The pandemic placed bat immunology in the spotlight and it has been suggested that further detailed study may help prevent future pandemics (48). Bats harbor many viruses and share human and domestic animal environments. They have a long-life expectancy and their diversity and winged locomotion favors the emergence and dispersal of new viral species (49). As bats are the only autonomous flying mammals, they are, possibly, the only non-human mammals to occupy almost all regions of the planet, except the Arctic and Antarctic poles (50).

Bats may (like any other mammal) become sick when infected by certain viral species, and the spread of new pathogens into bat colonies be fatal for them (51). However, previous studies on the diversity and evolution of viruses in bats have revealed that they exhibit unique immunological approaches to enable coexistence with viral infections, showing minimal or no overt clinical signs, while allowing enough replication for transmission (26, 52–57). Many of these bat-borne viruses induce several zoonotic human diseases (e.g. Ebola virus, Marburg virus, Nipah virus (NiV), Hendra virus (HeV), SARS-coronavirus (SARS-CoV), MERS coronavirus and (MERS-CoV) (38, 58–60) and all viral species associated with these diseases were found in apparently healthy bats (61).

Experimental infection with Egyptian rousette bats with Marburg virus (MARV) or Ebola virus followed by mRNA expression analysis, revealed the concentration of virus transcripts in the liver and many transcriptional changes in multiple tissues (62). These tissue changes included a robust overexpression of the ANXA1 gene, involved in the regulation of inflammation and cell signaling pathways (63), as well as of MRC1 (CD206) gene associated to a subset of tissue resident macrophages regulating inflammation (64). MARV severe disease includes inflammatory gene dysregulation (cytokine storm) and immune response suppression followed by systemic damage and death in humans, which is avoided in the Egyptian rousette bats by the upregulation of antiviral genes IFN-stimulated gene 15 (ISG15), IFN Induced Protein With Tetratricopeptide Repeats 1 (IFIT1) and 2′,5′-oligoadenylate synthase (OAS) 3 gene in the absence of any significant induction of proinflammatory genes, such as C-C Motif Chemokine Ligand 8 (CCL8), FAS (also called CD95 or APO-1 or TNFRSF6) and interleukin(IL)-6 (27). Because these viruses do not elicit a robust immune responses, it seems to prevent immunopathology, but it also prevents viral clearance that (65) leads to immunological tolerance. Thus, while it has been suggested that viral zoonotic risk seems to be homogeneous among mammalian and avian reservoirs hosts (66), it has been demonstrated that bats harbor significantly higher proportion of zoonotic viruses than all other mammalian orders (49, 67). A broad metagenomic analysis, recently conducted in China, to screen the virome in pharyngeal and anal swab samples of 4440 apparently healthy bats (no overt signs of disease) from 40 major different bat species revealed the presence of a diverse set of viruses (59). The most widely distributed families of mammalian viruses found were Herpesviridae, Papillomaviridae, Retroviridae, Adenoviridae and Astroviridae (~61% of the total viral sequence reads). Many reads related to other families were also found and included Circoviridae, Paramyxoviridae, Coronaviridae, Caliciviridae, Polyomaviridae, Rhabdoviridae, Hepeviridae, Bunyaviridae, Reoviridae, Flaviviridae and Picornaviridae, and the subfamily Parvovirinae. These authors identified in the swabs of a few genera, Rhinolophus spp. Miniopterus spp., and Myotis spp., 16, 10 and 13 virus families respectively, suggesting that these three genera are the major reservoirs for mammalian viruses in China (59).

These data confirmed previous findings that bats are indeed unique reservoirs of diverse virus with immune tolerance (15, 61, 68).

The ability of bats to control intracellular pathogens does not mean that they are not susceptible to extracellular infections (69). Indeed, environmental exposure of small, hibernating brown bats to Pseudogymnoascus destructans has been shown to cause infection and high mortality in colonies associated with white nose syndrome (70).

Free-living migratory birds can also be zoonotic reservoirs and can contribute to the dispersion of microorganisms as biological carriers, mechanical carriers, or carriers of infected hematophagous ectoparasites (71–73). As for bats, migratory birds also host viruses and can be the long-distance transmitters or local amplifiers of a variety of viruses that may induce severe disease in humans, domestic animals, and other wildlife (74–78).

Virome investigations of free-living birds and exploration of the impact of environmental factors on viral infection in the hosts may help to detect virus species with potential translation into human risk (79, 80). In keeping with these ideas, the virome of cloacal swab specimens collected from 3182 birds including more than 87 different species (mostly wild species), from 10 different avian orders was investigated (81). They identified 707 viral genomes from 18 defined families and 4 unclassified virus groups, with 265 virus genomes comprising new virus families, genera, or species. The authors provided evidence for the potential cross-species transmission of certain viruses in wild birds, showing > 95% amino acid sequence identity with previously reported viruses in domestic birds.

