Pathogenicity and virulence of Rodent-Borne Orthohantaviruses

ABSTRACT

The Orthohantavirus genus in the family Hantaviridae includes viruses that cause zoonotic diseases in humans known as hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Exposure of humans to these viruses occurs through inhalation of aerosols of urine, feces, and saliva of rodents, who are the reservoirs for pathogenic orthohantaviruses. The clinical courses of HFRS and HPS are characterized by initial high fever and body pain with severe HFRS or HPS leading to renal failure, pulmonary failure, or both. The underlying pathogenic mechanism of both diseases includes vascular dysregulation leading to vessel leakage and shock.

Introduction

Orthohantaviruses have been detected in many animal species including rodents, shrews, moles, bats, reptiles, and fish; however, only certain rodent-borne viruses in the Orthohantavirus genus of the family Hantaviridae are currently known to cause disease in humans. The occurrence of orthohantavirus-associated disease in humans correlates with the geographic distribution of the natural rodent hosts for these pathogenic viruses [1–5]. Orthohantavirus-infected rodent hosts display no obvious disease symptoms as compared to uninfected rodents and prolonged secretion of infectious virus has been reported for some persistently infected rodents despite the presence of high levels of neutralizing antibodies [6] (and as summarized in [7]). Spillover of virus from natural hosts to other rodent species is common but usually results in clearance of the virus [8–10]. Transmission of pathogenic orthohantaviruses to humans is also almost always a dead-end spillover event, although infrequent person-to-person transmission of a South American orthohantavirus, Andes virus (ANDV), has been reported [11–13].

Two orthohantavirus-associated disease syndromes are recognized: hemorrhagic fever with renal syndrome (HFRS) and hantavirus (cardio) pulmonary syndrome (HPS). Pathogenic orthohantaviruses are carried by rodents in the subfamilies Murinae (family Muridae) and Arvicolinae (family Cricetidae) and the subfamilies Sigmodontinae (Cricetidae) and Neotominae (Cricetidae). For HFRS, the fatality rate ranges from <1% to as high as 12% depending on which orthohantavirus species is causing the disease. Compared to HFRS, HPS has a much higher case fatality rate of up to 35% in some countries. However, it is difficult to determine the case fatality for each HPS-causing orthohantavirus species due to the rarity of HPS cases in some countries such as Paraguay [14,15].

At least five species of orthohantaviruses cause HFRS and at least seven species cause HPS [16]. The diversity and widespread distribution of orthohantaviruses correlates with the migration of rodents carrying the orthohantaviruses and their evolution over time within their rodent hosts. For example, distinct species and strains of the HFRS-associated orthohantaviruses occur across Europe and Asia, with the prototypic orthohantavirus, Hantaan virus (HTNV), residing in Apodemus agrarius east of the Ural Mountains, in China and Korea. A second species, Dobrava virus (DOBV), harbored by Apodemus agrarius and Apodemus flavicollis, causes HFRS in Serbia, Croatia, Slovenia, Bosnia-Herzegovina, Hungary, Greece, Lithuania, Czech Republic, Estonia, and Albania [17–24]. In Europe, a third species, Puumala virus (PUUV) causes a less lethal form of HFRS, sometimes referred to as nephropathia epidemica (NE) [4,25–28]. The primary rodent vector for PUUV is the bank vole, Myodes glareolus (previously known as Clethrionomys glareolus) which occurs throughout Europe [26,29]. A fourth orthohantavirus species able to cause HFRS is Seoul virus (SEOV) harbored by Rattus rattus and Rattus norvegicus. Although Rattus are old world rodents, they have migrated with travel and commerce throughout the world over the past ~500 years leading to the distribution of SEOV in rats, world-wide. Although it is rarely associated with HFRS cases, Tula virus, (TULV), found in common voles (Microtus arvalis) [30], is widespread throughout Eurasia. TULV infections may be underreported due to the strong cross-reactivity of antibodies against PUUV and TULV [31–33].

Prototype HPS-causing orthohantaviruses identified in the Americas include ANDV, Bayou virus (BAYV), Black Creek Canal virus (BCCV), El Moro Canyon virus (ELMCV), Choclo virus (CHOV), Laguna Negra virus (LANV) and Sin Nombre virus (SNV). In North America, the primary rodent-hosts carrying HPS-causing orthohantaviruses include the deermouse (Peromyscus maniculatus) that harbors SNV, the cotton rat (Sigmodon hispidus) that harbors BCCV, the rice rat (Oryzomys palustris) that harbors BAYV, and the white-footed mouse (Peromyscus leucopus) that harbors New York virus (a strain of SNV). In Latin America, HPS-causing orthohantaviruses have been identified in every country. This is not surprising given the immense diversity of the more than 400 species of Sigmodontinae rodents in Central and South America. Four of the 17 tribes within the Sigmodontinae harbor hantaviruses, the Sigmodontini, Oryzomyini, Phyllotini and Akodontini [34]. In addition to BAYV mentioned above, other Oryzomyalia-borne hantaviruses are clearly associated with outbreaks of HPS disease such as Choclo virus (Oligoryzomys fulvescens) in Panama, and LANV in vesper mice (Calomys laucha) in Paraguay and Bolivia [35–37]. Juquitiba virus and Juquitiba-like viruses have been identified in Uruguay [38], Brazil [39,40], and Paraguay [41] harbored by Oligoryzomys nigripes and are also associated with HPS. In the Southern cone, ANDV harbored by the long-tailed pygmy rice rats (Oligoryzomys longicaudatus) are mostly commonly associated with HPS; however, numerous lineages of closely related AND-like hantaviruses have been discovered in Argentina including Pergamino virus (PRGV) found in Akodon azarae, Maciel virus (MACV) found in Necromys benefactus, Lechiguanas virus (LECHV) found in Oligoryzomys flavescens, Bermejo virus (BMJV) found in Oligoryzomys chacoensis, and others [42–44].

Outbreaks of HFRS and HPS have been noted across the globe since the discovery of these diseases (Table 1). While the outbreaks of the disease drew the initial recognition to HFRS and HPS, many countries have notable endemic disease such as Argentina, Brazil, Chile, China, Croatia, Slovenia, Sweden, Russia, and the USA. Emerging orthohantaviruses pose a significant threat to global public health due to their potential to cause significant morbidity and fatal diseases, their wide geographic distribution, and their ability to spread from rodents to humans. As climate change, deforestation, and urbanization alter rodent habitats and increase their contact with human populations, the risk of outbreaks grows. Additionally, the lack of widely available vaccines and the challenge of early diagnosis due to nonspecific initial symptoms exacerbate the public health burden. Strengthening surveillance, developing effective treatments and vaccines, and promoting awareness about preventive measures are crucial steps in appreciating disease incidence and mitigating the global threat.

Table 1.

Selected outbreaks of HFRS or HPS and associated mortality.

Yearsa	Virus	Reservoir	Outbreak	Disease	Mortalityb	Reference
1950–4	Hantaan virus	Apodemus agrarius	Korean War	HFRS	7%	[45]
1960	Seoul virus	Rattus norvegicus	Japan	HFRS	1.7%	[46]
1967	Dobrava-Belgrade Virus	Apodemus flavicollis	Bosnia-Herzegovina	HFRS	2.6%	[46]
1984–5	Puumala virus	Myodes glareolus	Sweden	NE	0.7%	[47]
1991–2	Dobrava-Belgrade Virus	Apodemus flavicollis	Russia	HFRS	0%	[48]
1993	Sin nombre virus	Peromyscus maniculatus	United States, Canada	HPS	78%	[49]
1993–6	Andes virus	Oligoryzomys longicaudatus	Argentina	HPS	52%	[50]
1995–6	Laguna Negra virus	Calomys laucha	Paraguay	HPS	9%	[51]
1997–8	Andes virus	Oligoryzomys longicaudatus	Chile	HPS	54%	[52]
1999–2000	Choclo virus	Oligoryzomys fulvescens	Panama	HPS	0% confirmed 25% suspected cases	[53]
2002	Laguna Negra virus	Calomys laucha	Bolivia	HPS	unknown	[54]
2005–10	Puumala virus Dobrava-Belgrade and others	Myodes glareolus, Apodemus flavicollis, A. agrarius, others	Europe	HFRS	0.1–5%	[55]
2007	Puumala virus	Myodes glareolus	Sweden	NE	0.5%	[56]
2009–10	Castelo dos Sonhos (suspected)	Oligoryzomys utiaritensisrats	Brazil	HPS	10%	[57]
2012	Sin nombre virus	Peromyscus maniculatus	Yosemite, USA	HPS	30%	[58]
2018	Unknown	Unknown	Bolivia	HPS	15%	[59]
2018–9	Andes virus	Oligoryzomys longicaudatus *Person to Person	Argentina	HPS	32%	[12]

^aOutbreak duration; ^b approximate mortalities in humans if known during outbreak period.

