Keywords

1 Introduction

Several factors have contributed to the emergence and reemergence of viruses of medical importance: the increase in displacement of individuals in all areas of the globe, demographic increase and geographic expansion, destruction of biodiversity, and global warming are some examples. Additionally, the genome of most of the roboviruses (rodent-borne viruses ) consists of RNA molecule(s), which present high mutation and recombination rates. There is also the possibility of viral genomic segments reassortment between different viruses (in the case of segmented genomes) that are pivotal events for viral evolution but can also increase the risk of the emergence of more adapted and virulent strains.

Hantaviruses are members of the genus Hantavirus, family Bunyaviridae [7], which contains more than 350 members and represents a major class of zoonotic pathogens that cause two severe diseases in humans: hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus cardiopulmonary syndrome (HCPS) in the Americas. The name Hantavirus is after a river’s name in Korea where the Hantaan virus (HTNV) was first identified in a rodent by Ho-Wang Lee and colleagues in the 1970s [48].

The transmission of hantavirus to humans occurs through inhalation of aerosols generated from feces, urine, and saliva of infected rodents, but other small mammals such as shrews, moles, and bats can also harbor the virus [30, 36, 98]. The rodent-borne hantaviruses persistently infect rodents from the family Muridae and subfamilies Arvicolinae (Europe), Murinae (Europe and Asia), and Sigmodontinae/Neotominae (America) [71]. In general, each hantavirus is predominantly associated with a rodent-host species in a given geographic region (reviewed in Plyusnin 2002), although few cases of interhuman transmission have been described [64, 78, 80, 81].

HCPS is an immunopathology of rapid progression that begins with a mild fever and can progress to noncardiogenic pulmonary edema and shock. HCPS was initially described in 1993 in a cluster of patients presenting an acute respiratory distress disorder in the southwestern region of the United States [76]. The hantavirus implicated in this outbreak was named Sin Nombre (SNV) , harbored by the wild rodent reservoir Peromyscus maniculatus. In the Americas, hantaviruses are emergent, and more than 40 genotypes have been described, indicating that these agents are widely dispersed, and nearly half of them are pathogenic to humans [31].

Mortality rates related to hantavirus infection vary from 0.1% to 40%, depending on the specific virus involved. The emergence of hantavirus in human populations is correlated with rodent population density, which depends on several environmental factors, such as precipitation, temperature, habitat quality, and food availability, or, alternatively, anthropogenic behavior in the rodent–host environment [93]. Although hantavirus disease has been recognized for more than four decades, there is still no specific therapy available, and medical treatment is mainly palliative. The Syrian golden hamster recapitulates the human clinical picture and is a valuable model to study vital pathogenesis, viral evolution, and antiviral strategies for prevention and prophylactics of HCPS [37].

2 Virion Structure and Replicative Cycle

The viral particles are spherical with a diameter of 80–120 nm, with an envelope (derived from Golgi membranes of the host cell) and containing equimolar amounts of three negative single-stranded RNA genome segments designated large (L), medium (M), and small (S) [74]. The three RNA segments are coated with the nucleoprotein (N), forming ribonucleoproteins (RNPs) [20, 45].

The L segment of approximately 6.6 kb encodes a 250-kDa RNA-dependent RNA polymerase (viral replicase) that is associated to each viral segment in the virions and is required to initiate viral replication in the host cell cytosol. Comparative analyses of nucleotide and amino acid sequences of the L segment of different hantaviruses showed a conserved primary structure, despite the marked variability of its nucleotide sequence.

The M segment , about 3.7 kb, has a single open reading frame and encodes a precursor glycoprotein (GPC) that is processed by a cellular protease into two envelope glycoproteins, Gn and Gc (formerly named G1 and G2), 70 kDa and ~50 kDa, respectively. Gn and Gc glycosylation takes place in the Golgi complex during the maturation process [21]. Structural studies have demonstrated that Gn and Gc proteins form spikes protruding from the viral membrane and that these Gn/Gc glycoprotein heterodimers may interact with cellular receptors. Gn and Gc proteins present large globular and hydrophobic transmembrane domains and a C-terminal cytoplasmic tail. Because bunyaviruses lack a classical matrix protein, it is suggested that the Gn/Gc tail would interact with the N protein [22, 24]. The hantavirus glycoproteins are involved in the modulation of host innate immune response and virulence [59, 66].

