Interspecies transmission and emergence of novel viruses: lessons from bats and birds

Emergence of new viruses

Both mammalian and avian coronaviruses (CoV) have diverse host ranges. Phylogenetic dating of RNA-dependent RNA polymerase (RdRp) sequence divergence suggested that the most recent common ancestor (MRCA) of mammalian CoVs appeared around 7000–8000 years ago, whereas the MRCA of avian CoVs dates back to 10 000 years ago (Figure 1 ). These results are likely underestimations because they could not account for additional sequence diversity from undiscovered viruses. Nonetheless, the present estimates roughly coincide with the dispersal of the human population around the world about 50 000–100 000 years ago and greatly increased in the last 10 000 years during the first historic transition. During this transition, humans began various farming activities, such as forest clearing for agriculture and animal herding, leading to a significant shift in the ecology and population dynamics of viruses owing to the intrusion of wildlife habitats and intensive mixing of different animal hosts. Finally, the expansion of human travel and trading directly led to the spread of viruses to distant and isolated places. The migration of early humans over long distances was very limited and effectively a unidirectional ‘rare’ event. Eventually, improvements in transportation technology enabled distant trade missions in early Mesopotamia around 5000 years ago and possibly earlier in other regions [1]. These periodic yet infrequent visits might have enabled transmission of various disease agents to previously segregated non-immune populations, leading to a serial founder effect associated with a boom and bust cycle. However, further technological improvements, especially the development of aviation and the flight industry in the last century, have allowed this type of travel to occur at such high frequencies that multiple segregated host populations effectively have become a single large population. These changes coincided with increased breeding between different host populations (for both humans and domestic animals), which can significantly impact the genetic and immunological make-up of the host populations. When these occurrences are considered in the context of the high mutation rate of RNA viruses, they become a driving force for speciation and subsequent evolution of new viruses. Although phylogenetic analysis and dating of individual influenza genes and lineages have been reported previously, large sequence divergence and frequent reassortment led to difficulties in precisely dating and phylogenetic positioning the common ancestor of modern influenza viruses [2].

About 70% of the emerging pathogens infecting humans originate from animals. Most of these major outbreaks were due to RNA viruses as a result of their higher mutation rates compared with other types of microbes and their capability for unique genetic change, either by genetic recombination in positive-sense RNA viruses or genetic reassortment in RNA viruses with segmented genomes. Those with greatest impact on humans include the severe acute respiratory syndrome coronavirus (SARS-CoV), influenza virus, and HIV. Little was known about CoVs until the 2003 SARS epidemic, which caused 774 deaths among 8098 cases in over 30 countries [3]. The natural reservoir of the more ancestral bat SARS-CoV is the Chinese horseshoe bat (Rhinolophus sinicus), which may have transmitted the virus to other game mammals including Himalayan palm civets (Paguma larvata), raccoon dogs (Nyctereutes procyonoides), and Chinese ferret badgers (Melogale moschata) in wildlife markets in South China [4]. This finding sparked intense hunting for novel CoVs in humans and different animal species, especially in bats. The latest emerging novel human CoV, originally named human coronavirus EMC/2012 and later renamed Middle East respiratory syndrome coronavirus (MERS-CoV), which has caused 30 deaths among 54 cases in the Middle East, Europe, and Africa, is also phylogenetically closely related to the Tylonycteris bat CoV HKU4 (Ty-BatCoV-HKU4) and Pipistrellus bat CoV HKU5 (Pi-BatCoV-HKU5) discovered in bats in Hong Kong 5, 6, 7, 8 (http://www.who.int/csr/don/2013_06_05/en/index.html). The importance of birds as natural reservoirs of emerging influenza viruses is underscored by the persistent threat of avian influenza H5N1 since 1997 and the emergence of H7N9 in 2013 9, 10, 11, 12. The role of bats in the emergence of novel influenza viruses is less clear although influenza A H17 and H3N2 viruses have been discovered in Sturnira lilium recently and in Nyctalus noctula bats in Kazakhstan in 1970, respectively. We review the importance of bats and birds in the genesis of new virus mutants and interspecies jumping using CoVs and influenza viruses as examples.

