Molecular Detection of Orthohantavirus puumalaense in Plasma and Urine Samples from Hospitalized Patients Presenting with a Serologically Confirmed Acute Hantavirus Infection in France.

Molecular detection of Orthohantavirus puumalaense (PUUV) RNA during the course of the disease has been studied in blood of patients in Sweden and Slovenia. The use of urine has been poorly investigated. The aims of this work were to study PUUV RNA detection in plasma from a cohort of patients in France where a different PUUV lineage circulates and to assess the use of urine instead of plasma. Matched plasma and urine samples were collected daily from hospitalized patients presenting with fever, pain, and thrombocytopenia within the last 8 days and testing positive for IgM and IgG against PUUV in serum collected at inclusion and/or approximately 1 month after release. RNA was extracted from samples, and PUUV RNA was detected using real-time reverse transcription-PCR for plasma and urine samples. Sixty-seven patients presented a serologically confirmed acute hantavirus infection. At inclusion, PUUV RNA was detected in plasma from 55 of 62 patients (88.7%) sampled within the first week after disease onset, whereas it was detected in 15 of 60 (25.0%) of matched urine samples. It was then detected from 33 (71.7%) and 2 (4.4%) of 46 patients discharged from the hospital during the second week after disease onset, in plasma and urine, respectively. When PUUV RNA was detected in urine it was also detected in plasma, and not vice versa. Detection of PUUV RNA in plasma from hospitalized patients in France is similar to that observed in Sweden and Slovenia. Urine is not an appropriate sample for this detection.

Seroprevalence of Hantavirus in Forestry Workers, Northern France, 2019-2020.

We aimed to estimate the seroprevalence of Puumala orthohantavirus (PUUV) among forestry workers in northern France, and to explore sociodemographic risk factors. We conducted a random cross-sectional seroprevalence survey among 1777 forestry workers in 2019-2020. The presence of immunoglobulin G against PUUV antigens in serum was assessed using enzyme-linked immunosorbent assay and confirmed using immunofluorescence assay. Poisson regression models were used to explore factors associated with seropositivity. Weighted seroprevalence was 5% (3-6) in northeastern France, 4% (2-6) in north central France, and 1% in two regions located in the center of the country (Auvergne and Limousin). There were no seropositive workers detected in northwestern France. Seropositivity was associated with age, sex, and cumulative seniority in the forestry sector. Seroprevalence was highest in known endemic areas of the northeast and lowest in the northwest. Nevertheless, we found serological evidence of PUUV infection in two regions located in the center of the country, suggesting circulation of the virus in these regions, previously thought to be non-endemic.

Level of maternal antibodies against respiratory syncytial virus (RSV) nucleoprotein at birth and risk of RSV very severe lower respiratory tract infection.

BACKGROUND: The nucleoprotein (N protein) of respiratory syncytial virus (RSV) is a candidate antigen for new RSV vaccine development. The aim of the present study was to investigate the association between maternal antibody titers against the RSV N protein at birth and the newborns' risk of developing very severe lower respiratory tract infection (VS-LRTI). METHODS: In this single-center prospective cohort study, 578 infants born during the RSV epidemic season in France were included. Among these, 36 were hospitalized for RSV VS-LRTI. A generalized linear model was used to test the occurrence of a VS-LRTI in function of sex, mode of delivery, parity of the mother, type of pregnancy, date of birth in relation to the peak of the epidemic, and antibody titer against N protein. RESULTS: All cord blood samples had detectable antibodies against N protein. The mean titers were significantly lower in newborns with risk factors for RSV severe LRTI (preterm infants, birth before the peak epidemic, multiparous mother). There was no association between antibody titer against the N protein and a protection against VS-LRTI. CONCLUSIONS: Further studies are needed to support the hypothesis that transfer of maternal antibodies against the RSV N protein can provide a significant immune protection early in infancy and that N protein candidate vaccine may be a suitable target for maternal vaccine.

Eosinophilia during Hantavirus infection: a cohort study.