The S segment , approximately 1.09 kb, encodes the nucleoprotein (N) with a molecular mass of approximately 50 kDa, which associates with the viral RNA segments to form helicoidal filamentous structures called ribonucleocapsids (RNPs) [20, 98, 108]. Among the structural proteins, the nucleocapsid is the most abundant and antigenic protein in the virus particle and accumulates in the cytoplasm of infected cells early during infection. Immunodominant and cross-reactive epitopes at the N-terminal domain of the N protein make it suitable to be used as a recombinant antigen for diagnosis purposes. Hantavirus-infected patients present a very short-term viremia and have detectable IgM and IgG antibodies against the nucleocapsid antigen at the onset of clinical symptoms [11, 79]; thus, serological tests are often used for the detection of these antibody classes [109].

N is a multifunctional protein involved in the initiation of transcription and translation of the viral genome, binding selectively viral RNA (vRNA) and viral positive sense antigenomic RNA (cRNA), leading to the encapsidation of newly synthesized vRNA and direct virus assembly, and interacts with the MxA proteins interfering with the host type I interferon response [74].

The 5′- and 3′-noncoding regions (NCRs) of the viral genomic segments present complementary sequences at their ends that are paired to form “panhandle”-like structures, which presumably account for the circular form of the RNAs observed by electron microscopy. These complementary regions are likely to have an important role in replication, providing signals for recognition by RdRp synthesis or for the packaging of the viral genome [20] (Fig. 11.1).

Fig. 11.1
figure 1

Schematic representation of the hantavirus virion . The viral particles are spherical with a diameter of 80–120 nm, with an envelope derived from Golgi membranes of the host cell. The GPC glycoprotein forms spikes protruding from the envelope. The genome is composed of three segments of negative single-stranded RNA designated large (L), medium (M), and small (S). Each RNA segment is coated with the nucleoprotein (N) and is associated with RNA-dependent RNA polymerase (viral replicase)

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The virus enters the host cell through interaction of viral glycoproteins with specific cell receptors and subsequent endocytosis. Gavrilovskaya et al. [28] described that the use of vitronectin , a protein that binds to β3 integrin, inhibits the entry of the Sin Nombre virus (SNV) and New York virus (NYV) in Vero E6 cells , indicating that this would be a receptor on the host cell involved in viral penetration [28]. Potential receptors for Old World hantaviruses also include β1, β2, and β3 integrins (reviewed in Albornoz et al. [2]). In the acidic compartment of the late endosomes, RNPs are released in the cytoplasm after viral glycoprotein and endosome membrane fusion. Subsequently, the virion supplied RNA-dependent RNA polymerase-mediated primary transcription of negative-strand RNA in the cytoplasm, following viral mRNA translation; transcription shifts from mRNA to positive-strand complementary RNA (cRNA) and de novo negative-strand viral RNA synthesis with the concomitant formation of ribonucleoprotein structures. Transcription and translation can also take place in the endoplasmic reticulum–Golgi compartment. The RdRp possesses transcriptase, polymerase, and endonuclease activity [23]. Its endonuclease activity is involved in cap-snatching cytoplasmic cellular mRNAs to prime viral mRNA synthesis. The GPC synthesis (derived from M segment mRNA) occurs on endoplasmic reticulum (ER) membrane-bound ribosomes, with the simultaneous primary glycosylation of envelope proteins. Gn and Gc glycoproteins are translocated to the Golgi complex, and new virus particles are formed by a budding process at smooth-surface vesicles in the Golgi and are liberated from the cell by exocytosis [98].