Several avian species sharing the variants of the Influenza A virus, except H17N10 and H18N11 subtypes, have been isolated exclusively from bats (82). Influenza viruses of types A and B lead to seasonal influenza epidemics, but only type A is linked to pandemics (83, 84). Migratory birds directly contribute to the maintenance and dissemination of avian influenza A virus within the Northern Atlantic flyway of North America (85).

Owing to the detection of rearrangements and mutations of the Influenza virus in wild birds, public concern about the potential pandemic risk posed by this viral species and its mutations is growing significantly (77, 86, 87). Indeed, evidence of transmission from avian viral species to humans has previously been described for A/H7N9, A/H6N1 and A/H10N8 variants, and approximately 35% of patients infected with A/H7N9 succumbed to zoonotic infection (87).

Although most people with influenza exhibit acute respiratory symptoms and muscle aches, with or without fever, which disappear within 1 week (88), avian species do not always have symptomatic infection, facilitating human exposure to the virus. Wild birds also play significant roles in the ecology and circulation of Eastern and Western Equine Encephalomyelitis and Sindbis alphaviruses, West Nile, Usutu, and St. Louis Encephalitis flaviviruses (71, 89, 90).

The inflammatory response to viral infections in migratory birds has also been well documented in several different domestic and aquatic birds (91). Gallinaceous poultry species, domestic ducks and various aquatic and terrestrial birds are vulnerable to avian influenza viruses (92–96) and host inflammatory response seems to contribute to morbidity and mortality in all species investigated so far (96, 97). Avian influenza (AI) viruses for example, have been detected in more than 105 species of birds of different taxa but the species most frequently found harboring influenza virus belong to Charadriiforms (gulls, terns and shorebirds) and Anseriformes (ducks, geese and swans) orders, where no apparent disease signs or lesions were found (97). It has been suggested that the recognition and elimination of invading pathogens (resistance)(98, 99) or the control of the infection associated tissue damage (tolerance)(100) may explain asymptomatic or minimally symptomatic infections.

Virus infections were associated with liver diseases in chickens raised in scaled farms causing significant economic losses and this was associated with multiple virus infections. Indeed, panvirome profiling of livers, spleens, kidneys, and recta revealed coinfection of chicken infectious anemia virus, avian leukemia, avian encephalomyelitis virus (AEV) and multiple fowl adenoviruses, with a higher abundance of the last two virus species in the liver (101). Many other virus infections with novel strains and variants caused economic losses to the poultry industry worldwide and these included infectious bronchitis virus (102), bursal disease virus (103), H5Nx avian influenza viruses in Europe, Africa and the Americas (104–106), H5N8 and H5N1 avian influenza viruses (107–111). Similarly, the host response to pigeon paramyxovirus type I (PPMV-1) infection (associated with New Castle disease, a great threat to the pigeon industry), induces strong innate immune responses and intense inflammatory responses at an early stage and this is associated with viral pathogenesis (112). After infection, pigeons showed upregulated expression of toll-like receptors (TLRs), such as TLR2, TLR3 and TLR15, together with interferon (IFN) gamma (IFNɣ) and IL-6, whereas IL-18 expression was found down- regulated (112). PPMV-1 is now worldwide disseminated causing extensive infections in domestic and feral pigeons, wild birds and poultry (113).

Differential host responses to avian influenza viruses in avian species with differing susceptibilities were analyzed by transcriptomics, and susceptible birds showed strong neuro-inflammatory responses associated with greater viral load (114). Ducks are natural hosts and reservoirs of influenza A virus and transcriptomic analysis in infected individuals showed this condition associated with downregulation of a distinct set of proinflammatory cytokines in lung, key elements of leukocyte recruitment and complement in intestine (115).

Comparative analysis of chickens and ducks following high pathogenic avian influenza virus infection was associated with increased proinflammatory responses in chickens whereas downregulation of inflammatory response in ducks was associated with mild or asymptomatic profiles (116). In line with these observations mallard ducks are permissive to low pathogenic avian influenza viruses in their intestinal tissues without overt disease signs, thus limiting the duration of proinflammatory cytokine expression, whereas in chickens, respiratory and systemic disease is associated with enhanced virus replication and associated tissue damage (117). While the co-evolution of Mallard ducks with influenza virus have allowed host-pathogen interaction and resistance through retinoic acid-inducible gene I (RIG-I) pathway, providing asymptomatic or minimally symptomatic infections with robust IFN response, chickens lacking the key sensors of RIG-I pathway, with compromised host response, show significant inflammatory changes, associated clinical signs and lesions to the skin, respiratory, digestive, reproductive and nervous systems following high pathogenic influenza virus (HPAIV) infections (117–119).

