Reviewing the ecological evidence base for management of emerging tropical zoonoses: Kyasanur Forest Disease in India as a case study.

Zoonoses disproportionately affect tropical communities and are associated with human modification and use of ecosystems. Effective management is hampered by poor ecological understanding of disease transmission and often focuses on human vaccination or treatment. Better ecological understanding of multi-vector and multi-host transmission, social and environmental factors altering human exposure, might enable a broader suite of management options. Options may include "ecological interventions" that target vectors or hosts and require good knowledge of underlying transmission processes, which may be more effective, economical, and long lasting than conventional approaches. New frameworks identify the hierarchical series of barriers that a pathogen needs to overcome before human spillover occurs and demonstrate how ecological interventions may strengthen these barriers and complement human-focused disease control. We extend these frameworks for vector-borne zoonoses, focusing on Kyasanur Forest Disease Virus (KFDV), a tick-borne, neglected zoonosis affecting poor forest communities in India, involving complex communities of tick and host species. We identify the hierarchical barriers to pathogen transmission targeted by existing management. We show that existing interventions mainly focus on human barriers (via personal protection and vaccination) or at barriers relating to Kyasanur Forest Disease (KFD) vectors (tick control on cattle and at the sites of host (monkey) deaths). We review the validity of existing management guidance for KFD through literature review and interviews with disease managers. Efficacy of interventions was difficult to quantify due to poor empirical understanding of KFDV-vector-host ecology, particularly the role of cattle and monkeys in the disease transmission cycle. Cattle are hypothesised to amplify tick populations. Monkeys may act as sentinels of human infection or are hypothesised to act as amplifying hosts for KFDV, but the spatial scale of risk arising from ticks infected via monkeys versus small mammal reservoirs is unclear. We identified 19 urgent research priorities for refinement of current management strategies or development of ecological interventions targeting vectors and host barriers to prevent disease spillover in the future.

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.

Kyasanur Forest disease virus non-mouse animal models: a pilot study.

OBJECTIVES: Mouse models have delivered variable recapitulation of Kyasanur Forest disease (KFD) pathology and consistently demonstrated neurological involvement which may be a limited feature of human disease. With the purpose of more accurately modelling human disease progression we infected several small-mammalian models: guinea pigs, hamsters and ferrets with a titered infectious dose of Kyasanur Forest disease virus (KFDV). Clinical indicators of disease severity were observed for seventeen days, on day eighteen a visual post-mortem analysis of visceral organs was conducted. Viral load in selected tissues was measured to infer disease signs and the establishment of viral replication. DATA DESCRIPTION: Daily monitoring did not reveal any observable signs of illness; weight loss was minimal across species and gross pathology did not indicate severe viral infection. Tissue specific tropism and establishment of viral infection was monitored by quantitative real-time polymerase chain reaction (qRT-PCR). No viral replication was detected in ferrets (n = 0/3), but was present in the spleen of guinea pigs (n = 3/3) and the brain of hamsters (n = 3/3). Low levels of viral RNA were detected in multiple hamster tissues (kidney, liver, lung and spleen) suggesting the possibility of viral tropism and possible adaptation to the host. No serological tests were performed.

A preliminary ecological profile of Kyasanur Forest disease virus hosts among the mammalian wildlife of the Western Ghats, India.

