Hantavirus pulmonary syndrome in Latin America.

Andes virus (ANDV) is the predominant cause of hantavirus pulmonary syndrome (HPS) in South America and the only hantavirus known to be transmitted person-to-person. There are no vaccines, prophylactics, or therapeutics to prevent or treat this highly pathogenic disease (case-fatality 35–40%). Infection of Syrian hamsters with ANDV results in a disease that closely mimics human HPS in incubation time, symptoms of respiratory distress, and disease pathology. Here, we evaluated the feasibility of two postexposure prophylaxis strategies in the ANDV/hamster lethal disease model. First, we evaluated a natural product, human polyclonal antibody, obtained as fresh frozen plasma (FFP) from a HPS survivor. Second, we used DNA vaccine technology to manufacture a polyclonal immunoglobulin-based product that could be purified from the eggs of vaccinated ducks (Anas platyrhynchos). The natural “despeciation" of the duck IgY (i.e., Fc removed) results in an immunoglobulin predicted to be minimally reactogenic in humans. Administration of ≥5,000 neutralizing antibody units (NAU)/kg of FFP-protected hamsters from lethal disease when given up to 8 days after intranasal ANDV challenge. IgY/IgYΔFc antibodies purified from the eggs of DNA-vaccinated ducks effectively neutralized ANDV in vitro as measured by plaque reduction neutralization tests (PRNT). Administration of 12,000 NAU/kg of duck egg-derived IgY/IgYΔFc protected hamsters when administered up to 8 days after intranasal challenge and 5 days after intramuscular challenge. These experiments demonstrate that convalescent FFP shows promise as a postexposure HPS prophylactic. Moreover, these data demonstrate the feasibility of using DNA vaccine technology coupled with the duck/egg system to manufacture a product that could supplement or replace FFP. The DNA vaccine-duck/egg system can be scaled as needed and obviates the necessity of using limited blood products obtained from a small number of HPS survivors. This is the first report demonstrating the in vivo efficacy of any antiviral product produced using DNA vaccine-duck/egg system.

Human Orthohantavirus disease prevalence and genotype distribution in the U.S., 2008–2020: a retrospective observational study

Sin Nombre virus (SNV) and Andes virus (ANDV) cause hantavirus pulmonary syndrome (HPS) in humans. Both SNV and ANDV infect Syrian hamsters, but only ANDV causes lethal disease. A co-infection study was performed to determine which virus, SNV or ANDV, would dominate the survival outcome in hamsters. Infection of hamsters with SNV 1 day before ANDV challenge did not result in disease characteristic of the latter virus, and all animals survived challenge. Control animals infected solely with ANDV all succumbed by day 14. In contrast, when viruses were injected at the same site concurrently, all hamsters succumbed to HPS disease. Hantaviruses are segmented viruses; therefore we investigated which segment might be responsible for the protective phenotype of SNV by using two SNV/ANDV reassortant viruses, both with reciprocal M-segments from the other virus (denoted ASA and SAS). Both reassortants asymptomatically infect hamsters, similar to SNV. However, unlike SNV, 1 day prior preinfection with the reassortant virus did not prevent ANDV lethality. The ASA reassortant virus, but not SAS, protected hamsters from lethal ANDV infection when administered 3 days prior to ANDV challenge. Similar to SNV preinfection, the potent innate immune stimulator poly I:C administered to hamsters 1 day before ANDV challenge prevented lethal ANDV disease. Combined, these results suggest that the difference in pathogenicity of SNV and ANDV in hamsters involves differences in early host-pathogen interactions and resultant anti-viral immune responses of both the innate and adaptive immune system.

