Perspective Chapter: Viral Zoonoses – Pathways and Mechanisms

1. Introduction

Zoonotic viruses have caused fatal diseases in humans over the years (Table 1). Further, zoonotic viruses also continue to emerge and re-emerge, causing epidemics, pandemics and many of them establishing themselves as endemic in different regions across the globe (Figure 1) [2, 3]. Only a few of the zoonotic viruses have been controlled through measures such as vaccination and surveillance and others sustain to cause periodic outbreaks, leading to significant socioeconomic impact (Figure 1, Table 1). The phenomenon of viral zoonosis is, therefore, a serious public health concern. In addition, it affects the trade of animal-based products and some of the zoonotic viruses are also listed among the Biodefense Pathogens by the National Institute of Allergy and Infectious Diseases (NIAID) [4]. Despite challenges, particularly posed by the vast diversity of viral and animal species, studies have addressed various aspects of viral zoonosis. Research has shown that the animal viruses cross species boundaries through spillover events driven by various factors [5, 6]. Modes of transmission and transmission capability of many zoonotic viruses have also been established. Upon a host jump from an animal, humans are the dead-end hosts for some of the zoonotic viruses, such as the rabies virus, because they either have no or have limited capability of human-to-human transmission or transmission to any other host. Whereas, some zoonotic viruses, such as the SARS coronaviruses, zika virus, dengue virus and some others, have undergone genomic evolution and have emerged as new human viruses, capable of either human-to-human transmission (horizontal or vertical) or transmission through vectors [7]. The following sections describe our current understanding of reservoirs of the zoonotic viruses, various spillover pathways, molecular mechanisms involved in the evolution of viral zoonoses and a discussion on future perspectives for control of viral zoonoses.

2. Reservoirs of zoonotic viruses

A Pathogen reservoir is defined as the host where the infectious agent normally lives, multiplies, endemically circulates, co-evolves, is maintained permanently and gets transmitted to the target population [8, 9]. A pathogen could have multiple reservoirs and the disease may or may not be manifestated in the reservoir host. In addition to the reservoirs, intermediate/amplifying host, spillover host, susceptible host and non-susceptible host have also been described for some of the viruses, such as the SARS coronaviruses [9]. Appropriate methods to establish the existence of a reservoir, clear identification of the reservoir(s), determination of host range and managing them can help prevent fatal epidemics [10, 11]. Control of many diseases such as Ebola virus infection and rabies has been hampered due to an incomplete understanding of their reservoirs [12, 13, 14].

Majority of the zoonotic viruses belong to the families, Flaviviridae, Arenaviridae, Rhabdoviridae, Bunyaviridae, Orthomyxoviridae, Paramyxoviridae, Filoviridae and Togaviridae (Table 1) [15]. Among their identified animal reservoirs, mammalian (Rodentia, Primates, Chiroptera, Cetartiodactyla, Perissodactyla, Carnivora, Diprotodontia, Artiodactyla) and avian species (Passeriformes, Anseriformes, Galliformes) represent the majority [15, 16, 17]. Such studies have suggested that the risk of cross-species transmission for the emergence of viral zoonosis arises not only from the animal species that are evolutionarily closer to humans but also from distant groups like rodents, bats, ungulates and birds (Figure 2) [16, 18, 19, 20, 21]. Two popular hypotheses that have emerged, providing explanations for the noted patterns, are the “special reservoirs hypothesis” and “reservoir richness hypothesis”. According to the “Special reservoir hypothesis,” a high ecological overlap, due to the domestication of certain evolutionarily distant animal groups such as rodents and ungulates, provides opportunities for pathogen transmission to humans. Whereas, according to the “species richness hypothesis,” existence of a large number of certain animal species such as rodents and bats relative to other mammalian groups, is responsible for their high representation among the virus reservoirs [22].

