Charting the Proteins of Oropouche Virus

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Charting the Proteins of Oropouche Virus

https://doi.org/10.21203/rs.3.rs-5098470/v1

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The Oropouche virus (OROV) is responsible for the disease Oropouche fever, also known as sloth fever. This virus is currently endemic to the Americas and is transmitted primarily through midges and mosquitoes. Recently, an increase in cases has been noted among American and European tourists visiting Cuba, likely due to environmental changes that have led to a rise in midge populations. These climatic shifts have exacerbated the spread of the virus. OROV is classified as an enveloped virus with a tripartite genome, which consists of three distinct single-stranded, negative-sense RNA segments. Despite its growing significance, there are no vaccines available to protect against OROV, and human-to-human transmission has not been documented to date. However, the absence of effective diagnostics and preventive measures poses a significant public health challenge. This paper seeks to address this critical gap by providing a detailed analysis of the structure and epitopes of OROV's major proteins. By elucidating these molecular features, the study aims to lay the groundwork for the development of novel diagnostic tools and vaccines. Such advancements could play a crucial role in controlling the spread of OROV and reducing its impact on both human and animal populations.

Virology

Vaccine Development

General Microbiology

Oropouche virus

Oropouche virus disease

OROV

Sloth fever

protein mapping

antigenic proteins.

Oropouche virus disease is an arboviral illness caused by the Oropouche virus (OROV), which is a segmented, single-stranded RNA virus classified under the genus Orthobunyavirus within the Peribunyaviridae family. This virus was first identified in 1955 in the community of Oropouche in Trinidad and Tobago (Files et al. 2022). The Oropouche swamp and Oropouche River is located in the southwestern part of Trinidad. Since its discovery, the virus has been sporadically detected in various parts of South America. As of August 16, 2024, the Centers for Disease Control and Prevention (CDC) have reported 21 cases of Oropouche virus disease among U.S. travelers returning from Cuba (Morrison et al. 2024).

Oropouche fever is characterized by an acute febrile illness in humans, presenting with symptoms such as fever, headache, general malaise, muscle pain (myalgia), joint pain (arthralgia), sensitivity to light (photophobia), nausea, vomiting, and dizziness. In some cases, it can lead to more severe conditions like encephalitis and meningitis. The symptoms typically last from 2 to 7 days, though some patients may experience a recurrence. These symptoms can be particularly debilitating, leading to considerable discomfort and disruption of daily activities. In certain instances, the disease can escalate to more severe complications, including meningitis or encephalitis. These serious complications can pose significant health risks and may require more intensive medical intervention. While the majority of individuals with Oropouche fever recover fully without long-term effects, the absence of a specific antiviral treatment or a vaccine means that management of the illness largely focuses on alleviating symptoms and providing supportive care (Tilston-Lunel et al. 2015; Sakkas et al. 2018).

The Oropouche virus has a sylvatic (wild) transmission cycle, primarily involving pale-throated sloths, non-human primates, and birds as reservoirs. Transmission to humans is mainly mediated by midges of the species Culicoides paraensis, and possibly mosquitoes. Despite this sylvatic cycle, the virus has been known to infect people in urban areas far from forested environments, suggesting the presence of an urban transmission cycle as well. As of now, there is no documented evidence of human-to-human transmission of OROV (Sakkas et al. 2018).

The OROV is an enveloped virus with a tripartite genome, consisting of three single-stranded, negative-sense RNA segments. The small (S) segment encodes two overlapping open reading frames (ORFs), which produce the nucleocapsid protein (N) and the nonstructural (NS) protein NSs. NSs functions as a type I interferon inhibitor, helping the virus evade the host's immune response. The medium (M) segment encodes a polyprotein that is cleaved into structural glycoproteins Gn and Gc, as well as the NSm protein, whose role remains unclear. The Gc protein is a 939-amino-acid class II membrane fusion protein with 3–4 potential N-linked glycosylation sites, while Gn is a 290-amino-acid protein with one potential N-linked glycosylation site. Despite their importance, there is relatively little structural information available on Gc and Gn proteins in members of the Orthobunyavirus genus (Files et al. 2022).