To the Editor: Since the etiology of hantavirus cardiopulmonary syndrome (HCPS) was recognized in 1993 in the United States (1), various hantaviruses have been associated with the syndrome in South America (2,3). Depending on the viral genotype involved, hantavirus infection can take a wide variety of forms, from asymptomatic or oligosymptomatic to the classic clinical form (4,5). The first cases of HCPS in Brazil were reported in the state of Sao Paulo in 1993 (6,7). In 2000, an outbreak of HCPS was reported in the municipality of Anajatuba in the state of Maranhao in the Maranhao western lowlands, a microregion in the Brazilian Amazon (8,9). An ecologic study in this region identified antibodies against hantaviruses in wild rodents (Oligoryzomys fornesi and Holochilus sciureus). Analysis of RNA from the viruses isolated from these 2 rodent species showed 2 new hantaviruses, which were named Anajatuba and Rio Mearim, respectively (10). The Maranhao western lowlands is a swampy region that consists of 21 municipalities and ≈400,000 inhabitants. A cross-sectional study was conducted from August 2004 through September 2006 to identify exposures and activities associated with hantavirus infection. A convenience sample comprising 6 of the 21 municipalities in the region was selected: Pinheiro in the north, Vitoria do Mearim in the south, Sao Bento and Sao Joao Batista in the east, and Penalva and Viana in the west. In each municipality, a village in the countryside was randomly chosen. All members of those communities were invited to participate. Persons who agreed to take part in the study were interviewed, and a standardized questionnaire was used to collect demographic information. Participants provided blood samples after giving written informed consent. ELISA to detect immunoglobulin (Ig) G antibodies against hantaviruses was performed in the Arbovirology and Hemorrhagic Fevers Department at the Evandro Chagas Institute (Belem, PA, Brazil) by using antigen from the Sin Nombre virus supplied by the Centers for Disease Control and Prevention (Atlanta, GA, USA). Samples were initially screened at 1:100, and all positive results were confirmed in a serial dilution (starting at 1:400) test. Positive samples were those with titers >400 (10). Odds ratios (ORs) were estimated by logistic regression. Variables with p<0.20 in the univariate analysis were subjected to multivariate analysis. Our study comprised 1,386 persons. The seroprevalence of antihantavirus antibodies in the 6 municipalities was 4.7% (65/1,389), distributed as follows: Sao Joao Batista, 2.6% (8/307); Sao Bento, 3.1% (6/195); Penalva, 3.5% (5/145); Pinheiro, 4.8% (13/273); Viana, 5.6% (9/160); and Vitoria do Mearim, 7.8% (24/309). Most persons interviewed were farmers and illiterate and lived in rammed-earth huts. Killing rats in the fields was reported by 24.3% (337/1,389). According to univariate analysis, seeing rats in the dwelling or in the field, having killed rats in the dwelling or in the fields, being a farmer, being male, and being >20 years of age were associated with IgG against hantaviruses. No association was found between storage of grain inside a dwelling or presence of natural predators of rodents, e.g., domestic cats and snakes such as the red-tailed boa, and antibodies against hantaviruses (Table). Table Univariate analysis of risk factors associated with hantavirus infection in humans, Maranhao western lowlands, Brazil, 2006* Because of colinearity between rat exposure variables, only “having killed rats in the field” was submitted to adjusted analysis. In the multivariate analysis, being >20 years of age and having killed rats in the field remained associated with hantavirus infection. In the model that did not consider sex and age, being a farmer (OR 2.63, 95% confidence interval [CI] 1.56%–4.41%) and having killed rats in the field (OR 2.30, 95% CI 1.38%–3.84%) were independently associated with hantavirus infection. Hantavirus infection is endemic to the Maranhao western lowlands. It has characteristics of an occupational infection associated with agricultural work, but age mostly explained the effect of being a farmer (OR for being a farmer 1.29, 95% CI 0.74%–2.24%, adjusted for sex and age). Exposure to rodents appeared to occur both in fields and dwellings. Although serologic evidence indicated that hantaviruses were circulating in the 6 municipalities studied, no cases of HCPS were reported in any of them. This finding suggests that either only mild or asymptomatic cases were occurring and that these were not recognized as HCPS or that cases of hantavirus were not being detected and reported. However, in a study in 2000 in Anajatuba in a cohort of 234 persons who did not initially have IgG against hantaviruses, 4 seroconversions to IgG against these viruses were detected; 2 of these infections were asymptomatic and 2 were self-limiting febrile illnesses that occurred after 24 months of follow-up (9). The present study reinforces the suspicion that mild or atypical cases are occurring in this region and may be the main reason classical hantavirus infection has not been identified.