In addition to identifying the animal taxa that are more likely to maintain and/or transmit zoonotic viruses and recognizing any patterns in the relationship between viruses and their reservoirs, it is also of interest to understand if the zoonotic ability is determined by traits of the virus alone or the reservoir also has an active role. Studies have emerged on viral immunity in bats, which are reservoirs for many significant viruses. Bats are noted to have an immune system that tolerates many zoonotic viruses including the SARS-CoV 1 and 2, Ebola virus and Hendra virus [23]. Role of bat immune response on virus replication, clearance and persistence is not fully understood. However, an interesting hypothesis that has been proposed is that, distinct selective pressures in bats might have triggered the evolution of viral accessory proteins. It is conceived based on observations such as the observed truncations in the accessory proteins of zoonotic viruses hosted by bats [24]. Most of the viral accessory proteins are poorly characterized but have an important role in pathogenesis. Hence, the investigation of viral immunity in the animal reservoirs and it is impact on zoonoses could improve our understanding of the principles of viral zoonosis.

Involvement of vectors and maintenance of zoonoses by sylvatic cycle is another aspect of interest. Arthropods, including ticks, flies and mosquitoes, are important transmission vectors. Arboviruses such as Chikungunya virus, Dengue virus, zika virus and Yellow fever virus have originated in non-human primates. In the natural forest habitats of non-human primates, these viruses are transmitted by mosquitos from infected to naïve animals in what is known as a sylvatic transmission cycle [25]. Sylvatic cycles allow the maintenance of arboviruses and possibly also provide opportunities for development of new viral strains. The sylvatic transmission cycle can “spill over” to urban transmission cycle when humans infected in forests spread the virus among people via urban mosquitoes [26, 27, 28, 29]. Chikungunya virus, dengue virus and zika virus are fully adapted to urban cycles and do not really require sylvatic cycle for their maintenance [30]. Yellow fever virus is maintained by sylvatic (jungle), intermediate (savannah) and urban cycles (Figure 3). An amplification of Yellow fever virus in non-human primates, prior to short-lived outbreaks in urban population has been documented [31]. The Aedes or Haemagogus species of mosquitoes can transmit the Yellow fever virus to humans or non-human primates by feeding on infected primates (human or non-human). The sylvatic cycle involves virus transmission between non-human primates and species of mosquitoes found in the forest canopy (Figure 3). Occasionally, forest mosquitoes can also transmit the virus to humans when humans are working or visiting the forest. In the intermediate (savannah) cycle, in Africa, the virus is transmitted by mosquitoes from non-human primates to humans or from humans to humans when humans are working or living in areas bordering the jungle. In the urban cycle, virus (which is usually brought to the urban areas by a viremic human who was infected in the jungle or savannah) is transmitted between humans and urban mosquitoes, primarily Aedes aegypti. Further, based on serological analyses, several other zoonotic arboviruses including Mayaro virus, Oropouche virus, O’nyong’nyong virus, Spondweni virus and Lumbo virus are suspected to be maintained in nature by sylvatic cycles [32, 33].

3. Spillover of zoonotic viruses

Spillover is the transmission of a pathogen from a non-human vertebrate host to humans. The six main components in the chain of infection are pathogen, reservoir, portal of exit, mode of transport, portal of entry and susceptible host. In the spillover pathway of viral zoonoses, the virus leaves its animal reservoir through a portal of exit and enters the susceptible host(s), through an appropriate portal of entry, either by a direct or an indirect mode of transmission (Figure 4). In some of the pathways, amplifying or intermediate hosts are also involved. For instance, palm civet is identified as the intermediate/amplifying host in the emergence of SARS coronavirus zoonosis. Furthermore, apart from humans, multiple other spillover/susceptible hosts are identified for SARS coronavirus, which include Chinese ferret badger, domestic cat, dog, pig and racoon [34, 35]. Portal of exit refers to the site where the pathogen is localized and can be transmitted, such as body fluids, urine, fecal material, mucus, oral cavity and skin. Whereas, portal of entry refers to the site through which the pathogen can enter the susceptible host and gain access to the tissues in which it can multiply. Infectious agents may use the same or separate sites for exit and entry. For example, influenza virus uses the respiratory tract as the portal of exit as well as the portal of entry, whereas the polio virus, which is transmitted through “fecal-oral” route, uses separate portals for exit and entry. The direct transmission of zoonotic viruses involves contact of humans with the infected animals. The indirect transmission refers to the transfer of the infectious agent from the reservoir to a host either by suspended air particles (airborne transmission), inanimate objects (transmission by vehicles) or animate vectors (vector-borne transmission). Vehicles for indirect transmission of an infectious virus include food, water and biological samples such as blood and fomites. Vectors such as mosquitoes, ticks and fleas can transmit infectious viruses. Vectors may be mechanical carriers, support growth or cause changes in the infectious agents. Modes of transmission of various zoonotic viruses are summarized in Table 2.

