Target enrichment metaviromics enables comprehensive surveillance of coronaviruses in environmental and animal samples.

The COVID-19 pandemic has underscored the importance of understanding the role of animals in the transmission of coronaviruses (CoVs) and their impact on human health. A One Health approach, integrating human, animal, and environmental health, is essential for effective CoVs control. Next-generation sequencing has played a pivotal role in identifying and monitoring the evolution of novel CoVs strains, like SARS-CoV-2. However, viral occurrence and diversity studies in environmental and animal samples are challenging because of the complexity of viral communities and low abundance of viruses in these samples. Target enrichment sequencing (TES) has emerged as a valuable tool for investigating viral families in challenging samples. This approach involves the specific capture and enrichment of viral genomes using sequence-specific probes, thereby enhancing the efficiency of detection and characterization. In this study, we aimed to develop and validate a TES panel to study CoVs in various complex environmental and animal derived samples. The results demonstrated the panel's effectiveness in capturing and sequencing a wide diversity of CoVs providing valuable insights into their abundance and host diversity in urban wastewater, farm animal corpses lixiviates and bat guano samples. In sewage samples, CoVs were detected solely when TES was employed while in guano samples, sequencing of CoVs species was achieved in 2 out of 4 samples showing an almost three-logarithmic increase in the number of reads obtained in comparison with the untargeted approach. For animal lixiviates, only the TES application enabled the acquisition of CoVs reads. The information obtained can significantly contribute to early detection, surveillance, and control measures for CoVs, including viral discovery and potential spillover events. Additionally, this sequencing panel shows potential for studying other significant viral families and monitoring viral diversity in different animal populations.

High rates of antibodies against Toscana and Sicilian phleboviruses in common quail Coturnix coturnix birds.

Introduction: Birds are involved natural cycle of a number of vector-borne viruses in both rural and urban areas. Toscana (TOSV) and Sicilian (SFSV) phleboviruses are sandfly-borne viruses in the genus Phlebovirus that can cause diseases in human. However, there is limited information on the role of the birds in sandfly-borne phleboviruses natural cycle and reservoirs ofthese viruses remain unknown. Methods: In this study, we analyzed Common Quail (Coturnix coturnix) sera from Spain to identify the seroprevalence of these two phleboviruses. We tested respectively, 106 and 110 quail serum against TOSV and SFSV from 2018, 2019, and 2021 from two locations in northern Spain with using virus neutralization test. Results: We identified high neutralizing antibody rates for SFSV (45.45%) and TOSV (42.45%) with yearly fluctuation. Discussion: This is the first identification of SFSV and TOSV neutralizing antibodies in wild birds. High seroprevalence rates of TOSV and SFSV in quail birds raises the question whether birds have a role as amplifying hosts in the natural cycle of phleboviruses.

First Coronavirus Active Survey in Rodents From the Canary Islands.

Since the beginning of the 21st century five new coronaviruses inducing respiratory diseases in humans have been reported. These emergences has promoted research on coronaviruses in wildlife. We started the first eco-epidemiological study to screen the presence of coronaviruses circulating in mice and rats of four Canary Islands. Between 2015 and 2019, we obtained fecal samples of three rodent species (150 Mus musculus, 109 Rattus rattus and 1 Rattus norvegicus) captured in urban and rural areas. Fecal samples were analyzed by nRT-PCR and the resulting sequences were compared to known diversity using Bayesian phylogenetic methods. We only found coronavirus RNA in house mice from El Hierro (10.53%), Tenerife (7.02%) and Lanzarote (5.26%) islands. All coronaviruses detected belong to the species Murine coronavirus belonging to the genus Betacoronavirus and subgenus Embecovirus, being all positive house mice captured in anthropogenic environment. The phylogenetic analysis shows that murine coronaviruses from the Canary Islands are related to European murine coronaviruses. Albeit data are still scarce in the region, the most probable origin of M. coronavirus present in the Canary Islands is continental Europe. According to temporal Bayesian phylogenetics, the differentiation between Canary and continental viruses seems to be quite recent. Moreover, murine coronaviruses from El Hierro, Tenerife and Lanzarote islands tend to segregate in different clades. This enlightens the potential role of rodents or other possibly invasive species in disseminating infectious diseases to remote places through exchanges with the continent. It is important to consider these aspects in the sanitary control of islands, for health and biodiversity preservation concerns.

Seroprevalence of Toscana Virus and Sandfly Fever Sicilian Virus in European Bat Colonies Measured Using a Neutralization Test.

Toscana phlebovirus (TOSV) and Sicilian phlebovirus (SFSV) are endemic in the Mediterranean area where they are transmitted to humans by infected sandflies. Vertebrates of several species have been postulated to act as reservoirs of these viruses, but convincing evidence is still awaited. Among them, bats have been suggested, however documented evidence is lacking. Here we tested a total of 329 bats belonging to eight species collected from twelve localities in southern Spain for the presence of neutralizing antibodies specific to TOSV and SFSV. Positive sera were detected in Schreiber's long-fingered bat (Miniopterus schreibersii), mouse-eared Myotis (Myotis myotis), European free-tailed bat (Tadarida teniotis), and common serotine (Eptesicus serotinus) with the latter showing the highest prevalence rates for SFSV (22.6%) and TOSV (10%). There was no difference between females and males. Results suggest that bats are not likely to play a major role in the natural cycle of these two sandfly-borne phleboviruses. However, large breeding colonies of bats can be used as sentinels for surveillance of the presence of such viruses in a given locality. In addition, capture-recapture studies should be initiated in order to understand better the dynamics of TOSV and SFSV in bat populations.