Kyasanur Forest disease (KFD) virus is one of India's severe arboviruses capable of causing prolonged debilitating disease. It has been expanding beyond its historical endemic locus at an alarming rate over the last two decades. The natural nidus of this zoonosis is located in the monsoon rainforest of the Western Ghats, India, which is one of the world's most important biodiversity hotspots. Definitive reservoir hosts for KFD virus (KFDV) have yet to be delineated, and thus much of the infection ecology of this virus, and its consequent transmission dynamics, remains uncertain. Given its unique biogeographical context, identifying ecological parameters of KFDV relevant to the virus' epidemiology has been complex and challenging. The challenge has been exacerbated by diminished research efforts in wildlife surveillance over the last two decades, coinciding with the expansion of the range of KFD across the region. The current investigation sought to define a preliminary ecological profile of KFDV hosts based on their life history and feeding traits to aid in re-establishing targeted wildlife surveillance and to discern those ecological traits of wildlife hosts that may improve our understanding of KFD epidemiology. The importance of fast-living among KFDV hosts was of special interest with respect to the latter aim. We compared mammalian traits between host and non-host species using general additive models and phylogenetic generalised linear models. This study found that both body mass and forest forage were strongly associated with mammalian host infection status, but that reproductive life history traits were not. These findings will help in structuring ecologically based wildlife surveillance and field investigations, while also helping to parameterise novel epidemiological models of zoonotic infection risk that incorporate species functional traits in a region where biogeography, landscape ecology, and community ecology manifest extraordinary complexity, particularly under growing anthropogenic pressure.

Phylogeography of Kyasanur Forest Disease virus in India (1957-2017) reveals evolution and spread in the Western Ghats region.

The Kyasanur Forest Disease (KFD) has become a major public health problem in the State of Karnataka, India where the disease was first identified and in Tamil Nadu, Maharashtra, Kerala, and Goa covering the Western Ghats region of India. The incidence of positive cases and distribution of the Kyasanur Forest Disease virus (KFDV) in different geographical regions raises the need to understand the evolution and spatiotemporal transmission dynamics. Phylogeography analysis based on 48 whole genomes (46 from this study) and additionally 28 E-gene sequences of KFDV isolated from different regions spanning the period 1957-2017 was thus undertaken. The mean evolutionary rates based the E-gene was marginally higher than that based on the whole genomes. A subgroup of KFDV strains (2006-2017) differing from the early Karnataka strains (1957-1972) by ~2.76% in their whole genomes and representing spread to different geographical areas diverged around 1980. Dispersal from Karnataka to Goa and Maharashtra was indicated. Maharashtra represented a new source for transmission of KFDV since ~2013. Significant evidence of adaptive evolution at site 123 A/T located in the vicinity of the envelope protein dimer interface may have functional implications. The findings indicate the need to curtail the spread of KFDV by surveillance measures and improved vaccination strategies.

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.

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.

Epidemiology, Pathogenesis, and Control of a Tick-Borne Disease- Kyasanur Forest Disease: Current Status and Future Directions.

In South Asia, Haemaphysalis spinigera tick transmits Kyasanur Forest Disease Virus (KFDV), a flavivirus that causes severe hemorrhagic fever with neurological manifestations such as mental disturbances, severe headache, tremors, and vision deficits in infected human beings with a fatality rate of 3-10%. The disease was first reported in March 1957 from Kyasanur forest of Karnataka (India) from sick and dying monkeys. Since then, between 400 and 500 humans cases per year have been recorded; monkeys and small mammals are common hosts of this virus. KFDV can cause epizootics with high fatality in primates and is a level-4 virus according to the international biosafety rules. The density of tick vectors in a given year correlates with the incidence of human disease. The virus is a positive strand RNA virus and its genome was discovered to code for one polyprotein that is cleaved post-translationally into 3 structural proteins (Capsid protein, Envelope Glycoprotein M and Envelope Glycoprotein E) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). KFDV has a high degree of sequence homology with most members of the TBEV serocomplex. Alkhurma virus is a KFDV variant sharing a sequence similarity of 97%. KFDV is classified as a NIAID Category C priority pathogen due to its extreme pathogenicity and lack of US FDA approved vaccines and therapeutics; also, the infectious dose is currently unknown for KFD. In India, formalin-inactivated KFDV vaccine produced in chick embryo fibroblast is being used. Nevertheless, further efforts are required to enhance its long-term efficacy. KFDV remains an understudied virus and there remains a lack of insight into its pathogenesis; moreover, specific treatment to the disease is not available to date. Environmental and climatic factors involved in disseminating Kyasanur Forest Disease are required to be fully explored. There should be a mapping of endemic areas and cross-border veterinary surveillance needs to be developed in high-risk regions. The involvement of both animal and health sector is pivotal for circumscribing the spread of this disease to new areas.

