Molecular characterization of varicella zoster virus isolated from clinical samples in India.

Background & objectives: Varicella zoster virus (VZV) strains are classified into six different clades based on the sequencing of its genome. Clades 4 and 5 are reported from India based on the single-nucleotide polymorphism (SNP). Till now, multiple clade circulations using partial sequences have been reported from India due to the lack of availability of the full VZV genome sequence. This study conducted a genome sequencing of VZV in India to identify circulating clade. Methods: Four clinical samples obtained from symptomatic patients tested positive for VZV by real-time PCR were used. These four samples were preferred to retrieve the genomic VZV sequence using the next-generation sequencing method. A reference-based assembly method was used to retrieve the genome of VZV, which was further analyzed. Results: At the least, 98 per cent of the whole-genome sequences were recovered from the four samples. The VZV sequences obtained in this study formed a separate monophyletic branch with clade 5, indicating it to be evolved from a distinct ancestor. The nucleotide-based analysis revealed 13 different SNP mutations and one multiple nucleotide variation in the VZV sequences when compared to one of the clade 5 genomes having accession number: DQ457052.1. Interpretation & conclusions: The present study described approximately 98 per cent of the genome sequence of VZV from India. The availability of these genomic sequences will lead to enrichment in the clinical genomic data set from India. The available data would help in the development of diagnostic methods along with evolutionary analysis. We hypothesize the existence of a new sub-clade that belongs to clade 5 and propose further experiments to confirm these results.

Study of Kyasanur forest disease viremia, antibody kinetics, and virus infection in target organs of Macaca radiata.

The present manuscript deals with experimental infections of bonnet macaques (Macaca radiata) to study disease progression for better insights into the Kyasanur Forest Disease (KFD) pathogenesis and transmission. Experimentally, 10 monkeys were inoculated with KFD virus (KFDV) (high or low dose) and were regularly monitored and sampled for various body fluids and tissues at preset time points. We found that only 2 out of the 10 animals showed marked clinical signs becoming moribund, both in the low dose group, even though viremia, virus shedding in the secretions and excretions were evident in all inoculated monkeys. Anti-KFDV immunoglobulin (Ig)M antibody response was observed around a week after inoculation and anti-KFDV IgG antibody response after two weeks. Anaemia, leucopenia, thrombocytopenia, monocytosis, increase in average clotting time, and reduction in the serum protein levels were evident. The virus could be re-isolated from the skin during the viremic period. The persistence of viral RNA in the gastrointestinal tract and lymph nodes was seen up to 53 and 81 days respectively. Neuro-invasion was observed only in moribund macaques. Re-challenge with the virus after 21 days of initial inoculation in a monkey did not result in virus shedding or immune response boosting.

Assessment of NS1 protein as an early diagnostic marker for Kyasanur forest disease virus.

BACKGROUND & OBJECTIVES: Due to the emergence of Kyasanur forest disease (KFD) virus to new regions in India, there is an urgent need to develop an early diagnostic system, which is cost-effective and can be efficiently used with minimum paraphernalia. The non-structural-1 (NS1) protein is known to be an early diagnostic marker for flaviviruses. Furthermore, NS1 antigen capture ELISA kits developed using bacterially expressed dengue NS1 protein are commercially available. METHODS: Based on the data available on dengue virus, West Nile virus and other flaviviruses, bacterially expressed Kyasanur forest disease virus (KFDV) NS1 protein and polyclonal serum raised against the NS1 protein in mice and rabbit were used to develop an antigen capture ELISA for early diagnosis of the virus. The feasibility of this ELISA was further tested using in silico predictions. RESULTS: KFDV NS1 gene was cloned, expressed and confirmed by SDS-PAGE and western blotting. An antigen detection ELISA was standardized and sensitivity and specificity was tested with other flaviviruses. KFDV acute phase 43 samples were tested and only two were found to be positive for KFDV NS1 antigen. Superimposition of KFDV NS1 and TBEV NS1 revealed a root mean square distance (RMSD) of ~0.79 Å covering 1220 backbone atoms. This implies that the structures are very similar in terms of 3D fold. The identity of amino acid composition between these proteins was 73.4% and similarity was 92.9%, as revealed from the pairwise comparison. INTERPRETATION & CONCLUSION: The study points out that the half-life, expression and secretion levels of KFDV NS1 protein are not sufficient enough for its use as early diagnostic marker. The protein may have to be expressed in eukaryotic host to counter the lack of glycosylation in bacterial plasmid based expression of proteins. Hence, bacterially expressed KFDV NS1 protein may not be an ideal early diagnostic marker for the virus.