Sin Nombre virus (SNV) and Andes virus (ANDV) cause most of the hantavirus pulmonary syndrome (HPS) cases in North and South America, respectively. The chances of a patient surviving HPS are only two in three. Previously, we demonstrated that SNV and ANDV DNA vaccines encoding the virus envelope glycoproteins elicit high-titer neutralizing antibodies in laboratory animals, and (for ANDV) in nonhuman primates (NHPs). In those studies, the vaccines were delivered by gene gun or muscle electroporation. Here, we tested whether a combined SNV/ANDV DNA vaccine (HPS DNA vaccine) could be delivered effectively using a disposable syringe jet injection (DSJI) system (PharmaJet, Inc). PharmaJet intramuscular (IM) and intradermal (ID) needle-free devices are FDA 510(k)-cleared, simple to use, and do not require electricity or pressurized gas. First, we tested the SNV DNA vaccine delivered by PharmaJet IM or ID devices in rabbits and NHPs. Both IM and ID devices produced high-titer anti-SNV neutralizing antibody responses in rabbits and NHPs. However, the ID device required at least two vaccinations in NHP to detect neutralizing antibodies in most animals, whereas all animals vaccinated once with the IM device seroconverted. Because the IM device was more effective in NHP, the Stratis® (PharmaJet IM device) was selected for follow-up studies. We evaluated the HPS DNA vaccine delivered using Stratis® and found that it produced high-titer anti-SNV and anti-ANDV neutralizing antibodies in rabbits (n=8/group) as measured by a classic plaque reduction neutralization test and a new pseudovirion neutralization assay. We were interested in determining if the differences between DSJI delivery (e.g., high-velocity liquid penetration through tissue) and other methods of vaccine injection, such as needle/syringe, might result in a more immunogenic DNA vaccine. To accomplish this, we compared the HPS DNA vaccine delivered by DSJI versus needle/syringe in NHPs (n=8/group). We found that both the anti-SNV and anti-ANDV neutralizing antibody titers were significantly higher (p-value 0.0115) in the DSJI-vaccinated groups than the needle/syringe group. For example, the anti-SNV and anti-ANDV PRNT50 geometric mean titers (GMTs) were 1,974 and 349 in the DSJI-vaccinated group versus 87 and 42 in the needle/syringe group. These data demonstrate, for the first time, that a spring-powered DSJI device is capable of effectively delivering a DNA vaccine to NHPs. Whether this HPS DNA vaccine, or any DNA vaccine, delivered by spring-powered DSJI will elicit a strong immune response in humans, requires clinical trials.

Nosocomial transmission of Andes virus has been documented in Argentina, but has not yet been proven in Chile. We studied 215 contacts (106 family member contacts and 109 health care worker contacts) of 20 index cases of hantavirus cardiopulmonary syndrome (HCPS) in Chile. The seroprevalence of IgG antibodies against Andes virus was 1.9% (95% confidence interval [CI] = 0.34-6.3%) among the family members and 0.0% (95% CI = 0-3.2%) among the health care workers. Our data suggest that there is no evidence for nosocomial transmission of Andes virus in region IX of Chile.

Expression and purification of the surface proteins from Andes virus

In the past 20 years of surveillance for hantavirus in humans in the United States, 624 cases of hantavirus pulmonary syndrome (HPS) have been reported, 96% of which occurred in states west of the Mississippi River. Most hantavirus infections are caused by Sin Nombre virus, but cases of HPS caused by Bayou, Black Creek Canal, Monongahela, and New York viruses have been reported, and cases of domestically acquired hemorrhagic fever and renal syndrome caused by Seoul virus have also occurred. Rarely, hantavirus infections result in mild illness that does not progress to HPS. Continued testing and surveillance of clinical cases in humans will improve our understanding of the etiologic agents involved and the spectrum of diseases.

