Puumala orthohantavirus (PUUV) is an emerging zoonotic virus endemic to Europe and Russia that causes nephropathia epidemica, a mild form of hemorrhagic fever with renal syndrome (HFRS). There are limited options for treatment and diagnosis of orthohantavirus infection, making the search for potential immunogenic candidates crucial. In the present work, various bioinformatics tools were employed to design conserved immunogenic peptides containing multiple epitopes of PUUV nucleocapsid protein. Eleven conserved peptides (90% conservancy) of the PUUV nucleocapsid protein were identified. Three conserved peptides containing multiple T and B cell epitopes were selected using a consensus epitope prediction algorithm. Molecular docking using the HPEP dock server demonstrated strong binding interactions between the epitopes and HLA molecules (ten alleles for each class I and II HLA). Moreover, an analysis of population coverage using the IEDB database revealed that the identified peptides have over 90% average population coverage across six continents. Molecular docking and simulation analysis reveal a stable interaction with peptide constructs of chosen immunogenic peptides and Toll-like receptor-4. These computational analyses demonstrate selected peptides\' immunogenic potential, which needs to be validated in different experimental systems.

Hantaviruses cause the acute zoonotic diseases hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Infected patients show strong systemic inflammation and immune cell activation. NK cells are highly activated in HFRS, suggesting that also other innate lymphoid cells (ILCs) might be responding to infection. Here, we characterized peripheral ILC responses, and measured plasma levels of soluble factors and plasma viral load, in 17 Puumala virus (PUUV)-infected HFRS patients. This revealed an increased frequency of ILC2 in patients, in particular the ILC2 lineage-committed c-Kitlo ILC2 subset. Patients\' ILCs showed an activated profile with increased proliferation and displayed altered expression of several homing markers. How ILCs are activated during viral infection is largely unknown. When analyzing PUUV-mediated activation of ILCs in vitro we observed that this was dependent on type I interferons, suggesting a role for type I interferons-produced in response to virus infection-in the activation of ILCs. Further, stimulation of naïve ILC2s with IFN-β affected ILC2 cytokine responses in vitro, causing decreased IL-5 and IL-13, and increased IL-10, CXCL10, and GM-CSF secretion. These results show that ILCs are activated in HFRS patients and suggest that the classical antiviral type I IFNs are involved in shaping ILC functions.

Puumala orthohantavirus-caused hemorrhagic fever with renal syndrome (PUUV-HFRS) is characterized by strong neutrophil activation. Neutrophils are the most abundant immune cell type in the circulation and are specially equipped to rapidly respond to infections. They are more heterogenous than previously appreciated, with specific neutrophil subsets recently implicated in inflammation and immunosuppression. Furthermore, neutrophils can be divided based on their density to either low-density granulocytes (LDGs) or \"normal density\" polymorphonuclear cell (PMN) fractions. In the current study we aimed to identify and characterize the different neutrophil subsets in the circulation of PUUV-HFRS patients. PMNs exhibited an activation of antiviral pathways, while circulating LDGs were increased in frequency following acute PUUV-HFRS. Furthermore, cell surface marker expression analysis revealed that PUUV-associated LDGs are primarily immature and most likely reflect an increased neutrophil production from the bone marrow. Interestingly, both the frequency of LDGs and the presence of a \"left shift\" in blood associated with the extent of thrombocytopenia, one of the hallmarks of severe HFRS, suggesting that maturing neutrophils could play a role in disease pathogenesis. These results imply that elevated circulating LDGs might be a general finding in acute viral infections. However, in contrast to the COVID-19 associated LDGs described previously, the secretome of PUUV LDGs did not show significant immunosuppressive ability, which suggests inherent biological differences in the LDG responses that can be dependent on the causative virus or differing infection kinetics.

