house/close contact, B: Neighborhood contacts, C: Animal handlers, D: General population, E: Farmers, F: Abattoir workers, G: Veterinarian, H: Healthcare workers. A total of 4978 human serum samples were collected from 33 districts of Gujarat during year 2015, 2016 and 2017. All the samples were screened for the presence of anti-CCHFV IgG using indigenously developed anti-CCHFV IgG ELISA. Univariate regression analysis was performed to recognize significant risk factors for CCHF seropositivity. RESULTS: Twenty-five serum samples were found to be positive with an overall CCHF human seropositivity of 0.5% (95% CI 0.30-0.74%). Gender predisposition to CCHF prevalence was observed in males (OR: 2.80; p-value: 0.020). The risk for seropositivity increased sevenfold when a person was in contact or neighbor with a CCHF case (OR 7.02; p-value: < 0.0001). No significant difference in seropositivity was observed within different age groups. Veterinarians, healthcare workers, and control group were found to be seronegative for CCHF. CONCLUSIONS: In-spite of CCHF sporadic outbreaks reported in Gujarat, the seropositivity for CCHF in the state was low as compared to other endemic countries. Males, close contacts and neighbors were identified as a high-risk population for CCHF infection. To recognize the high-risk area, tick screening and animal serosurvey would be a wiser choice. The study also suggests circulation and under diagnoses of CCHFV in the naïve regions of Gujarat.

A virus precipitation method for concentration & detection of avian influenza viruses from environmental water resources & its possible application in outbreak investigations.

Background & objectives: Avian influenza (AI) viruses have been a major cause of public health concern. Wild migratory birds and contaminated environmental sources such as waterbodies soiled with bird droppings play a significant role in the transmission of AI viruses. The objective of the present study was to develop a sensitive and user-friendly method for the concentration and detection of AI viruses from environmental water sources. Methods: Municipal potable water, surface water from reservoirs and sea were spiked with low pathogenic AI viruses. To concentrate the viruses by precipitation, a combination of potassium aluminium sulphate with milk powder was used. Real-time reverse transcription-polymerase chain reaction was performed for virus detection, and the results were compared with a virus concentration method using erythrocytes. Drinking water specimens from poultry markets were also tested for the presence of AI viruses. Results: A minimum of 101.0 EID50(50% egg infectious dose)/ml spiked H5N1 and 101.7 EID50/ml spiked H9N2 viruses were detected from spiked potable water; 101.0 and 102.0 EID50/ml spiked H5N1 virus was detected from surface water and seawater samples, respectively. The present method was more sensitive than the erythrocyte-binding method as approximately 10-fold higher infectious virus titres were obtained. AI H9N2 viruses were detected and isolated from water from local poultry markets, using this method. Interpretation & conclusions: Viability and recovery of the spiked viruses were not affected by precipitation. The present method may be suitable for the detection of AI viruses from different environmental water sources and can also be applied during outbreak investigations.

Emerging/re-emerging viral diseases & new viruses on the Indian horizon.

Infectious diseases remain as the major causes of human and animal morbidity and mortality leading to significant healthcare expenditure in India. The country has experienced the outbreaks and epidemics of many infectious diseases. However, enormous successes have been obtained against the control of major epidemic diseases, such as malaria, plague, leprosy and cholera, in the past. The country's vast terrains of extreme geo-climatic differences and uneven population distribution present unique patterns of distribution of viral diseases. Dynamic interplays of biological, socio-cultural and ecological factors, together with novel aspects of human-animal interphase, pose additional challenges with respect to the emergence of infectious diseases. The important challenges faced in the control and prevention of emerging and re-emerging infectious diseases range from understanding the impact of factors that are necessary for the emergence, to development of strengthened surveillance systems that can mitigate human suffering and death. In this article, the major emerging and re-emerging viral infections of public health importance have been reviewed that have already been included in the Integrated Disease Surveillance Programme.

Kinetics of viral RNA, immunoglobulin-M & G antibodies in Kyasanur forest disease.

Background & objectives: Kyasanur forest disease (KFD) is an infectious disease discovered in Karnataka State of India in 1957; since then, the State has been known to be enzootic for KFD. In the last few years, its presence was observed in the adjoining five States of the Western Ghats of India. The present study was conducted to understand the kinetics of viral RNA, immunoglobulin M (IgM) and IgG antibody in KFD-infected humans for developing a diagnostic algorithm for KFD. Methods: A prospective follow up study was performed among KFD patients in Sindhudurg district of Maharashtra State, India. A total of 1046 suspected patients were tested, and 72 KFD patients were enrolled and followed for 17 months (January 2016 to May 2017). Serum samples of KFD patients were screened for viral RNA, and IgM and IgG antibodies. Results: KFD viral positivity was observed from 1st to 18th post-onset day (POD). Positivity of anti-KFD virus (KFDV) IgM antibodies was detected from 4th till 122nd POD and anti-KFDV IgG antibodies detected from 5th till 474th POD. A prediction probability was determined from statistical analysis using the generalized additive model in R-software to support the laboratory findings regarding viral kinetics. Interpretation & conclusions: This study demonstrated the presence of KFD viral RNA till 18th POD, IgM antibodies till 122nd POD and IgG till the last sample collected. Based on our study an algorithm was recommended for accurate laboratory diagnosis of KFDV infection. A sample collected between 1 and 3 POD can be tested using KFDV real-time reverse transcriptase polymerase chain reaction (RT-PCR); between 4 and 24 POD, the combination of real-time RT-PCR and anti-KFDV IgM enzyme-linked immunosorbent assay (ELISA) tests can be used; between POD 25 and 132, anti-KFDV IgM and IgG ELISA are recommended.