Identification and phylogenetic analysis of herpes simplex virus-1 from clinical isolates in India.

Human herpes simplex virus (HSV)-1 infection is acquired in childhood and persists throughout a person's lifetime. Here, we present two cases from India; one showing symptoms of postpartum haemorrhage with disseminated intravascular coagulation, and the second one showing signs of acute encephalitis syndrome. The aetiological agent in both cases was identified as HSV-1 using the PCR method. The next-generation sequencing method retrieved ~97 % of the viral genome from the isolates of the clinical samples. The phylogenetic analysis of the retrieved genomes revealed that they belong to clade II of HSV-1. This study identifies a few sequence variations in the glycoprotein region of HSV-1 during two different clinical manifestations. There are a couple of papers that analyse variations in the glycoprotein region of clinical samples. Further, this study also highlights the importance of considering HSV-1 during differential diagnosis when analysing the nosocomial infection.

Clinico-epidemiological investigation on Varicella Zoster Virus indicates multiple clade circulation in Maharashtra state, India.

BACKGROUND: Varicella Zoster Virus (VZV) is consistently in circulation and shows an increase in disease burden during the spring season. Due to a wide range of clinical presentation from a vesicular rash to bleeding or neurological complications, it makes the clinical diagnosis difficult. The present study aims to understand whether the same strain of virus is responsible for the increase in the seasonal outbreaks occurring in different parts of the country with reference to the samples from Maharashtra, Rajasthan and Gujarat states of India. MATERIALS AND METHODS: This study reports the clinico-epidemiological and laboratory findings of suspected Varicella cases. To understand the circulating clade few representative real-time Polymerase Chain Reaction (PCR) positive were analyzed by conventional PCR and partial Open Reading Frame (ORF) 22, partial ORF 38 and partial ORF 54 were sequenced to identify single nucleotide polymorphisms responsible for clade determination. Further partial glycoprotein B gene was sequenced, and a phylogenetic tree was generated. RESULTS: A total of 50 cases from Maharashtra (Mumbai district) and referred clinical samples of Rajasthan (Barmer district; n = 12) and Gujarat States (Gandhi Nagar, Surat districts; n = 17) were tested for the presence of VZV. Vesicular rash with fever was a common clinical presentation with 82% cases having contact history with VZV positive cases, suggesting higher secondary attack rate. The vesicular fluid of all 50 cases from Mumbai revealed the presence of VZV by real-time PCR. Urine, serum and throat swab samples showed positivity by real-time PCR. Healthcare provider's samples from Rajasthan showed 36.4% [4/11] positivity. Clinical samples from Gujarat had positivity of 41.2% [7/17]. CONCLUSIONS: This study analyses the clade based circulation of VZV in three states in India and suggests different clades circulating in Maharashtra state. Health education amongst the general population is suggested to reduce the secondary cases by early diagnosis, effective isolation policies and vaccination to reduce the burden of disease.

Characterization of Unknown Orthobunya-Like Viruses from India.

Next-generation sequencing (NGS) of agents causing idiopathic human diseases has been crucial in the identification of novel viruses. This study describes the isolation and characterization of two novel orthobunyaviruses obtained from a jungle myna and a paddy bird from Karnataka State, India. Using an NGS approach, these isolates were classified as Cat Que and Balagodu viruses belonging to the Manzanilla clade of the Simbu serogroup. Closely related viruses in the Manzanilla clade have been isolated from mosquitos, humans, birds, and pigs across a wide geographic region. Since Orthobunyaviruses exhibit high reassortment frequency and can cause acute, self-limiting febrile illness, these data suggest that human and livestock infections of the Oya/Cat Que/Manzanilla virus may be more widespread and/or under-reported than anticipated. It therefore becomes imperative to identify novel and unknown viruses in order to understand their role in human and animal pathogenesis. The current study is a step forward in this regard and would act as a prototype method for isolation, identification and detection of several other emerging viruses.