3 Origin, Evolution , and Classification of Hantaviruses

For several years, hantaviruses were only associated with rodents (Murinae and Cricetidae), with the exception only of Thottapalayam virus, which was isolated in 1964 from the Asian house shrew Suncus murinus [13]. From 2007 to present, more than 20 new hantaviruses were discovered in association with insectivore mammals such as shrews and moles (order Eulipotyphla; families Soricidae and Talpidae) [5, 29, 30, 43, 52, 99]. To add more complexity to the picture, recent findings report new hantaviruses associated with bats (order Chiroptera; families Vespertilionidae, Rhinolophidae, Nycteridae, and Hipposideridae) in Asia and Africa [4, 102, 113, 114]. Up to now, no human disease has been associated with these newfound hantaviruses.

Evolutive studies on the origin of placental mammals propose that the superorder Laurasiatheria (which include bats, shrews, and moles) diverged from Euarchontoglires (where order Rodentia is placed) at 100 million years ago [8, 68]. The discovery of new hantaviruses in such divergent reservoir hosts leads to revising the origin of the Hantavirus genus and the main forces driving its evolution.

Early studies on hantaviruses and their rodent hosts showed a high degree of congruence in their respective phylogenies , reinforcing the idea that coevolution between hosts and viruses was the main force influencing hantavirus evolution. Basically, each hantavirus was carried by a unique rodent species, and in turn, genetically similar viruses were hosted by closely related rodents. Incongruent topologies in phylogenies were explained by the occurrence of sporadic host switching [38, 82]. From 2007 to present, the finding of a growing number of new hantaviruses harbored by insectivores questioned this paradigm. Co-phylogenetic reconciliation analyses and estimations on the evolutionary rates showed that divergence times between hantavirus lineages from rodents, insectivores, and bats were far more recent than the divergence times between their respective hosts. Further, host switching events appeared more frequently than expected under a co-evolutionary theory. So, as a result of these approaches, the similarities between hantaviruses and their mammalian hosts phylogenies may be the result of preferential host switching followed by local adaptation instead of co-evolution [89, 90].

Nowadays the debate remains open. New phylogenetic approaches and more comprehensive analyses lead to reevaluating the role of co-divergence in hantavirus evolution. Sequence analyses of complete and partial L genes from all known rodent, insectivore, and bat hantaviruses together with phylogenetic fossil host hypothesis testing showed that mammals in the superorder Laurasiatheria could have been the potential hosts of ancestral hantaviruses at most basal tree nodes. According to these inferences, hantaviruses from Muridae and Cricetidae rodents appear as paraphyletic groups, originated by two independent host switches from hantaviruses carried by laurasiatherian mammals. Overall, the main hantavirus groups show typical systematics of co-speciation, where virus phylogeny primarily resembles the phylogeny of host mammals and only secondarily their geographic dispersion [83, 114, 116].

Another point of controversy is the criterion for species delimitation in hantaviruses. According to the International Committee on Taxonomy of Viruses (ICTV) , a hantavirus species should fulfill the following criteria: a hantavirus species should (i) occupy a unique ecological niche (i.e., a clear association of a new hantavirus with a different primary rodent reservoir species or subspecies), (ii) have at least a 7% difference in the amino acid sequences of the complete nucleocapsid (N) and glycoprotein precursor (GPC) proteins to all known species, (iii) show an at least fourfold difference in a two-way cross-neutralization test, and (iv) show the absence of genetic reassortment with other species in nature [81, 82].

For the majority of known hantaviruses it has been difficult to fulfill the four criteria. Genetic reassortment of closely related hantaviruses has been reported in vitro, but also in nature [34, 46, 92, 117]. Additionally, several South American hantaviruses have been detected in more than one rodent species, and the species involved may vary according to the geographic areas studied. As an example, the genetically related Juquitiba, Araucaria, and Itapúa hantaviruses were found in five different species (Oligoryzomys nigripes, Oxymycterus judex, Oxymycterus nasutus, Akodon montensis, and Akodon paranaensis) in South Brazil, Paraguay, and Uruguay [15, 19, 86]. This finding also raises difficulties in determining the primary reservoir host for a given hantavirus and the role of other sympatric rodents in maintaining the virus in the environment.