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.

Prevalence and spatial distribution of Ixodid tick populations in the forest fringes of Western Ghats reported with human cases of Kyasanur forest disease and monkey deaths in South India.

Kyasanur forest disease (KFD) is a major tick-borne viral haemorrhagic fever caused by KFD virus (KFDV) (Flaviviridae). The disease was reported to be confined to five districts of Karnataka state India until 2011. During 2012-2016, emergence of KFD has been reported in newer areas of Karnataka and adjoining states. Therefore, survey of tick vectors was carried out in these new areas of Karnataka and adjoining states reported with monkey deaths and human cases of KFD. In all selected sites, ticks from the forest floor were collected by lint clothes using flagging method. Tick samples were tested for KFDV nucleic acid by real-time RT-PCR. A total of 4772 ticks, comprising eight species of genus Haemaphysalis and one species each of genus Amblyomma, Ixodes and Rhipicephalus was collected. Haemaphysalis spinigera, the principal vector of KFDV was the predominant tick species (59.5%) collected followed by H. turturis (8.6%). The abundance of H. spinigera ranged from 9.2 to 33.9 per man-hour in the six districts surveyed. Of 214 (4418 tick samples) pools screened by real-time RT-PCR, two pools of H. spinigera were positive for KFDV. High abundance of Haemaphysalis vectors in the six districts indicated that the districts are receptive for KFD outbreaks. KFDV was detected in the tick vectors in the new foci of the KFD. Data on tick distribution will be useful in creating KFD risk map for strengthening the ongoing preventive measures such as vaccination and supply of insect repellents to the high risk groups and intensive health education.

Kyasanur forest disease virus breaking the endemic barrier: An investigation into ecological effects on disease emergence and future outlook.

Kyasanur Forest disease (KFD) is found in a limited range of India, but is epidemiologically understudied. The seasonal patterns of KFD are well known; however, the significant concern is on the extent to which changes in epidemiology happen especially under the influence of ecological destructions and by the eventual effects of resulting climate change. Presently, a southward and northward spread of the Kyasanur Forest disease virus (KFDV) along the Western Ghats has been reported in the adjoining states of Kerala, Tamil Nadu, Goa and Maharashtra. In this review, we investigate the cascade of factors that might have facilitated the resurgence of KFDV among the endemic regions in higher frequency and its recent emergence in the area previously not reported. Utilizing published data, we additionally endeavour to exhibit a portion of the impediments of control systems and embody the powerful option strategies for developing KFDV control.

Use of hydrogen peroxide vapour & plasma irradiation in combination for quick decontamination of closed chambers.

BACKGROUND & OBJECTIVES: Various conventional methods such as gaseous, vapour and misting systems, fogging, manual spray and wipe techniques employing a number of chemical agents are used for decontamination of enclosed spaces. Among all these methods, use of aerosolized formaldehyde is the most preferred method due to cost-effectiveness and practical aspects. However, being extremely corrosive in nature generating very irritating fumes and difficulty in maintaining a high level of gas concentration, many laboratories prefer the vaporization of hydrogen peroxide (H 2 O 2 ) as an alternative. We present here the results of using H 2 O 2 vapour in combination with plasma irradiation for quick decontamination of closed chambers. METHODS: The present study describes a decontamination method, using plasma irradiation in combination with H 2 O 2 (5%). Effect of plasma irradiation and H 2 O 2 on the viability of bacterial spores (Bacillus subtilis), Chikungunya and Kyasanur Forest Disease viruses was assessed. RESULTS: Data suggest that with the combination of H 2 O 2 vapour and plasma irradiation, within short time (three minutes), decontamination of surfaces and space volume could be achieved. Although it showed damage of spores present on the strips, it did not show any penetration power. INTERPRETATION & CONCLUSIONS: The results were encouraging, and this method was found to be efficient for achieving surface sterilization in a short time. This application may be useful in laboratories and industries particularly, those working on clean facility concept following good laboratory and manufacturing practices.

