Hamster and ferret experimental infection with intranasal low dose of a single strain of SARS-CoV-2.

Understanding the pathogenesis of the SARS-CoV-2 infection is key to developing preventive and therapeutic strategies against COVID-19, in the case of severe illness but also when the disease is mild. The use of appropriate experimental animal models remains central in the in vivo exploration of the physiopathology of infection and antiviral strategies. This study describes SARS-CoV-2 intranasal infection in ferrets and hamsters with low doses of low-passage SARS-CoV-2 clinical French isolate UCN19, describing infection levels, excretion, immune responses and pathological patterns in both animal species. Individual infection with 103 p.f.u. SARS-CoV-2 induced a more severe disease in hamsters than in ferrets. Viral RNA was detected in the lungs of hamsters but not of ferrets and in the brain (olfactory bulb and/or medulla oblongata) of both species. Overall, the clinical disease remained mild, with serological responses detected from 7 days and 10 days post-inoculation in hamsters and ferrets respectively. The virus became undetectable and pathology resolved within 14 days. The kinetics and levels of infection can be used in ferrets and hamsters as experimental models for understanding the pathogenicity of SARS-CoV-2, and testing the protective effect of drugs.

Massive transient damage of the olfactory epithelium associated with infection of sustentacular cells by SARS-CoV-2 in golden Syrian hamsters.

Anosmia is one of the most prevalent symptoms of SARS-CoV-2 infection during the COVID-19 pandemic. However, the cellular mechanism behind the sudden loss of smell has not yet been investigated. The initial step of odour detection takes place in the pseudostratified olfactory epithelium (OE) mainly composed of olfactory sensory neurons surrounded by supporting cells known as sustentacular cells. The olfactory neurons project their axons to the olfactory bulb in the central nervous system offering a potential pathway for pathogens to enter the central nervous system by bypassing the blood brain barrier. In the present study, we explored the impact of SARS-CoV-2 infection on the olfactory system in golden Syrian hamsters. We observed massive damage of the OE as early as 2 days post nasal instillation of SARS-CoV-2, resulting in a major loss of cilia necessary for odour detection. These damages were associated with infection of a large proportion of sustentacular cells but not of olfactory neurons, and we did not detect any presence of the virus in the olfactory bulbs. We observed massive infiltration of immune cells in the OE and lamina propria of infected animals, which may contribute to the desquamation of the OE. The OE was partially restored 14 days post infection. Anosmia observed in COVID-19 patient is therefore likely to be linked to a massive and fast desquamation of the OE following sustentacular cells infection with SARS-CoV-2 and subsequent recruitment of immune cells in the OE and lamina propria.

Discovery of hantavirus circulating among Rattus rattus in French Mayotte island, Indian Ocean.

Hantaviruses are emerging zoonotic viruses that cause human diseases. In this study, sera from 642 mammals from La Réunion and Mayotte islands (Indian Ocean) were screened for the presence of hantaviruses by molecular analysis. None of the mammals from La Réunion island was positive, but hantavirus genomic RNA was discovered in 29/160 (18 %) Rattus rattus from Mayotte island. The nucleoprotein coding region was sequenced from the liver and spleen of all positive individuals allowing epidemiological and intra-strain variability analyses. Phylogenetic analysis based on complete coding genomic sequences showed that this Murinae-associated hantavirus is a new variant of Thailand virus. Further studies are needed to investigate hantaviruses in rodent hosts and in Haemorrhagic Fever with Renal Syndrome (HFRS) human cases.

Reduction in SARS-CoV-2 Virus Infectivity in Human and Hamster Feces.

