Characterization of Hantavirus N Protein Intracellular Dynamics and Localization

Hantaviruses are enveloped viruses that possess a tri-segmented, negative-sense RNA genome. The viral S-segment encodes the multifunctional nucleocapsid protein (N), which is involved in genome packaging, intracellular protein transport, immunoregulation and several other crucial processes during hantavirus infection. In this study we have generated fluorescently tagged N protein constructs derived from Puumalavirus, the dominant hantavirus species in Central, Northern and Eastern Europe. We have comprehensively characterized this protein in the rodent cell line CHO-K1, monitoring the dynamics of N protein complex formation and investigating co-localization with host proteins as well as the viral glycoproteins Gc and Gn. We found a significant spatial correlation of N with vimentin, actin and P-bodies, but not with microtubules. N constructs also co-localized with Gn and Gc, albeit not as strong as the glycoproteins associated with each other. Moreover, we as-sessed oligomerization of N constructs, observing efficient and concentration-dependent multi-merization, with complexes comprising more than 10 individual proteins.

Severity Biomarkers in Puumala Hantavirus Infection

Annually, over 10,000 cases of hemorrhagic fever with renal syndrome (HFRS) are diagnosed in Europe. Puumala hantavirus (PUUV) causes most of the European HFRS cases. PUUV causes usually a relatively mild disease, which is rarely fatal. However, the severity of the infection varies greatly, and factors affecting the severity are mostly unrevealed. Host genes are known to have an effect. The typical clinical features in PUUV infection include acute kidney injury, thrombocytopenia, and increased vascular permeability. The primary target of hantavirus is the endothelium of the vessels of different organs. Although PUUV does not cause direct cytopathology of the endothelial cells, remarkable changes in both the barrier function of the endothelium and the function of the infected endothelial cells occur. Host immune or inflammatory mechanisms are probably important in the development of the capillary leakage. Several immunoinflammatory biomarkers have been studied in the context of assessing the severity of HFRS caused by PUUV. Most of them are not used in clinical practice, but the increasing knowledge about the biomarkers has elucidated the pathogenesis of PUUV infection.

Coumarin Derivative N6 as a Novel anti-hantavirus Infection Agent Targeting AKT

Hantaviruses are globally emerging zoonotic viruses that can cause hemorrhagic fever with renal syndrome (HFRS) in Asia and Europe, which is primarily caused by Hantaan virus (HTNV) infection, results in profound morbidity and mortality. However, no specific treatment is available for this disease. Coumarin derivatives have been reported as antiviral molecules, while studies about the bioactivity of coumarin derivatives against HTNV infection are limited. To study the potential antiviral activity of coumarin derivatives, 126 coumarin derivatives are synthesized, and their inhibitory activity against HTNV is analyzed in vitro. Among these compounds, N6 inhibits HTNV with relatively high selectivity index at 10.9, and the viral titer of HTNV is reduced significantly after 5, 10, and 20 μM N6 treatments. Furthermore, the administration of N6 at the early stage of HTNV infection can inhibit the replication and production of infectious HTNV in host cell, this therapeutic efficacy is confirmed in HTNV-infected newborn mice at the early stage of infection. The molecular docking results show that N6 forms interactions with the key amino acid residues at its active site, and reveals several molecular interactions responsible for the observed affinity, and the treatment of N6 can inhibit the expression of p (Ser473)Akt and HTNV nucleocapsid protein significantly. As such, these observations demonstrate that coumarin derivative N6 might be used as a potential agent against HTNV infection.

Ecology of Hantaviruses in Rodent Reservoirs and Their Early Innate Immune Responses in Human Model Systems