The rodent-borne hantavirus Puumala virus (PUUV) and related agents cause hemorrhagic fever with renal syndrome (HFRS) in humans. Other hantaviruses, including Andes virus (ANDV) and Sin Nombre virus, cause a distinct zoonotic disease, hantavirus cardiopulmonary syndrome (HCPS). Although these infections are severe and have substantial case fatality rates, no FDA-approved hantavirus countermeasures are available. Recent work suggests that monoclonal antibodies may have therapeutic utility. We describe here the isolation of human neutralizing antibodies (nAbs) against tetrameric Gn/Gc glycoprotein spikes from PUUV-experienced donors. We define a dominant class of nAbs recognizing the "capping loop" of Gn that masks the hydrophobic fusion loops in Gc. A subset of nAbs in this class, including ADI-42898, bound Gn/Gc complexes but not Gn alone, strongly suggesting that they recognize a quaternary epitope encompassing both Gn and Gc. ADI-42898 blocked the cell entry of seven HCPS- and HFRS-associated hantaviruses, and single doses of this nAb could protect Syrian hamsters and bank voles challenged with the highly virulent HCPS-causing ANDV and HFRS-causing PUUV, respectively. ADI-42898 is a promising candidate for clinical development as a countermeasure for both HCPS and HFRS, and its mode of Gn/Gc recognition informs the development of broadly protective hantavirus vaccines.

Andes virus (ANDV) selectively infects pulmonary microvascular endothelial cells (PMECs), causing a 35% fatal acute pulmonary edema termed hantavirus pulmonary syndrome (HPS). However, the mechanism of ANDV-induced vascular permeability remains an enigma. Gorbunova and Mackow (e00396-21) found that the ANDV N-protein binds to RhoGDI, the primary repressor of RhoA-directed vascular permeability. N-protein binding to S34-phosphorylated RhoGDI inhibited RhoGDI binding and repression of RhoA, revealing a novel edemagenic mechanism that permits ANDV to cause HPS. These findings rationalize targeting PKCα phosphorylation of RhoGDI to therapeutically inhibit HPS patient edema and may be broadly applicable to other causes of acute respiratory distress.

Andes virus (ANDV) is a South American hantavirus that causes hantavirus cardiopulmonary syndrome (HCPS), a severe respiratory infection with a case fatality rate as high as 40%. A critical model for studying HCPS is the Syrian hamster model, which faithfully recapitulates key aspects of the disease. Recent studies have shown that strains of ANDV from different origins, i.e., from rodent or human hosts, can produce widely different outcomes in the hamster model. CHI-7913, a human isolate, does not cause lethal disease and replicates poorly in hamster tissues compared with the classical rodent isolate Chile-9717869. Here, we passaged CHI-7913 in vivo in Syrian hamsters 25 times and infected groups of hamsters with virus from passages 2, 12, and 24 to assess whether viral adaptation occurs that might ultimately drive the phenotype of the virus during infection in this model. Very few mutations occurred during passage in hamsters, with only a single coding mutation discovered after 25 passages. Nearly all mutations occurring in hamsters occurred early, within two passages, and none of the passaged virus caused overt disease or pathology in infected hamsters, consistent with minimal changes in the viral genome. Early in infection, CHI-7913 viral RNA levels were still well below those seen in lethal Chile-9717869 infection. Overall, our data offer insights into the lack of selective pressure on hantaviruses in certain hosts and further evidence of distinct outcomes between rodent and human-derived viral isolates that should be studied further in additional viral species.IMPORTANCEInfection of Syrian hamsters with Andes virus (ANDV) can result in disparate outcomes, depending on the source of the virus used for the infection. The ANDV strain CHI-7913 does not cause lethal disease in hamsters but is able to replicate in hamster tissues. We successively passaged CHI-7913 in vivo to study how continued infection influences adaptation in hamsters and whether passaging would lead to the development of a lethal model, as sometimes occurs with other viruses. Surprisingly, even after 25 passages, minimal mutations occurred in ANDV CHI-7913 genomes, with only one coding mutation present above consensus by passage 25, in the viral glycoprotein. Our data suggest that depending on both viral origin and the host, hantaviruses may face minimal selective pressure to mutate toward a disease phenotype. Studies with additional ANDV strains and other hantavirus species are warranted to further study this phenomenon.