BACKGROUND: Hemorrhagic fever with renal syndrome (HFRS) is the most common zoonotic human viral disease in the Russian Federation. More than 98% of the HFRS cases are caused by Puumala orthohantavirus (PUU). Effective serological tests are required for laboratory diagnosis of HFRS.
OBJECTIVE: Construction of an enzyme immunoassay (ELISA) test system for detection of specific antibodies using standard antigen in the form of highly purified inactivated PUU virus as immunosorbent.
METHODS: Preparation of PUU virus antigen, designing the ELISA for detection of specific antibodies, developing parameters of the ELISA system, parallel titration of HFRS patients sera by fluorescent antibody technique (FAT) and the new ELISA.
CONCLUSIONS: For the first time, ELISA based on purified inactivated PUU virus as standard antigen directly absorbed onto immunoplate was developed. Parallel titration of 50 samples from HFRS patients blood sera using FAT and the developed ELISA showed high sensitivity and specificity of this ELISA, with 100% concordance of testing results and significant level of correlation between the titers of specific antibodies in the two assays.
CONCLUSIONS: The ELISA based on purified inactivated PUU virus as an immunosorbent can be effectively used for HFRS serological diagnosis and for mass seroepidemiological studies.
Введение. Геморрагическая лихорадка с почечным синдромом (ГЛПС) является наиболее распространенным зоонозным вирусным заболеванием человека на территории Российской Федерации. Более 98% заболеваемости ГЛПС вызвано ортохантавирусом Пуумала (ПУУ). Для лабораторной диагностики ГЛПС, в частности серодиагностики клинических случаев, требуются эффективные (высокочувствительные, специфичные, максимально объективные и быстрые в исполнении) серологические тесты, разработка которых является важнейшим элементом контроля данного вирусного заболевания. Цель исследования. Конструирование системы иммуноферментного анализа (ИФА) для определения специфических антител с использованием стандартного антигена в виде высокоочищенного инактивированного вируса ПУУ в качестве иммуносорбента. Материалы и методы. Получение препаратов высокоочищенного антигена вируса ПУУ, конструирование системы ИФА для определения специфических антител, отработка параметров системы ИФА, параллельное титрование сывороток крови больных ГЛПС методом флуоресцирующих антител (МФА) и новым вариантом ИФА. Результаты и обсуждение. Впервые в лабораторной практике исследования ГЛПС была сконструирована система ИФА на основе очищенного инактивированного вируса ПУУ (целевой компонент экспериментальной вакцины против ГЛПС) в качестве стандартного антигена при прямой сорбции на твердую фазу (иммунопанель). Параллельное титрование 50 образцов сывороток крови больных ГЛПС методами МФА и разработанного варианта ИФА показало высокую чувствительность и специфичность данного варианта ИФА, отмечены 100% совпадение результатов тестов (на уровне положительный/отрицательный результат) и значительный уровень корреляции величин титров специфических антител двух тестов. Заключение. Разработанный вариант ИФА для определения антител к вирусу ПУУ на основе очищенного инактивированного вируса ПУУ – целевого компонента вакцинного препарата против ГЛПС в качестве иммуносорбента – может быть эффективно использован для серодиагностики ГЛПС и массовых серо-эпидемиологических исследований.

Viruses in the genus Orthohantavirus within the family Hantaviridae cause human hantavirus infections and represent a threat to public health. Hokkaido virus (HOKV), a genotype of Orthohantavirus puumalaense (Puumala virus; PUUV), was first identified in Tobetsu, Hokkaido, Japan. Although it is genetically related to the prototype of PUUV, the evolutionary pathway of HOKV is unclear. We conducted a field survey in a forest in Tobetsu in 2022 and captured 44 rodents. Complete coding genome sequences of HOKVs were obtained from five viral-RNA-positive rodents (four Myodes rufocanus bedfordiae and one Apodemus speciosus). Phylogenetic analysis revealed a close relationship between the phylogenies and geographical origins of M. rufocanus-related orthohantaviruses. Comparison of the phylogenetic trees of the S segments of orthohantaviruses and the cytochrome b genes of Myodes species suggested that Myodes-related orthohantaviruses evolved in Myodes rodent species as a result of genetic isolation and host switching.