Rodentia is the most diverse order of mammals, representing more than 40% of mammalian species including 33 families and 2,600 species (120). They are natural virus reservoirs of a variety of species associated with human severe diseases (121–124).

At the molecular level, Cohn and collaborators performed transcriptional and physiological monitoring across 33 mouse strains during in vivo infection with the influenza virus (125). They identified two host-defense gene programs associated with disease tolerance and resistance respectively, providing a paradigm for exploring these immune responses in different species.

At the cellular level, a key subset of specialized T cells for regulation of immune responses and maintenance of the immune tolerance in the periphery, the Treg cells, are recruited and expanded, undergoing functional and molecular reprogramming (126). These changes may protect against exacerbated immune responses while providing tissue homeostasis (127).

Dual RNAseq differential expression analysis following HIN1 infection in two mouse strains with contrasting resistance to influenza virus infections enabled investigation of both the viral and host gene expression profile in the same individual and revealed that host genes were involved in the development of severe pathology, viral replication and immune responses (128). From this report it emerges that IFN Regulatory Factor 7 (Irf7) gene deletion (obtained in the knock-out line on a C57BL/6J background) was associated with greater susceptibility to H1N1 infection with significant body weight losses and increased mortality compared to wild controls (128).

Furthermore, it has been demonstrated using lymphocytic choriomeningitis and Influenza A virus models in mice, that the Treg-dependent immunosuppression is associated with the effector state of Treg cells, which maintain stability and functionality while exposed to IFNɣ. The stability of regulatory T cells may be diminished or reinforced by different Type-1 cytokines (127). The deletion restricted to regulatory T (Treg) cells of the IFNɣ receptor, but not to the IL-12 receptor, prevents Th1-type polarization and promotes Th2-type polarization, avoiding long inflammatory process, and adequate response from memory T cells (129).

It is important to highlight, however, that although many significant immunological discoveries have been obtained in laboratory mice, they do not directly mimic the physiology of wild mice living in their natural environment. To circumscribe this limitation, a new murine model named “wilding” was generated by transferring embryos of C57BL/6 into wild mice (130). This approach revealed that the immune profiles of wilding mice, laboratory mice, and wild-type mice showed that wilding individuals and laboratory mice exhibit a different epithelial barrier microbiome, gut microbiome, virome, and repertoire of pathogens. Interestingly, immunological phenotypes of wildings and wild-type mice were closer to and seemingly a better mimic of the human immune system. In agreement, the first phase 1 clinical trial in multiple laboratory mouse models of autoimmune/inflammatory diseases, using CD28 monoclonal antibodies, which are able to activate regulatory T cells, resulted in human life threatening activation of inflammatory T cells and cytokine storms (131). Similarly, laboratory mice born from wild mice (wilding mice) after treatment with a CD28-superagonist showed an inflammatory cytokine response and an absence of Treg expansion (130).

In addition to the sustained virome surveillance of bats, rodents and migratory birds, arthropods are another essential source of information for the future control of pandemics (132–134). As in mammals and birds, arthropods also react to potential threatens with an innate immune system response comprising both cellular and humoral components (135). Arthropods harbor numerous arboviruses that may spread through migratory birds and bats, at stopover sites, to other species, or to other individuals of the same species, and many of these arboviruses are of great epidemiological interest (136, 137). For example, mosquito-borne and tick-borne viruses cause some of the most severe diseases with high fatality rates in humans and animals and continue to threat human and livestock health globally (135, 138, 139).

To understand the emergence of arboviruses and the dynamics of potential outbreaks, it is essential to reconstruct the virome of the species of interest and understand its composition and potential modulatory actions on arbovirus transmission (140, 141). For example, mosquito vector competence for dengue is modulated by insect-specific viruses (142). Using virome analysis of 815 urban Aedes mosquitoes collected from 12 countries worldwide it was demonstrated that two mosquito-specific viruses were the most abundant (Phasi Charoen-like virus and Humaita Tubiacanga virus). The spatiotemporal analysis of virus circulation in an endemic urban area revealed a 200% increase in chances of having DENV in wild A. aegypti mosquitoes when both Phasi Charoen-like virus and Humaita Tubiacanga virus were present (142).

Metagenomic analysis of mosquitoes can provide valuable information, including new or unsuspected arboviruses, as well as non-arboviral pathogens ingested from hosts on which they feed (143). Next generation sequencing of RNA extracted from excreta of Aedes vigilax and Culex annulirostris reversed for cDNA and sequenced, allowed the assembly of near-full-length viromes of Australian Anopheles totivirus, Wuhan insect virus 33, and Hubei odonate virus 5 and 7 new viruses, closely related to members of the order Picornavirales and to previously described, but unclassified RNA viruses (143).