Another difficulty is the requirement to perform cross-neutralization test assays to define species, given that hantaviruses have proven to be very hard to isolate in tissue culture. The majority of the rodent- and insectivore-borne and all the bat-borne hantaviruses have not been isolated. In fact, most of them were identified through phylogenetic analyses on (mostly) partial sequences of the N, GPC, or L protein genes.

The last ICTV report for the Hantavirus genus recognizes only 24 species, 23 of which are rodent borne and 1 shrew borne, so the majority of the hantaviruses remain taxonomically unclassified [1]. Recently, another scheme for the demarcation of hantavirus species has been suggested. Maes et al. propose an amino acidic sequence distance >10% for N protein or >12% for GPC to limit species and an amino acidic sequence distance >24% for N protein or >32% for GPC to delimitate hantavirus groups [62].

4 Hantavirus Studies in Latin America

HCPS is a serious health problem in Latin America. Growing urbanization, together with the expansion of agriculture and cattle-breeding areas into natural ecosystems, has increased the chances of close contact between infected rodents and humans. Several studies have pointed out that habitat fragmentation caused by human activities tends to reduce rodent diversity, which in turn results in an increase of hantavirus prevalence in endemic areas [47, 58, 103, 104].

The rodent family Cricetidae is the second largest mammalian family (more than 500 species), including all the New World mice, and it is divided into three subfamilies: Sigmodontinae (predominantly South American), Neotominae (almost exclusively North American), and the Arvicolinae. All these groups are morphologically and ecologically diverse. Sigmodontinae rodents of South America present high diversity because of recent invasion of the continent followed by a rapid adaptive radiation [101]. In accordance with this diversity, many different hantavirus lineages were characterized in Latin America (Fig. 11.2); most of them are associated with Sigmodontinae rodents and its three main tribes: Akodontini, Oryzomyini, and Phyllotini.

Fig. 11.2
figure 2

Hantavirus lineages characterized in Latin America. Hantaviruses are transmitted directly to humans by small mammals , which are their natural reservoir. In general, each hantavirus has a unique rodent host. The majority of the Latin American hantaviruses are associated with rodents of the subfamily Sigmodontinae and its three main tribes: Akodontini, Oryzomyini, and Phyllotini

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The first HCPS outbreak reported in Latin America occurred in 1993. The three cases were diagnosed in the rural locality of Juquitiba, São Paulo State, Brazil. Further molecular studies identified the virus responsible for the outbreak; this new hantavirus was named Juquitiba [41, 110].

In 1994–1995, several outbreaks of HCPS were reported in Central and Southern Argentina. Genome amplification, sequencing, and phylogenetic analyses allowed the identification of two novel hantaviruses: Andes and Lechiguanas. Soon after that, rodent trapping in the likely places of exposure for human cases led to the identification of the long-tailed rice rat and the yellow pigmy rice rat (Oligoryzomys longicaudatus and O. flavescens, respectively) as the primary reservoir hosts for Andes and Lechiguanas hantavirus [50, 51, 55]. Isolation of the aforementioned hantavirus from rodent tissues and the molecular cloning and protein expression of Araucaria and Araraquara N protein were of capital importance to locally develop diagnostic tools. These techniques allowed detecting antibodies to autochthonous hantavirus in human and rodent samples with better sensitivity and specificity [50, 57, 77, 87]. From 1996 and on, outbreaks were reported in Argentina, Chile, Brazil, Uruguay, and Paraguay, and numerous field studies were carried out to determine the reservoir hosts in each geographic area [25, 40, 86, 88, 106].