The lack of available vaccines makes it challenging to prevent OROV infections. This paper provides a comprehensive analysis of the protein structure of OROV, detailing the spatial arrangement of its constituent proteins. In addition to mapping the overall protein architecture, the study identifies and characterizes both B-cell and T-cell epitopes within the viral proteins. These epitopes are crucial as they are recognized by the immune system and play a significant role in the development of immune responses. Understanding the precise structure and location of these epitopes allows for more targeted approaches in the creation of diagnostics and vaccines. By leveraging this detailed protein mapping, researchers can design more effective diagnostic tools to detect OROV infections and develop vaccines that elicit a robust immune response, providing enhanced protection against this virus. This structural information is therefore pivotal for advancing both the prevention and management of OROV-related diseases.

Protein sequence of Oropouche virus

The sequences of the OROV were downloaded from the NCBI (https://www.ncbi.nlm.nih.gov/protein/) protein database.

Protein modeling

A comprehensive understanding of biological systems depends on grasping how protein complexes and networks operate, which requires a detailed examination of protein interactions and their quaternary structure. Protein plots, or snake diagrams, offer a two-dimensional view of a protein sequence, highlighting features like secondary structure (Skrabanek et al., 2003). To create these diagrams, we used Protter (http://wlab.ethz.ch/protter), an interactive web-based tool. Protter allows users to integrate and visualize annotated and predicted protein sequence features, experimental proteomic data, and post-translational modifications onto the protein's transmembrane topology. Users can select from various annotation sources, upload their own proteomics data files, choose peptides for targeted quantitative proteomics, and generate high-quality visualizations (Omasits et al., 2014).

Phobius was used for the prediction of transmembrane topology and signal peptides from the amino acid sequence of proteins (https://phobius.sbc.su.se/) (Thomas, 2023).

For the three-dimensional homology modeling we employed AlphaFold ver.2 (Jumper et al. 2021) and Phyre2 (Thomas, 2023) and visualized by RasMol (Jumper et al. 2021). The protein sequences of OROV were entered in FASTA format.

The B-cell epitope and T -cell epitope (MHC-II allele, HLA-DRB1) was predicted using IEDB Analysis Resource. (http://tools.iedb.org)

Oropouche virus (OROV) is the causative agent of Oropouche fever, a disease characterized by symptoms similar to those of dengue fever and known for its sporadic outbreaks. The virus is characterized by its tripartite RNA genome. This genome consists of three distinct segments: S (small), M (medium), and L (large). Each segment encodes different structural proteins: the S segment codes for the nucleocapsid protein, the M segment encodes two external glycoproteins, and the L segment encodes the RNA polymerase enzyme (Romero-Alvarez and Escobar, 2018).

Currently, there are no vaccines available to protect against OROV. The complete protein profile of this virus has not yet been fully characterized. Understanding the proteins in greater detail is crucial for developing effective vaccines and therapeutic strategies against OROV.

The major proteins of OROV includes: Gn glycoprotein, Gc glycoprotein, NSs non-structural protein (NSs), NSm non-structural protein, nucleocapsid protein, RNA polymerase. Gn glycoprotein and Gc glycoprotein are the structural proteins of OROV and they are integral to the viral envelope and play a significant role in the virus's ability to infect host cells. The proteins of OROV plays a role in its structure and function.

Gn glycoprotein:

The medium (M) segment of RNA of OROV encodes a polyprotein that is cleaved into structural glycoproteins Gn and Gc, as well as the NSm protein. Gn glycoprotein is a structural protein of OROV and is the smallest glycoprotein. It is one of the envelope glycoproteins responsible for facilitating viral entry into host cells as well as release from host cells. Bioinformatic analysis using Protter demonstrated that the Gn glycoprotein has two transmembrane domains; the structure was confirmed using Phobius (Fig. 1A, B). AlphaFold 2 predicted the 3D structure of the Gn glycoprotein (Fig. 1C). The B-cell and T-cell epitopes are depicted in Fig. 1D. Some of the B-cell and T-cell epitopes overlap and may be considered vaccine candidates.