Kyasanur Forest Disease - First Reported Case in Kerala.

Kyasanur Forest disease is a tick-borne arboviral fever with biphasic course of illness with prominent hemorrhagic features in the first phase and encephalitic picture in the second phase. So far it has been described in the southern Karnataka only. Here we report a case of Kyasanur Forest Disease for the first time from Kerala in an 18 year old male from Noolpuzha - Alathoor colony of Wayanad district.

An Early Passage Human Isolate of Kyasanur Forest Disease Virus Shows Acute Neuropathology in Experimentally Infected CD-1 Mice.

BACKGROUND: Kyasanur Forest disease virus (KFDV) is a tick-borne Flavivirus that causes a severe illness in humans. Disease spectrum can vary from subclinical infection to fatal cases with hemorrhagic complications. The pathology of KFDV remains incompletely understood. METHODS: This study describes the histopathologic and immunohistochemical findings in experimentally infected infant CD-1 mice with an early passage human KFDV isolate. RESULTS: Acute histological changes were primarily seen in the brain. The spectrum of changes included gliosis, inflammatory response, necrosis, neural loss, and syncytium formation in mid and hind brain structures. Microscopic lesions observed in the liver were mainly necrosis and vacuolation of hepatocytes and in small intestine, prominent epithelial cell necrosis. KFDV antigens could be stained by a sensitive immunohistochemical labeling in the same organs. CONCLUSIONS: Findings from this study are suggestive of neuropathology as the main manifestation of an early passaged human KFDV isolate. Importantly, this suggests that KFDV may be causing primarily a neurologic disease and secondary organ damage could be because of disease pathology per se. The use of primary low passage human isolates and neuropathology profile could also be more apt in developing a challenge model for testing potential antivirals and therapeutic agents.

Comparative pathogenesis of Alkhumra hemorrhagic fever and Kyasanur forest disease viruses in a mouse model.

Kyasanur Forest disease virus (KFDV) and Alkhumra hemorrhagic fever virus (AHFV) are genetically closely-related, tick-borne flaviviruses that cause severe, often fatal disease in humans. Flaviviruses in the tick-borne encephalitis (TBE) complex typically cause neurological disease in humans whereas patients infected with KFDV and AHFV predominately present with hemorrhagic fever. A small animal model for KFDV and AHFV to study the pathogenesis and evaluate countermeasures has been lacking mostly due to the need of a high biocontainment laboratory to work with the viruses. To evaluate the utility of an existing mouse model for tick-borne flavivirus pathogenesis, we performed serial sacrifice studies in BALB/c mice infected with either KFDV strain P9605 or AHFV strain Zaki-1. Strikingly, infection with KFDV was completely lethal in mice, while AHFV caused no clinical signs of disease and no animals succumbed to infection. KFDV and high levels of pro-inflammatory cytokines were detected in the brain at later time points, but no virus was found in visceral organs; conversely, AHFV Zaki-1 and elevated levels of cytokines were found in the visceral organs at earlier time points, but were not detected in the brain. While infection with either virus caused a generalized leukopenia, only AHFV Zaki-1 induced hematologic abnormalities in infected animals. Our data suggest that KFDV P9605 may have lost its ability to cause hemorrhagic disease as the result of multiple passages in suckling mouse brains. However, likely by virtue of fewer mouse passages, AHFV Zaki-1 has retained the ability to replicate in visceral organs, cause hematologic abnormalities, and induce pro-inflammatory cytokines without causing overt disease. Given these striking differences, the use of inbred mice and the virus passage history need to be carefully considered in the interpretation of animal studies using these viruses.

Kyasanur forest disease.