A four-year survey (2011-2014) of West Nile virus infection in humans, mosquitoes and birds, including the 2012 meningoencephalitis outbreak in Tunisia.

A West Nile virus (WNV) outbreak occurred in Tunisia between mid-July and December 2012. To assess the epidemiological features of the WNV transmission cycle, human cerebrospinal fluid samples from patients with suspected cases (n = 79), Culex pipiens mosquitoes (n = 583) and serum specimens from domestic and migratory birds (n = 70) were collected for 4 years (2011-2014) in the Tunisian Sahel region. Viral testing was performed by polymerase chain reaction (PCR). The WNV genome was detected in 7 patients (8.8%), 4 Culex pipiens pools, and a domestic mallard (Anas platyrhynchos). All PCR-positive samples were from the Monastir region. Phylogenetic analysis revealed that two different WNV strain groups circulated, and isolates from the reservoir (bird), vector (Culex pipiens), and dead-end hosts (humans) were closely related. The Monastir region is a hot-spot for WNV infection, and the reiterative presence of WNV over the years has increased the risk of viral reemergence in Tunisia, which highlights the need for more enhanced and effective WNV surveillance in humans with public awareness campaigns strengthened by monitoring mosquitoes and maintaining avian surveillance for early detection of WNV circulation.

Molecular characterization and phylogenetic analysis of highly pathogenic Vibrio alginolyticus strains isolated during mortality outbreaks in cultured Ruditapes decussatus juvenile.

In the summer of 2008 and 2009, a series of mortalities in growing out seeds of R. decussatus juveniles were occurred in the eastern Tunisian littoral. Nine predominant bacterial strains were isolated from dead and moribund juveniles and characterized as Vibrio alginolyticus. These isolates were subjected to biochemical and molecular characterization. All the Vibrio strains were tested for their susceptibility against the most widely used antibiotic in aquaculture as well as, the assessment of the presence of erythromycin (emrB) and tetracycline (tetS) resistance genes among the tested bacteria. The degree of genetic relatedness between V. alginolyticus strains was evaluated on the basis of the Entero-Bacterial Repetitive Intergenic Consensus (ERIC) and the Random Amplification of Polymorphic DNA-PCR (RAPD-PCR) approaches. We also looked for siderophore activity and the ability to grow under iron limitation. Furthermore, the pathogenic potential of the tested isolates was evaluated using R. decussatus larva and juveniles as infection models. On antimicrobial susceptibility test, Vibrio strains exhibited total resistance to at least four antibiotics. The MICs data revealed that flumequine and oxolinic acid were the most effective antibiotics to control the studied bacteria. Results also showed that studied antibiotics resistance genes were widely disseminated in the genome of V. alginolyticus strains. Both ERIC and RAPD-PCR fingerprinting showed the presence of genetic variation among Vibrio isolates. However, RAPD typing exhibited a higher discriminative potential than ERIC-PCR. Besides, we reported here for the first time the co-production of catechol and hydroxamte by V. alginolyticus species. The challenge experiment showed that most of Vibrio isolates caused high mortality rates for both larva and juveniles at 48-h post-exposure to a bacterial concentration of 106 CFU/ml.

Clinical surveillance for human astrovirus in Monastir region, Tunisia.

BACKGROUND/AIMS: Astroviruses (AstVs) are enteric viruses that can cause gastroenteritis in children. This study is part of monitoring the circulation of astroviruses in children hospitalized and/or outpatients for acute gastroenteritis at the primary care center of Ouerdanine or at the Pediatric Department of the University Hospital Fattouma-Bourguiba (Monastir, Tunisia). The aims of our study were to know the prevalence of human astrovirus in clinical samples of children, characterize the strains and evaluate the infectivity of isolated strains on cell culture. METHODS: Fifty stool samples were collected from children under five years old in the region of Monastir (Tunisia) from October 2010 to June 2011. All specimens were subjected to RT-PCR amplification followed by sequencing and phylogenetic analysis. RESULTS: The study shows a low prevalence of astrovirus (4 %) in children. The two positive samples obtained were HAstV type 3. Samples that were RT-PCR positive were cultured in CaCO-2 cells and the presence of infectious viral particles was confirmed. The phylogenetic analysis shows that the different HAstV-3 strains isolated in Tunisia are grouped into two clusters. The first cluster includes strains obtained in 2004, which belong to lineage HAstV-3a, while strains isolated in 2010 belong to lineage HAstV-3c. CONCLUSIONS: This study is part of monitoring the circulation of astroviruses in children younger than five years old from Monastir region, Tunisia. The results show low prevalence (4 %). All genotyped samples belonged to lineage HAstV-3c, which could be presently emerging. Two different lineages have been isolated in Tunisia: HAstV-3a in 2004 and HAstV-3c in 2010.