In this review, we will introduce the reader to the taxonomy and replication of orthohantaviruses and summarize what is currently known about the epidemiology, clinical history, and disease for HFRS and HPS. We also describe cellular and molecular mechanisms hypothesized to contribute to pathogenesis and clinical manifestations. Finally, we review animal models, diagnostics, and barriers to current treatment. To the extent possible, we highlight notable gaps in the field that if addressed would advance our understanding of the biology of orthohantaviruses and their treatment.

Taxonomy

Taxonomy provides a standardized means to keep track of an ever-expanding knowledge base of viruses. For orthohantaviruses, taxonomic designations have radically changed since 1987, when the International Committee on Taxonomy of Viruses accepted a proposal to add the Hantavirus genus to the Bunyaviridae family. The discovery of hundreds of new orthohantaviruses after that contributed to the family being promoted first to the order Bunyavirales, and then to the class Bunyaviricetes [60] which resulted in the Hantavirus genus being reclassified as the family Hantaviridae. The family Hantaviridae was further divided into four subfamilies including Mammantavirinae, Repantavirinae, Actantavirinae and Agantavirinae. In addition to the Orthohantavirus genus, the subfamily mammantavirinae includes the genera Loanvirus, Mobatvirus, and Thottimvirus, which incorporate viruses detected in bats, moles, shrews, and other rodents [61,62].

For most orthohantaviruses, phylogenetic relationships (and taxonomic classifications) were historically deduced from partial or complete nucleotide sequences of the S and/or M segments as the L-segment is more difficult to detect. Phylogenetic analyses of the partial genomes of rodent-borne orthohantaviruses support a taxonomic organization that largely correlates with the specific rodent subfamilies that carry these viruses; i.e. Murinae (old world rats, mice), Arvicolinae (e.g. northern hemisphere voles, lemmings), Neotominae (mostly North American, new-world rats, mice) and Sigmodontinae (mostly South American, new-world rats, mice) [42,63,64]. Thus, gene sequences of known or novel orthohantaviruses detected in HFRS or HPS patients can be used to predict the rodent subfamily likely to carry that orthohantavirus species. It is not possible, however, to predict whether a rodent-borne orthohantavirus will cause human disease. Likewise, it is not currently possible to predict disease severity from gene sequences of the virus.

A taxonomic change in 2023 was implemented for viruses in the family Hantaviridae, which requires that all three genome segments’ sequences be considered to assign a taxonomic designation of “species” [62,65]. This has led to at least short-term disruptions to the nomenclature commonly used for known orthohantaviruses. For example, the complete genome sequences of LANV are not available, but they were available for the related Rio Mamoré virus (RIOMV). Consequently, the previously proposed species designation Orthohantavirus negraense was changed to Orthohantavirus mamorense. Of course, the viruses still exist, so only the official classification name of the species has changed. As more complete genome sequences are obtained and more viruses are discovered, their taxonomic designations will continue to change as well.

It is worth noting that the classification of rodents harboring the pathogenic orthohantaviruses is also in flux [66]. For example, genetic studies have indicated that the North American deermouse, P. Maniculatus, is not a single species, but rather is part of a species complex (reviewed in [67]). The Mammal Diversity Database v1.13, released 13 July 2024, lists four species of the P. Maniculatus complex with distinct geographical distributions that could be hosts for pathogenic orthohantaviruses. As more sampling and genetic analysis of rodents and the viruses they carry are completed, the taxonomic pictures of both will undoubtedly change.

Replication of orthohantaviruses

Morphology and genome organization

Orthohantavirus virions are asymmetric, pleiomorphic particles, and until recently were thought to have an average diameter of approximately 80–154 nm. Cryoelectron microscopy (Cryo-EM) and Cryo-EM tomography studies of sucrose gradient purified HTNV virions show the particles range in size from 120 to 154 nm ⁶⁸. Structural studies of purified virions revealed a surface structure composed of an unusual square, grid-like pattern distinct from other bunyaviruses and a lack of icosahedral symmetry typical of many other virus families [68,69]. Surprisingly, CryoEM structures show patches of open membrane [68–70]. The square spike on the outer surface contains the glycoprotein (GP) projections, which extend ~12 nm from the lipid bilayer and comprise 4 molecules of Gn and Gc [68].

Each virion contains three, negative-sense, single-stranded RNA gene segments designated as large (L), medium (M) and small (S) which, respectively, encode an RNA-dependent RNA polymerase (L), the Gn and Gc proteins embedded in the viral envelope, and a nucleocapsid protein (N) [71–73]. Structural and molecular Virology studies indicate that the L, M, and S genome segments complex with N to form three separate ribonucleoprotein (RNP) structures. Base pairing of complementary terminal nucleotides on each segment results in noncovalently closed circular RNPs [74,75].

Replication cycle

The replication cycle of orthohantaviruses has seven key steps, attachment, entry, transcriptions, translation, genome replication, assembly, and egress. Following infection of the lung, the virus first enters epithelial cells and then traverses the interstitial space or through the infection of patrolling immune cells such as macrophages to infect microvascular endothelial cells (Figure 1) [2]. Entry begins when the virus GP binds to one or more host cell receptors that allow for viral attachment. Early research showed that orthohantaviruses selectively bind to the host β₁ or β₃ integrins [76–78]. Pathogenic orthohantaviruses such as ANDV, HTNV, and SNV attach through α_vβ₃ and α_IIbβ₃ and the nonpathogenic orthohantavirus, Prospect Hill virus (PHV), binds to β₁ integrins. Decay accelerating receptor (DAF/CD55) has also been identified as a receptor or co-receptor for orthohantavirus attachment and entry [79,80]. A third membrane protein, protocadherin-1 (PCDH1) serves as a receptor for the new-world orthohantaviruses but not for the old-world orthohantaviruses [81]. The importance of PCDH1 during attachment and entry of new-world orthohantaviruses is supported by receptor knockout studies, where the depletion of β1, β3 integrins, and/or DAF did not affect the infectivity of old-world and new-world orthohantaviruses while the PCDH1 KO resulted in lowered the infectivity of new-world orthohantaviruses [82].

Figure 1.

Replication cycle of orthohantaviruses.

The replication cycle of orthohantaviruses comprises seven steps as follows. (1) Attachment of viral Gn proteins to a host cell receptor such as β-integrins, DAF, and/or PCDH1. (2) Entry through clathrin-mediated or clathrin-independent endocytosis in a pH-dependent manner. (3) Transcription of viral mRNAs via the viral RdRp requires a primer from the host mRNA (cap snatching- not shown). (4) Translation of viral proteins on free ribosomes (L and S segment mRNA) produce RdRp and N protein, respectively, and cotranslation of the M-segment on ER-bound ribosomes to produce GnGc. (5) Replication of the viral genome (vRNA) replication using a cRNA template, (6) Further maturation and assembly of virions from the cis-Golgi trans-Golgi and budding from trans-Golgi. (7) Egress of virus via exocytosis.

Entry of the virus following viral attachment occurs through various mechanisms of endocytosis. The route of entry differs for old- and new-world orthohantaviruses [83]. HTNV enters through clathrin-dependent receptor-mediated endocytosis or micropinocytosis, depending on the cell type [84,85]. ANDV, on the other hand, uses various mechanisms ranging from clathrin-dependent and -independent, dynamin- and cholesterol-dependent pathways as well as micropinocytosis [83,86]. The route of entry of large pleomorphic particles [70] is yet to be confirmed but one may assume that the larger particles enter only through macropinocytosis.

As orthohantavirus glycoproteins are class II fusion proteins their fusogenic activity is pH dependent. The lowering of pH as the endosome traffics throughout the cell from early to late endosome activates the fusogenic activity of the GP and may cause conformational changes to the GP that allow for membrane fusion and virus uncoating [87,88]. The fusogenic activity is controlled by the “head” of Gn that is released at low pH levels which drives conformation changes to domain II of Gc that allows fusion to occur [89]. The pH at which membrane fusion occurs differs between the old and new world orthohantaviruses with the pH threshold reported at 6.3 for HTNV [90,91, 6.6–6.8] for SEOV [92] and 5.8 for ANDV [93]. Following fusion, the three viral RNA (vRNA) segments, the RNA-dependent RNA polymerase, (L protein, RdRP) and N proteins are released into the cytoplasm and the RdRp initiates replication of complementary RNA (cRNA) and the three viral mRNAs. Transcription of viral mRNAs occurs via a prime-and-realign mechanism [94] that begins when the RdRp and an unidentified host protein [95] cleaves 12–15 nt of the 5´ end of the host mRNAs that have a 7-methylguanosine cap structure, a process referred to as cap snatching. The short host-derived RNA primer aligns to a cysteine residue at the 3´ ends of viral genomic RNAs and initiates synthesis of a short stretch of nucleotides. The RdRp slips backwards and realigns nascent RNA to genomic viral RNA. Following the slippage and realignment, final elongation occurs to form complete viral mRNAs which are not encapsidated by the N protein. The viral mRNAs are not polyadenylated; however, one study found that the M segment mRNA of SNV was polyadenylated [96]. The gap in our understanding of viral RNA composition and modifications in cell culture and in wild rodent populations and how this translates into persistent infections is one area of research that could be further explored with the advancements in sequencing technology.