Development of single step RT-PCR for detection of Kyasanur forest disease virus from clinical samples.

BACKGROUND: Kyasanur Forest Disease (KFD), a tick borne flavivirus, which was earlier endemic to Karnataka state, India, has been confirmed and detected from neighboring states of Tamil Nadu, Maharashtra, Goa and Kerala states in India. Increased human and vector surveillance therefore becomes essential for the identification of KFD affected regions and control of further spread of the disease. Currently, available KFD detection assays include realtime RT-PCR and nested RT-PCR assays. Here we describe the development of a sensitive single step RT-PCR assay for the detection of KFD viral RNA. This can be easily used in any BSL-2 laboratory for screening of KFD suspected cases or for differential diagnosis of viral hemorrhagic fever panel. METHOD: Three primer sets were designed and checked for sensitivity using known dilutions of KFD viral RNA (Ranging from 106 copies to 10 copies). The primer set (2) was found to be most sensitive was selected and tested for specificity for Kyasanur forest disease virus (KFDV) by testing against zika, dengue, chikungunya, crimean congo hemorrhagic fever (CCHF), yellow fever, japanese encephalitis (JE) and west nile viruses. A total of 104 samples (human, monkey and tick positive and negative samples) were tested using this assay. RESULT: No false positive or false negative results were seen for human, monkey or tick samples. The assay was specific for KFD and could detect upto 100 copies of KFD viral RNA. DISCUSSION AND CONCLUSION: The previously published sensitive real time RT-PCR assay requires higher cost in terms of reagents and machine setup and technical expertise has been the primary reason for development of this assay. A single step RT-PCR is relatively easy to perform and more cost effective than real time RT-PCR in smaller setups in the absence of Biosafety Level-3 facility. This study reports the development and optimization of single step RT-PCR assay which is more sensitive and less time-consuming than nested RT-PCR and cost effective for rapid diagnosis of KFD viral RNA.

A mini-review of Bunyaviruses recorded in India.

Newly emerging and re-emerging viral infections are of major public health concern. Bunyaviridae family of viruses comprises a large group of animal viruses. Clinical symptoms exhibited by persons infected by viruses belonging to this family vary from mild-to-severe diseases i.e., febrile illness, encephalitis, haemorrhagic fever and acute respiratory illness. Several arthropods-borne viruses have been discovered and classified at serological level in India in the past. Some of these are highly pathogenic as the recent emergence and spread of Crimean-Congo haemorrhagic fever virus and presence of antibodies against Hantavirus in humans in India have provided evidences that it may become one of the emerging diseases in this country. For many of the discovered viruses, we still need to study their relevance to human and animal health. Chittoor virus, a variant of Batai virus; Ganjam virus, an Asian variant of Nairobi sheep disease virus; tick-borne viruses such as Bhanja, Palma and mosquito-borne viruses such as Sathuperi, Thimiri, Umbre and Ingwavuma viruses have been identified as the members of this family. As Bunyaviruses are three segmented RNA viruses, they can reassort the segments into genetically distinct viruses in target cells. This ability is believed to play a major role in evolution, pathogenesis and epidemiology of the viruses. Here, we provide a comprehensive overview of discovery, emergence and distribution of Bunyaviruses in India.

Cross-sectional Serosurvey of Crimean-Congo Hemorrhagic Fever Virus IgG in Livestock, India, 2013-2014.

We conducted a cross-sectional serosurvey of Crimean-Congo hemorrhagic fever (CCHF) among livestock in 22 states and 1 union territory of India. A total of 5,636 samples from bovines, sheep, and goats were screened for CCHF virus IgG. IgG was detected in 354 samples, indicating that this virus is widespread in this country.