Gc glycoprotein:

Gc glycoprotein is the largest of the membrane glycoprotein and along with Gn, forms the envelope of the virus and is crucial for its structural integrity and interaction with host cells. Gc glycoprotein facilitates viral entry into host cells as well as release from host cells. Bioinformatic analysis using Protter demonstrated that the Gc glycoprotein has one transmembrane domain (Fig. 2A). The structure was confirmed using Phobius (Fig. 2B). AlphaFold 2 predicted the 3D structure of the Gc glycoprotein (Fig. 2C). The B-cell and T-cell epitopes are depicted in Fig. 2D. Some of the B-cell and T-cell epitopes overlap and may be considered vaccine candidates.

NSm protein:

NSm is a non-structural protein and is encoded in the medium (M) segment of RNA of OROV. The NSm protein is not essential for virus replication in the mammalian and mosquito cell lines that were evaluated. NSm is also less prone to mutation (Tilston-Lunel et al. 2015). Bioinformatic analysis using Protter demonstrated that the NSm protein has two transmembrane domains (Fig. 3A). The structure was confirmed using Phobius (Fig. 3B). AlphaFold 2 predicted the 3D structure of the NSm protein (Fig. 3C). There are no T-cell epitopes for the NSm protein. The B-cell epitopes are depicted in Fig. 3D.

NSs protein:

The small (S) segment of the virus encodes two overlapping open reading frames (ORFs), which are responsible for producing the nucleocapsid protein (N) and the nonstructural protein NSs. The nucleocapsid protein (N) plays a key role in forming the viral core, while the NSs protein has a crucial function in the virus's ability to evade the host’s immune response. Specifically, NSs acts as a type I interferon inhibitor, effectively suppressing the host's antiviral defenses. This inhibition of interferon response is vital for the virus, as it enhances viral replication and contributes significantly to the virus's pathogenicity. Thus, the NSs protein is essential not only for evading immune detection but also for the overall replication and disease process of the virus (Tilston-Lunel et al. 2015). Bioinformatic analysis using Protter demonstrated that the NSs protein has no transmembrane domains (Fig. 4A). The structure was confirmed using Phobius (Fig. 4B). AlphaFold 2 predicted the 3D structure of the NSm protein (Fig. 4C). The NSm protein does not contain any T-cell epitopes. The B-cell epitopes are depicted in Fig. 4D.

Nucleocapsid protein:

This protein encapsulates the viral RNA, forming the nucleocapsid that protects the genetic material and aids in the viral replication process. Protter demonstrated that the nucleocapsid protein has no transmembrane domains (Fig. 5A). The structure was confirmed using Phobius (Fig. 5B). The predicted 3D structure of the nucleocapsid protein as determined by AlphaFold 2 is shown in Fig. 5C. The nucleocapsid protein has both T-cell epitopes and B-cell epitopes that partially overlap and are depicted in Fig. 5D.

RNA polymerase:

This RNA polymerase enzyme is essential for transcribing the viral RNA and is involved in the replication of the virus's genetic material. The RNA polymerase is the largest protein of OROV. Protter demonstrated that the nucleocapsid protein has no transmembrane domains (Fig. 6A). The structure was confirmed using Phobius (Fig. 6B). The predicted 3D structure of the nucleocapsid protein as determined by Phyre 2 is shown in Fig. 6C. The nucleocapsid protein has both T-cell epitopes and B-cell epitopes that overlap and are depicted in Fig. 6D.