BACKGROUND: There are emerging eosinophil-related considerations concerning viral infections. The role of eosinophils has poorly been evaluated during Hantavirus infection. METHODS: The aim of this study was to determine the prevalence of eosinophilia (defined as an eosinophil count above 500 cells/mm3) during haemorrhagic fever with renal syndrome (HFRS) in a large cohort of patients, and to identify factors associated with eosinophilia. RESULTS: Among 387 patients hospitalized for HFRS, 98 (25.3%) had eosinophilia. By univariate analysis, eosinophilia was significantly associated with more severe thrombocytopenia, high C-reactive protein level, white blood cell count and neutrophil count and lower nephrotoxic drug intake. As there was a collinearity between white blood cell count and C-reactive protein level, only C-reactive protein level with platelet count and nephrotoxic drug intake were entered in the multivariable analysis. Elevated C-reactive protein concentrations remained independently associated with eosinophilia. CONCLUSION: Eosinophilia during HFRS affects one quarter of patients, and supports the role of eosinophils in antiviral immunity against hantavirus infection.

Transcriptome Profile During Rabies Virus Infection: Identification of Human CXCL16 as a Potential New Viral Target.

Rabies virus (RABV), the causative agent for rabies disease is still presenting a major public health concern causing approximately 60,000 deaths annually. This neurotropic virus (genus Lyssavirus, family Rhabdoviridae) induces an acute and almost always fatal form of encephalomyelitis in humans. Despite the lethal consequences associated with clinical symptoms of rabies, RABV limits neuro-inflammation without causing major histopathological lesions in humans. Nevertheless, information about the mechanisms of infection and cellular response in the central nervous system (CNS) remain scarce. Here, we investigated the expression of inflammatory genes involved in immune response to RABV (dog-adapted strain Tha) in mice, the most common animal model used to study rabies. To better elucidate the pathophysiological mechanisms during natural RABV infection, we compared the inflammatory transcriptome profile observed at the late stage of infection in the mouse brain (cortex and brain stem/cerebellum) with the ortholog gene expression in post-mortem brain biopsies of rabid patients. Our data indicate that the inflammatory response associated with rabies is more pronounced in the murine brain compared to the human brain. In contrast to murine transcription profiles, we identified CXC motif chemokine ligand 16 (CXCL16) as the only significant differentially expressed gene in post-mortem brains of rabid patients. This result was confirmed in vitro, in which Tha suppressed interferon alpha (IFN-α)-induced CXCL16 expression in human CNS cell lines but induced CXCL16 expression in IFN-α-stimulated murine astrocytes. We hypothesize that RABV-induced modulation of the CXCL16 pathway in the brain possibly affects neurotransmission, natural killer (NK) and T cell recruitment and activation. Overall, we show species-specific differences in the inflammatory response of the brain, highlighted the importance of understanding the potential limitations of extrapolating data from animal models to humans.

Imported haemorrhagic fever with renal syndrome caused by Dobrava-Belgrade hantavirus in France.

Acute kidney injury (AKI) caused by hantavirus infections is rare but should be suspected in any patient presenting with flu-like symptoms, signs of haemolytic-uraemic syndrome or presence of anti-glomerular basement membrane (anti-GBM) antibodies. We report the first case of Dobrava-Belgrade virus in France imported from southeastern Europe. The characteristic macroscopic appearance of the fresh renal biopsy specimen, displaying a haemorrhagic appearance of the medulla, suggested hantavirus infection. AKI caused by hantavirus infections remains a diagnostic challenge, especially outside endemic areas.

Human Exposure to Hantaviruses Associated with Rodents of the Murinae Subfamily, Madagascar.

We conducted a national human serologic study of a hantavirus detected in Madagascar rodents using a commercial kit and a new ELISA targeting the virus. Our results suggest a conservative estimate of 2.7% (46/1,680) IgG seroprevalence. A second single-district study using the new ELISA revealed a higher prevalence (7.2%; 10/139).

Fast, Sensitive and Specific Detection of Thailand orthohantavirus and its Variants Using One-Step Real-Time Reverse-Transcription Polymerase Chain Reaction Assay.