At least 500 arboviruses have been identified so far, 100 of which may induce human diseases and 40 of them cause domestic animal diseases (144, 145), and the Amazon is a spotlight for emergence of new arboviruses and this has been associated with disturbance of the natural ecosystem (146).

Emergent viruses may involve urbanization - in which humans have become the amplification hosts - and peridomestic mosquitoes (mainly Aedes aegypti) mediate human-to-human transmission (dengue, yellow fever, chikungunya and Zika viruses) (147, 148). Secondary amplification, for example, has occurred in outbreaks of Japanese encephalitis, Venezuelan equine encephalitis, and Rift Valley fever viruses (149, 150). Alternatively, simple spillover from enzootic cycles may occur as it is observed in West Nile fever (151), Crimean-Congo hemorrhagic fever (152, 153), tick-borne encephalitis (154, 155) and Alkhurma hemorrhagic fever virus (156, 157).

Vector-borne viruses enter the vector, infect, and replicate. Escaping from the gut and reaching the salivary glands, they infect vertebrate host through saliva during blood feeding (136, 158, 159). Sialotranscriptomes of unfed and fed ticks showed that blood feeding alters the expression profiles to facilitate the feeding process and pathogen transmission (160).

More than two years after its emergence, the SARS-CoV-2 virus continues to affect a subgroup of patients. These patients, who have recovered from the acute phase of the disease, report a diverse repertoire of symptoms that characterize chronic or long COVID-19 (204, 205). During the acute phase, as part of the virus replicative cycle, SARS-CoV-2 induces the death and injury of virus-infected cells and tissues, followed by a wave of local inflammation associated with increased secretion of the pro-inflammatory cytokines and chemokines IL-6, IFNɣ, MCP1 (monocyte chemoattractant protein-1) and IP-10 (interferon gamma inducible protein) (206). In most individuals, the recruited cells clear the infection in the lung, the immune response subsides, and patients recover (207). However, in some patients, a dysfunctional immune response occurs, that is characterized by augmented cytokine production, that leads to widespread lung inflammation and multi-organ failure (207–209).

Indeed, severe acute respiratory syndrome induced by SARS-CoV-2 induces proinflammatory immune responses in the periphery and/or in the brain via classical TL receptor inflammatory pathway and this event is crucial for the onset of acute and chronic neuroinflammation and sickness-related behavior (210). Dysregulated immune responses resulting in lymphopenia and increased proinflammatory cytokine production seems to be the principal cause of the pathophysiology in COVID-19 and patients. High concentrations of IL-6 accompany the most severe cases (211).

The humoral immune response by B-lymphocytes is part of the adaptive immune mechanisms, controlling viral infections through the neutralizing antibodies and antibody-dependent cellular toxicity (212, 213). The elimination of viral infection and the apoptosis of virally infected cells is also provided by B lymphocytes through effector molecules, exhibiting antibody-independent functions (214). However, lack of B cells has been associated with a mild form of COVID-19 suggesting that inflammatory cytokines, especially IL-6, may have a central role in the disease severity (213). In fact, patients with agammaglobulinemia (caused by a lack of B lymphocytes), show mild symptoms, short duration, and no need of treatment by IL-6 inhibitors, while patients exhibiting variable immune deficiencies with dysfunctional B lymphocytes, (also showing significant low levels of immunoglobulins in the serum), show severe outcomes and require mechanical ventilation, antiretroviral agents and IL-6-blocking agents (215).

Survivors from acute phase may exhibit a form of post-acute sequelae characterized by atypical chronic persistence of symptoms that may last for months resulted in the long-COVID syndrome (204). Long COVID appears to be a multi-organ disease and with a spectrum of symptoms (systemic, neuropsychiatric, cardiac, and respiratory) have been associated with persistent inflammation, induced autoimmunity, and putative organ reservoirs of SARS-CoV-2 RNA or antigens (216–219). Recent evidence supports, however, the hypothesis that the persistence of the inflammatory response for a long period after the acute phase is closely correlated with the post-acute sequelae of COVID-19 (PASC). Actually, the combination of digital epidemiology with selective biobanking recent analysis suggested that instead of autoantibodies, the elevated plasma levels of IL-6, IL-1β and TNF-α is in the core of the clinical symptoms of PASC COVID-19 (220). While rheumatoid factor, antinuclear antibodies, and antiphospholipid antibodies showed no correlation with post-acute COVID-19 sequelae, analysis of blood samples from participants with ongoing post-COVID19 sequelae, revealed at 6 and 8 months after infection, increased levels of the pro-inflammatory cytokines TNF-α, IL-1β and IL-6 in close correlation with symptoms of the post-acute COVID-19 syndrome (220).