OROV, or the Oropouche virus, was first detected in 1955 in a febrile forest worker in a village in Trinidad and Tobago called Vega de Oropouche, near the Oropouche River. The virus later gained attention in 1960 when it was detected in a sloth in Brazil, which led to its colloquial name, "sloth fever." A significant outbreak occurred in Brazil in 1961, resulting in around 11,000 reported cases. Since then, more than 500,000 cases of OROV have been documented across the Americas. However, this number likely underrepresents the true extent of the virus's impact, as many cases may go unreported or undiagnosed, suggesting that the actual prevalence and transmission of OROV could be considerably higher (Henry and Murphy, 2018; Sah et al. 2024). In 2024 several deaths were reported due to OROV (Source: WHO).

In June 2024 the first cases of the Oropouche virus disease were reported in Cuba. As of August 16, 2024, the Centers for Disease Control and Prevention (CDC) have reported 21 cases of Oropouche virus disease among U.S. travelers returning from Cuba (Morrison et al. 2024). In June and July 2024, the EU reported 19 imported cases of Oropouche virus disease for the first time. The Oropouche virus disease involved travel to Cuba and Brazil (https://www.ecdc.europa.eu/en/publications-data/threat-assessment-brief-oropouche-virus-disease-cases-imported-european-union).

OROV is maintained in the wild by circulating in nonhuman primates, such as the pale-throated three-toed sloth (Bradypus tridactylus) and the black-tufted marmoset (Callithrix penicillata). In addition to midges of the species Culicoides paraensis that are the vectors of the virus, laboratory experiments and epidemiological surveys have reported that mosquitoes Aedes serratus, Aedes scapularis, Aedes albopictus, Culex fatigans, Culex quiquefaciatus, Coquilettidia venezuelensis, and Psorophora ferox are susceptible to OROV infection (Tilston-Lunel et al. 2015). Prevention relies on personal protective measures to avoid insect bites.

Preliminary diagnosis of Oropouche virus disease relies on assessing the patient's clinical symptoms, potential exposure locations (including travel history), and activities that may have increased the risk of infection. During the first week of infection, the virus can be detected in serum samples. It is typically cultivable within the initial days of illness but is rarely detected beyond day 5. However, viral RNA may be found for several days after the virus has been cleared. IgM antibodies generally appear toward the end of the first week, followed by IgG antibodies. In cases of neuroinvasive disease, viral RNA might be present but is often absent from cerebrospinal fluid (CSF). Consequently, serologic testing is preferred for detecting evidence of infection in the CSF. Additionally, viral RNA has been identified in a patient's saliva and urine by the fifth day of illness (Ref. https://www.cdc.gov/oropouche/hcp/clinical-overview/index.html). Clinical presentation may be mistaken for other arboviruses such as dengue, chikungunya, and Zika viruses, and malaria. Currently there are no vaccines to protect against OROV. Due to the potential for disease spread driven by climate change and increased international travel for business and tourism, there is a growing need for an effective vaccine to protect against OROV.

Epitope-based vaccines offer several significant advantages over conventional vaccines. One of their most notable benefits is their specificity, which minimizes off-target effects and reduces the likelihood of adverse immune responses. This specificity not only enhances the effectiveness of the vaccine but also helps to avoid potential side effects associated with broader, less targeted immune activation. Moreover, epitope-based vaccines are designed to stimulate a targeted immune response that can lead to the development of long-lasting immunity. By focusing on key components of the pathogen, these vaccines can provide enduring protection against diseases, often with fewer booster doses required compared to traditional vaccines (Ahmad et al. 2016).

Combining B-cell and T-cell vaccine regimens has been shown to significantly enhance the durability of protection against infectious diseases. This approach leverages the complementary roles of B-cells and T-cells in the immune response to provide more comprehensive and long-lasting immunity. B-cells are primarily responsible for producing antibodies that target and neutralize pathogens. On the other hand, T-cells play a crucial role in recognizing and destroying infected cells. They can directly kill cells that are infected with a pathogen or help coordinate the overall immune response. T-cell responses are vital for clearing infections that are not effectively managed by antibodies alone and for maintaining long-term immunity. By incorporating both B-cell and T-cell responses into a vaccine regimen, researchers can create a more robust and enduring immune defense. This combination not only enhances the immediate protective effects of the vaccine but also helps ensure that the immune system remains vigilant and capable of responding to future exposures to the pathogen. Studies have demonstrated that such combined vaccine strategies can lead to longer-lasting immunity and improved protection against diseases, making them a promising approach for developing more effective vaccines. This comprehensive immune response is particularly beneficial for tackling diseases that pose significant public health challenges and for situations where long-term protection is crucial (Maciel et al. 2024).