Genetic variants of Thailand orthohantavirus (THAIV) have been recently reported from rodents in South-East Asia and in islands from the South-West part of the Indian Ocean. In order to detect THAIV and its variants, we developed a sensitive and specific real-time RT-PCR targeting the S segment. Our assay was developed in two different RT-PCR systems that gave similar results in terms of sensitivity. Moreover, our results demonstrated a specificity of 100%.

Wild Rats, Laboratory Rats, Pet Rats: Global Seoul Hantavirus Disease Revisited.

Recent reports from Europe and the USA described Seoul orthohantavirus infection in pet rats and their breeders/owners, suggesting the potential emergence of a "new" public health problem. Wild and laboratory rat-induced Seoul infections have, however, been described since the early eighties, due to the omnipresence of the rodent reservoir, the brown rat Rattus norvegicus. Recent studies showed no fundamental differences between the pathogenicity and phylogeny of pet rat-induced Seoul orthohantaviruses and their formerly described wild or laboratory rat counterparts. The paucity of diagnosed Seoul virus-induced disease in the West is in striking contrast to the thousands of cases recorded since the 1980s in the Far East, particularly in China. This review of four continents (Asia, Europe, America, and Africa) puts this "emerging infection" into a historical perspective, concluding there is an urgent need for greater medical awareness of Seoul virus-induced human pathology in many parts of the world Given the mostly milder and atypical clinical presentation, sometimes even with preserved normal kidney function, the importance of simple but repeated urine examination is stressed, since initial but transient proteinuria and microhematuria are rarely lacking.

Clinical Characteristics of Ratborne Seoul Hantavirus Disease.

Although Seoul orthohantavirus is the only globally spread hantavirus pathogen, few confirmed human infections with this virus have been reported in Western countries, suggesting lower medical awareness of the milder, transient, and often chameleon-like symptoms of this zoonosis. We describe lesser known clinical and laboratory characteristics to help improve underreporting of this virus.

Puumala Hantavirus Genotypes in Humans, France, 2012-2016.

The analysis of the nucleoprotein gene of 77 Puumala hantavirus strains detected in human samples in France during 2012-2016 showed that all belonged to the Central European lineage. We observed 2 main clusters, geographically structured; one included strains with the Q64 signature and the other strains with the R64 signature.

Bioclinical Test to Predict Nephropathia Epidemica Severity at Hospital Admission.

We conducted a multicenter, retrospective cohort study of hospitalized patients with serologically proven nephropathia epidemica (NE) living in Ardennes Department, France, during 2000-2014 to develop a bioclinical test predictive of severe disease. Among 205 patients, 45 (22.0%) had severe NE. We found the following factors predictive of severe NE: nephrotoxic drug exposure (p = 0.005, point value 10); visual disorders (p = 0.02, point value 8); microscopic or macroscopic hematuria (p = 0.04, point value 7); leukocyte count >10 × 109 cells/L (p = 0.01, point value 9); and thrombocytopenia <90 × 109/L (p = 0.003, point value 11). When point values for each factor were summed, we found a score of <10 identified low-risk patients (3.3% had severe disease), and a score >20 identified high-risk patients (45.3% had severe disease). If validated in future studies, this test could be used to stratify patients by severity in research studies and in clinical practice.

Seoul Virus Infection in Humans, France, 2014-2016.

We report detection of Seoul virus in 3 patients in France over a 2-year period. These patients accounted for 3 of the 4 Seoul virus infections among 434 hantavirus infections (1.7%) reported during this time. More attention should be given to this virus in Europe where surveillance has been focused mostly on Puumala and Dobrava-Belgrade hantaviruses.

Evidence of two distinct phylogenetic lineages of dog rabies virus circulating in Cambodia.