OBJECTIVE: There is extensive evidence that SARS-CoV-2 replicates in the gastrointestinal tract. However, the infectivity of virions in feces is poorly documented. Although the primary mode of transmission is airborne, the risk of transmission from contaminated feces remains to be assessed. DESIGN: The persistence of SARS-CoV-2 (infectivity and RNA) in human and animal feces was evaluated by virus isolation on cell culture and RT-qPCR, respectively. The exposure of golden Syrian hamsters to experimentally contaminated feces through intranasal inoculation has also been tested to assess the fecal-oral transmission route. RESULTS: For periods that are compatible with average intestinal transit, the SARS-CoV-2 genome was noticeably stable in human and animal feces, contrary to the virus infectivity that was reduced in a time- and temperature-dependent manner. In human stools, this reduction was variable depending on the donors. Viral RNA was excreted in the feces of infected hamsters, but exposure of naïve hamsters to feces of infected animals did not lead to any productive infection. Conversely, hamsters could be experimentally infected following exposure to spiked fresh feces. CONCLUSION: Infection following exposure to naturally contaminated feces has been suspected but has not been established so far. The present work demonstrates that SARS-CoV-2 rapidly lost infectivity in spiked or naturally infected feces. Although the possibility of persistent viral particles in human or animal feces cannot be fully ruled out, SARS-CoV-2 transmission after exposure to contaminated feces is unlikely.

Isolation and Genetic Characterization of Puumala Orthohantavirus Strains from France.

Puumala orthohantavirus (PUUV) causes a mild form of haemorrhagic fever with renal syndrome (HFRS) called nephropathia epidemica (NE), regularly diagnosed in Europe. France represents the western frontier of the expansion of NE in Europe with two distinct areas: an endemic area (north-eastern France) where PUUV circulates in rodent populations, with the detection of many human NE cases, and a non-endemic area (south-western France) where the virus is not detected, with only a few human cases being reported. In this study, we describe the different stages of the isolation of two PUUV strains from two distinct French geographical areas: Ardennes (endemic area) and Loiret (non-endemic area). To isolate PUUV efficiently, we selected wild bank voles (Myodes glareolus, the specific reservoir of PUUV) captured in these areas and that were seronegative for anti-PUUV IgG (ELISA) but showed a non-negligible viral RNA load in their lung tissue (qRT-PCR). With this study design, we were able to cultivate and maintain these two strains in Vero E6 cells and also propagate both strains in immunologically neutral bank voles efficiently and rapidly. High-throughput and Sanger sequencing results provided a better assessment of the impact of isolation methods on viral diversity.

Puumala Virus Variants Circulating in Forests of Ardennes, France: Ten Years of Genetic Evolution.

In Europe, Puumala virus (PUUV) transmitted by the bank vole (Myodes glareolus) is the causative agent of nephropathia epidemica (NE), a mild form of haemorrhagic fever with renal syndrome. In France, very little is known about the spatial and temporal variability of the virus circulating within bank vole populations. The present study involved monitoring of bank vole population dynamics and PUUV microdiversity over a ten-year period (2000-2009) in two forests of the Ardennes region: Elan and Croix-Scaille. Ardennes region is characterised by different environmental conditions associated with different NE epidemiology. Bank vole density and population parameters were estimated using the capture/marking/recapture method, and blood samples were collected to monitor the overall seroprevalence of PUUV in rodent populations. Phylogenetic analyses of fifty-five sequences were performed to illustrate the genetic diversity of PUUV variants between forests. The pattern of the two forests differed clearly. In the Elan forest, the rodent survival was higher, and this limited turn-over resulted in a lower seroprevalence and diversity of PUUV sequences than in the Croix-Scaille forest. Uncovering the links between host dynamics and virus microevolution is improving our understanding of PUUV distribution in rodents and the NE risk.

Experimental infection of squirrel monkeys with nipah virus.

We infected squirrel monkeys (Saimiri sciureus) with Nipah virus to determine the monkeys' suitability for use as primate models in preclinical testing of preventive and therapeutic treatments. Infection of squirrel monkeys through intravenous injection was followed by high death rates associated with acute neurologic and respiratory illness and viral RNA and antigen production.

How Bank Vole-PUUV Interactions Influence the Eco-Evolutionary Processes Driving Nephropathia Epidemica Epidemiology-An Experimental and Genomic Approach.

