Novel Strategies for Tracing Animal and Human Sources of Envelope Virus Outbreaks

Introduction

Understanding the circumstances that lead to virus outbreaks is critical for deterring future zoonotic pandemics, discovering new drugs, developing vaccines (Li et at., 2020; Worobey ety al., 2022, Pekar et al., 2022). However, this can prove to be a very difficult task, if not almost impossible. To provide an example, the origins of SARS-CoV-2 are still hotly debated (Zhou et al., 2020). Once established that bats are the natural reservoirs of SARS-related coronaviruses (Yu et al., 2019), a feverish search has sought to identify the likely source of cases in early reports. It has been suggested that the earliest known COVID-19 cases, geographically centered on the Huanan Seafood Wholesale Market in Wuhan, occurred through the live wildlife trade in China (Worobey ety al., 2022), being the result of at least two separate cross-species transmission events into humans (Pekar et al., 2022). Zoonotic spillovers have been hypothesized from various SARS-CoV-2-susceptible mammals identified as potential intermediate hosts, including civets, bamboo rats, raccoon dogs, pangolins, etc (Jaimes et al., 2020; Li et at., 2020; Crits-Christoph et al., 2024).

From a methodological standpoint, genotypes of potential animal hosts are recovered, analyzed and compared with those from humans and environmental samples (Goodrum et al, 2023; Crits-Christoph et al., 2024). The comparison is usually made by using metagenomic and phylogenetic approaches combined with structural modeling, phylodynamic rooting methods and epidemic simulations (Jaimes et al., 2020; Pekar et al., 2022). These approaches establish the genetic foundation for identifying a shortlist of potential intermediate hosts to prioritize for serological and viral sampling (Crits-Christoph et al., 2024). They also help pinpoint the critical time frame between the initial zoonotic spillover into humans and the emergence of the first reported human cases (Pekar et al., 2022).

Here we suggest a novel method to detect and analyze the origin, spillover, intermediate hosts and human reservoirs in case of enveloped viruses. Instead of relying solely on traditional genetic studies, we propose examining the viral envelopes, which contain phospholipids and proteins derived from host cell membranes. Envelope viruses encompass both DNA and RNA viruses such as Orthomyxoviridae (e.g., influenza virus), Poxviridae (e.g., smallpox virus), and Paramyxoviridae (e.g., human parainfluenza viruses). We will focus on viral families that hijack host cellular compartments such as the endoplasmic reticulum (ER), the Golgi apparatus (GA) or the ER-Golgi intermediate compartment (ERGIC) to ensure their replication cycle, viral assembly, envelope formation, budding and release by exocytosis (Risco et al., 2020; Chen et al., 2020). These families include, among others, Flaviviridae such as Dengue virus and Zika virus that acquire their envelopes during budding through ER (Li et al., 2015; Das et al., 2023) and Coronaviridae such as SARS-CoV-2 that assemble in ERGIC (Brian and Baric, 2005; Boson et al., 2021; Scherer et al., 2022). In many of these viruses, virions are transported to the cell surface following intracellular assembly and are subsequently released through exocytosis.

A virus infecting a human can originate from two sources: either an animal host through spillover, or another infected human. Given the detectable inter-species and inter-individual variations in ER, GA and ERGIC, we contend that the study of viral envelopes could offer valuable clues about the animal source of human spillover and the identity of patient zero, i.e., the first human infected during an outbreak. Specifically, we will focus on the lipid components of the ER and GA that are incorporated into the viral envelope, which can vary depending on the virus’s most recent host. We argue that researchers should identify specific host-derived lipidic biomarkers within viral envelopes to provide critical insights into determining the virus's most recent host, whether animal or human.

Lipidomic Differences in Golgi Apparatus and Endoplasmic Reticulum in Animals and Humans

Lipidomics is a rapidly expanding field focused on uncovering the unique lipid profiles of cellular organelles (Symons et al., 2021). Mass spectrometry-based lipidomics can rapidly identify as well as quantify >1,000 lipid species at the same time, facilitating robust analyses of lipids in tissues, cells and body fluids (Song et al., 2022). Accordingly, lipidomics is now being widely applied in various fields, including nutrition science to assess food obtained from livestock and poultry as well as fish food products (Song et al., 2022; Harlina et al., 2023). Detailed studies have mapped the lipid compositions of various mammalian organelles, including ER and GA (Sarmento et al., 2023). A systematic analysis of the overall variation in the mammalian lipidome, with a particular focus on Mus musculus, has been conducted to evaluate the effects of diet, sex, and genotype (Surma det al., 2021). The cellular lipidome is highly adaptable, shifting in response to numerous physiological processes such as aging and a range of pathological states (Onal et al., 2017). Alterations in the organelles’ lipid profiles may lead to disrupted lipid metabolism, persistent inflammation and oxidative stress that have been documented across cancer, metabolic diseases and neurodegenerative disorders.

