Fungal Spores in Insect Trapping Fluids: Simultaneous Sampling for Insects and Pathogens

Surveillance for early detection of non-native, invasive pathogens requires simple, sturdy, and easy to use collecting devices. In this study, we compared the fungal species detected in wet collection cups of Lindgren traps versus those detected on slides with oiled cheesecloth as aerial spore collectors. DNA was extracted and amplified from both using the primers ITS1F -ITS7G, and Illumina sequencing was used for metabarcoding of fungi present in samples. In 90 samples there were 1277 fungal OTUs. For fungal OTUs only detected by one collection method, insect traps had three times the number of fungal OTUs compared to slides, and this pattern persisted when analyses were restricted to pathogens and forest pathogens. Annually, thousands of insect traps are deployed in North America and the associated trap fluids have added value in forest disease research and monitoring.

A pigtailed macaque model of Kyasanur Forest disease virus and Alkhurma hemorrhagic disease virus pathogenesis

Kyasanur Forest disease virus (KFDV) and the closely related Alkhurma hemorrhagic disease virus (AHFV) are emerging flaviviruses that cause severe viral hemorrhagic fevers in humans. Increasing geographical expansion and case numbers, particularly of KFDV in southwest India, class these viruses as a public health threat. Viral pathogenesis is not well understood and additional vaccines and antivirals are needed to effectively counter the impact of these viruses. However, current animal models of KFDV pathogenesis do not accurately reproduce viral tissue tropism or clinical outcomes observed in humans. Here, we show that pigtailed macaques (Macaca nemestrina) infected with KFDV or AHFV develop viremia that peaks 2 to 4 days following inoculation. Over the course of infection, animals developed lymphocytopenia, thrombocytopenia, and elevated liver enzymes. Infected animals exhibited hallmark signs of human disease characterized by a flushed appearance, piloerection, dehydration, loss of appetite, weakness, and hemorrhagic signs including epistaxis. Virus was commonly present in the gastrointestinal tract, consistent with human disease caused by KFDV and AHFV where gastrointestinal symptoms (hemorrhage, vomiting, diarrhea) are common. Importantly, RNAseq of whole blood revealed that KFDV downregulated gene expression of key clotting factors that was not observed during AHFV infection, consistent with increased severity of KFDV disease observed in this model. This work characterizes a nonhuman primate model for KFDV and AHFV that closely resembles human disease for further utilization in understanding host immunity and development of antiviral countermeasures.

Identification of bio-climatic determinants and potential risk areas for Kyasanur forest disease in Southern India using MaxEnt modelling approach

Background Kyasanur forest disease (KFD), known as monkey fever, was for the first time reported in 1957 from the Shivamogga district of Karnataka. But since 2011, it has been spreading to the neighbouring state of Kerala, Goa, Maharashtra, and Tamil Nadu. The disease is transmitted to humans, monkeys and by the infected bite of ticks Haemaphysalis spinigera. It is known that deforestation and ecological changes are the main reasons for KFD emergence, but the bio-climatic understanding and emerging pathways remain unknown. Methods The present study aims to understand the bio-climatic determinants of distribution of tick vector of KFD in southern India using the Maximum Entropy (MaxEnt) model. The analysis was done using 34 locations of Haemaphysalis spinigera occurrence and nineteen bio-climatic variables from WorldClim. Climatic variables contribution was assessed using the Jackknife test and mean AUC 0.859, indicating the model performs with very high accuracy. Results Most influential variables affecting the spatial distribution of Haemaphysalis spinigera were the average temperature of the warmest quarter (bio10, contributed 32.5%), average diurnal temperature range (bio2, contributed 21%), precipitation of wettest period (bio13, contributed 17.6%), and annual precipitation (bio12, contributed 11.1%). The highest probability of Haemaphysalis spinigera presence was found when the mean warmest quarter temperature ranged between 25.4 and 30 °C. The risk of availability of the tick increased noticeably when the mean diurnal temperature ranged between 8 and 10 °C. The tick also preferred habitat having an annual mean temperature (bio1) between 23 and 26.2 °C, mean temperature of the driest quarter (bio9) between 20 and 28 °C, and mean temperature of the wettest quarter (bio8) between 22.5 and 25 °C. Conclusions The results have established the relationship between bioclimatic variables and KFD tick distribution and mapped the potential areas for KFD in adjacent areas wherein surveillance for the disease is warranted for early preparedness before the occurrence of outbreaks etc. The modelling approach helps link bio-climatic variables with the present and predicted distribution of Haemaphysalis spinigera tick.