The spillover of zoonotic RNA viruses is responsible for a great deal of the disease outbreaks in human populations. These spillover events are set to continue due to anthropogenic and environmental changes that impact the distribution of these viruses. The viruses in the family Hantaviridae are classified as one of these emerging zoonotic RNA viruses. The spillover of the viruses in this family are responsible for two severe human diseases, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). These viruses are distributed across the globe and are responsible for a large number of human disease cases with potentially high mortality rates each year. Unfortunately, there is a lack of surveillance efforts to identify hantaviruses in most countries making accurate diagnosis or recognition of hantavirus cases complicated. To address the potential public health impact of hantaviruses, we surveyed rodents in a rural region of Ukraine, and identified a high prevalence which underscores the potential for human disease in this country. As it is challenging to address how hantaviruses infect humans, I established approaches to evaluate the early innate immune response in primary lung microvascular endothelial cells (HLMVECs) with pathogenic and nonpathogenic hantaviruses. Surprisingly, my findings challenged some of the current dogma in that there were not dramatic difference between pathogenic and nonpathogenic viruses. This work highlights the critical need for advancement of cell culture models to probe the immune response. To understand the ecology of hantaviruses in their reservoirs their prevalence was studied in northwestern Ukraine. A field capture study was conducted at two sites which each had distinct habitats and contained nine capture lines. During this survey, we captured 424 small mammals, consisting of species across three orders. The most abundant species were Myodes glareolus, the bank vole (45%); Apodemus flavicollis, the yellow-necked mouse (29%); and Apodemus agrarius, the striped field mouse (14.6%). Out of the collection, it was determined that 79 animals were seropositive by immunofluorescent assay (IFA), from which 15.7% were M. glareolus, 20.5% A. flavicollis, and 33.9% A. agrarius. These finding were of interest as M. glareolus and Apodemus spp. harbor Puumala orthohantavirus and Dobrava-Belgrade orthohantavirus viruses, respectively, which are responsible for causing HFRS in humans. IFA reciprocal titer showed a wide distribution indicating new infections are occurring. No relationship was found between species diversity and the proportion of hantavirus seropositive animals captured at these sites. Population analysis on M. glareolus and Apodemus spp. revealed that neither sex nor age was associated with being seropositive. To define the early innate immune responses during human infection by hantaviruses, this research studies the responses in HLMVECs, the primary cells of infection in humans, infected by the pathogenic viruses, Andes orthohantavirus (ANDV) and Hantaan orthohantavirus (HTNV), and the nonpathogenic virus, Prospect Hill orthohantavirus (PHV). A curated list of 39 host genes were studied across multiple time points during the first 72 hours of infection of HLMVECs from a male donor by these three viruses. mRNA level analysis revealed the mRNA levels of only CCL5, CXCL10, CXCL11, IDO1, IFNB1, IRF7, and TLR3 we increased during infection of each viruses. The measurement of CCL5, CXCL10, CXCL11, IDO, and IFN-β secreted protein levels in the same HLMVEC donor during infection confirmed gene expression findings. The study of host immune responses to hantavirus infection was expanded to include HLMVECs from an additional male and two female donors. Measurement of secreted protein levels of CCL5, CXCL10, CXCL11, IDO, and IFN-β by each of the four donors revealed that levels of these proteins are upregulated during infection by each of the viruses. Pair wise analysis on these secreted protein levels by each of the donors during hantavirus infection suggests that donor characteristics and virus species together drive different outcomes. However, female donors had higher levels of CXCL10, IDO, and IFN-β and these increased protein levels were species specific. Lastly, the suppression of immune response involved in cell death were examined and it was found that ANDV is capable of inhibiting cell death in HLMVECs. In summary, the findings presented, show the critical need to understand and define the early innate immune responses to hantaviral infection in human models as well as the necessity of understanding the ecology of hantaviruses in their reservoir hosts.

ABO and Rhesus Blood Groups in Acute Puumala Hantavirus Infection

Puumala hantavirus (PUUV) causes hemorrhagic fever with renal syndrome. We aimed to evaluate whether ABO and rhesus blood groups associate with the susceptibility or the severity of PUUV infection. We analyzed blood groups in 289 adult patients treated in Tampere University hospital due to PUUV infection during the years 1982–2017. Patients’ blood group distribution was compared to that of healthy, voluntary blood donors living in the Tampere University Hospital responsibility area (n = 21,833). The severity of PUUV infection, as judged by the severity of acute kidney injury (AKI), thrombocytopenia, inflammation, capillary leakage, and the length of hospital care, was analyzed across the groups. The ABO and rhesus blood group distributions did not differ between the patients and blood donors. Patients with non-O blood groups had lower systolic blood pressure compared to patients with blood group O, but there was no difference in other markers of capillary leakage or in the severity of AKI. Minor deviations in the number of platelets and leukocytes were detected between the O and non-O blood groups. To conclude, patients with blood group O may be less susceptible to hypotension, but otherwise blood groups have no major influences on disease susceptibility or severity during acute PUUV infection.

