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

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

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