Currently, almost all Latin American countries have reported HCPS cases or outbreaks. Countries with no reports on HCPS cases or rodent survey are Ecuador, Guyana, Surinam, Cuba, Nicaragua, El Salvador, Guatemala, and Belize. Since 1993, about 4000 accumulated cases have been reported in the Americas, and almost half of them occurred in Brazil [26].

Up to today, 28 hantavirus lineages are present in Latin America (Table 11.1). Most of them have been identified through genome amplification plus sequencing and phylogeny , based on viral S and/or M segments [10, 18, 27, 49, 69, 70, 85, 94, 96]. In turn, field studies allowed the identification of 14 rodent-only viral lineages that still have not been associated with human disease [16, 44, 54, 65, 72, 75, 95, 107, 112].

Table 11.1 Hantavirus lineages present in Latin America
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Except for Andes, Lechiguanas, and Maciel hantavirus, viral isolation was not achieved for these hantaviruses , hampering the determination of their taxonomic status according to the ICTV criteria. Despite this, availability of new sequence information on Latin American hantavirus lineages together with accurate rodent identification will contribute to clarifying the virus reservoir–host relationships and the epidemiology of this relevant zoonosis.

5 Pathogenesis and Implication on Treatment

Hantavirus infection induces serious microvascular leakage, and clinically, patients present with hemorrhages, hemoconcentration, and hypotension [35]. Hantavirus infections are associated with two diseases: hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) , which present similar pathological findings, being the consequence of both innate and adaptive and humoral and cellular immune mechanisms, but the contribution of these factors to disease development remains indeterminate [60].

Previous studies showed that CD4+ and CD8+ T-cell responses to hantavirus infection in humans are associated with immunoprotection, including the magnitude of the cellular immune response and the frequency of the CD8+ T-cell response, which were much higher in patients with mild/moderate HFRS than in those with severe/critical disease at the acute stage of the disease [53, 105]. Moreover, Hantavirus glycoprotein produces a strong CD4+ T-cell response, which elicits greater defense against the infection and is inversely correlated with plasma viral load and disease outcome [56]. Conversely, a report has shown the importance of monocyte activation to hantavirus infection . Increased monocyte counts have been positively correlated with elevated plasma-soluble CD14 levels, as well as increased tumor necrosis factor (TNF)-α and soluble CD163 levels, which are associated with severe acute kidney injury in HFRS patients [105].

Primarily, it was supposed that the endothelial cell (EC) dysfunction demonstrated in HFRS and HCPS patients was related to a strong cellular immune response, elicited by cytotoxic CD8+ T and NK cells, although no clear endothelial cell damage has been clearly observed. Recent studies in ANDV-infected Syrian hamsters showed that depletion of T cells did not impact disease onset or outcome of HCPS [33, 84]. The infected EC is probably protected from cytotoxic lymphocyte-mediated killing through an inhibition of apoptosis induction in infected cells in combination with an increased expression of HLA class [12]. Contrasting with previous findings, in a recently reported macaque model for Sin Nombre hantavirus disease, an association between the expansion of T-lymphocyte-activated and disease severity was observed [91]. The importance and function of T cells activated in human hantavirus infections are still poorly understood, and further studies are needed.

The increased vascular permeability occurs without any cytopathic evidence, suggesting that the pathogenesis is associated with immunopathological mechanisms. It results from the interaction between EC surface-receptor and cytoplasmic signaling responses such as pro-inflammatory cytokine: interleukin (IL)-6, IL-8, IL-33, interferon (IFN)-γ, and tumor necrosis factor-α (TNF-α) secreted by activated innate immune cells, which are upregulated in hantavirus infections [9, 105, 115], as well as EC interactions with immune cells [17].

In hantavirus acute infection, a significant upregulation of cytokines has been shown, some associated with leukocyte migration and repair of lung tissue, and other factors linked to increasing the endothelial monolayer permeability that facilitates leukocyte transendothelial migration. In addition, a downregulation of cytokines also can be observed and has been associated with platelet numbers, reduction, and dysfunction [73].