Differences in the lipid composition of the ER and GA have been observed not only across different mammalian species but also among individual humans. In the following two paragraphs, we will explore these variations in more detail.

Lipidomic differences in various animals. Extended lipid profiles of several animal species have been compared via lipidomic analysis conducted by liquid chromatography-high-resolution mass spectrometry, allowing the identification of about a hundred of molecular species of lipids (Kaabia et al., 2018). ER focuses on synthesizing lipids tailored to environmental and metabolic challenges, while GA specializes in modifying and transporting these lipids for specific cellular and systemic functions. Both exhibit species-specific lipid compositions that fulfill critical biological purposes, reflecting adaptations across mammals to genetic factors, environmental conditions, dietary habits, metabolic needs (Di Conza et al., 2021). For instance, humans and rodents share high proportions of phosphatidylcholine and phosphatidylethanolamine essential for membrane structure and cellular signaling (Adamson et al., 2024). Carnivorous mammals like raccoon dogs have lipidomic compositions tailored to protein-heavy diets, whereas omnivorous and insectivorous species like bats show distinct profiles suited to their high metabolic demands (Xenoulis et al, 2020; Takatsuki and Inaba, 2024). Marine mammals like seals and whales have ER membranes rich in phosphatidylserine and long-chain PUFAs to maintain fluidity under the extreme conditions of cold, high-pressure aquatic environments (Fayolle et al., 2000). In turn, desert mammals like camels display higher concentrations of unsaturated phospholipids to cope with dehydration and heat stress. In primates, cholesterol biosynthesis is optimized for neural and immune functions (Zio et al., 2024). Herbivorous species like cows and sheep can efficiently convert cholesterol into bile acids for digesting plant-based diets. By contrast, carnivorous mammals like cats and dogs exhibit less diverse bile acid profiles and simpler cholesterol synthesis pathways, consistent with their protein-rich diets (Xenoulis et al, 2020).

Marine mammals’ ER produce elevated levels of ceramides and sphingomyelins with long-chain bases conferring resistance to salt and temperature fluctuations. Primates and rodents synthesize an array of gangliosides and glycosphingolipids for neural signaling and immune interactions (Allende and Proia, 2014; Agliarulo and Parashuraman, 2022; Kobayashi 2023). Primates maintain a balance between saturated and unsaturated fatty acids, while rodents favor linoleic acid derivatives (Burr et al., 2023). Marine mammals emphasize omega-3 fatty acids such as DHA and eicosapentaenoic acid to adapt to cold environments (Yudin et al., 2017). Lipid droplet formation in the ER reflects species-specific metabolic needs. Hibernating mammals like bears accumulate triglycerides for energy storage during pre-hibernation, while marine mammals produce blubber lipids characterized by high triglyceride and wax ester content (Nelson 1980).

The lipid composition of the GA is equally diverse and tailored to species-specific functions. The lipid remodeling processes in the GA support specific secretory functions, such as the packaging of milk fat globules in lactating mammals or the production of blubber in marine mammals (Dai et al., 2022). Cholesterol transport and modification in the GA are more prominent in herbivores to facilitate bile acid production, while carnivores exhibit simpler pathways (Hocquette and Bauchart, 1999). Phosphoinositides, particularly phosphatidylinositol and its derivatives, play a vital role in GA vesicle trafficking, with species-specific variations reflecting distinct membrane transport requirements (D’Angelo et al., 2012). Also, environmental challenges drive adaptations in GA lipid metabolism to thrive under diverse environmental pressures. Cold-adapted mammals like Arctic foxes produce a higher proportion of unsaturated lipids to maintain vesicle and membrane fluidity in low temperatures (Nowicki et al., 2014). Conversely, desert mammals such as camels synthesize lipids (also in their milk) that resist dehydration and oxidative stress (Gorban and Izzeldin, 2001).

In sum, lipidomic differences in the ER and GA among mammals demonstrate the remarkable versatility of these organelles. By tailoring their lipid composition and metabolic pathways, the ER and GA meet the unique dietary, ecological, and physiological demands of each species. These variations underscore the evolutionary adaptations that enable mammals to maintain cellular functions crucial for survival in diverse habitats and under varying biological demands.