Background: The largest documented outbreak of hemorrhagic fever with renal syndrome occurred in Primorje-Gorski Kotar County, Croatia, in 2021, marking the first-time cases of hantavirus infection recorded outside of the known endemic region in the north of the county. Aim: To identify the factors contributing to the spread of the outbreak and to compare risk factors for acquiring hantavirus infection in the endemic and newly affected regions. Methods and Results: A total of 189 cases were confirmed by positive Puumala IgM/IgG antibodies (93.6%), and 13 probable cases were identified by clinical and epidemiological data (6.4%) using a structured questionnaire. Of the 179 cases with available clinical data, 59 (33.0%) were hospitalized. Three cases received hemodialysis, and no deaths were reported. Among 170 cases with information on exposures, 66 (38.8%) reported occupational risk. Cases in the northern part of county were more likely to have been infected in early spring (OR 27.1, 95% CI 2.93-250.7), to report seeing a rodent (OR 6.5; 95%CI 2.3-18.4), and to know someone with hemorrhagic fever with renal syndrome (HFRS) (OR 3.0; 95%CI 1.2-8.0) than cases from the southern part of the county. Data from Croatian Forests Ltd. suggested that an unusually good production of beech seeds in 2020 may have contributed to an increased rodent population in 2021. However, average temperature, rainfall, and humidity data from 2021 did not illustrate a significant difference from previous years (Kruskal-Wallis p = 0.837, p = 0.999, p = 0.108). Conclusion: The 2021 HFRS outbreak was likely fueled by an abundant rodent population and virus transmission in rodent hosts. Human activity, environmental factors, and the ensuing animal-human interactions have spread hantavirus infection from Croatia\'s mountainous region to a previously nonendemic coastal area with a Mediterranean climate.

Pathogenic Eurasian hantaviruses cause hemorrhagic fever with renal syndrome (HFRS), which is characterized by acute kidney injury. The clinical course shows a broad range of severity and is influenced by direct and immune-mediated effects. The neutrophil-to-lymphocyte ratio (NLR) is a marker of systemic inflammation and predicts severity and outcome in various diseases. Therefore, we examined the role of NLR in HFRS caused by hantavirus Puumala (PUUV) and its association with disease severity and kidney injury. We detected elevated NLR levels on admission (NLRadm: median 3.82, range 1.75-7.59), which increased during acute HFRS. Maximum NLR levels (NLRmax: median 4.19, range 1.75-13.16) were 2.38-fold higher compared to the reference NLR level of 1.76 in the general population. NLR levels on admission correlate with markers of severity (length of hospital stay, serum creatinine) but not with other markers of severity (leukocytes, platelets, C-reactive protein, lactate dehydrogenase, serum albumin, proteinuria). Interestingly, levels of nephrin, which is a specific marker of podocyte damage in kidney injury, are highest on admission and correlate with NLRmax, but not with NLRadm. Together, we observed a correlation between systemic inflammation and the severity of HFRS, but our results also revealed that podocyte damage precedes these inflammatory processes.

Laboratory diagnosis of orthohantavirus infection is primarily based on serology. However, for a confirmed serological diagnosis, evaluation of a follow-up serum sample is essential, which is time consuming and causes delay. Real-time reverse transcription polymerase chain reaction (RT-PCR) tests, if positive, provide an immediate and definitive diagnosis, and accurately identify the causative agent, where the discriminative nature of serology is suboptimal. We re-evaluated sera from orthohantavirus-suspected clinical cases in the Dutch regions of Twente and Achterhoek from July 2014 to April 2016 for the presence of Puumala orthohantavirus (PUUV), Tula orthohantavirus (TULV), and Seoul orthohantavirus (SEOV) RNA. PUUV RNA was detected in 11% of the total number (n = 85) of sera tested, in 50% of sera positive for anti-PUUV/TULV IgM (n = 16), and in 1.4% of sera negative or indeterminate for anti-PUUV/TULV IgM (n = 69). No evidence was found for the presence of TULV or SEOV viral RNA. Based on these findings, we propose two algorithms to implement real-time RT-PCR testing in routine orthohantavirus diagnostics, which optimally provide clinicians with early confirmed diagnoses and could prevent possible further invasive testing and treatment.
OBJECTIVE: The addition of a real-time reverse transcription polymerase chain reaction test to routine orthohantavirus diagnostics may better aid clinical decision making than the use of standard serology tests alone. Awareness by clinicians and clinical microbiologists of this advantage may ultimately lead to a reduction in over-hospitalization and unnecessary invasive diagnostic procedures.

