Research Project: Viral Ecology of Henipaviruses in Endemic Settings and Intervention Strategies to Prevent their Spread to Domestic Animals

Location: Zoonotic and Emerging Disease Research

2024 Annual Report

Objectives
Objective 1. Characterize the ecology of henipahviruses with a focus on the One-Health concept. - Conduct the molecular characterization of new and emerging henipahviruses, including phylogenetics, and network analysis. - Examine known or emerging henipahviruses that may have an impact on animal agriculture, including their potential impact on public health. Objective 2. Elucidate the host-pathogen interactions of henipahviruses infections. - Investigate virus-specific factors and viral molecular markers associated with infectivity, pathogenicity and transmissibility of henipahviruses in susceptible animal species including virus tissue tropism and replication. - Investigate host-specific factors associated with the infectivity, pathogenicity and transmissibility in different animal species. - Characterize the innate and adaptive immune response to henipahviruses infections in animal models that are either susceptible, tolerant, or resistant to infection. Objective 3. Develop surveillance strategies and early warning systems for henipahviruses. - Improve surveillance strategies to detect henipaviruses in high-risk countries. - Establish a formal laboratory network for henipavirus surveillance that includes standardized specimen collection, laboratory testing scheme, quality control, specimen referral and accreditation.

Approach
Henipahviruses are members of the family Paramyxoviridae, order Mononegavirales. The name henipavirus was recommended for the genus of both Hendra virus and Nipah virus. Henipaviruses have a large host range, unlike other members of the Paramyxoviridae, which generally have a very narrow host range. The cell attachment protein, unlike many other members for the paramyxovirus subfamily, does not have haemagglutinating activity and as a consequence does not bind sialic acid on the surface of cells. The natural reservoir of the henipaviruses are fruit bats mainly from the genus Pteropus (flying foxes). Nucleic acid and antibody signatures of exposure to Nipah virus or Nipah-like viruses has been documented in a diversity of bat species across the globe. The threat for a natural introduction of henipaviruses in the United States is low, but there is significant concern that henipaviruses could be used for nefarious purposes to harm agriculture and people. Both Hendra virus and Nipah virus are on the HHS and USDA list of overlap Select Agents and Toxins. Henipaviruses are listed as APHIS Tier 3 high-consequence foreign animal diseases and pests. Henipaviruses are promiscuous in their ability to cause severe morbidity in several animal species, including people, and human infections result in a very high mortality rate. The mortality rate associated with Nipah virus infections in pigs has been reported to be approximately 2.5% in adult pigs – high morbidity, but low mortality. Mortality rates in humans however are significantly higher and range from 40% (Malaysia) to 75% (up to 100%) in Bangladesh. The animal reservoir includes several species of bats, and henipaviruses may thus be readily available in these wildlife reservoirs.