Viral cRNA replication begins by generation of a complementary or antigenomic RNA (cRNA) that is also complexed with N protein. This cRNA may use a similar prime and realign mechanism, in which a guanosine triphosphate that aligns to the cysteine residue but after final elongation the overhanging guanosine triphosphate is cleaved leaving a uridine monophosphate terminal nucleotide [94,96]. Base pairing of the characteristic terminal nucleotides may result in panhandle structure formation.

The N protein has numerous functions in the viral replication cycle such as encapsidation of vRNA and cRNA, cap snatching and translation. Purified N protein from HTNV or SNV binds specifically to synthetic panhandle structures of the same orthohantavirus and may facilitate encapsidation of the vRNA and cRNA [97–99]. P bodies accumulate N protein bound to caps and are thought to be involved in cap snatching for orthohantavirus mRNAs [100]. To date, there are no in vitro biochemical assays to confirm the role of N in transcriptional mechanisms, and the lack of biochemical assays to study replication remains an important gap in the field. In addition to binding of viral vRNA and cRNA, the N protein supports translation of viral mRNA over host mRNA by mimicking host’s cap-binding complex of eukaryotic initiation factor 4F (eIF4F) which is responsible for host mRNA translation [101]. The dearth of biochemical assays to define the mechanisms of replication remains a fundamental gap in hantavirus research.

Following transcription, L- and S-segment mRNAs are translated by free ribosomes and M-segment mRNAs by ribosomes on the rough endoplasmic reticulum [102]. Translated HTNV N protein traffics to the ER-Golgi intermediate complex (ERGIC) through microtubule dynein for virus assembly [103]. In a study of the intracellular trafficking of PUUV N using fluorescently tagged PUUV N fusion proteins [104], the PUUV N colocalized with cytoskeleton components including vimentin, actin, and P-bodies. Cotranslation of the M-segment mRNAs on the rough endoplasmic reticulum produces a precursor glycoprotein that is cleaved in the ER at a conserved amino acid motif, WAASA, which for HTNV is located at amino acids 264–268 [105]. Cleaved proteins undergo further post-translation modification through N- and O-linked glycosylation [106,107].

Virus assembly occurs through an unknown mechanism in which the RNP complex interacts with the glycoproteins and buds into the Golgi to form the enveloped virions that are transported in vesicles to the cell’s plasma membrane, where they fuse with the membrane and release the virions through exocytosis [108,109]. While virus assembly and egress occur through the Golgi, it has been proposed that new-world orthohantaviruses sometimes are able to assemble at the plasma membrane [110,111]. Future research that characterizes the molecular details of assembly and egress are essential to the advancement in biological understanding and will give important critical information for the development of new therapeutics and vaccines.

Ecology and epidemiology

Hemorrhagic fever with renal syndrome

HTNV and SEOV are responsible for most cases of HFRS in Asia. Case fatality rates for HTNV in China average around 2.89% while South Korea HRFS case fatalities range from1–2% [3,112,113]. Fatal cases of SEOV are usually less than 1% [3,112,113]. While only a few acute cases of HFRS caused by SEOV have been identified in the New World, it is likely that in certain parts of the world, including the USA, renal disease [114] caused by these viruses, remains completely undiagnosed [115–117].

HFRS caused by HTNV occurs in rural environments where Apodemus field mice are common and has seasonal peaks of disease (Spring and Autumn) [113]. These peaks correlate with agricultural processes and increases in rodent activity [118]. Increase of HFRS cases has been long associated with military field activities, such as digging, which unearths Apodemus burrows [119–123].

Like HTNV in Asia, DOBV in Europe causes severe HFRS with a case fatality rate approaching 10% although the four genotypes of DOBV appear to cause differing disease severity. The case fatality rate reported is highest for the DOBV genotype (10–12%), followed by Sochi virus ( >6%), Kurkino virus (0.3–9%) and Saaremaa virus ( <1%) [3,124]. Surveillance in Bulgaria of patients with acute undifferentiated febrile illness suggests that DOBV and PUUV may cause illness without hemorrhages or renal impairment [125].

HFRS caused by SEOV infection mainly occurs in urban settings where rats are present. Because SEOV-carrying rats are found in domesticated settings, there is less seasonal occurrence of or climactic impact on SEOV-associated HFRS as compared to HFRS caused by field rodent associated orthohantaviruses. One of the primary carriers of SEOV, the brown rat (R. norvegicus) cohabitates with humans around the world and is not impacted by climatic factors to the extent that rodents living in nature are [126]. Although SEOV has historically been thought to have highly conserved genes [127], the results of a 2023 phylogenetic and bioinformatics study of 80 complete genomes of SEOV and 146 complete genomes of HTNV indicated that SEOV M and S segments have higher evolutionary rates and rapidly increasing genetic diversity as compared to HTNV. The SEOV L segment as well as all three genome segments of HTNV displayed slowly decreasing relative genetic diversity over the past 60 years [128].

By purposeful selective breeding, a domesticated subspecies of the brown rat was derived (R. norvegicus domestica). This subspecies includes both laboratory rats used for research, as well as pet rats known as fancy rats. HFRS caused by infection with SEOV in fancy rat breeders or owners has been reported in the UK, Canada, the USA, France, and the Netherlands [129–131]. Following several such cases of acute kidney injury in England, a seroprevalence study was conducted in 2013–2014 in 844 individuals with exposure to domesticated or wild rats. The study found that orthohantavirus seroprevalence among the pet fancy rat owner group was ~ 34% compared with ~ 3% in a control group and ~ 2% with occupational exposure to wild rats. Phylogenetic analysis of SEOV gene sequences derived from rats associated with these fancy rat-related disease outbreaks revealed a monophyletic clade distinct from SEOV sequences recovered from wild rodents in Europe and the Americas. The data suggest that SEOV was not introduced in separate exposures of breeding colonies to wild rats, but instead a single introduction of SEOV into a rat breeding colony resulted in persistent infection that was then expanded by trading of animals among ratteries [132].

Outbreaks of PUUV in Europe have been associated with an increase in natural rodent population due to extrinsic factors and climate occurrences that increase food sources [133]. The prevalence of illness associated with PUUV is very high in Northern Europe with an incidence of 20 cases per 100,000 people per year [134,135]; although in some years the number of cases has been 10-fold higher [56]. The case fatality of PUUV-associated HFRS is low showing 0.5% mortality in some regions [136]. While renal failure is common, hemorrhagic manifestations are mild and only associated with a third of the cases [137]. PUUV has a high genetic variability in its endemic areas [138,139], which have been shown to correspond to at least eight lineages [140].

In 2006, the Sangassou virus (SANGV) was discovered in an African wood mouse (Hylomyscus simus) in Guinea [141]. Examination of patients in Sangassou village with fever of unknown origin detected antibodies in 4.4% of people [142]. Additionally, population surveillance in the area identified a seroprevalence rate of 1.2% against SANGV [142]. SANGV appears to be genetically distinct from other orthohantaviruses, but phylogenetic analysis of genome segments revealed that it is most closely related to the Dobrava–Belgrade strain [143]. Additionally, RNA was detected in a Therese’s shrew (Crocidura theresae) and led to classification of the novel virus, Tanganya (TNGV) [144].

Trapping and screening small mammals in other regions of Africa identified orthohantavirus RNA in the Cape pipistrelle bat (Neoromicia capensis) in Ethiopia and an African wood mouse (Hylomyscus endorobae) from Kenya [145]. In the Kenyan Rift Valley region, Somali shrews (Crocidura somalica) were also found to harbor orthohantavirus RNA [146]. Amino acid analysis determined these viruses had homology to other orthohantaviruses found in shrews such as TNGV. Other shrew-borne orthohantaviruses identified include Azagny virus (AZGV), detected in a West African pygmy shrew (Crocidura obscurior) from Côte d’Ivoire [147] as well as the Kilimanjaro virus (KMJV) and Uluguru virus (ULUV), both found in the Kilimanjaro mouse shrew (Myosorex) [147].

Although the presence of HFRS is not commonly recognized in West or Central Africa, the presence of SANGV antibodies raises concerns about the potential to cause human disease. The similarity of symptoms between HFRS and other febrile illnesses may lead to underdiagnosis or misdiagnosis. Further research is essential to assess the clinical significance, the full extent of their distribution, potential for human disease, and public health impact. Ongoing surveillance and studies are crucial to enhance understanding and inform preventive measures against these emerging pathogens.