In Europe, Puumala virus (PUUV) is responsible for nephropathia epidemica (NE), a mild form of hemorrhagic fever with renal syndrome (HFRS). Despite the presence of its reservoir, the bank vole, on most of French territory, the geographic distribution of NE cases is heterogeneous and NE endemic and non-endemic areas have been reported. In this study we analyzed whether bank vole-PUUV interactions could partly shape these epidemiological differences. We performed crossed-experimental infections using wild bank voles from French endemic (Ardennes) and non-endemic (Loiret) areas and two French PUUV strains isolated from these areas. The serological response and dynamics of PUUV infection were compared between the four cross-infection combinations. Due to logistical constraints, this study was based on a small number of animals. Based on this experimental design, we saw a stronger serological response and presence of PUUV in excretory organs (bladder) in bank voles infected with the PUUV endemic strain. Moreover, the within-host viral diversity in excretory organs seemed to be higher than in other non-excretory organs for the NE endemic cross-infection but not for the NE non-endemic cross-infection. Despite the small number of rodents included, our results showed that genetically different PUUV strains and in a lesser extent their interaction with sympatric bank voles, could affect virus replication and diversity. This could impact PUUV excretion/transmission between rodents and to humans and in turn at least partly shape NE epidemiology in France.

Detection and Genetic Characterization of Puumala Orthohantavirus S-Segment in Areas of France Non-Endemic for Nephropathia Epidemica.

Puumala virus (PUUV) in Europe causes nephropathia epidemica (NE), a mild form of hemorrhagic fever with renal syndrome (HFRS). The incidence of NE is highly heterogeneous spatially, whereas the geographic distribution of the wild reservoir of PUUV, the bank vole, is essentially homogeneous. Our understanding of the processes driving this heterogeneity remains incomplete due to gaps in knowledge. Little is known about the current distribution and genetic variation of PUUV in the areas outside the well-identified zones of NE endemicity. We trapped bank voles in four forests in French regions in which NE is considered non-endemic, but sporadic NE cases have been reported recently. We tested bank voles for anti-PUUV IgG and characterized the S segment sequences of PUUV from seropositive animals. Phylogenetic analyses revealed specific amino-acid signatures and genetic differences between PUUV circulating in non-endemic and nearby NE-endemic areas. We also showed, in temporal surveys, that the amino-acid sequences of PUUV had undergone fewer recent changes in areas non-endemic for NE than in endemic areas. The evolutionary history of the current French PUUV clusters was investigated by phylogeographic approaches, and the results were considered in the context of the history of French forests. Our findings highlight the need to monitor the circulation and genetics of PUUV in a larger array of bank vole populations, to improve our understanding of the risk of NE.

The Needs for Developing Experiments on Reservoirs in Hantavirus Research: Accomplishments, Challenges and Promises for the Future.

Due to their large geographic distribution and potential high mortality rates in human infections, hantaviruses constitute a worldwide threat to public health. As such, they have been the subject of a large array of clinical, virological and eco-evolutionary studies. Many experiments have been conducted in vitro or on animal models to identify the mechanisms leading to pathogenesis in humans and to develop treatments of hantavirus diseases. Experimental research has also been dedicated to the understanding of the relationship between hantaviruses and their reservoirs. However, these studies remain too scarce considering the diversity of hantavirus/reservoir pairs identified, and the wide range of issues that need to be addressed. In this review, we present a synthesis of the experimental studies that have been conducted on hantaviruses and their reservoirs. We aim at summarizing the knowledge gathered from this research, and to emphasize the gaps that need to be filled. Despite the many difficulties encountered to carry hantavirus experiments, we advocate for the need of such studies in the future, at the interface of evolutionary ecology and virology. They are critical to address emerging areas of research, including hantavirus evolution and the epidemiological consequences of individual variation in infection outcomes.

Phylogeography of Puumala orthohantavirus in Europe.

Puumala virus is an RNA virus hosted by the bank vole (Myodes glareolus) and is today present in most European countries. Whilst it is generally accepted that hantaviruses have been tightly co-evolving with their hosts, Puumala virus (PUUV) evolutionary history is still controversial and so far has not been studied at the whole European level. This study attempts to reconstruct the phylogeographical spread of modern PUUV throughout Europe during the last postglacial period in the light of an upgraded dataset of complete PUUV small (S) segment sequences and by using most recent computational approaches. Taking advantage of the knowledge on the past migrations of its host, we identified at least three potential independent dispersal routes of PUUV during postglacial recolonization of Europe by the bank vole. From the Alpe-Adrian region (Balkan, Austria, and Hungary) to Western European countries (Germany, France, Belgium, and Netherland), and South Scandinavia. From the vicinity of Carpathian Mountains to the Baltic countries and to Poland, Russia, and Finland. The dissemination towards Denmark and North Scandinavia is more hypothetical and probably involved several independent streams from south and north Fennoscandia.