Equine Encephalosis Virus in India, 2008.

A virus isolated from a sick horse from India in 2008 was confirmed by next-generation sequencing analysis to be equine encephalosis virus (EEV). EEV in India is concerning because several species of Culicoides midge, which play a major role in EEV natural maintenance and transmission, are present in this country.

Identification and characterization of novel mosquito-borne (Kammavanpettai virus) and tick-borne (Wad Medani) reoviruses isolated in India.

In 1954, a virus named Wad Medani virus (WMV) was isolated from Hyalomma marginatum ticks from Maharashtra State, India. In 1963, another virus was isolated from Sturnia pagodarum birds in Tamil Nadu, India, and named Kammavanpettai virus (KVPTV) based on the site of its isolation. Originally these virus isolates could not be identified with conventional methods. Here we describe next-generation sequencing studies leading to the determination of their complete genome sequences, and identification of both virus isolates as orbiviruses (family Reoviridae). Sequencing data showed that KVPTV has an AT-rich genome, whereas the genome of WMV is GC-rich. The size of the KVPTV genome is 18 234 nucleotides encoding proteins ranging 238-1290 amino acids (aa) in length. Similarly, the size of the WMV genome is 16 941 nucleotides encoding proteins ranging 214-1305 amino acids in length. Phylogenetic analysis of the VP1 gene, along with the capsid genes VP5 and VP7, revealed that KVPTV is likely a novel mosquito-borne virus and WMV is a tick-borne orbivirus. This study focuses on the phylogenetic comparison of these newly identified orbiviruses with mosquito-, tick- and Culicoides-borne orbiviruses isolated in India and other countries.

Clinical & epidemiological significance of Kyasanur forest disease.

Kyasanur forest disease (KFD) is a known viral haemorrhagic fever in India, for the last 60 years. However, in recent years, the change in epidemiological profile of the disease has suggested that it is now time to consider KFD as an emerging tropical disease in India. The preference should be to educate not only the villagers where it is being reported or detected but also to public health experts, veterinarians, forest officials and medical professionals to pay attention while seeing a patient overlapping with endemic diseases such as Japanese encephalitis, West Nile, dengue, chikungunya, malaria and tuberculosis. Although the existence of KFD is known for a long time, updated understanding of its clinical profile in humans is still limited. This article describes in detail the clinical presentation of KFD reported till date. It also highlights geographical distribution of the disease, risk factors for virus transmission, biochemical/haematological findings and control measures. There is an urgent need for research on KFD, particularly for understanding biphasic nature of illness, development of cost-effective diagnostic tools, utility of non-invasive samples for diagnosis and development of new vaccines.

Bioinformatics characterization of envelope glycoprotein from Kyasanur Forest disease virus.

BACKGROUND & OBJECTIVES: Kyasanur Forest disease (KFD) is a febrile illness characterized by haemorrhages and caused by KFD virus (KFDV), which belongs to the Flaviviridae family. It is reported to be an endemic disease in Shimoga district of Karnataka State, India, especially in forested and adjoining areas. Several outbreaks have been reported in newer areas, which raised queries regarding the changing nature of structural proteins if any. The objective of the study was to investigate amino acid composition and antigenic variability if any, among the envelope glycoprotein (E-proteins) from old and new strains of KFDV. METHODS: Bioinformatic tools and techniques were used to predict B-cell epitopes and three-dimensional structures and to compare envelope glycoprotein (E-proteins) between the old strains of KFDV and those from emerging outbreaks till 2015. RESULTS: The strain from recent outbreak in Thirthahalli, Karnataka State (2014), was similar to the older strain of KFDV (99.2%). Although mutations existed in strains from 2015 in Kerala KFD sequences, these did not alter the epitopes. INTERPRETATION & CONCLUSIONS: The study revealed that though mutations existed, there were no drastic changes in the structure or antigenicity of the E-proteins from recent outbreaks. Hence, no correlation could be established between the mutations and detection in new geographical areas. It seems that KFDV must be present earlier also in many States and due to availability of testing system and alertness coming into notice now.