Hantavirus pulmonary syndrome

Cases of HPS were first recognized in 1993, when two young, healthy adults living in the Navajo Nation fell ill and died from an unexplained adult respiratory distress syndrome (ARDS) [148]. Unexplained deaths are reported to the Office of the Medical Investigator (OMI) in New Mexico and between the OMI and the Indian Health Service, it was quickly recognized there were at least five cases of ARDS. The outbreak led to collaborative investigations by state health departments in Arizona, Colorado, New Mexico, and Utah, the Indian Health Service, the OMI, the University of New Mexico, and the CDC, with the assistance of the Navajo Nation Division of Health to identify cases, rodent reservoirs, and develop diagnostic and treatment approaches. Within a year’s time, the CDC identified the causative agent as SNV harbored by P. maniculatus [149], a new world rodent species. Other groups also reported the identification of SNV [150] and SNV-like viruses from patients and a number of other rodent reservoirs for related viruses (e.g. BAYV, BCCV) in the USA and Canada [150–157]. The ability of these SNV-like viruses to reassort their gene segments in nature suggested the potential for the emergence of new orthohantaviruses [158].

In the USA, HPS cases have been reported to the Nationally Notifiable Disease Surveillance System since 1995. The average case fatality of SNV in the USA is 35% (1993–2012) with a total of 833 cases as of 2020 [159]. Compared to HFRS, HPS progresses rapidly in patients, resulting in noncardiogenic pulmonary edema, respiratory failure, and shock [160–162]. In the USA, seroprevalence studies show that few persons, even those with elevated risk (e.g. mammalogists, agricultural workers), show prior exposure to orthohantaviruses [163–169]. These findings suggest that SNV rarely, if ever, causes subclinical disease; in contrast to PUUV in Northern Europe [170], and orthohantaviruses endemic to Paraguay [171] and Panama [172].

Just 2 years after the outbreak in North America, two more orthohantaviruses, ANDV in Argentina and Chile [52,173,174], and LANV in Paraguay were associated with geographically localized outbreaks of HPS [35,51]. In Paraguay, the outbreak was associated with those living in the agricultural communities within the Chaco in the western part of the country. In contrast to the severity of disease and high mortality (40–50%) caused by ANDV, the disease caused by LANV showed a lower mortality ( <15%) [175]. In the third outbreak in El Bolson, Argentina in 1996 [50], one physician in Buenos Aires fell ill 27 days after taking care of an HPS patient [176]. This was the first recognition that these viruses such as ANDV may cause person-to-person transmission. Since that report, studies have shown that person-to-person transmission can occur between sexual partners, persons who sleep in the same bed or room of index patients with ANDV infection, or sustained contact during travel (e.g. on a bus) [11,176–178]. However, person-to-person transmission has been limited geographically to Chile and Argentina [12,13,179].

Following these initial outbreaks in the Americas, cases of HPS were quickly recognized in many other countries in Latin American due to clusters of cases in focal geographical regions (Table 1). In Brazil, HPS cases have been associated with several different species or genotypes of orthohantaviruses; e.g. Araraquara (ARQV), Juquitiba-like/Araucária virus, LANV-like viruses, Castelo dos Sonhos virus, and Anajatuba virus [180–183]. From the first report of the disease in Brazil in 1993 to 2019, about 2100 cases of HPS have been reported, with a lethality of approximately 39% [184]. In certain parts of Brazil, exposure to rodent-borne orthohantaviruses are high as noted in a serological survey carried out in the city of Jardinópolis in which 14.3% of the population had antibodies to orthohantaviruses [185]. It is not clear how many infections resulted in asymptomatic or mild disease.

Clinical manifestations of HFRS and HPS pathogenesis

Orthohantavirus disease includes a spectrum of vascular-leak syndromes in humans, ranging from proteinuria to pulmonary edema and hemorrhage [186]. Despite apparent differences in their pathogenesis and clinical evolution, all the hemorrhagic fever causing viruses (e.g. Crimean Congo hemorrhagic fever virus (CCHFV), Lassa fever virus) show vascular involvement and can induce vascular injury leading to thrombosis, hemorrhage, and organ failure. The clinical manifestations of Leptospira spp., and CCHFV have many symptoms in common with orthohantavirus infections (Table 2). In regions of the world where these pathogens cocirculate, it is critical to rule out these infections during clinical diagnosis. For example, bacterial infections, such as leptospirosis, can be confused with old-world hantavirus infections. This is exemplified by a 2009 report of an orthohantavirus-infected 31-year-old male resident of Tbilisi, Georgia, who presented with generalized symptoms of an acute febrile disease. Differential diagnosis included other infectious and noninfectious causes of renal failure including leptospirosis, but assay of antibodies against a panel of pathogen antigens showed reactivity only with the orthohantavirus antigens [187]. Likewise, other hemorrhagic fever viruses have overlapping symptoms with HFRS. For example, in one study of 28 patients with CCHF, the onset of illness was extremely sudden, with severe headache frequently accompanied by dizziness, neck pain and stiffness, sore eyes, photophobia, and general myalgia and malaise with intense backache or leg pains, fever, rigors, and chills [188]. In one-half of the patients, nausea, sore throat, vomiting and nonlocalized abdominal pain occurred [188]. In patients with HFRS, many of these symptoms are also noted [28,42]. Similarly, in Latin American countries with HPS and epidemics of leptospirosis, dengue, yellow fever, and/or atypical pneumonias, differential diagnosis based on symptoms is challenging.

Table 2.

Common clinical symptoms associated with HFRS, NE, and HPS.

Clinical Symptoms	Commonly Observed in Patients With
	HFRS			HPS
	DOBV Belgrade	DOBV Kurkino	NE PUUV	SNV	ANDV
Headache	✓	✓	✓	✓	✓
Backache	✓	✓	✓	✓	✓
Abdominal pain	✓	✓	✓	✓	✓
Nausea	✓	✓	✓	✓	✓
Vomiting	✓	✓	✓	✓	✓
Petechiae	✓	✓	✓ (rare)
Dizziness	✓	✓	✓
Oliguria	✓	✓	✓		✓a
Hemorrhageb	✓			✓c	✓c
Internal hemorrhage	✓				✓
Fever	✓	✓	✓	✓	✓
Dyspnea				✓	✓
Tachycardia	✓			✓	✓
Photophobia				✓	✓

^arare, reported in Brazilian patients; ^bConjunctival, skin, mucous membranes; ^chemorrhage is reported more often in ANDV in Chile than in USA or Brazil, frank hemorrhage in gastrointestinal tract; ^dvery high.

Therefore, clinical diagnosis must discriminate against a potentially wide background of more common pathogens (Table 2) and include the results from laboratory findings (Table 3). Molecular diagnostic testing is also important and will be discussed later in this review. As the disease progresses, the evolution of symptoms, clinical findings, and results of routine laboratory testing can provide additional clinical diagnostic insight [42,189–192]. As there are numerous reviews in the past decade that detail in depth the clinical features of HFRS and HPS (reviewed in [4,28,183,184,190,193]), we will summarize the common features associated with each syndrome.

Table 3.

Common laboratory findings associated with HFRS, NE, and HPS.

Clinical/Laboratory Findings	Commonly Observed in Patients With:
	HFRS		NE	HPS
	DOBV Belgrade	DOBV Kurkino	PUUV	SNV	ANDV
Thrombocytopenia	✓	✓	✓	✓	✓
Elevated Urea, Creatinine	✓	✓		✓	✓
Leukocytosis	✓	✓
Proteinuria	✓	✓	✓	✓	✓
Hematuria	✓
Hypotension	✓	✓
Increased vascular permeability	✓	✓	✓	✓	✓
Hepatitis [1]
Polyuria	✓	✓	✓
Hepatomegaly
Lymphadenopathy
Anemia
Signs of shock	✓			✓	✓
High WBC counts				✓b	✓ b
Bilateral pulmonary infiltrates (x-ray)				✓	✓

^ahas been noted in SEOV cases; ^bvery high in severe cases, often associated with lethality.

Clinical progression and unique features of HFRS

HFRS ranges from mild-to-severe disease that is characterized by fever, vascular leakage resulting in hemorrhagic manifestations, and renal failure (Table 2). HFRS includes a cluster of symptoms with five phases following an initial incubation period of 2–4 weeks. These phases are as follows: Febrile (lasting 3–7 days), Hypotensive (hours-2 days), Oliguric (3–7 days), Diuretic (1–2 weeks) and Convalescence (3–6 weeks). Typically, in HFRS, the symptoms and laboratory findings (Table 3) and the absence of upper or lower respiratory tract symptoms and lack of pulmonary infiltrates at the onset of illness helps differentiate orthohantaviruses from common upper and lower respiratory tract illnesses, such as influenza or bacterial pneumonia. There are reported cases of pulmonary involvement in HFRS; however, it is typically mild and rare [194–196]. In one case report, the patient with pulmonary syndrome had no prominent shock phase [197].