The objective is to determine the complete nucleotide sequence and conduct a phylogenetic analysis of genome variants of the Puumala virus isolated in the Saratov region.
METHODS: The samples for the study were field material collected in the Gagarinsky (formerly Saratovsky), Engelssky, Novoburassky and Khvalynsky districts of the Saratov region in the period from 2019 to 2022. To specifically enrich the Puumala virus genome in the samples, were used PCR and developed a specific primer panel. Next, the resulting PCR products were sequenced and the fragments were assembled into one sequence for each segment of the virus genome. To construct phylogenetic trees, the maximum parsimony algorithm was used.
RESULTS: Genetic variants of the Puumala virus isolated in the Saratov region have a high degree of genome similarity to each other, which indicates their unity of origin. According to phylogenetic analysis, they all form a separate branch in the cluster formed by hantaviruses from other subjects of the Volga Federal District. The virus variants from the Republics of Udmurtia and Tatarstan, as well as from the Samara and Ulyanovsk regions, are closest to the samples from the Saratov region.
CONCLUSIONS: The data obtained show the presence of a pronounced territorial confinement of strains to certain regions or areas that are the natural biotopes of their carriers. This makes it possible to fairly accurately determine the territory of possible infection of patients and/or the circulation of carriers of these virus variants based on the sequence of individual segments of their genome.
Цель работы – определение полной нуклеотидной последовательности и проведение филогенетического анализа вариантов геномов вируса Пуумала, выделенных на территории Саратовской области. Материалы и методы. Образцами для исследования послужил полевой материал, собранный в Гагаринском (бывшем Саратовском), Энгельсском, Новобурасском и Хвалынском районах Саратовской области в период с 2019 по 2022 г. Для специфического обогащения генома вируса Пуумала в образцах использовали ПЦР и панель праймеров, подготовленную для данного исследования. Далее проводили секвенирование полученных продуктов реакции и сборку фрагментов в одну последовательность для каждого из сегментов генома вируса. При построении филогенетических деревьев применяли алгоритм maximum parsimony. Результаты. Показано, что генетические варианты вируса Пуумала, выделенные в Саратовской области, имеют высокую степень подобия генома, что говорит о единстве их происхождения. По данным филогенетического анализа, все выделенные варианты вируса (за исключением изолятов вируса из Хвалынского района) образуют обособленную ветвь в кластере, сформированном хантавирусами из других субъектов Приволжского федерального округа. Самыми близкими к образцам из Саратовской области являются варианты вируса из республик Удмуртия и Татарстан, а также из Самарской и Ульяновской областей. Заключение. Полученные данные указывают на наличие выраженной территориальной приуроченности штаммов к определенным регионам или областям, являющимся природными биотопами их носителей. Этот факт позволяет довольно точно определять территорию возможного инфицирования заболевших и/или циркуляцию переносчиков данных вариантов вируса по последовательности отдельных сегментов их генома..

BACKGROUND: Puumala hantavirus (PUUV) causes nephropathia epidemica (NE), an endemic form of transient acute renal injury (AKI). Serological testing is the mainstay of diagnosis. It was the aim of the present study to assist decision-making for serological testing by constructing a simple tool that predicts the likelihood of PUUV positivity.
METHODS: We conducted a comparative cohort study of all PUUV-tested cases at Aachen University tertiary care center in Germany between mid-2013 and mid-2021. N = 293 qualified for inclusion; N = 30 had a positive test result and clinical NE; N = 263 were negative. Two predictive point scores, the Aachen PUUV Score (APS) 1 and 2, respectively, were derived with the aid of logistic regression and receiver operating characteristic (ROC) analysis by determining the presence of four admission parameters. For internal validation, the internal Monte Carlo method was applied. In addition, partial external validation was performed using an independent historic cohort of N = 41 positive cases of NE.
RESULTS: APS1 is recommended for clinical use as it estimated the probability of PUUV positivity in the entire medical population tested. With a range from 0 to 6 points, it yielded an area under the curve of 0.94 by allotting 2 points each for fever or headache and 1 point each for AKI or LDH>300 U/L. A point sum of 0-2 safely predicted negativity for PUUV, as was confirmed in the NE validation cohort.
CONCLUSIONS: Here, we present a novel, easy-to-use tool to guide the diagnostic management of suspected PUUV infection/NE and to safely avoid unnecessary serological testing, as indicated by point sum class 0-2. Since 67% of the cohort fell into this stratum, half of the testing should be avoidable in the future.