Lipidomic differences in various human individuals. Lipidomic variations in ER and GA among human individuals arise from interplay of genetic factors, diet, lifestyle, environmental exposures and health conditions. Phosphatidylcholine and phosphatidylethanolamine levels, for instance, are influenced by polymorphisms in the PEMT gene (Sun et al., 2023). Diets rich in omega-3 fatty acids contribute to higher levels of docosahexaenoic acid in ER’s phospholipids, particularly in individuals consuming fish-heavy diets (Yamagata 2021). Variations in the PTDSS1 gene affect phosphatidylserine levels, which are associated with cognitive and neural functions (Long et al., 2024). Ceramide levels in the ER are elevated in insulin resistance and type 2 diabetes, with genetic variations in DEGS1 playing a key role in modulating ceramide biosynthesis (Blackburn et al., 2019). Diet, particularly high saturated fat intake, contributes to increased sphingomyelin levels. Genetic polymorphisms in the FADS1 and FADS2 genes impact the synthesis of long-chain polyunsaturated fatty acids such as arachidonic acid and DHA, leading to manifold fatty acid profiles (Koletzko et al., 2019). Individuals with obesity or metabolic syndrome often have a higher saturation of ER membrane lipids, which increases susceptibility to ER stress. Cholesterol biosynthesis in the ER varies depending on genetic polymorphisms, such as those in the HMGCR gene (Perrone et al., 2023). Individuals with familial hypercholesterolemia exhibit altered cholesterol synthesis and handling. Lipid droplets in individuals with high dietary fat intake or metabolic disorders are enriched with triglycerides, an adaptation to chronic ER stress.

GA exhibits pronounced individual variability in lipid composition (Agliarulo and Parashuraman, 2022). Glycosphingolipid profiles vary due to genetic differences, including polymorphisms in the B4GALNT1 gene (Sipione et al., 2020). These variations impact the synthesis of gangliosides and globosides, which play critical roles in the function of neural and immune cells. Elevated levels of lactosylceramides in individuals with lipid storage diseases or metabolic disorders suggest impaired GA lipid processing. Variability in phosphoinositide metabolism impacts GA vesicle formation and trafficking, while dietary fat intake affects phosphatidylinositol levels and associated signaling pathways.

Differences in ABCG1 activity influence plasma lipid profiles, with altered GA lipid composition observed in individuals with high cholesterol levels (Matsuo 2022). The remodeling of GA lipids for secretion is particularly evident during specialized physiological states such as lactation. Sphingolipid and ceramide trafficking in GA show variability based on CERT gene polymorphisms, leading to differences in sphingomyelin and glycosphingolipid levels (Zhang et al., 2019). Elevated ceramide levels in inflammatory conditions suggest altered sphingolipid metabolism contributing to individual differences in disease susceptibility. The GA stress response varies across individuals due to genetic differences in GOLPH3, which impact the organelle’s ability to manage lipid overload or trafficking defects (Kuna and Field, 2019). GA dysfunctions are particularly evident in individuals with cancer or neurodegenerative diseases. Diet also influence the GA lipid composition and its ability to respond to stress, contributing to variability in immune responses, neural signaling and disease susceptibility.

In summary, lipidomic differences in the ER and GA among human individuals reflect a dynamic interplay of genetic, dietary and environmental factors, as well as health status. These variations influence lipid synthesis, processing and trafficking, impacting a wide range of physiological and pathological processes.

Conclusions

We suggest a novel research methodology to assess viral spread and infection sources. Our approach takes advantage of the fact that viruses acquire their envelopes from host organelles like ER, GA and ERGIC. To provide an example, recent lipidomic analyses have elucidated the molecular composition of the SARS-CoV-2 lipid envelope. The virus envelope exhibits exposed phosphatidylethanolamine and phosphatidylserine and is predominantly composed of phospholipids, with minimal cholesterol and sphingolipids (Saud et al., 2022). This implies that, despite significant differences between the viral envelope and host cell membranes, the lipid constituents are the same.

This study focuses on lipidomic differences in viral envelopes stemming from their ER and/or GA origins. However, incorporating analyses of other biomolecules could offer a broader understanding. To further investigate unique inter-species and inter-individual biomolecular signatures in ER and GA compositions, proteomics and glycomics analyses could be valuable tools to uncover specific differences in protein and carbohydrate structures. Additionally, transcriptomics and genomics could offer insights into gene expression patterns and structural variations that influence the functions of the ER and GA. Since these differences could significantly impact the composition of viral envelopes derived from host membranes, identifying molecular disparities might provide crucial clues about the origins of infection and the interplay between viral mechanisms and host cellular machinery.