The clinical severity and case-fatality rate of DOBV infection in humans appear to be intricately linked to genotype. The molecular basis of virulence of different genotypes has yet to be understood. There are three DOBV genotypes known to cause disease in humans: Belgrade, Kurkino, and Sochi [198–201]. While there are many similarities in their clinical manifestations in humans, one of the main differences between the strains DOBV-Belgrade and DOBV-Kurkino, considered together, and the DOBV-Sochi strain is the greater virulence of the Belgrade and Kurkino forms, and, therefore, their severity and case-fatality rate in infected patients.

PUUV causes NE, a less lethal form of HFRS as compared to HTNV or DOBV [191,202], and is characterized by fever, abdominal pain and/or back pain and/or headache, and signs of renal involvement. While the initial symptoms have a sudden onset and are often severe (fever, chills, vomiting, headache, and abdominal pain), the remaining clinical course is usually mild and self-limiting [202] although long-term sequelae are sometimes also observed to include long-lasting hematuria [203].

Clinical progression and unique features of HPS

HPS is characterized by fever and vascular leakage resulting in non-cardiogenic pulmonary edema followed by shock, with a case-fatality rate of ~ 40% depending on the region within the Americas [5]. As with HFRS, the clinical course of HPS can be broken into distinct phases, with some variation in incidence and severity of symptoms among patients which is preceded by an incubation period ranging from 9 to 33 days and may be as long as 46–51 days [184,204–207]. These include: Prodrome (1–6 days), Cardiopulmonary (2–7 days), and Convalescence (months-years). The prodromal phase is characterized by mild febrile illness with fever, myalgia, headache, back pain, abdominal pain, and diarrhea. Laboratory findings typically show progressive thrombocytopenia, a hallmark of HPS [162]. The cardiopulmonary phase begins with cough and shortness of breath due to the development of bilateral pulmonary infiltrates, another hallmark of HPS typically diagnosed via X-rays, and is the phase associated with mortality from respiratory failure. In some patients, respiratory distress may occur in 24 hours after being admitted and usually requires mechanical ventilation. Patients that survive the cardiopulmonary-phase progress to a diuretic stage of disease. The last phase, convalescence, may last from months to years and in some individuals chronic fatigue and lung function may impact quality of life [184,205]. Additionally, HPS patients that undergo prolonged periods of hypoxemia requiring mechanical ventilation and treatment in intensive care units may show cognitive defects during convalescence [208]. Although exposure to HPS-causing viruses can result in significant morbidity, there are reports of silent infections in Brazil occurring in humans [209].

As with HFRS-causing orthohantaviruses, some patients may experience additional uncommon symptoms in addition to those noted above for HPS. An acute disseminated encephalomyelitis-like syndrome has been reported for cases with SNV and ANDV in the USA [210] and Argentina [211], respectively. In patients with BCCV [157] and BAYV [154] infections in the USA, renal compromise have been reported. Likewise, during the outbreak of LANV in the Chaco region of Paraguay [51], HPS cases also had kidney involvement.

Cellular and molecular mechanisms of orthohantavirus pathogenesis

The hallmark and mystery of orthohantavirus-induced pathology is the presence of vascular and coagulation abnormalities that occur without lytic destruction of endothelial cells. Although orthohantaviruses have been studied extensively, no single mechanistic feature has been identified to explain the dysfunction that develops after infection. Collectively, there are several key findings in which orthohantaviruses alter cellular and molecular pathways thought to contribute to pathology. For this review article, the focus will be on 1) viral protein molecular mechanisms to overcome initial detection (Figure 2), and 2) cellular mechanisms that mediate vascular leakage, coagulation, and thrombosis abnormalities during infection (Figure 3).

Figure 2.

Molecular mechanisms of immune evasion by orthohantavirus proteins. Orthohantavirus GP and N can inhibit multiple arms of the innate immune response, but mechanisms differ among viruses tested. Gn and N proteins of HPS-causing viruses target IFN by binding TRAF3, preventing TBK1 complex formation, and inhibiting IRF-3. Gn can also HFRS-causing N blocks NF-κB by interacting with importin-α and preventing translocation to the nucleus. HPS-causing N or Gn block JAK/STAT signaling to inhibit induction of IFN-β.

Figure 3.

Cellular mechanisms of orthohantavirus pathogenesis. Orthohantaviruses primarily infect endothelial cells without significant pathology suggesting pathogenesis is the result of indirect dysregulation of cellular pathways. There are currently several hypotheses that focus on vascular permeability and coagulopathy to explain pathogenesis. Excessive activation and secretion of cytokines and VEGF-A are seen during infection with increased paracellular permeability. PKa and FXIIa activity is increased upon orthohantavirus-infected cell exposures allowing for liberation of BK and increased permeability. Increased TF, thrombin, and fibrin are thought to contribute to thrombocytopenia and DIC. Fibrinolysis is increased during infection, however, the inhibitor PAI-1 is only elevated in HPS and may contribute to differences in hemorrhaging manifestations.

Molecular mechanisms of evasion

One mechanism by which orthohantaviruses dampen the host’s responses is to inhibit the host’s pattern recognition receptors (PRR), signaling through orthohantavirus GP interactions with retinoic acid-inducible gene (RIG-I). Of the two proteins that make up the orthohantavirus glycoprotein, it is the Gn, specifically its C-terminus, that regulates RIG-I driven interferon (IFN) responses by inhibiting tank-binding kinase 1 (TBK1) complex formation through binding to tumor necrosis factor associated factor 3 (TRAF3) [212]. A domain with the 42 C-terminal residues within the cytoplasmic tail of Gn interacts with and regulate TRAF3 [213]. Interestingly, orthohantavirus species differ in their ability to regulate TRAF3, as only ANDV, SNV, and TULV but not PHV are able to bind TRAF3 [212–215]. In another study, Gallo et al. reported that the cytosolic tail of the Gn protein of PHV, PUUV, and TULV are not able to inhibit RIG-I driven expression of IFN-β, but that the Gn precursor of only PUUV inhibits this induction [216].

In addition, orthohantaviruses regulate host responses through interactions with the viral N protein. The N protein blocks tumor necrosis factor-α (TNF-α) driven activation of nuclear factor kappa B (NF-κB). The inhibition of this activation is through viral N protein interacting with importin-α which is responsible for the translocation of NF-κB into the nucleus. Interestingly, not all orthohantavirus species can inhibit this activation, of the species tested only DOBV, HTNV, and SEOV exhibited this ability, but not ANDV, PUUV, and SNV [217,218].

ANDV and SNV regulate IFN-β induction and janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling, but the viral proteins that inhibit differ between the two viruses as both the N protein and GP are required for ANDV but for SNV only GP is required [219]. The N protein of ANDV, but not those of PHV or SNV regulates RIG-I and melanoma differentiated associated protein 5 (MDA5) driven responses [220]. The site within ANDV N protein that is responsible for inhibiting these responses is mapped to a single amino acid, serine 386. This single amino acid residue is not observed in Maporal virus (MAPV), which does not regulate IFN-β [221]. The nonstructural (NS) proteins of PHV, PUUV, and TULV inhibit the induction of IFN-β driven by RIG-I activation [216]. Orthohantaviruses also suppress immune responses associated with apoptosis, leading to the inhibition of cell death. The inhibition of apoptosis occurs through viral N protein sequestering NF-κB in the cytoplasm and degrading inhibitor of nuclear factor kappa B (IκB) in the cytoplasm blocking TNF directed NF-κB and apoptosis induction. This function mapped to aa 60 amino acid region of the N protein (AA 270 to 330) of HTNV [222].

Orthohantaviruses have a remarkable ability to evade the host immune response, which plays a role in disease progression and severity. By blocking early innate and inflammatory signaling cascades, orthohantaviruses delay the activation of antiviral defenses, allowing the virus to replicate and spread more effectively. This immune dysregulation, combined with increased vascular leakage, results in life-threatening complications like respiratory failure, shock, and organ dysfunction. Understanding how these viruses evade and exploit the immune response could be crucial for developing targeted therapies to prevent severe disease progression. Potential intervention points for consideration are therapies that stimulate interferon production or mimic their activity to strengthen the host’s antiviral defenses and limit viral replication in the initial stages. Modulating the immune system’s inflammatory response could also be crucial and strategies like using anti-inflammatory agents, cytokine inhibitors, or immune-modulating therapies could help reduce excessive organ dysfunction.