Analyzing protein structures and identifying specific epitopes is a critical step in the development of effective vaccines and diagnostic tools (Thomas et al. 2011). In our study, we performed an in-depth investigation to reveal the structure and epitopes of OROV's proteins. We identified both B-cell and T-cell epitopes of the proteins of OROV. The two known structural proteins of OROV are the membrane glycoproteins Gn and Gc. The Gn and Gc glycoproteins play a crucial role in both viral entry into host cells and the release of newly formed viral particles. Additionally, they are essential for maintaining the structural integrity of the virus. Our bioinformatics analysis revealed that these glycoproteins contain transmembrane domains, which are critical for their function. Furthermore, we identified overlapping B-cell and T-cell epitopes within these glycoproteins, suggesting that they could serve as promising candidates for vaccine development. These epitopes have the potential to stimulate both humoral and cellular immune responses, making Gn and Gc glycoproteins highly suitable targets for protective immunity against the virus.

Both the structural proteins Gn and Gc, along with non-structural proteins, hold significant potential for use in diagnostic applications. The structural proteins Gn and Gc, which are vital for viral entry and assembly, offer specific antigenic sites that can be targeted for accurate detection of the virus in infected individuals. These proteins can serve as biomarkers in serological assays, aiding in the identification of immune responses and the detection of active infections.

In addition to the structural proteins, non-structural proteins, which are involved in viral replication and modulation of host-cell processes, also present valuable diagnostic targets. These proteins can help in distinguishing between acute and past infections, as they are typically expressed at different stages of the viral life cycle. By incorporating both structural and non-structural proteins into diagnostic tools, more comprehensive and sensitive assays can be developed, improving the ability to detect early infections, monitor disease progression, and assess immune responses in vaccinated or previously exposed individuals.

In this study, we focused on elucidating the structural and non-structural proteins of OROV, providing a detailed analysis of their molecular architecture. By mapping these proteins, we gained crucial insights into their functions and interactions, which are essential for understanding the virus's biology and pathogenic mechanisms. Additionally, we identified both B-cell and T-cell epitopes within these proteins. B-cell epitopes are specific regions that are recognized by antibodies, while T-cell epitopes are targeted by T cells during the immune response. Characterizing these epitopes is vital for advancing the development of diagnostic tools and vaccines. Our findings will aid in designing more precise and effective diagnostics by targeting the identified epitopes to improve the detection of OROV infections. Furthermore, these epitopes can be used to engineer vaccines that stimulate a robust and targeted immune response, enhancing protection against the virus. This comprehensive approach not only contributes to a deeper understanding of OROV but also paves the way for better preventive and diagnostic strategies.

Data availability statement

The programs used to generate the data is stated in the Materials and Methods. The complete data generated in the study is shown in the results.

Conflict of Interest

The author declares no conflict of interest.

Acknowledgements

The author acknowledges Abraham Thomas Foundation for providing the resources for this work.

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World Health Organization https://www.who.int/emergencies/disease-outbreak-news/item/2024-DON521#:~:text=Oropouche%20virus%20disease%20is%20an,to%2Dhuman%20Oropouche%20virus%20transmission

The authors declare no competing interests.

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Charting the Proteins of Oropouche Virus

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Abstract

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INTRODUCTION

MATERIALS AND METHODS

Protein sequence of Oropouche virus

Protein modeling

RESULTS

Gn glycoprotein:

Gc glycoprotein:

NSm protein:

NSs protein:

Nucleocapsid protein:

RNA polymerase:

DISCUSSION

Declarations

Data availability statement

Conflict of Interest

Acknowledgements

References

Additional Declarations

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