Endothelial cells (ECs) are fundamental to preserve the fluid barrier and have a vital role in maintaining the balance between tissues and vascular compartments, through a complex system of surface–receptor interactions. Microvascular and lymphatic EC surface receptors and the endothelial glycocalyx are keys to fluid management and vascular homeostasis [6]. Furthermore, lymphatic tissues and lymphatic endothelial cells (LECs) are exclusively regulated by cell-surface receptors and are responsible for edema regulation by clearing fluid from tissues, and LECs are sentinel antigen-presenting cells that determine tolerance and viral clearance [17]. Studies showed that hantavirus infection is associated with LEC dysfunction and, consequently, alteration of liquid pulmonary clearance in patients with HCPS [61].

Nonpathogenic hantaviruses use αvβ1 integrin receptors, whereas pathogenic hantavirus binds and inactivates αvβ3 integrin conformers, which usually form complexes with VEGF receptors, leading to dysregulation of VEGF-induced permeability; this may lead to impairment of vascular endothelial cadherin expression and subsequent loss of endothelial barrier function [17, 63].

Currently, there are no antiviral drugs or immunotherapeutic or effective post-exposure prophylactics available for hantavirus infection [3, 32], and only supportive interventions, such as early diagnosis and aggressive support in a cardiac and pulmonary intensive care unit, are recommended [42]. In vitro studies have demonstrated ribavirin as a drug capable of preventing hantavirus infection, including in vivo studies carried out in golden Syrian hamsters which showed that drug administration before or up to 3 days after infection was able to prevent infection in all animals [97]. However, despite randomized studies conducted in HRFS individuals who showed response to ribavirin when it was administrated up to 7 days of infection, in HCPS patients no benefit of treatment could be observed, probably in consequence of the long incubation period of the disease, which can last up to 5 weeks [63].

The use of molecules to block receptor- and pathway-specific regulation of VEGFR2 or downstream signaling pathway responses that control EC barrier functions has also been evaluated as a therapeutic intervention. However, the effectiveness of this approach in patients who are already symptomatic remains to be determined [63].

Based on the immune response exacerbation observed during the course of hantavirus infection, studies using corticosteroids to prevent severe HCPS in acute disease were performed, but no benefits were demonstrated and it has not been recommended [111]. An alternative approach to HCPS treatment was the passive administration of neutralizing antibodies by serum transfusion from convalescent patients. Preliminary results carried out in Chile have indicated a reduced mortality in the treated patients [111], although the final evaluation of this study is not yet available.

In conclusion, during hantavirus infection, several factors contribute simultaneously, culminating with vascular leakage, and the underlying mechanisms associated with disease pathogenesis are not completely understood. Prospective studies with animal models are crucial to evaluate individual immune cell types involved in the disease process. Knowing their different mechanisms of action is fundamental to the development of drugs that will act on specific targets, seeking to reduce disease severity and, consequently, to reduce mortality rates.

6 Prevention

Small interfering RNA (siRNA) against Andes virus (ANDV) genes has been tested as a potential antiviral strategy [14]. Although with promising results, such therapy would take years to be available for human use.

A phase III inactivated viral vaccine against the Old World hantavirus Hantaan has been tested in healthy adults, but the results of the plaque reduction neutralization test (PRNT) after a three-dose vaccination are modest [100]. Some DNA vaccines were developed and tested in animal models, but the effects are still pending [39, 67].

Despite the long time since the association of hantavirus infection and HFRS and HCPS in humans has been known, no specific treatment and prophylaxis have been obtained, although efforts have been committed. Several aspects of hantavirus biology and pathogenesis remain unclear . As an example, it has been assumed that the primary target cells during HCPS infection were human alveolar macrophages. Recently, Hammerbeck and colleagues have demonstrated that depletion of alveolar macrophages in golden Syrian hamsters does not prevent hantavirus disease [32]. These findings open new perspectives on hantavirus infection strategies on human beings.