Cellular mechanisms of vascular leakage and coagulopathy

Vascular leakage

Of the proposed pathogenic mechanisms, hypercytokinemia has been studied the most in cell culture-based models and patient populations (Figure 3). Once the virus infects epithelial cells and microvascular endothelial cells, replication intermediates, i.e. the viral double-stranded RNA replication intermediate, triggers the host’s PRR, which include toll-like receptor 3 (TLR3) as well as RIG-I and MDA5 [214,223]. Orthohantaviruses induce these signaling cascades with the result being the expression of IFN and interferon stimulated genes (ISGs). Clinical studies of HFRS and HPS patients show that both diseases induce a strong immune response with the high protein levels of several cytokines and chemokines, interleukin (IL)-1, IL-6, IL-8, IL-10, IL-15, IL-18, IFN-γ, TNF-α, indoleamine 2,3-dioxygenase (IDO), vascular endothelial growth factor (VEGF), CCL5, and CXCL10; suggesting the role of hypercytokinemia [224–228]. Several cytokines correlate with disease severity in HFRS and/or HPS patients. High IL-6 levels in HPS patients’ sera correlate with disease severity but in bronchoalveolar lavage (BAL) samples of HFRS patients the protein levels are not increased [224,229]. In HFRS patients, IDO is highly activated in patients’ sera and these high levels correlate with disease severity as well as high levels of Forkhead Box P3 (FOXP3) expression in regulatory T cells indicating that IDO potentially suppresses immune responses that hinder virus clearance [230]. An upregulation of IDO has been reported in primary human lung microvascular endothelial cells from human male and female donors infected with orthohantaviruses [231] suggesting the utility of this model for probing mechanisms of pathogenesis.

Pathogenic strains such as HTNV, ANDV, SNV, and NY-1 utilize β3 integrin for entry, while nonpathogenic strains like PHV use β1 integrin [77]. This distinction may be significant in understanding the pathogenic mechanisms of orthohantavirus infections. Integrin αvβ3 interacts with vascular endothelial growth factor receptor 2 (VEGFR2) on endothelial cell surfaces to regulate various cellular functions, including permeability, angiogenesis, migration, and survival [232]. The formation of a complex between β3 integrin and VEGFR2 leads to synergistic signaling, resulting in cytoskeletal reorganization and increased permeability (Figure 3) [232]. Upon infection with pathogenic orthohantaviruses, endothelial cells become highly sensitized to the effects of VEGF, and this sensitization results in increased permeability [233,234]. The mechanism of this sensitization is hyperphosphorylation of VEGFR2 and subsequent internalization and degradation of VE-Cadherin [235]. This effect is observed in cells infected with pathogenic HTNV, ANDV, and NY-1, but not in TULV-infected cells [234,236]. The enhanced permeability in response to VEGF exposure can be blocked using antibodies against VEGFR2 or VEGFR2 kinase inhibitors like pazopanib [233,235]. The association between VEGF and increased permeability in orthohantavirus-infected cells has been primarily studied through in vitro experiments involving exogenous VEGF addition. However, the relevance in a clinical setting assumes VEGF levels are altered in infected patients. Clinical data from HFRS and HPS patients support this hypothesis by demonstrating significant changes in VEGF levels during orthohantavirus infections. In HFRS patients infected with the HTNV virus, VEGF is detectable at the onset of fever and shows a rapid increase, with levels positively correlating with disease severity [237]. This observation is further corroborated by another study where HTNV-infected patients had elevated levels of VEGF and reduced Ang-1 and sVEGFR2 levels, suggesting a broader dysregulation of vascular-related factors [238]. Similarly, in SNV-infected patients, elevated VEGF levels were observed in both pulmonary edema fluid and PBMC samples, with the highest levels associated with fatal cases [239]. In ANDV-infected patients, VEGF levels did not differ from controls. Conflicting findings in the clinical setting should be evaluated further to address potential virus strain differences.

Another pathway has garnered attention as a mediator of vascular and coagulation dysfunction during viral infections. The kallirein-kinin system (KKS) and the inflammatory peptide, bradykinin (BK) have been implicated in the pathogenesis of Dengue virus, SARS-CoV-2, and orthohantaviruses (Figure 3, reviewed in [240]). The liberation of bradykinin is the product of a multi-step cascade whereby damaged endothelial surfaces or coagulation triggers the conversion of prekallikrein to an active enzyme that cleaves kininogen to generate bradykinin [241]. BK is a potent inflammatory mediator and vasodilator that increases permeability and contributes to local inflammation and edema in tissues [241]. in vitro, HTNV and ANDV-infected cells trigger activation of the KKS and liberation of BK after exposure to purified plasma proteins [242]. Specifically, the data suggests that activated FXII (FXIIa) drives this cascade resulting in increased permeability [242]. Furthermore, the effect on permeability was deemed specific to KKS activation and BK liberation since it could be blocked by the addition of inhibitors targeting various components of the pathway [242]. In ANDV-infected hamsters displaying HPS-like disease, the kininogen gene is significantly upregulated with peak levels correlating with viremia [243]. In patients, detailed studies of KKS activation and BK liberation have not been reported but elevated plasma kallikrein (PKa) has been detected in HFRS patients [244,245]. However, there is some indirect evidence since the BK inhibitor, Icatibant, has been administered to a few patients infected with PUUV. After treatment, patients stabilized and gradually showed improvement [246–248]. The effectiveness of treatment may be dependent on early treatment since one patient with late stage HFRS did not respond [249].

The complement system is comprised of many proteins that serve to regulate cell activation, migration, and inflammatory responses [250,251]. C3 is the central effector molecule on which the lectin, classical, and alternate pathways converge to generate an active fragment [250,251]. C3 shows reduced levels during PUUV infection, suggesting consumption of factors and activation of the complement system [252–255]. SC5b-9, also known as the terminal complement complex (TCC) has several functions related to endothelial homeostasis. The TCC is well known for cytolysis; however, there are two other forms known as the sublytic MAC and the iTCC. The sublytic MAC can bind to endothelial cells and induce gene expression to generate proteins that regulate vascular tone [256]. Additionally, iTCCs (inactive) which are found circulating in plasma, partially contribute to increased permeability through crosstalk with the KKS and liberation of BK [257]. SC5b-9 levels are significantly increased during the acute phase of PUUV infection and the levels of soluble SC5b-9 predict the occurrence of complement activation and correlate with the clinical course of the disease [254]. In a study with ANDV-infected patients, C5/C5a levels were higher in survivors as compared to fatal cases [227]. However, C5a is not part of the TCC so further studies would be beneficial. A study performed in golden Syrian hamsters infected with ANDV and displaying HPS-like disease demonstrated significant transcriptional upregulation of C4BPA, C4A, C1R, C1S, and C1QA ²⁴⁷.

Coagulopathy

Thrombocytopenia is also a prominent feature in HFRS and HPS (Figure 3). The complex systems that regulate platelet formation require tight control to avoid spontaneous bleeding. Orthohantavirus-induced thrombocytopenia is well documented in HFRS and HPS cases [258–261]. Virus-induced thrombocytopenia mechanisms are attributed to decreased production or increased elimination of platelets [262]. Previous evidence suggested that orthohantaviruses might enhance the elimination of platelets through hyperactivation and exhaustion. In PUUV-infected patients, blood levels of immature platelet fraction and thrombopoietin were elevated even during the acute phase suggesting platelet production is not impaired [263,264]. β3 integrins on the surface of infected endothelial cells, ANDV, HTNV, and PUUV but not TULV, can mediate binding of naïve platelets [265,266]. However, binding alone is insufficient for activating platelets and thrombosis. A study with platelets from PUUV-infected patients did not demonstrate activation or elevation of integrin receptor glycoprotein IIb/IIIa, platelet degranulation markers, and desialytion [266]. Additionally, platelets are not preactivated in PUUV-infected patients, suggesting that exhaustion is not a mechanism of depletion [263]. Instead, PUUV-infected cells may sequester platelets to the endothelium [266].

In HFRS and HPS patients, laboratory profiles commonly demonstrate increased coagulation and fibrinolysis, which are thought to contribute to the presence of hemorrhages and disseminated intravascular coagulopathy (DIC) [258–261]. The architecture of a clot contains platelets and fibrin; both are required for hemostasis. During normal physiologic conditions, the endothelium plays an important role in preventing thrombosis, and pathogens can directly or indirectly disrupt the lining of the vessel wall, exposing “hidden” proteins in the subendothelial layer and initiate binding and activation [262]. The exposure of tissue factor (TF) to blood proteins initiates the sequential activation of a series of coagulation system proteins, leading to thrombin generation and conversion of fibrinogen to fibrin. TF, thrombin, and fibrin are elevated during HFRS and HPS [267–270]. However, the mechanism of how this pathway is activated without significant endothelial cell damage is unclear. Current theory and evidence suggest that the function of thrombosis is not only hemostasis but also a mechanism of the innate immune response. Pathogen-associated molecular patterns (PAMPs) can trigger monocytes to release microvesicles containing TF allowing activated TF to be delivered directly to the area of infection [271]. HTNV RNA can trigger the innate immune response through PAMPs [272]. In SNV and PUUV-infected patients, extracellular vesicle TF is significantly elevated and is strongly associated with DIC in patients [269,270]. Neutrophil extracellular traps (NETs) have been shown to promote coagulation through several mechanisms. They can bind and activate platelets, degrade TF pathway inhibitor, and facilitate activation of the intrinsic coagulation protein, FXII [271]. HTNV can stimulate the release of NETs and patients have high levels during infection [273]. Complement components C3a, C5a, and SC5b-9 also contribute to immunothrombosis by directly triggering platelet activation, enhance coagulation, and upregulate TF expression [256,271,274]. In HFRS patients, individual complement components are decreased indicating activation and consumption of factors [254,255,275].