A lethal disease model for hantavirus pulmonary syndrome.

Cases of Hantavirus cardiopulmonary syndrome (HCPS) have been reported in Chile between Region V and XI. In our country HCPS is caused by Andes virus. Hantavirus infection is acquired mainly through inhalation of aerosolized rodent fluids. Person to person transmission is considered exceptional. The identified rodent reservoir of the virus extends throughout Region III to XII, and no cases have been reported in the north of Chile. The incubation period is prolonged. We report the first diagnosed patient with HCPS in the north of Chile. In this case we suggest the possibility of person to person transmission.

The small messenger RNA (SmRNA) of the Andes orthohantavirus (ANDV), a rodent-borne member of the Hantaviridae family of viruses of the Bunyavirales order, encodes a multifunctional nucleocapsid (N) protein and for a nonstructural (NSs) protein of unknown function. We have previously shown the expression of the ANDV-NSs, but only in infected cell cultures. In this study, we extend our early findings by confirming the expression of the ANDV-NSs protein in the lungs of experimentally infected golden Syrian hamsters. Next, we show, using a virus-free system, that the ANDV-NSs protein antagonizes the type I interferon (IFN) induction pathway by suppressing signals downstream of the melanoma differentiation-associated protein 5 (MDA5) and the retinoic acid-inducible gene 1 (RIG-I) and upstream of TBK1. Consistent with this observation, the ANDV-NSs protein antagonized mitochondrial antiviral-signaling protein (MAVS)-induced IFN-β, NF-κB, IFN-regulatory factor 3 (IRF3), and IFN-sensitive response element (ISRE) promoter activity. Results demonstrate that ANDV-NSs binds to MAVS in cells without disrupting the MAVS-TBK-1 interaction. However, in the presence of the ANDV-NSs ubiquitination of MAVS is reduced. In summary, this study provides evidence showing that the ANDV-NSs protein acts as an antagonist of the cellular innate immune system by suppressing MAVS downstream signaling by a yet not fully understand mechanism. Our findings reveal new insights into the molecular regulation of the hosts' innate immune response by the Andes orthohantavirus.IMPORTANCEAndes orthohantavirus (ANDV) is endemic in Argentina and Chile and is the primary etiological agent of hantavirus cardiopulmonary syndrome (HCPS) in South America. ANDV is distinguished from other hantaviruses by its unique ability to spread from person to person. In a previous report, we identified a novel ANDV protein, ANDV-NSs. Until now, ANDV-NSs had no known function. In this new study, we established that ANDV-NSs acts as an antagonist of cellular innate immunity, the first line of defense against invading pathogens, hindering the cellular antiviral response during infection. This study provides novel insights into the mechanisms used by ANDV to establish its infection.

Rodent trapping was conducted at seven hantavirus pulmonary syndrome (HPS) case sites from June 1993 to March 1994 during the HPS outbreak in the southwestern United States. To determine if there were changes in the rodent population or the hantavirus seroprevalence in rodents since the HPS outbreak, rodents were trapped at the same sites three years later using the same trapping protocol. The trap success decreased from the numbers trapped during the outbreak, however, the number of Peromyscus, as a percentage of the total rodents captured, did not noticeably decrease. In addition, the seroprevalence of hantavirus antibodies in Peromyscus decreased significantly (P < 0.0001).