Humans are regularly exposed to many potentially infectious pathogens of animal origin. However, most of them cannot spillover and cause disease in humans. It is a relatively rare event because the probability of a spillover is determined by the ability of the virus to cross a series of nonlinear and dynamic barriers outside – as well as within the spillover host [36, 37]. Among the factors that determine the probability of a successful spillover are the distribution and density of the reservoir; prevalence of the zoonotic virus and intensity of the infection; release, survival and spread of the virus; human encounter and a productive infection of the spillover host [5]. In addition to the inherent properties of the reservoir, the virus and the spillover host, spillover events are also driven by factors such as anthropogenic activities, demography, climate, environment and ecological conditions [5, 38, 39]. For instance, in the year 1999, a severe outbreak of the Nipah virus took place in Malaysia, due to virus spillover from bats to pigs [40]. Ecological changes such as encroachment into the bat habitat encouraging an encounter, intensification of pig farming resulting in a high density of hosts and international trade leading to the spread of infection are suggested as significant contributors [41, 42]. Climate and environmental changes are speculated to have played a role in the emergence of hantavirus and spillover of Hendravirus. An escalation in the population of rodents as a result of environmental changes is suggested to have led to the emergence of hantavirus in 1993 [43]. Similarly, climatic changes are predicted to have contributed to southward movement of black flying fox, a reservoir of Hendra virus, leading to the spillover of the virus into the southern horse populations, which subsequently infected humans [44, 45]. New opportunities for spillover and emergence of zoonoses are created by anthropogenic activities as well. Rapid urbanization an increase in populations residing in low-quality and crowded dwellings have promoted the emergence and spread of arboviruses such as chikungunya virus, dengue virus and zika virus. Studies have shown that the vectors for these viruses, Aedes aegypti and Aedes albopictus mosquitoes, are well adapted to urban areas [46, 47, 48]. Dense and highly connected urban areas are transmission centers for rapid spread of viruses such as SARS coronaviruses and influenza viruses [49, 50]. Further, demographic changes are also noted for contribution to the emergence of viral zoonoses. It has been postulated that globally aging populations and aging immune landscapes heighten the risk for spillover [51, 52, 53].

4. Molecular mechanisms in the emergence of zoonoses

There are multiple ways by which humans can encounter animal viruses. However, encounter alone is not sufficient for them to emerge as human pathogens. For the establishment of the disease, a virus also needs to be internalized, replicate efficiently in the human host, evade host defenses and be disseminated [54, 55]. A zoonotic virus becomes a threat to the human population when it has evolved the capacity for human-to-human transmission and is able to spread beyond the spillover zone. SARS-CoV 2 and H1N1 influenza virus (swine flu) are examples of zoonotic viruses, which have overcome multiple molecular and physiological barriers for a successful jump to humans, acquired the ability of human-to-human transmission and have spread beyond the spillover zone, causing pandemics. Rabies, caused by a Lyssavirus from the Rhabdoviridae family; West Nile encephalitis, caused by a Flavivirus from the Flaviviridae family; and monkeypox, caused by an Orthopoxvirus, are examples of zoonotic viruses with limited or no human-to-human transmission, resulting in outbreaks that are typically endemic in nature. Spillover could also lead to emergence of new human viruses. AIDS (HIV) and smallpox (variola virus) are viral diseases that started as a zoonosis, but the causative viruses eventually evolved into human-only strains.

Virus evolution is an important driver in the emergence of zoonoses and the appearance of novel human viruses. Using the approaches of genomics and comparative genomics, studies have shown that the virus evolution is a continuous process occurring both, before and after the emergence of zoonosis [7]. Under selective pressure, adaptive mutations are rapidly acquired by viruses due to their inherent properties of high mutation rates, short generation times and large populations [56, 57, 58, 59]. The mechanisms of viral genome evolution could involve either small but significant changes through point mutations, small deletions and/or insertions or major genome remodeling through events such as recombination and/or reassortment [60, 61]. Alterations in viral genomes are known to influence their adaptation to new host by modulating key features, such as the specificity of receptor binding, dynamics of virus fusion, protein synthesis, immune evasion strategies and transmission efficiency (Table 3).