Bats as natural reservoirs for emerging viruses

A number of unique ecological, biological, immunological, and genetic features make bats a favorable animal reservoir for the emergence of novel viruses. Bats have remarkable species diversity, with over 1240 species (20% of the nearly 5000 known species within Mammalia and only second to rodents) [9]. Because viruses are obligatory intracellular microbes, it is assumed that viruses have multiple independent evolutionary origins not separable from co-evolution of their hosts. This high biodiversity of bats makes them an important source of new viruses for interspecies jumping.

Bats are widely distributed in all continents except the polar regions and a few oceanic islands. Their roosting or hibernation environment ranges from natural habitats such as caves, rock crevices, bird nests, and tree cavities, to man-made structures like mines, tombs, buildings, and bridges, which bring them closer to humans and companion animals or livestock. Their unique ability among mammals to fly long distances (up to 2000 km) to locate suitable habitats also allows them to acquire or disseminate viruses [9]. Their habit of roosting in large colonies ranging from 10–200 000 bats and relative longevity of up to 35 years also provide abundant mating opportunities and thus exchange of viral genetic material and further viral spread to other species. Exposures to infected urine and aerosols generated during defecation have been suggested as possible routes of intraspecies and interspecies transmission of viruses from bats [9]. Bats may also transmit viruses to human and other animals via bites and scratches as in the case of rabies. Consumption and handling of undercooked bat meat is still practiced in China, Guam, and some parts of Asia.

Besides ecological and biological traits, bats also possess special immunological attributes that enhance their ability to serve as gene pools for emerging viruses (Table 1 ). Asymptomatic or persistent viral shedding with little evidence of pathology in bats is well reported [13]. One possible mechanism is the very early control of viral replication by their innate immune response involving the early recognition by pattern recognition receptors (PRRs) and interferons (IFNs) followed by partial control by the adaptive immune response [14]. Similar to other mammalian species, some bats possess the two major families of virus-sensing PRRs, namely the Toll-like receptors (TLRs), which are membrane-bound PRRs that detect single-stranded RNA (ssRNA) or double-stranded RNA (dsRNA), and retinoic acid inducible gene I (RIG-I)-like helicases (RLHs), which are cytosolic PRRs that detect dsRNA. Bat TLRs have been described in many fruit bat species and are highly similar to other mammalian TLRs 15, 16. In addition, cytoplasmic RLHs including RIG-I, melanoma differentiation-associated protein 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2) have been found in Pteropus alecto and their homologs in Myotis davidii [16]. These PRRs allow bats to recognize a similar range of pathogens as other mammalian species and in turn these pathogens are controlled by IFNs. Type I IFNs have been described in numerous fruit bat species [17], and type III IFNs have been found in Myotis lucifugus, P. alecto, and Pteropus vampyrus [18]. Notably, the type I IFNω family of genes are expanded in some bat species with up to a dozen members, and the type III IFN receptors are more widely distributed in tissues of P. alecto than in humans and mice [19]. Bats may also demonstrate a delayed or differential IFN response during in vitro infection by different kinds of viruses 17, 18.

However, important differences between the adaptive immune response of bats and other mammals have been observed. In the humoral response, bats have an antibody repertoire as diverse as those of humans and mice. At least 23 genes for immunoglobulin (Ig) V_H and the λ and κ loci of Ig V_L have been identified in P. alecto, and diverse Ig D_H and Ig J_H elements and the λ locus of Ig V_L have been found in M. lucifugus and other microbats (Table 1). However, the amino acid sequence composition of the antigen-binding site (CDR3 region) of the expressed V_H region in bats is different from those of humans and mice with a higher proportion of arginine and alanine residues, and lower proportion of tyrosine residues, and thus possibly a lower poly-reactivity [20]. Such antibodies have lower avidity and form a weaker association with antigens. Furthermore, the primary antibody response may be delayed from reaching a peak and the secondary antibody response may also be slow or delayed in bats. In adverse environmental situations such as prolonged exposure to low temperature at 8 °C simulating the state of hibernation, bats may temporarily fail to mount an antibody response but resume this ability 1 week after transferring to 24 °C. Importantly, bats may clear viral infection even in the absence of neutralizing antibody by other unknown mechanisms [14]. Bats appeared asymptomatic during bat–SARS-CoV infection despite a high viral load in their anal swabs with absent or low serum neutralizing antibody although their body weights were generally lower than the uninfected bats 4, 21. As for a cell-mediated immune response, those of bats are generally slower to peak after stimulation by T-cell mitogens. Environmental changes such as roost ecology and physiological alterations such as pregnancy also affect cell-mediated immunity of bats [14]. Further studies should be performed to ascertain if these quantitative and qualitative differences in the adaptive immune response of bats may account for their unique interaction with viruses leading to asymptomatic infection and viral persistence.