Occupational Hantavirus Infections in Agricultural and Forestry Workers: A Systematic Review and Metanalysis

Hantaviruses are zoonotic pathogens that can cause serious human disorders, including hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome. As the main risk factor for human infections is the interaction with rodents, occupational groups such as farmers and forestry workers are reportedly at high risk, but no summary evidence has been collected to date. Therefore, we searched two different databases (PubMed and EMBASE), focusing on studies reporting the prevalence of hantaviruses in farmers and forestry workers. Data were extracted using a standardized assessment form, and results of such analyses were systematically reported, summarized and compared. We identified a total of 42 articles, including a total of 28 estimates on farmers, and 22 on forestry workers, with a total workforce of 15,043 cases (821 positive cases, 5.5%). A pooled seroprevalence of 3.7% (95% confidence interval [95% CI] 2.2–6.2) was identified in farmers, compared to 3.8% (95% CI 2.6–5.7) in forestry workers. Compared to the reference population, an increased occurrence was reported for both occupational groups (odds ratio [OR] 1.875, 95% CI 1.438–2.445 and OR 2.892, 95% CI 2.079–4.023 for farmers and forestry workers, respectively). In summary, our analyses stress the actual occurrence of hantaviruses in selected occupational groups. Improved understanding of appropriate preventive measures, as well as further studies on hantavirus infection rates in reservoir host species (rodents, shrews, and bats) and virus transmission to humans, is needed to prevent future outbreaks.

Urinary genome detection and tracking of Hantaan virus from hemorrhagic fever with renal syndrome patients using multiplex PCR-based next-generation sequencing

Background Hantavirus infection occurs through the inhalation of aerosolized excreta, including urine, feces, and saliva of infected rodents. The presence of Hantaan virus (HTNV) RNA or infectious particles in urine specimens of patient with hemorrhagic fever with renal syndrome (HFRS) remains to be investigated. Methodology/Principal findings We collected four urine and serum specimens of Republic of Korea Army (ROKA) patients with HFRS. We performed multiplex PCR-based next-generation sequencing (NGS) to obtain the genome sequences of clinical HTNV in urine specimens containing ultra-low amounts of viral genomes. The epidemiological and phylogenetic analyses of HTNV demonstrated geographically homogenous clustering with those in Apodemus agrarius captured in highly endemic areas, indicating that phylogeographic tracing of HTNV genomes reveals the potential infection sites of patients with HFRS. Genetic exchange analyses showed a genetic configuration compatible with HTNV L segment exchange in nature. Conclusion/Significance Our results suggest that whole or partial genome sequences of HTNV from the urine enabled to track the putative infection sites of patients with HFRS by phylogeographically linking to the zoonotic HTNV from the reservoir host captured at endemic regions. This report raises awareness among physicians for the presence of HTNV in the urine of patients with HFRS.

Hantavirus Cardiopulmonary Syndrome and Diffuse Alveolar Hemorrhage in the Era of COVID-19

Hantavirus Cardiopulmonary Syndrome (HCPS) can occur after infection with Hantavirus which can occur by inhaling aerosolized rodent urine, feces, and saliva contaminated with the virus. It presents with the rapid development of pulmonary edema, respiratory failure, and cardiogenic shock with the hallmark being microvascular leakage. We report a patient with a history of alcohol abuse and recent exposure to mice and sick kittens who presented with cough with sputum production, shortness of breath, orthopnea, and new-onset lower extremity edema. Imaging revealed bilateral infiltrates more common on the left with an unremarkable echocardiogram. Testing for COVID-19, Human Immunodeficiency Virus (HIV), influenza, bacterial pneumonia including tuberculosis and methicillin-resistant Staphylococcus aureus (MRSA), aspergillosis, histoplasmosis, Blastomyces, and Coccidiodes was negative. Bronchoscopy and bronchoalveolar lavage revealed diffuse alveolar hemorrhage (DAH) and were negative for acid-fast bacilli and Nocardia cultures. He was further tested for Hantavirus, Q fever, leptospirosis, toxoplasmosis, and empiric treatment with doxycycline initiated. His Hantavirus IgM antibody came back positive. Human Hantavirus infection occurs after inhalation of infected rodent excreta; fortunately, human-to-human transmission has not been documented. HCPS most commonly occurs due to the Sin Nombre virus (SNV), has a case fatality rate of 50%, and is a notifiable disease in the United States. It has 3 distinct phases, prodromal, cardiopulmonary, and convalescent/recovery. The cardiopulmonary phase occurs from increased permeability of pulmonary capillaries and in severe cases can progress to cardiogenic shock. Diagnosis is based on the presence of IgM and IgG Hantavirus antibodies. Treatment is mainly supportive; however, patients are usually treated with broad-spectrum antibiotics while workup is underway. In animal models, ribavirin and favipiravir are only effective when administered in the prodromal phase. If suspicion of Hantavirus infection exists, early mobilization to the intensive care unit for treatment is recommended. Extracorporeal membrane oxygenation (ECMO) has been suggested to improve outcomes in severe HCPS with refractory shock.