In the spring of 1957, an outbreak of severe disease was documented in people living near the Kyasanur forest in Karnataka state, India, which also affected wild nonhuman primates. Collection of samples from dead animals and the use of classical virological techniques led to the isolation of a previously unrecognized virus, named Kyasanur forest disease virus (KFDV), which was found to be related to the Russian spring-summer encephalitis (RSSE) complex of tick-borne viruses. Further evaluation found that KFD, which frequently took the form of a hemorrhagic syndrome, differed from most other RSSE virus infections, which were characterized by neurologic disease. Its association with illness in wild primates was also unique. Hemaphysalis spinigera was identified as the probable tick vector. Despite an estimated annual incidence in India of 400-500 cases, KFD is historically understudied. Most of what is known about the disease comes from studies in the late 1950s and early 1960s by the Virus Research Center in Pune, India and their collaborators at the Rockefeller Foundation. A report in ProMED in early 2012 indicated that the number of cases of KFD this year is possibly the largest since 2005, reminding us that there are significant gaps in our knowledge of the disease, including many aspects of its pathogenesis, the host response to infection and potential therapeutic options. A vaccine is currently in use in India, but efforts could be made to improve its long-term efficacy.

Seroprevalence of Alkhurma and other hemorrhagic fever viruses, Saudi Arabia.

A 2009 deployment of military units from several Saudi Arabian provinces to Jazan Province, Saudi Arabia, enabled us to evaluate exposure to Alkhurma, Crimean-Congo, dengue, and Rift Valley hemorrhagic fever viruses. Seroprevalence to all viruses was low; however, Alkhurma virus seroprevalence was higher (1.3%) and less geographically restricted than previously thought.

Recent ancestry of Kyasanur Forest disease virus.

Kyasanur Forest disease virus (KFDV) is enzootic to India and maintained in ticks, mammals, and birds. It causes severe febrile illness in humans and was first recognized in 1957 associated with a high number of deaths among monkeys in Kyasanur Forest. Genetic analysis of 48 viruses isolated in India during 1957-2006 showed low diversity (1.2%). Bayesian coalescence analysis of these sequences and those of KFDVs from Saudi Arabia and the People's Republic of China estimated that KFDVs have evolved at a mean rate of approximately 6.4 x 10(-4) substitutions/site/year, which is similar to rates estimated for mosquito-borne flaviviruses. KFDVs were estimated to have shared a common ancestor in approximately 1942, fifteen years before identification of the disease in India. These data are consistent with the view that KFD represented a newly emerged disease when first recognized. Recent common ancestry of KFDVs from India and Saudi Arabia, despite their large geographic separation, indicates long-range movement of virus, possibly by birds.

Kyasanur forest disease: an epidemiological view in India.

Kyasanur forest disease (KFD) was first recognised as a febrile illness in the Shimoga district of Karnataka state of India. The causative agent, KFD virus (KFDV), is a highly pathogenic member in the family Flaviviridae, producing a haemorrhagic disease in infected human beings. KFD is a zoonotic disease and has so far been localised only in a southern part of India. The exact cause of its emergence in the mid 1950s is not known. A variant of KFDV, characterised serologically and genetically as Alkhurma haemorrhagic fever virus (AHFV), has been recently identified in Saudi Arabia. KFDV and AHFV share 89% sequence homology, suggesting common ancestral origin. Homology modelling of KFDV envelope (E) protein exhibited a structure similar to those of other flaviviruses, suggesting a common mechanism of virus-cell fusion. The possible mechanism of receptor-ligand interaction involved in infection by KFDV may resemble that of other flavivirses. Present understanding is that KFDV may be persisting silently in several regions of India and that antigenic and structural differences from other tick borne viruses may be related to the unique host specificity and pathogenicity of KFDV. From January 1999 through January 2005, an increasing number of KFD cases have been detected in Karnataka state of Indian subcontinent despite routine vaccination, suggesting insufficient efficacy of the current vaccine protocol. However, the exact cause of the increase of KFD cases needs further investigation. Considering the requirement of safer and more effective vaccines in general, there is clearly a need for developing an alternative vaccine as well as a rapid diagnostic system for KFD. The changing ecology of the prime focus of the KFD also warrants attention, as it may lead to establishment of the disease in newer localities, never reported before.