Orthohantaviruses also affect the fibrinolysis system, which dissolves clots and clears them from circulation (Figure 3). Plasmin is the key regulator of this system and converts fibrinogen to soluble forms (D-dimers) [276]. Activation to plasmin requires conversion from the inactive form plasminogen, a process regulated by urokinase plasminogen activator (uPA), tissue plasminogen activators (tPA), and to a lower extent, PKa and FXIIa [276]. Once conversion occurs, plasminogen activity is regulated by plasminogen activator inhibitor-1 (PAI-1). During infection, uPA, tPA, and PAI-1 levels are altered in infected patients. In PUUV-infected patients and macaques, tPA, but not PAI-1 levels are upregulated, and this positively correlates with disease severity [277]. Conversely, during SNV infection, there is evidence of elevated uPA [278]. Furthermore, PAI-1 is significantly elevated with persistent uPA activity suggesting a refractory mechanism [278,279]. The disparity of PAI-1 levels in HFRS and HPS-infected patients was corroborated in another study where disease severity of ANDV but not DOBV was positively associated with levels of PAI-1 [280]. Additionally, tPA elevation was detected in DOBV and ANDV samples. Future studies could examine the significance of PAI-1 and hemorrhaging since this is not as severe in HPS cases.

In summary, orthohantavirus-induced vascular and coagulation abnormalities are a result of the complex interplay of endothelial cells, immune responses, and coagulation system activation. However, the specific molecular pathways involved in this process are not fully understood and require further research to potentially develop targeted therapies for managing orthohantavirus-induced manifestations.

Animal models

Animal models of HRFS

The first reported infection and disease model for HFRS, a suckling mouse model, came shortly after the discovery of HTNV in the mid 1980’s [281–283]. Newborn outbred ICR suckling mice infected with HTNV by intracerebral (1C), intraperitoneal (IP), intramuscular (IM), or subcutaneous (SC) routes were found to develop a fatal neurological illness with inflammatory and destructive lesions in brain, lung and spleen and die within 2–3 weeks of infection [283–285]. The highest titers of virus occurred in the brain and lung with corresponding meningoencephalitis and pneumonitis lesions, respectively. High titers of virus were also observed in the kidney, but with no apparent pathology. In addition, HTNV was detected in the spleen along with lymphoid hyperplasia and in the liver which showed peri-cholangiohepatitis. HTNV was 100% lethal in mice up to 3-days-old, but mortality rates steadily decreased thereafter so that by the time mice were 2 weeks old, no lethality was observed. Like the suckling mouse model, suckling rats were found to be an age-dependent lethal model for two rat-borne orthohantaviruses, with 1-day-old rats showing 100% lethality and 10-day-old-rats showing 0% mortality when injected IP with the orthohantaviruses [286].

As suckling rodents do not have competent immune systems and because the lethal diseases observed after orthohantavirus infections are not reflective of HFRS in humans, these models have limited utility for evaluating medical countermeasures. Immunocompetent mice and rats also have limited use as models, as they do not develop noticeable disease after infection with HFRS-causing viruses. Instead, adult mice or rats infected with HFRS-causing viruses will typically develop transient viremias followed by clearance or persistence of the virus in the lungs [287]. One report of lethal disease in adult mice infected with an orthohantavirus has appeared, in which IP injection of 8-week-old C57BL/6, SJL/J, or BALB/c mice with 10 [5] PFU of HTNV resulted in a neurological disease similar to that seen in suckling mice [288]. Type 1 IFN knockout mice were also found to develop neurological disease, which occurred sooner than observed in the immunocompetent mice. Studies by others could not reproduce these results but instead found that infection of adult mice with HTNV and three other orthohantaviruses resulted in asymptomatic infections [287]. In a separate study, type 1 IFN knockout mice infected with SEOV remained healthy despite having disseminated virus in the spleen, kidney, and lung [289]. Although not a good model for pathogenesis studies, the availability of commercial reagents and inbred mouse strains to study immune responses have made mice a valuable resource for the initial assessment of immune responses to HFRS [290,291].

Another small animal model for HFRS is the outbred golden Syrian hamster model [287,290,292]. Although hamsters do not develop overt disease when infected with known HFRS-causing viruses, they develop disseminated infections with viral antigen present in several organs, including target organs such as lungs and kidneys. The hamster infection model has been used extensively for evaluation of vaccines based on the M-segment of orthohantaviruses. That is, vaccination with M-segment-based vaccines expressing the glycoprotein genes of the virus followed by challenge with an HFRS-causing orthohantavirus can be evaluated based on the development of antibodies to N. Antibodies to N indicate that the vaccine did not prevent infection, as the S-segment expressing the N gene was not present in the vaccine [293].

Nonhuman primates (NHP) have not been found to be useful disease models for severe HFRS. However, a disease akin to mild HFRS caused by PUUV was reported using cynomolgus (crab-eating) macaques [294]. After infection with PUUV, the NHP became lethargic and developed mild proteinuria and histopathology noted in the medullary tubular cells of the kidneys. Analysis of cytokines and chemokines from the PUUV-infected NHP also showed changes analogous to those in humans with mild HFRS [294].

Animal models of HPS

Mice and rats are also not good disease models for HPS. Like the HFRS-causing viruses, most HPS-causing viruses do not cause disease in hamsters. However, the discovery that ANDV causes lethal disease in hamsters reflective of human HPS provided the first useful disease model for testing medical countermeasures [295]. Similarities between the ANDV-associated disease in hamsters and HPS in humans include the severe dyspnea, rapid progression from first symptoms to death, fluid in the pleural cavity and the histopathology in the lungs and spleen. The incubation period in hamsters ranged from 10 to 24 days versus 12 to 27 days in humans [296]. Differences between the hamster and human disease include significant histopathology in the liver and readily detectable infectious virus in hamsters, which are not generally present in HPS patients. For those HPS-causing viruses, such as SNV, that do not cause disease in hamsters an immunosuppressive model was developed for testing antiviral drugs and vaccines [297]. Hamsters treated with a combination of two immunosuppressive drugs (dexamethasone and cyclophosphamide) were found to be a 100% lethal model for SNV, with viral dissemination and pathology of the lung displaying marked inflammation and edema within the alveolar septa of the infected hamsters.

As found for HFRS-causing viruses, most studies with HPS-causing viruses in NHP have resulted in the absence of disease, despite infections. For example, cynomolgus macaques infected with ANDV either intravenously or by aerosol did not develop disease symptoms. However, they did have significant decreases in lymphocytes 8–11 days after infection, and they all developed neutralizing antibodies as well as both IgM and IgG antibodies [298]. One study reported HPS-like symptoms in rhesus macaques infected with SNV that had been passaged only through deer mice [299]. The NHP developed thrombocytopenia and leukocytosis, with severe pulmonary edema characteristic of human HPS. This model has not been replicated by others, so to date, it has not been used extensively for studies of medical countermeasures.

Diagnostic detection of orthohantaviruses

More than half of the recognized human infectious diseases are zoonotic or originated as zoonoses through the spillover of viruses from wildlife to humans [300–307]. The global prevalence and health burden of new and reemerging RNA viruses from zoonotic infections poses a constant challenge in accurate diagnosis for the clinician including diagnosis of HFRS and HPS.

Highly sensitive molecular tests have been developed based on detection of the viral genomes for orthohantaviruses [3,178,308]; however, it is often difficult to detect viral RNA in patient samples because of the transient, low viremia generally observed. Consequently, detection of genome sequences from patient samples often requires nested RT-PCR techniques, which employ primers targeted to regions with high homology (reviewed in [309]). Molecular diagnostic approaches typically focus on detection of the S-segment, but it is important to also include the M-segment since re-assortment of the viruses occurs. However, improved TaqMan assays show excellent specificity and sensitivity for several species [310]. Because of the difficulty of detecting the virus in patient samples, serological-based diagnostic tests are more common for screening for the prevalence of orthohantaviruses in areas where we have no knowledge of the gene sequences of the viral strains circulating (reviewed in [309,311,312]).

For serodiagnosis of HFRS, detection of orthohantavirus-specific IgM antibodies in serum is the most common approach because, at the onset of symptoms, virtually all acute HFRS serum samples contain IgM antibodies to the Gn, Gc, and N ³¹⁶. Thus, one of the first serologic tests used for diagnosis of HFRS in Europe and Asia was an indirect fluorescence assay (IFA), using orthohantavirus-infected Vero E6 cells fixed as antigen on diagnostic microscope slides. This test has high sensitivity and specificity. The use of virus-infected cells for serologic tests for IFA and ELISA is not widely employed because preparation of slides requires cell culture infections in BSL-3 laboratories. Alternatively, modern diagnostic assays increasingly use recombinant antigens that are expressed in cells and isolated in cell mixtures. All three structural proteins (Gn, Gc, N) induce virus-specific IgM antibodies detectable at the onset of symptoms [26,313–315]. Recombinant viral proteins have been tested as semi-purified antigens for ELISA, Western blot, or dip-stick assays with various outcomes in terms of sensitivity and specificity.