This first extensive retrospective study of the molecular epidemiology of dog rabies in Cambodia included 149 rabies virus (RABV) entire nucleoprotein sequences obtained from 1998-2011. The sequences were analyzed in conjunction with RABVs from other Asian countries. Phylogenetic reconstruction confirmed the South-East Asian phylogenetic clade comprising viruses from Cambodia, Vietnam, Thailand, Laos and Myanmar. The present study represents the first attempt to classify the phylogenetic lineages inside this clade, resulting in the confirmation that all the Cambodian viruses belonged to the South-East Asian (SEA) clade. Three distinct phylogenetic lineages in the region were established with the majority of viruses from Cambodia closely related to viruses from Thailand, Laos and Vietnam, forming the geographically widespread phylogenetic lineage SEA1. A South-East Asian lineage SEA2 comprised two viruses from Cambodia was identified, which shared a common ancestor with RABVs originating from Laos. Viruses from Myanmar formed separate phylogenetic lineages within the major SEA clade. Bayesian molecular clock analysis suggested that the time to most recent common ancestor (TMRCA) of all Cambodian RABVs dated to around 1950. The TMRCA of the Cambodian SEA1 lineage was around 1964 and that of the SEA2 lineage was around 1953. The results identified three phylogenetically distinct and geographically separated lineages inside the earlier identified major SEA clade, covering at least five countries in the region. A greater understanding of the molecular epidemiology of rabies in South-East Asia is an important step to monitor progress on the efforts to control canine rabies in the region.

Tula hantavirus infection in a hospitalised patient, France, June 2015.

We report an infection with Tula virus in June 2015, leading to hospitalisation, in a patient living approximately 60 km east of Paris with no previous remarkable medical history. Clinical symptoms were limited to a fever syndrome with severe headache. The main laboratory findings included thrombocytopenia and elevated transaminase levels. Based on S (small) gene sequence analysis, the strain affecting the patient was closely related to strains detected in Central Europe, especially to a south-east German strain.

Landscape and regional environmental analysis of the spatial distribution of hantavirus human cases in europe.

BACKGROUND: In Europe, the most prevalent hantavirus, Puumala virus, is transmitted by bank voles and causes nephropathia epidemica in human. The European spatial distribution of nephropathia epidemica is investigated here for the first time with a rich set of environmental variables. METHODS: The influence of variables at the landscape and regional level is studied through multilevel logistic regression, and further information on their effects across the different European ecoregions is obtained by comparing an overall niche model (boosted regression trees) with regressions by ecoregion. RESULTS: The presence of nephropathia epidemica is likely in populated regions with well-connected forests, more intense vegetation activity, low soil water content, mild summers, and cold winters. In these regions, landscapes with a higher proportion of built-up areas in forest ecotones and lower minimum temperature in winter are expected to be more at risk. Climate and forest connectivity have a stronger effect at the regional level. If variables are staying at their current values, the models predict that nephropathia epidemica may know intensification but should not spread (although southern Sweden, the Norwegian coast, and the Netherlands should be kept under watch). CONCLUSION: Models indicate that large-scale modeling can lead to a very high predictive power. At large scale, the effect of one variable on disease may follow three response scenarios: the effect may be the same across the entire study area, the effect can change according to the variable value, and the effect can change depending on local specificities. Each of these scenarios impacts large-scale modeling differently.

Detection in and circulation of Bluetongue virus among domestic ruminants in Madagascar.

So far, no published data was available concerning the circulation of Bluetongue virus (BTV) in Madagascar. During a survey on Rift Valley Fever, we were able to detect a virus belonging to BTV. Therefore, we conducted a study aiming at characterizing molecularly the BTV isolated and assess the importance of circulation of BTV in Madagascar. A total of 4393 sera from ruminants selected randomly by stratification and sampled in 30 districts of Madagascar were tested for BTV. Moreover, 175 cattle were followed during 11 months. Phylogenetic analyses were performed from virus isolated from unfed pools of mosquitoes. Overall, the estimated mean seroprevalence of infection at the national level was 95.9% (95% CI: [95.2-96.5]) in cattle and 83.7% (95% CI: [81.4-85.9]) in small ruminants. Estimation of incidence rate was 54 per 100 cattle-years assuming that the incidence rate is constant all year along. Phylogenetic analyses revealed that BTV detected belong to serotype 2. In conclusion, our results showed that BTV is endemic in Madagascar and highly prevalent among cattle. In our study we did not work on the vector involved in transmission of BTV in cattle. Thus, research should be conducted to better describe epidemiology of BTV in Madagascar including vectors and assess economic impact of the disease associated to BTV infections.

Characterization of Enteroviruses from non-human primates in cameroon revealed virus types widespread in humans along with candidate new types and species.

