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Research Project: Prediction of Viral Zoonoses and Reverse Zoonoses Using Next Generation Serodiagnostics (NGSeroDx)

Location: Zoonotic and Emerging Disease Research

Project Number: 3022-32000-021-005-S
Project Type: Non-Assistance Cooperative Agreement

Start Date: Aug 22, 2022
End Date: Aug 21, 2025

Viral zoonoses have a significant impact on human health, economy, and intertwined animal health. Several factors are responsible for these outbreaks including farming practices, wildlife habitat destruction, wildlife food sources, illegal animal trading, and global warming (Karesh et al, Lancet. 2012). Ticks, bats, rodents, and ungulates have been associated with several events of emergence and re-emergence of zoonotic viruses. With rapid economic developments, urbanization and climate change impacts, humans, livestock, and wild animals now come into contact more frequently, resulting in increased prevalence of interspecies transmission of pathogens, ultimately leading to disease outbreaks. Identifying these animal groups and associated viruses with zoonotic potential remains a major challenge due to genomic divergence and a major proportion of viruses yet to be discovered. High throughput sequencing (HTS) approaches for pathogen discovery and cost reduction have been key factors for viral surveillance, novel virus discovery in humans, animals, poultry, livestock, and wildlife. Although respiratory viruses pose the highest risk for larger outbreaks that may lead to epidemics or pandemics, viral zoonoses is common among blood-borne viruses, arboviruses, and hemorrhagic fever viruses that can lead to severe outbreaks with high fatality. Crimean-Congo hemorrhagic fever (CCHF) group and Henipavirus group are expanding geographically at alarming rates. CCHF is a fatal viral infection transmitted by ticks with a mortality rate of 3-40% in humans (Whitehouse CA, J. Antiviral. 2004). Viruses from genus Henipavirus are also negative-sense RNA viruses, including Nipah, Mojiang virus, Hendra, Cedar, and Ghana virus. Hendra and Nipah viruses were responsible for severe outbreaks in pigs, horses and humans with mortality rate ranging to 50%-100% (Marsh et al, J. Curr Opin Virol.2012). In contrast to molecular diagnostics where advances in technology such as PCR and high throughput sequencing have dramatically improved sensitivity, specificity and breadth over the past 20 years, serologic methods remain largely unchanged. This lag is concerning given the critical role of serology in establishing the distribution and frequency of infection, evaluating the association between an agent and disease, and in focusing efforts on pathogen discovery. Direct detection molecular methods of pathogen discovery aren’t useful once genetic material is undetectable, then indirect methods by detecting antibodies become crucial. Furthermore, the risk of disease post-exposure to an infectious agent is modulated by previous exposures to similar agents or vaccines. Such exposures may confer some degree of protection or may increase the risk for severe disease. Research for viral zoonotic diseases often focuses on infectious viruses transferring from animals to humans, however, an increasing number of recent reports indicate that humans are also transmitting pathogens to animals, Influenza A viruses from animal handlers to livestock and poultry in slaughterhouses and recently SARS-CoV-2 transmission from humans to minks, deer, and felines.

It is critical to identify antibodies present in human and animals in the events of viral zoonosis or reverse zoonosis. Therefore, a pan-viral platform is needed for serodiagnosis of infections with known and novel viral agents for studying potential threats, that can also be used to identify targets for point of care assays that can be used on farms or clinics. To address the challenge, we have built higher-density pan-viral Next Generation Serodiagnostics (NGSeroDx) platform that can detect and discriminate antibodies to group of vertebrate viruses may cause viral zoonoses, and that pose a threat to public health. NGSeroDx platform is a programmable microarray for epitope discovery that can accommodate up to 6-million distinct peptides on a 75-mm x 26-mm glass slide. Twelve amino acid (aa) peptides are printed on arrays covering whole viral proteomes/immunogenic proteins with 11 aa overlapped tiling (Mishra et al, MBio, 2018, 2019). The 12-mer format is based on the observation that antibodies efficiently bind linear peptides of 5 to 9 aa (Buss et. al, Mol. Cell Proteomics 2012). We identified specific immunoreactive epitopes that were neither predicted through modeling nor discovered using other existing serological assays. Support through this collaboration will allow us to: Aim 1) Build a specific peptide array to focus on broad range nairoviruses and henipaviruses to determine zoonotic and reverse zoonotic threat of these viruses by identifying immunoreactive linear peptides for human viruses in animal sources and vice-versa. As part of this effort samples will be collected from areas with known or documented transmission. In addition, NBAF partners will provide samples collected under other collaborative partnerships. Aim 2) Develop a “pan ag” zoonotic array to characterize the spillover of high consequence zoonotic agricultural agents and to identify potential hot spots for future investigations. Similar to Aim 1, samples will be provided by NBAF through other collaborative partnerships. Where necessary additional samples will be tested. Aim 3) Transition lead targets for development for ELISA and / or rapid point of care assays. Peptide-arrays are highly granular, expensive, and complicated tools, used for discovery of immunogenic epitope sequences. We will transfer immunoreactive discriminatory peptides to other commonly used serodiagnostic platforms in clinical microbiology laboratories including Phage display, ELISA and Luminex. Examples that illustrate the power of this strategy include a zika virus peptide ELISA for detection of IgG antibodies to zika virus in pregnant women, identification of rickettsia in children with encephalitis in Uttar Pradesh, and the implication of EVD68 in acute flaccid myelitis.