The discovery of influenza virus in bats challenges the notion that aquatic birds are the only source of all influenza A gene segments 9, 10. The recently discovered H17 influenza virus from Guatemala bats is unique in that all eight gene segments appear to be distinct from any known influenza A gene segments [10]. The hemagglutinin (HA) of this virus has unique structural features and exhibits receptor binding and fusogenic activities that are distinct from its counterparts in other influenza viruses [22]. Likewise, the neuraminidase (NA) of this virus, called N10, is phylogenetically distinct from the NAs of all influenza A and B viruses. The N10 is also structurally distinct with no enzymatic activity [23]. Additional surveillance is necessary to understand the ecology of influenza viruses in bats.

Waterfowl and shorebirds as a source of influenza virus genes

The high biodiversity of birds (over 10 000 species) allows avian influenza viruses to evolve in different host environments. Birds living near wetlands or aquatic environments, especially in the orders Anseriformes (aquatic waterfowls) and Charadriiformes (shorebirds), are considered to be the sources or gene pool for all influenza viruses 24, 25. Waterfowl feed on submerged water plants in marsh water that are readily contaminated by virus from excreta of infected waterfowls or other small animals. Birds are frequently infected by influenza A viruses. Re-infection during the same season with the same virus and coinfection with different viruses can occur [26]. Despite frequent infections, waterfowl usually remain asymptomatic even when infected by avian influenza viruses highly pathogenic in chicken [27]. Asymptomatic infections in ducks and geese often preceded devastating outbreaks in chickens. Influenza viruses persisting in asymptomatic waterfowls allow accumulation of mutations that may facilitate interspecies transmission [28]. Moreover, wild waterfowls often aggregate with domestic ducks and geese during feeding and roosting and therefore allow efficient mixing of different influenza viruses to form new subtypes. In the backyard farm setting, domestic ducks and geese are often mixed with chickens and other domestic poultries allowing interspecies transmission, which may eventually transmit into humans. Finally, migratory waterfowls can disseminate novel influenza viruses to very distant locations [9].

Similar to bats, birds have a unique immune system that is best studied in ducks (Table 1). Genes related to antiviral immunity, including major histocompatibility complex type I (MHC-I), interferon-induced protein with tetratricopeptide repeats 5 (IFIT5), oligoadenylate synthetase-like (OASL), CC chemokine ligand (CCL)19, CCL21, MDA5, TLR7, and IFN-α, are expressed in duck lungs as early as 1 day after H5N1 virus inoculation 29, 30, 31. Proinflammatory cytokines were lower in duck than in chicken embryonic fibroblasts infected with H5N1 virus [32]. Major differences in the immune system between ducks and chickens may account for the differential susceptibility to highly pathogenic avian influenza. Whereas chicken cells naturally lack RIG-I or RIG-I activity, duck RIG-I has a similar structure and function to its human ortholog. It was shown that transfection of duck RIG-I into a chicken embryonic fibroblast cell line augments the IFN response and suppresses viral replication [33]. Chicken cells apparently rely upon MDA5 and LGP2 to sense influenza virus infection [34]. Furthermore, the duck TLR7 differs from chicken in the ligand binding LRR domains [35]. When infected by influenza virus or stimulated with TLR7 agonists, chickens and ducks differentially activate the production of MDA5, TLR7, and IFN-α [31]. Notably, TLR8 and IFNω, which are present in bats, are either disrupted or not found in birds.

The adaptive immune system is particularly unique in ducks. Influenza virus elicits a short-lived antibody response in ducks [36]. Unlike mammals, ducks have three types of antibody, namely IgA, IgM, and IgY (human IgG counterpart). The predominant antibody response in infected ducks is the truncated form of IgY, which lacks the Fc region and does not exhibit hemagglutination inhibition, complement fixation, opsonization, and antibody-dependent cellular cytotoxicity. IgA is not produced until ducks are 2 weeks old and is expressed late after an infection [37]. T lymphocytes, although present, are not fully functional in young birds [38]. Although ducks have five MHC-I genes for antigen presentation, only two are expressed [39], which limits the repertoire of presentable peptides. These immunological features may allow waterfowl to shed large amount of viruses for a prolonged period [40]. In ducks infected by low pathogenic avian influenza virus, RIG-I, CCL19, and CCL21 are only slightly upregulated in intestinal tissues 29, 33. These unique features in the immune system of birds, especially in ducks, allow them to be the reservoir for influenza viruses.