Influenza A virus has segmented genomes allowing rapid reassortment of genomic segments among viruses co-infecting a single cell [62]. Reassortments can generate chimeric viruses with mix of genes useful for virus adaptation to multiple hosts [63]. Hence, influenced by cross-species transmissions between humans, pigs and birds, the flu landscape has changed multiple times [64, 65]. The influenza virus that caused the epidemic of 1989, likely originated from an avian reservoir host and descended to another virus, H2N2, which caused an epidemic in 1957. Reassortment of H2N2 and an avian virus resulted in the H3N2 virus, which caused the epidemic in 1968. Along with H3N2 in circulation, H1N1 was reintroduced in the year 1977, and by 1998, a chimera of the three viruses was circulating in pigs, which contained HA, NA and PB1 genes of the human H3N2 origin, M, NP and NS of the swine H1N1 and PA and PB2 of the avian origin virus. Currently circulating influenza virus, H1N1 S-OIV, emerged in humans in the year 2009, as a result of reassortment of an “avian-like” Eurasian H1N1 of swine lineage with the triple reassorted swine virus. Point mutations leading to changes in the receptor-binding pocket of the hemagglutinin protein of the virus have helped its adaptation to the human receptor, SAα2,6Gal [60, 66]. Other adaptive mutations of influenza virus include a substitution in the PB2 protein and truncation in the gene encoding a major virulence factor, PB1-F2.

Ebolavirus infection is characterized by extremely severe symptoms and with more than 40% case fatality rate [67]. It has caused several outbreaks, likely through multiple zoonoses and unknown intermediate hosts, which might have played a role in transmission from its major natural host, bats [67, 68, 69]. Many amino acid substitutions in the surface glycoprotein, some with epistatic effects, have emerged during the ebolavirus outbreaks, resulting in an enhanced human cell entry and fusion [70, 71]. These mutations could also promote antibody escape and contribute to increased mortality since glycoprotein is the only surface protein on ebolavirus particles [72, 73]. Ebolavirus persistence and the possibility of latency in immune-privileged sites are also noted [74]. These selective environment conditions could provide unique opportunities for virus evolution [73, 75, 76].

Three of the betacoronaviruses, SARS-CoV, SARS-CoV2 and MERS-CoV likely originated in bats and were passed to humans through intermediate/amplifying hosts. Genome of the SARS-CoV, with horseshoe bat as the reservoir host and palm civets as the intermediate host, harbors six amino acid residues in the Receptor Binding Domain (RBD) of spike protein that are different between human and civet isolates. The human receptor, ACE2, but not the civet ACE2, appears sensitive toward substitution of the residues at positions 479 and 489 in RBD, indicating that the virus evolved through acquired mutations for adaptation to human host. Spike inserts add flexibility and aid fusion (Table 3). Genome of the SARS-CoV2 has more than 96% similarity to a bat coronavirus, RaTG13, indicating it is origin in bats. Unlike RaTG13, the SARS-CoV2 genome contains an insertion with furin-like cleavage site in S protein, which could promote virion dissemination. The evolution of SARS-CoV2 continued through the course of its epidemic. Within the first year of the epidemic, spike protein with a mutation D614G which, had an impact on ACE2 binding, appeared as a dominant isolate. The first four significant variants, Alpha, Beta, Gamma and Delta are characterized by spike protein mutations, 501Y, 417N:484K:501Y, 417T:484K:501Y and 452R:478K, respectively. Mutation 501Y imparts a dramatic effect on binding affinity. Whereas, 484K and 417N/T disrupt neutralizing antibody binding. The omicron variant which, is highly infectious and immune evasive as compared to prior variants, contains 15 mutations in RBD and 9 nucleotide insertions in the N-terminus domain in the S protein. It also has adaptive mutation in the N protein, affecting its nuclear localization and is associated with increased pathogenicity potential [77]. Additionally, large deletions are present in the accessory genes, particularly ORF8 [78, 79]. Emergence of omicron variant which has a significantly different genome sequence and measurably different immune response, indicates that SARS-CoV2 could potentially evolve into various regional subtypes and distinct serotypes with limited cross-protection [75]. Unlike the two SARS coronaviruses, the primary animal reservoir for MERS-CoV is well established, which is the dromedary camels [80]. Further, the receptor for MERS-CoV is Dipeptidyl-peptidase 4 (DPP4), which is different than the other two betacoronaviruses, but similar signatures in the spike protein are observed. Entry of MERS-CoV is gained by spike cleavage-mediated membrane fusion. Bat cellular proteases are compatible with both, MERS-CoV and HKU4-CoV spike proteins but the human protease can mediate the entry of MERS-CoV and not of HKU4-CoV [81]. MERS-CoV spike has an insertion of four amino acids, next to the fusion peptide, which likely contributes to the observed compatibility and generally, MERS-CoV spike is able to adapt to species variation in the receptor DPP4 [82]. Another distinguishing feature of MERS coronavirus is that, unlike the SARS coronaviruses, recombination rates among MERS isolates are high in camels and several independent MERS-CoV zoonoses are predicted [83]. Further, the human transmission of MERS coronavirus is limited, suggesting its constraints on infectivity and low host tolerance [84]. Two distinct clades of MERS coronavirus are found in the Arabian and the North African region and similar to the SARS coronaviruses, deletions in their genes encoding accessory proteins, ORF3, ORF4a and ORF4b have been found in both [24, 85].