Revisiting the genetic diversity of emerging hantaviruses circulating in Europe using a pan-viral resequencing microarray.

Hantaviruses are zoonotic agents transmitted from small mammals, mainly rodents, to humans, where they provoke diseases such as Hemorrhagic fever with Renal Syndrome (HFRS) and its mild form, Nephropathia Epidemica (NE), or Hantavirus Cardio-Pulmonary Syndrome (HCPS). Hantaviruses are spread worldwide and monitoring animal reservoirs is of primary importance to control the zoonotic risk. Here, we describe the development of a pan-viral resequencing microarray (PathogenID v3.0) able to explore the genetic diversity of rodent-borne hantaviruses endemic in Europe. Among about 800 sequences tiled on the microarray, 52 correspond to a tight molecular sieve of hantavirus probes covering a large genetic landscape. RNAs from infected animal tissues or from laboratory strains have been reverse transcribed, amplified, then hybridized to the microarray. A classical BLASTN analysis applied to the sequence delivered through the microarray allows to identify the hantavirus species up to the exact geographical variant present in the tested samples. Geographical variants of the most common European hantaviruses from France, Germany, Slovenia and Finland, such as Puumala virus, Dobrava virus and Tula virus, were genetically discriminated. Furthermore, we precisely characterized geographical variants still unknown when the chip was conceived, such as Seoul virus isolates, recently emerged in France and the United Kingdom.

Prevalence of West Nile virus neutralizing antibodies in wild birds from the Camargue area, southern France.

The Camargue area of southern France experienced the re-emergence of West Nile Virus (WNV) in the late summer of 2000 and 2004. Immediately preceding the 2004 outbreak, samples were collected from 432 birds of 32 different species captured in mist nets and from 201 Cattle Egret (Bubulcus ibis) nestlings sampled in their nests between 1 April and 12 June 2004. West Nile virus neutralizing titers of >/=40 were detected in 4.8% (95% confidence limit, 2.9-7.5%) of the adult birds and in 1.6% (0.3-4.6%) of the egret nestlings. Migratory passerines had a higher prevalence of WNV neutralizing antibodies (7.0%) than did resident and short-distance migratory passerines (0.8%), suggesting exposure to WNV or a related flavivirus during overwintering in Africa.

Experimental infections of wild bank voles (Myodes glareolus) from nephropatia epidemica endemic and non-endemic regions revealed slight differences in Puumala virological course and immunological responses.

In Europe, the occurrence of nephropathia epidemica (NE), a human disease caused by Puumala virus (PUUV), exhibits considerable geographical heterogeneity despite the continuous distribution of its reservoir, the bank vole Myodes glareolus. To better understand the causes of this heterogeneity, wild voles sampled in two adjacent NE endemic and non-endemic regions of France were infected experimentally with PUUV. The responses of bank voles to PUUV infection, based on the levels of anti-PUUV IgG and viral RNA, were compared. Slight regional differences were highlighted despite the high inter-individual variability. Voles from the NE non-endemic region showed greater immune responsiveness to PUUV infection, but lower levels of RNA in their organs than voles from the endemic region. These results suggest the existence of regional variations in the sensitivity of bank voles that could contribute to the apparent absence of PUUV circulation among voles and the absence of NE in the non-endemic region.

Genome microevolution of chikungunya viruses causing the Indian Ocean outbreak.