Biochemical differences among mammals reflect adaptations to diet, metabolism, immune response, stress and environmental demands. Mammals with greater protein synthesis demands like dairy cows and whales upregulate ER chaperones to handle the increased folding load (Pobre et al., 2019). Carnivorous mammals like cats and dogs exhibit GA adaptations favoring high-protein diets, while hibernating mammals like bears display reduced GA activity to preserve energy. Primates exhibit different chaperone proteins to support the complex glycoproteins associated with their nervous and immune systems (Nakamura et al., 2001; Wisniewska et al., 2010). Humans possess a unique array of P450 enzymes in the ER, enabling them to adapt to diverse diets and efficiently metabolize drugs (Gorina et al., 2022). Rodents exhibit simpler glycosylation pathways, while primates synthesize more complex glycans for neural and immune functions (Cherepanova et al., 2016; Li et al., 2019). These variations influence also interactions with zoonotic viruses, as seen in bats and humans (Voigt et al., 2019). Bats, with their high metabolic rates, exhibit a robust unfolded protein response (UPR) system to withstand oxidative stress (Huang et al., 2020). In contrast, humans and primates rely on sensitive UPR pathways, involving proteins like IRE1, ATF6, and PERK, to mitigate neurodegenerative stress (Chadwick and Lajoie, 2019; Wiseman et al., 2022). Meanwhile, marine mammals like whales and seals adapt their ER protein-folding machinery for hypoxia tolerance during deep dives. Further, calcium-binding proteins exhibit species-specific differences aligned with metabolic needs (Michalak 2024). While cheetahs’ muscle-specific ER enables rapid calcium cycling for high-speed muscle contractions, diving mammals like dolphins optimize for slower calcium release to conserve energy.

The biochemical composition and function of ER and GA also vary between human individuals due to genetic, environmental and health-related factors. Polymorphisms in genes encoding chaperones and enzymes like glycosyltransferases within ER and GA affect their ability in protein folding and glycosylation patterns, contributing to individual susceptibility to diseases like congenital glycosylation disorders. Diet significantly affects the lipid composition of ER and GA, as fatty acids and carbohydrates required for membrane formation and glycosylation are derived from food. Exposure to toxins, specific drugs or alcohol can further modify their function (Shen et al., 2023). Chronic illnesses like diabetes or inflammatory disorders alter glycosylation and protein processing in GA, while neurodegenerative diseases such as Alzheimer’s and Parkinson’s impair protein folding in ER and glycosylation in GA (Gandhi et al., 2019).

The biochemical repertoire of ER and GA also changes with age, reflecting the varying physiological demands tied to growth, metabolic activity and cellular maintenance. In children, ER and GA are highly active to support rapid growth, facilitating the synthesis of proteins essential for tissue formation and contributing to support energy storage and membrane development (Grevendonk et al., 2021). In adults, ER and GA shift their focus toward maintaining cellular homeostasis and supporting specialized functions like detoxification, drug processing, enhancing immune defense, enabling antibody function. In the elderly, ER shows a diminished ability to ensure the quality of proteins, leading to an accumulation of misfolded proteins (Hartl 2017). Altered glycan structures impair the activity and stability of glycoproteins, including antibodies, which compromise immune function. GA dispersion and glycosylation changes in senescent cells further contribute to aging-related dysfunctions and neurodegenerative diseases (Udono et al., 2015). Additionally, age-related reductions in proteins processing in GA have been linked to storage deficits, particularly in diseases like diabetes (Calvo-Rodríguez et al., 2016; Janikiewicz et al., 2018).

In sum, by integrating advanced omics approaches that involve not just lipids, but also other biological molecules, researchers can build a comprehensive framework for understanding how host cellular composition affects viral envelope characteristics.

We focused here on the potential to detect the viral source, but our approach might also uncover other significant findings. For instance, during the SARS-CoV-2 pandemic, older individuals experienced more severe COVID-19 symptoms compared to children (Parri et al., 2020). Several mechanisms have been suggested to explain the milder clinical syndrome observed in children, including higher pediatric innate interferon responses, increases in naive lymphocytes and depletion of natural killer cells (Yoshida et al., 2022). Since human ERGIC membranes undergo age-related changes, SARS-CoV-2 particles produced in the tissues of children could exhibit phenotypic differences compared to those produced in older individuals. This means that the variations in the ERGIC features of SARS-CoV-2 emerging from human cells of individuals of different ages might contribute to differences in viral load, infectivity and clinical severity. Lastly, we propose another theoretical possibility that merits further exploration. Gaining a deeper understanding of these biochemical differences in ER and GA composition could significantly enhance personalized medicine, paving the way for targeted therapies tailored to individual biochemical profiles.