Zika virus has shown a geographical directionality of migration with a likely origin in Africa, prior to 1950 and moving sequentially through Asia by 1966, Pacific islands by 2007 and the Americas by 2015 [86]. Over this period, the virus has evolved significantly and is reflected by changes in its epidemiological properties. It began with endemic circulation and mild symptoms but progressed to causing epidemic cycles and manifestations of major neurological complications [87, 88]. Zika virus is transmitted through the mosquito Aedes aegypti and sexual and vertical transmissions [89]. Zika virus was first isolated from non-human primates but the early molecular events important for human infection or the reservoirs prior to host jump to humans are not clearly identified [87]. As the virus migrated eastward, several mutations that it has acquired have been identified (Table 3). The key changes lie within the envelop protein receptor motif, prM. Substitutions within prM can impact cytotoxicity and tissue tropism [88, 89]. Substitution in prM has been associated with congenital Zika virus syndrome and Guillain-Barre syndrome [89, 90, 91]. V473M substitution within the envelop protein is shown to increase viremia [92]. A188V is another important substitution within NS1, which results in enhanced interferon inhibition and increased viral titers in brain [89, 93]. T106A substitution in the capsid protein enhances its cleavage by the viral protease and thus virion maturation and infectivity [94].

5. Conclusion and future perspectives

The pathways and mechanisms underlying the emergence of viral zoonosis involve a complex interplay of viral, host, environmental, climatic and anthropogenic factors. Identification of viral reservoirs, understanding of the spillover principles and pathways and recognition of the transmission modes provide opportunities for developing effective public health interventions and reducing the risk of spillovers. For instance, an understanding of the spillover principles and molecular evolution of the SARS-CoV 2 proved to be of significant value in the control of COVID 19. Some zoonotic viruses may not require molecular evolution for transmission/spillover into humans. The emergence of the Hantavirus and Hendra virus is likely due to climatic and environmental factors. Similarly, urbanization could have promoted the emergence of arboviruses such as chikungunya virus, dengue virus and Zika virus. The observed changes in the spread and clinical symptoms of monkeypox virus suggest that there is a need to establish clear epidemiological relationships and the chains of transmission [95]. Our current understanding of viral zoonoses has been useful for developing ways to control some of the diseases. The EYE (Eliminate Yellow Fever Epidemics) strategy to eliminate the Yellow fever virus is comprehensive, multi-component and multi-partner. Endemic zoonotic viruses including the influenza virus and the Dengue virus are among the global priority pathogens for the development of vaccines [96]. Rabies is included in WHO’s 2021–2030 roadmap for global control of Neglected Tropical Diseases. The framework for the control of viral zoonoses is based on the concept of “One Health.” Efforts such as the launch of a global virome project will be beneficial for the control of outbreaks in the future [97, 98, 99, 100].