A live-attenuated viral vector vaccine protects mice against lethal challenge with Kyasanur Forest disease virus

Kyasanur Forest disease virus (KFDV) is a tick-borne flavivirus endemic in India known to cause severe hemorrhagic and encephalitic disease in humans. In recent years, KFDV has spread beyond its original endemic zone raising public health concerns. Currently, there is no treatment available for KFDV but a vaccine with limited efficacy is used in India. Here, we generated two new KFDV vaccine candidates based on the vesicular stomatitis virus (VSV) platform. We chose the VSV-Ebola virus (VSV-EBOV) vector either with the full-length or a truncated EBOV glycoprotein as the vehicle to express the precursor membrane (prM) and envelope (E) proteins of KFDV (VSV-KFDV). For efficacy testing, we established a mouse disease model by comparing KFDV infections in three immunocompetent mouse strains (BALB/c, C57Bl/6, and CD1). Both vaccine vectors provided promising protection against lethal KFDV challenge in the BALB/c model following prime-only prime-boost and immunizations. Only prime-boost immunization with VSV-KFDV expressing full-length EBOV GP resulted in uniform protection. Hyperimmune serum derived from prime-boost immunized mice protected naïve BALB/c mice from lethal KFDV challenge indicating the importance of antibodies for protection. The new VSV-KFDV vectors are promising vaccine candidates to combat an emerging, neglected public health problem in a densely populated part of the world.

20Forest loss shapes the landscape suitability of Kyasanur Forest disease in the Western Ghats, India

Background Anthropogenic pressure in biodiversity hotspots is increasingly recognised as a major driver of the spillover and expansion of zoonotic disease. In the Western Ghats region of India, a devastating tick-borne zoonosis, Kyasanur Forest disease (KFD), has been expanding rapidly beyond its endemic range in recent decades. While it has been suggested that anthropogenic pressure in the form of land use changes that lead to the loss of native forest may be directly contributing to the expanding range of KFD, clear evidence has not yet established the association between forest loss and KFD risk. Methods The current study sought to investigate the relationship between KFD landscape suitability and both forest loss and mammalian species richness to inform its epidemiology and infection ecology. Forty-seven outbreaks of KFD between 1 January, 2012 and 30 June, 2019 were modelled as an inhomogeneous Poisson process. Results Both forest loss (relative risk (RR) = 1.83; 95% C.I. 1.33 – 2.51) and mammalian species richness (RR = 1.29; 95% C.I. 1.16 – 1.42) were strongly associated with increased risk of KFD. Conclusions These results provide the first evidence of a clear association between increasing forest loss and risk for KFD. Moreover, the findings also highlight the importance of forest loss in areas of high biodiversity. This evidence supports integrative approaches to public health that incorporate conservation strategies simultaneously protective of humans, animals, and the environment. Key messages The association between deforestation and KFD risk suggest potential benefit in leveraging conservation efforts in the service of public health.

In-silico design of envelope based multi-epitope vaccine candidate against Kyasanur forest disease virus

Kyasanur forest disease virus (KFDV) causing tick-borne hemorrhagic fever which was earlier endemic to western Ghats, southern India, it is now encroaching into new geographic regions, but there is no approved medicine or effective vaccine against this deadly disease. In this study, we did in-silico design of multi-epitope subunit vaccine for KFDV. B-cell and T-cell epitopes were predicted from conserved regions of KFDV envelope protein and two vaccine candidates (VC1 and VC2) were constructed, those were found to be non-allergic and possess good antigenic properties, also gives cross-protection against Alkhurma hemorrhagic fever virus. The 3D structures of vaccine candidates were built and validated. Docking analysis of vaccine candidates with toll-like receptor-2 (TLR-2) by Cluspro and PatchDock revealed strong affinity between VC1 and TLR2. Ligplot tool was identified the intermolecular hydrogen bonds between vaccine candidates and TLR-2, iMOD server confirmed the stability of the docking complexes. JCAT sever ensured cloning efficiency of both vaccine constructs and in-silico cloning into pET30a (+) vector by SnapGene showed successful translation of epitope region. IMMSIM server was identified increased immunological responses. Finally, multi-epitope vaccine candidates were designed and validated their efficiency, it may pave the way for up-coming vaccine and diagnostic kit development.