Barriers to effective medical countermeasures for HFRS and HPS

Rodent control remains the most effective means of preventing infections with pathogenic orthohantaviruses. Supportive care of patients with orthohantavirus diseases is currently the primary means of treatment. For HFRS patients, supportive care can include replacing lost electrolytes and fluids to stabilize blood pressure and transfusion with platelets to manage thrombocytopenia (reviewed in [193,202]). In severe cases of HFRS renal or peritoneal dialysis is sometimes needed. For HPS patients, supportive care can include mechanical ventilation, hemofiltration, and extracorporeal membrane oxygenation (ECMO) (reviewed in [28,184,207]). Although additional medical countermeasures are clearly needed for orthohantaviral diseases, numerous barriers have prevented the deployment of effective prophylactic or therapeutic products.

Since the discovery of HTNV more than 40 years ago, many laboratory studies on development and testing of medical countermeasures for HFRS or HPS have been reported (reviewed in [316]). Despite these efforts, none have led to product licensure. Two of the most significant barriers to medical countermeasures for HFRS and HPS are the absence of financial incentives for drug and vaccine manufacturers and no clear path to regulatory licensure. These barriers are related to the absence of suitable animal models reflecting disease (especially for HFRS), and difficulties in conducting large-scale clinical trials both due to an inability for reliably predicting where sufficient cases of HFRS or HPS will occur as well as the absence of funding mechanisms to support such studies.

Because of the barriers, only a few therapeutic and candidate vaccines have been clinically tested. Except for ribavirin (1-B-D-ribofuranosyl-1,2,4-triazole-3-carboxamide, virazole), no other small molecule antiviral drugs have been evaluated for treatment of HFRS or HPS patients in controlled studies. Ribavirin is a broad spectrum antiviral drug with demonstrated activity against several RNA viruses including orthohantaviruses [317]. A trial in China showed efficacy of the drug against HFRS, especially when administered early after symptoms onset [318]. In contrast, ribavirin showed no benefit in patients with HFRS caused by PUUV [319] or against HPS [320,321]. The investigators noted that the drug might be more effective if administered before onset of symptoms.

Other treatments evaluated in clinical studies include high-dose intravenous methylprednisolone, which was ineffective against HPS in Chile [322] and icatabant, a bradykinin inhibitor, that was tested in a few patients with severe HFRS caused by PUUV infections with mixed results [323]. Antibody therapy has not been widely tested in clinical studies with HFRS or HPS, however, in a non-randomized multicenter study in Chile, mortality in 32 HPS patients treated with convalescent plasma was lower than in comparison groups patients [324]. Several other antiviral drugs and immune therapies have been evaluated in laboratory studies but have not progressed to clinical trials (reviewed in [325]). It is appreciated that antiviral treatments would be most effective if given prophylactically rather than therapeutically; however, controlled clinical studies would be needed for this and the sporadic and unpredictable occurrence of orthohantaviral diseases presents a major barrier for such studies.

Clinical studies of vaccines for HFRS and HPS have been reported and some of them have shown great promise. In general, elicitation of neutralizing antibodies has been the most accepted marker of protective immunity following vaccination and is the most readily available comparator of several types of vaccines. Vaccines tested clinically range from inactivated HFRS vaccines, a vaccinia virus vectored HFRS vaccine and nucleic acid-based vaccines for HFRS and/or HPS.

In addition to the general barriers noted for medical countermeasures for HFRS and HPS, specific barriers for several types of vaccines have also impeded their use in humans. For inactivated vaccines, obstacles include the need to propagate the viruses in a BSL-3 biocontainment environment, low viral titers, and absence of cytopathic effects in cultured cells. In addition, inactivated vaccines require multiple vaccinations and the inclusion of an adjuvant to elicit adequate immunity and even then, only low levels or no neutralizing antibodies and limited protection have been observed [326,327].

A vaccinia vectored vaccine tested in Phase 1 and Phase 2 clinical studies did elicit neutralizing antibodies in most subjects who had not previously received a smallpox vaccine, but not in those who had [328]. Although smallpox vaccinations are no longer routine, this approach was felt to have too many obstacles to be continued, thus was abandoned.

Several DNA vaccines and multiple delivery methods have been tested clinically and were found to elicit long-lasting neutralizing antibodies that could be enhanced by a boosting dose [329–331]. Nevertheless, multiple vaccinations and complicated delivery methods were required to achieve satisfactory immune responses, and support for large-scale trials could not be obtained. With the success of the DNA vaccine approach, along with the newer mRNA vaccine technology, nucleic acid vaccines offer the best path toward vaccines for HFRS and HPS.

Summary and conclusions

The first report of the Hantaan virus, the etiologic agent of Korean hemorrhagic fever, appeared in 1978. Within the next 10 years, molecular, serological, epidemiological, and ecological studies led to the recognition of at least four distinct viruses that cause clinically similar diseases now known as HFRS in the Old World. The unexpected discovery of several HPS-causing orthohantaviruses in North America in the early 1990s demonstrated that orthohantaviruses pose a world-wide disease threat. Since then, the number and diversity of recognized hantaviruses and their hosts have greatly expanded and the pandemic potential for a known or unknown orthohantavirus has been raised. As of now, orthohantavirus zoonosis is believed to be typically limited to rodent-to-human transmission, although in rare cases, human-to-human transmission of certain orthohantaviruses can occur. The ubiquity of rodent-borne orthohantaviruses and the high mortalities associated with HFRS and HPS indicate a need for effective medical countermeasures for prevention and treatment even if they do not pose a pandemic threat. Nevertheless, the numerous studies showing that orthohantaviruses have experienced genetic reassortment, host-spillover, and host switching many times over their long evolutionary history support the remote but concerning possibility that these viruses could further evolve to become more transmissible or more pathogenic in humans. Our current knowledge of the recognized zoonotic orthohantaviruses suggests that it is possible, but unlikely, that one of them could be the next “disease x” with pandemic potential. More likely, however, is that an unknown orthohantavirus, perhaps one residing in a non-rodent host such as a bat, could evolve to that status. Consequently, further research and development efforts are needed to ensure that known orthohantaviruses are effectively managed and to mitigate the threat of a novel orthohantavirus.

Among the areas of study still needed are those that can elucidate the natural host-virus dynamics to include determining why the viruses can persistently infect their rodent hosts with little to no health impact, but cause severe and sometimes fatal disease in humans, and only humans. Pathogenic orthohantaviruses have co-evolved with their natural hosts over many centuries, and it is likely that both host and viral factors are involved.

For humans, vascular leakage and coagulopathy contribute to severe disease for both HFRS and HPS. Many descriptive studies of immune responses (i.e. cytokine and chemokine responses) of HFRS and HPS patients have been published. Still, there is no clear therapeutic target for intervention in disease progression. A focus on the discovery of host targets could advance our treatment of orthohantaviruses. Particularly, gaining a better understanding of pathways and host proteins that contribute to coagulopathy and vascular leakage might reveal key targets for drug discovery. Research could focus on examining viral protein interactions with host signaling pathways through techniques like CRISPR-based gene editing and proteomics. To address the question why some orthohantavirus infections lead to a more severe disease, while others cause milder symptoms, host genetic factors and immune response variability through genome-wide association studies or single-cell RNA sequencing could provide additional insights. Longitudinal studies tracking cellular responses in infected individuals could also clarify crucial factors in disease progression. Combining these methods with enhanced rodent surveillance and ecological studies would provide a comprehensive approach to combating orthohantavirus infections.

In addition to therapeutic interventions, additional research on preventing HFRS and HPS is needed. Currently, rodent control and vaccines offer the best strategies for preventing human disease caused by orthohantaviruses. DNA vaccine studies suggest that it is possible to elicit long lasting neutralizing antibodies HFRS and HPS. Subunit vaccines for HFRS have been developed and tested extensively in China and Korea. Although similar vaccines for HPS are not available, molecular vaccines for both HFRS and HPS have been developed and tested in early clinical studies and show great promise.

In summary, both the continued threat of HFRS and HPS as well as the potential for further emergence of novel pathogenic orthohantaviruses suggest that it would be wise to continue to seek better insight into their ecology and epidemiology. Fortunately, as was the case for SARS CoV-2, the past 50 years of research has provided a solid foundation for quickly responding to the emergence of an orthohantavirus with increased disease potential.

Funding Statement

This work was supported in part by the Harriet Van Vleet Endowed Chair of Excellence in Virology to CBJ and NIH Research Grant number UC7AI180313 to SLT and CBJ; National Institutes of Health [UC7AI180313].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Data sharing is not applicable to this article as no new data was created or analyzed in this review.