Progress Report
Substantial progress was made in addressing the objectives of ARS Project 3022-32000-027-000D. To address Objective 1 partnerships were established in regions where bats that have been demonstrated to carry the henipahviruses are found. These partnerships were established with collaborators in both Indonesia and Bangladesh. A pre-kick off meeting and survey of the initial wet markets was conducted in Indonesia. Additionally, all permissions have been obtained from the government of Indonesia. A survey of wet markets in Bali recorded a long-tailed macaque, three common palm civets, one small Indian civet, seven Javan short-nosed fruit bats, and a dozen or so plantain squirrels (and a few wild-caught birds) all housed within the market. Approximately 200 primate skulls (macaques, langurs, gibbons, proboscis monkeys) as well as some other mammal skulls were observed indicating robust trade from other parts of Indonesia (Java, Borneo, Sulawesi) to Bali. In Bangladesh, in partnership with Johns Hopkins University, research protocols for capturing and sampling of wild animal species have been developed. The current targeted species within two study sites in Faridpur and the Chittagong Hill Tracts include rats (Rattus spp. and Bandicota spp.), mice (Mus musculus), shrews (Suncus murinus), jungle cats (Felis chaus), jackals (Canis aureus), foxes (Vulpes bengalensis), palm civets (Paradoxurus hermaphroditis), mongoose (Urva spp.), and bats (Rhinolophus spp.). In addition, the research protocols will include sampling of mosquitoes in habitats where primates (e.g., gibbons, langurs, macaques) are found and potentially have taken blood meals from these primates, as well as opportunistic sampling of dead animals within both field sites and at wet markets in the Chittagong Hill Tracts. These protocols were reviewed by two external experts in April 2024 and then submitted to the Research Review Committee (RRC) and approved in June 2024. The protocols are under review by the Ethical Review Committee (ERC) and the Animal Experimentation Ethics Committee (AEEC). Once the protocols have been approved by RRC, ERC, and AEEC our partners will submit the protocols for expedited review by the Johns Hopkins University Institutional Review Board (IRB). We anticipate that this process will be finished by early Fall 2024. We have also been pursuing the necessary approvals from the Bangladesh Forest Department and Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). The team has been constructing traps and will pilot these traps in the Faridpur district in July 2024. With partners in Bangladesh and Columbia University, we have been exploring zoonotic transmission of viruses through contaminated date palm sap consumption causing respiratory outbreak and encephalitis in Bangladesh. Viral discovery was performed on cases with undiagnosed infections in regions of Bangladesh where Nipah virus (NiV) outbreaks are prevalent. Samples were collected under the Nipah surveillance program from patients exhibiting symptoms of NiV infection, including fever, severe respiratory, and neurological issues. Additional testing of the samples that were negative for Nipah virus identified a number of viruses including Adenoviruses, Coronaviruses, Rhinoviruses, Enteroviruses, Metapneumoviruses, Herpesviruses, and Parvoviruses. Four samples were also found to have evidence of Pteropine orthoreoviruses (PRVs). PRVs are emerging bat-borne virus and have been previously linked to sporadic acute respiratory infections in humans in Southeast Asia. Analysis of virus isolated from these samples revealed a very close similarity to a virus isolated from Pteropus policephalus bats in 1968 in Australia and to Nelson Bay virus. A partnership was established with the biosafety level four (BSL4) laboratory in Geelong, Australia. Scientists travelled to Australia to meet with partners. As part of these efforts, bat collections will be performed in Australia and Indonesia. Protocols are under development for evaluation of novel henipahviruses and for evaluation of novel diagnostics in the BSL4. These partnerships will also facilitate addressing Objective 3. Sensitive, specific and easy-to-use diagnostics are essential for surveillance activities. Multiple assays for detection of henipahviruses and for evidence of previous exposure to henipahvirus are under development. Priority has been on assays that are easy to use and require little to no infrastructure or that can readily distinguish between exposure to different henipahviruses. To mitigate risk, multiple approaches are being utilized. To begin to address Objective 2, strategies to perform single-cell sequencing in maximum containment have been developed. Single-cell sequencing will allow scientists to map changes in the host responses and changes in the virus. Single-cell sequencing will allow scientists to work around the challenges of limited reagents for different species. This approach will be implemented with partners to characterize a nonhuman primate model's host and viral changes. This model was selected as primates have been demonstrated to be highly susceptible to the virus, and partner labs are limited to the size of the animals that can be worked with safely in biosafety level 4 (BSL4) in other laboratories. The serial sampling study in African Greens has been performed, samples have been collected and are currently being processed. As part of this work, samples will be collected to allow detailed mapping of immune responses. This information will inform vaccine development and identification of critical epitopes. To explore the risk of novel paramyxovirus, partners at the University of Texas Medical Branch have received an isolate of this virus from the Centers for Disease Control (CDC) Special Pathogens branch and are preparing a challenge seed to begin characterizing in vivo. In addition, 30 bat samples PCR (polymerase chain reaction positive for Angavokely virus genomes were obtained. Partners are attempting to isolate virus from these samples. If successful, a pilot challenge study will be performed to assess disease potential of this novel henipahvirus. To better understand Nipah virus pathogenesis, a partnership was developed to use artificial intelligence to facilitate pathologic analysis. The algorithms developed will potentially identify commonalities and differences in susceptible models. Lastly, in anticipation of work with swine at the facility, experimental work with swine at biosafety level 3 (BSL3) has been initiated with partners. Monkeypox (MPOX) was selected as the virus to be evaluated as it is a current zoonotic pathogen of concern. Investigation of MPOX in villages of the Democratic Republic of Congo identified pigs with pox-like lesions. There is little known about the susceptibility of agricultural animals. As the number of cases of MPOX are continuing to expand globally there is growing concern that the virus may find new ecological niches. Initial studies were performed with pigs at BSL3 Agriculture and MPOX. These studies demonstrated that pigs are susceptible to the virus and allowed staff to gain experience in containment and challenges they may encounter in the BSL4. The optimized protocols and standard operating procedures are forming the basis of NBAF specific protocols. In partnership with scientists in Australia and USDA Animal and Plant Health Inspection Service (APHIS), a protocol for a swine BSL4 study is under development. The Marburg virus was selected for this study based on interest and priority of partners and the availability of potential countermeasures should an accident occur.

Accomplishments
1. Establishment of agreements in countries with previously reported cases of Nipah virus. ARS researchers in Manhattan, Kansas, established agreements with partners in Australia, Bangladesh and Indonesia. These projects will allow the collection of bats and species in close contact to investigate the presence of Nipah or closely related viruses. These partnerships will focus on characterization of genetic diversity of the viruses as well as examining the risk of spillover to animals and those that work closely with animals.

2. Countries at risk for Nipah virus. ARS researchers in Manhattan, Kansas, established formal agreements in countries where they have not been previously reported cases of Nipah virus but may be at risk due to the presence of bat species known to harbor the virus. West and Central Africa are areas where bats known to carry henipahviruses are found.

3. Development of single cell sequencing techniques in biosafety level 4. ARS researchers in Manhattan, Kansas, developed and optimized strategies with Lassa Fever virus, a zoonotic virus requiring biosafety level 4, to allow single cell sequencing to be safely performed without data loss. This technique will allow scientists to understand how cells infected with the virus respond. The technique also allows investigators to map responses to viral infection and provides a solution to limited immunologic reagents for many agriculture species. The lack of immunologic reagents has been a significant barrier to characterizing disease pathogenesis and developing countermeasure. The techniques were transferred to partner laboratories and are being implemented in animal studies.

4. Initiation of animal studies with partners. ARS researchers in Manhattan, Kansas, in collaboration with researchers at the University of Texas Medical Branch, initiated the first studies to characterize the host response, including the immune response, to Nipah virus infections in a susceptible animal species.

5. Development of improved assays for the detection of henipahviruses and evidence of prior exposure. ARS researchers in Manhattan, Kansas, working with scientists at Broad University, developed a rapid, sensitive assay using combinatorial arrayed reactions for multiplexed evaluation of nucleic acids (CRISPR)-based technology and the combinatorial arrayed reactions for multiplexed evaluation of nucleic acids (CARMEN) platform. The assay will be further tested this fall in partner laboratories and has been incorporated into syndromic panel. A Loop-mediated isothermal amplification (LAMP) assay has also been developed and is currently being evaluated using synthetic materials. Three different assays for detecting previous exposure to Nipah and Hendra virus are under development. These assays will be significantly less expensive, require less infrastructure and can be used in almost any environment. The reduced cost and infrastructure requirements will dramatically impact the ability to perform surveillance activities for these agents.