BACKGROUND: A chikungunya virus outbreak of unprecedented magnitude is currently ongoing in Indian Ocean territories. In Réunion Island, this alphavirus has already infected about one-third of the human population. The main clinical symptom of the disease is a painful and invalidating poly-arthralgia. Besides the arthralgic form, 123 patients with a confirmed chikungunya infection have developed severe clinical signs, i.e., neurological signs or fulminant hepatitis. METHODS AND FINDINGS: We report the nearly complete genome sequence of six selected viral isolates (isolated from five sera and one cerebrospinal fluid), along with partial sequences of glycoprotein E1 from a total of 127 patients from Réunion, Seychelles, Mauritius, Madagascar, and Mayotte islands. Our results indicate that the outbreak was initiated by a strain related to East-African isolates, from which viral variants have evolved following a traceable microevolution history. Unique molecular features of the outbreak isolates were identified. Notably, in the region coding for the non-structural proteins, ten amino acid changes were found, four of which were located in alphavirus-conserved positions of nsP2 (which contains helicase, protease, and RNA triphosphatase activities) and of the polymerase nsP4. The sole isolate obtained from the cerebrospinal fluid showed unique changes in nsP1 (T301I), nsP2 (Y642N), and nsP3 (E460 deletion), not obtained from isolates from sera. In the structural proteins region, two noteworthy changes (A226V and D284E) were observed in the membrane fusion glycoprotein E1. Homology 3D modelling allowed mapping of these two changes to regions that are important for membrane fusion and virion assembly. Change E1-A226V was absent in the initial strains but was observed in >90% of subsequent viral sequences from Réunion, denoting evolutionary success possibly due to adaptation to the mosquito vector. CONCLUSIONS: The unique molecular features of the analyzed Indian Ocean isolates of chikungunya virus demonstrate their high evolutionary potential and suggest possible clues for understanding the atypical magnitude and virulence of this outbreak.

Development of a TaqMan RT-PCR assay without RNA extraction step for the detection and quantification of African Chikungunya viruses.

Chikungunya virus (CHIKV), a member of the alphavirus genus, is of considerable public health concern in Southeast Asian and African countries. However, despite serological evidence, the diagnosis of this arthropod-borne human disease is confirmed infrequently and needs to be improved. In fact, illness caused by CHIKV can be confused with diseases such as dengue or yellow fever, based on the similarity of the symptoms, and laboratory confirmation of suspected cases is required to launch control measures during an epidemic. Moreover, no quantitative molecular tool is described to study CHIKV replication or detection in clinical samples and cell culture supernatants. In this study, a specific and sensitive CHIKV one-step TaqMan RT-PCR assay was developed as a tool for the diagnosis of African CHIKV as well as a rapid indicator of active infection by quantifying viral load. This study also showed that a simple heat viral RNA release during the reverse transcription step constituted an alternative to the conventional RNA extraction method.

Complete Genome and Phylogeny of Puumala Hantavirus Isolates Circulating in France.

Puumala virus (PUUV) is the agent of nephropathia epidemica (NE), a mild form of hemorrhagic fever with renal syndrome (HFRS) in Europe. NE incidence presents a high spatial variation throughout France, while the geographical distribution of the wild reservoir of PUUV, the bank vole, is rather continuous. A missing piece of the puzzle is the current distribution and the genetic variation of PUUV in France, which has been overlooked until now and remains poorly understood. During a population survey, from 2008 to 2011, bank voles were trapped in eight different forests of France located in areas known to be endemic for NE or in area from where no NE case has been reported until now. Bank voles were tested for immunoglobulin (Ig)G ELISA serology and two seropositive animals for each of three different areas (Ardennes, Jura and Orleans) were then subjected to laboratory analyses in order to sequence the whole S, M and L segments of PUUV. Phylogenetic analyses revealed that French PUUV isolates globally belong to the central European (CE) lineage although isolates from Ardennes are clearly distinct from those in Jura and Orleans, suggesting a different evolutionary history and origin of PUUV introduction in France. Sequence analyses revealed specific amino acid signatures along the N protein, including in PUUV from the Orleans region from where NE in humans has never been reported. The relevance of these mutations in term of pathophysiology is discussed.

West Nile virus in Morocco, 2003.

West Nile virus (WNV) reemerged in Morocco in September 2003, causing an equine outbreak. A WNV strain isolated from a brain biopsy was completely sequenced. On the basis of phylogenetic analyses, Moroccan WNV strains isolated during the 1996 and 2003 outbreaks were closely related to other strains responsible for equine outbreaks in the western Mediterranean basin.