Interspecies transmission of CoVs

Before the discovery of SARS-CoV, CoVs were considered as causative agents of respiratory tract infections, gastroenteritis, hepatitis, and encephalomyelitis in birds and mammals. The first two human CoVs, HCoV-229E and HCoV-OC43, primarily caused self-limiting upper respiratory tract infections such as the common cold [41]. The emergence of SARS-CoV represented a new era in the history of CoV research. The significant global medical, economical, and social impact of the 2003 outbreak regenerated research interest on CoVs. One important finding was the identification of bats as the natural animal reservoir and civet and other mammals as the intermediate amplifying hosts of SARS-CoV [4]. These discoveries revolutionized the ‘hunting’ strategy for novel CoVs and redefined the classification of CoVs based on their updated phylogeny and the critical role of bats in inter- and intra-species transmissions of CoVs. With the new wealth of knowledge on CoV phylogeny generated after the SARS epidemic, the Coronavirus Study Group of the International Committee for Taxonomy of Viruses changed the classification into four genera, Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus to replace the traditional antigenic groups of 1, 2, and 3 (Figure 2A) (http://www.ictvonline.org/virusTaxonomy.asp?bhcp=1). This remarkable diversity of species and genotypes, as well as the emergence of novel species capable of adapting to new hosts and ecological niches, is the result of environmental, viral, and host factors that favor interspecies transmission.

Interspecies transmission of influenza viruses

Most influenza viruses are host specific. H5N1 influenza viruses failed to be transmitted from experimentally infected poultry to pigs [58]. Naturally occurring avian influenza viruses in mammals are rare. Even different avian species have different susceptibilities to the same influenza virus. However, interspecies jumping occurs frequently (Figure 2B). Reassortant pandemic influenza viruses often have significant mortality and morbidity. The 1957 H2N2 and the 1968 H3N2 pandemic influenza A viruses are reassortments between circulating avian and human influenza viruses, whereas the A(H1N1)pdm09 virus is a reassortant between the circulating swine influenza viruses [59]. For the 1918 pandemic H1N1 virus, some investigators suggested that the 1918 pandemic influenza virus directly jumped from an avian source to human, whereas others postulated that the virus circulated in swine before human. All gene segments from these pandemic influenza viruses have been ultimately traced to an avian source.

Direct transmission of avian influenza viruses from birds and poultry to humans have been documented for H5N1, H7N2, H7N3, H7N7, H7N9, H9N2, H10N7 11, 12. Swine influenza viruses including the swine-origin influenza H3N2 [(H3N2)v] [60] and triple reassortant swine H1N1 [61] have been transmitted directly from pigs to humans. Based on phylogenetic and epidemiological analysis, many influenza viruses have also been directly transmitted between avian species, between avian and non-human mammals, and between different mammalian species (Figure 2B). Outbreaks due to interspecies jumping include the avian H5N1 outbreaks in zoos involving different mammalian species [62] and in cats [63], equine H3N8 and avian H3N2 outbreaks in dogs [64], and the avian H3N8 outbreak in seals [65].

Concluding remarks

Both bats and birds are warm-blooded sustained flying vertebrates with a very high metabolic rate, biodiversity, and also roosting or migratory behavior [85]. The development and functioning of their immune systems are far less studied than that of humans or rodents. Apart from restrictions posed by host immune response and host cell receptors, many other cellular factors governing the viral life cycles of emerging viral mutants are unknown. The recent discovery of a unique influenza virus in bats may open up a new dimension in the genesis of influenza virus beyond birds while many more CoVs remain to be discovered in bats and birds. These flying vertebrates are the reservoir of many viral families from which pathogenic human and mammalian viruses are derived. It will be of great interest to elucidate the structural and mechanistic basis for the differences between these animals and humans in their innate and adaptive immune systems using state-of-the-art technologies including next-generation sequencing, gene targeting, and reverse genetics (Box 1 ).