Enteroviruses (EVs) infecting African Non-Human Primates (NHP) are still poorly documented. This study was designed to characterize the genetic diversity of EVs among captive and wild NHP in Cameroon and to compare this diversity with that found in humans. Stool specimens were collected in April 2008 in NHP housed in sanctuaries in Yaounde and neighborhoods. Moreover, stool specimens collected from wild NHP from June 2006 to October 2008 in the southern rain forest of Cameroon were considered. RNAs purified directly from stool samples were screened for EVs using a sensitive RT-nested PCR targeting the VP1 capsid coding gene whose nucleotide sequence was used for molecular typing. Captive chimpanzees (Pan troglodytes) and gorillas (Gorilla gorilla) were primarily infected by EV types already reported in humans in Cameroon and elsewhere: Coxsackievirus A13 and A24, Echovirus 15 and 29, and EV-B82. Moreover EV-A119, a novel virus type recently described in humans in central and west Africa, was also found in a captive Chimpanzee. EV-A76, which is a widespread virus in humans, was identified in wild chimpanzees, thus suggesting its adaptation and parallel circulation in human and NHP populations in Cameroon. Interestingly, some EVs harbored by wild NHP were genetically distinct from all existing types and were thus assigned as new types. One chimpanzee-derived virus was tentatively assigned as EV-J121 in the EV-J species. In addition, two EVs from wild monkeys provisionally registered as EV-122 and EV-123 were found to belong to a candidate new species. Overall, this study indicates that the genetic diversity of EVs among NHP is more important than previously known and could be the source of future new emerging human viral diseases.

Anjozorobe hantavirus, a new genetic variant of Thailand virus detected in rodents from Madagascar.

Until now, there was only serological evidence that hantaviruses were circulating in rodents and infecting humans from Madagascar. To assess the presence of a hantavirus on the island, between October, 2008, and March, 2010, we sampled 585 rodents belonging to seven species in the Anjozorobe-Angavo forest corridor, 70 km north from the capital city Antananarivo. A hantavirus was detected from organs of the ubiquist roof rat (Rattus rattus) and of the endemic Major's tufted-tailed rat (Eliurus majori). Amazingly, sequence analysis of the S (small), M (medium), and L (large) coding DNA sequence of this virus showed that the Anjozorobe strain (proposed name) was a new genetic variant of Thailand virus (THAIV) that comprises other variants found in Southeast Asia. Because THAIV is suspected of causing hemorrhagic fever with renal syndrome in humans, ongoing studies are addressing the risk of infection by this new variant in the Malagasy population.

Early-warning health and process indicators for sentinel surveillance in Madagascar 2007-2011.

BACKGROUND: Epidemics pose major threats in resource-poor countries, and surveillance tools for their early detection and response are often inadequate. In 2007, a sentinel surveillance system was established in Madagascar, with the aim of rapidly identifying potential epidemics of febrile or diarrhoeal syndromes and issuing alerts. We present the health and process indicators for the five years during which this system was constructed, showing the spatiotemporal trends, early-warning sign detection capability and process evaluation through timely analyses of high-quality data. METHODS: The Malagasy sentinel surveillance network is currently based on data for fever and diarrhoeal syndromes collected from 34 primary health centres and reported daily via the transmission of short messages from mobile telephones. Data are analysed daily at the Institut Pasteur de Madagascar to make it possible to issue alerts more rapidly, and integrated process indicators (timeliness, data quality) are used to monitor the system. RESULTS: From 2007 to 2011, 917,798 visits were reported. Febrile syndromes accounted for about 11% of visits annually, but the trends observed differed between years and sentinel sites. From 2007 to 2011, 21 epidemic alerts were confirmed. However, delays in data transmission were observed (88% transmitted within 24 hours in 2008; 67% in 2011) and the percentage of forms transmitted each week for validity control decreased from 99.9% in 2007 to 63.5% in 2011. CONCLUSION: A sentinel surveillance scheme should take into account both epidemiological and process indicators. It must also be governed by the main purpose of the surveillance and by local factors, such as the motivation of healthcare workers and telecommunication infrastructure. Permanent evaluation indicators are required for regular improvement of the system.