Experimental Susceptibility of North American Raccoons (Procyon lotor) and Striped Skunks (Mephitis mephitis) to SARS-CoV-2

Recent spillback events of SARS-CoV-2 from humans to animals has raised concerns about it becoming endemic in wildlife. A sylvatic cycle of SARS-CoV-2 could present multiple opportunities for repeated spillback into human populations and other susceptible wildlife. Based on their taxonomy and natural history, two native North American wildlife species —the striped skunk (Mephitis mephitis) and the raccoon (Procyon lotor) —represent a high likelihood of susceptibility and ecological opportunity of becoming infected with SARS-CoV-2. Eight skunks and raccoons were each intranasally inoculated with one of two doses of the virus (103 PFU and 105 PFU) and housed in pairs. To evaluate direct transmission, a naïve animal was added to each inoculated pair 48 h post-inoculation. Four control animals of each species were handled like the experimental groups. At predetermined intervals, we collected nasal and rectal swabs to quantify virus shed via virus isolation and detect viral RNA via rRT-PCR and blood for serum neutralization. Lastly, animals were euthanized at staggered intervals to describe disease progression through histopathology and immunohistochemistry. No animals developed clinical disease. All intranasally inoculated animals seroconverted, suggesting both species are susceptible to SARS-CoV-2 infection. The highest titers in skunks and raccoons were 1:128 and 1:64, respectively. Low quantities of virus were isolated from 2/8 inoculated skunks for up to day 5 post-inoculation, however no virus was isolated from inoculated raccoons or direct contacts of either species. Neither species had gross lesions, but recovering mild chronic pneumonia consistent with viral insult was recorded histologically in 5/8 inoculated skunks. Unlike another SARS-CoV-2 infection trial in these species, we detected neutralizing antibodies in inoculated raccoons; thus, future wildlife serologic surveillance results must be interpreted with caution. Due to the inability to isolate virus from raccoons, the lack of evidence of direct transmission between both species, and low amount of virus shed by skunks, it seems unlikely for SARS-CoV-2 to become established in raccoon and skunk populations and for virus to spillback into humans. Continued outbreaks in non-domestic species, wild and captive, highlight that additional research on the susceptibility of SARS-CoV-2 in wildlife, especially musteloidea, and of conservation concern, is needed.

Serological Positivity against Selected Flaviviruses and Alphaviruses in Free-Ranging Bats and Birds from Costa Rica Evidence Exposure to Arboviruses Seldom Reported Locally in Humans

Arboviruses have two ecological transmission cycles: sylvatic and urban. For some, the sylvatic cycle has not been thoroughly described in America. To study the role of wildlife in a putative sylvatic cycle, we sampled free-ranging bats and birds in two arbovirus endemic locations and analyzed them using molecular, serological, and histological methods. No current infection was detected, and no significant arbovirus-associated histological changes were observed. Neutralizing antibodies were detected against selected arboviruses. In bats, positivity in 34.95% for DENV-1, 16.26% for DENV-2, 5.69% for DENV-3, 4.87% for DENV-4, 2.43% for WNV, 4.87% for SLEV, 0.81% for YFV, 7.31% for EEEV, and 0.81% for VEEV was found. Antibodies against ZIKV were not detected. In birds, PRNT results were positive against WNV in 0.80%, SLEV in 5.64%, EEEV in 8.4%, and VEEV in 5.63%. An additional retrospective PRNT analysis was performed using bat samples from three additional DENV endemic sites resulting in a 3.27% prevalence for WNV and 1.63% for SLEV. Interestingly, one sample resulted unequivocally WNV positive confirmed by serum titration. These results suggest that free-ranging bats and birds are exposed to not currently reported hyperendemic-human infecting Flavivirus and Alphavirus; however, their role as reservoirs or hosts is still undetermined.

Seasonality of Coxiella burnetii among Wild Rabbits (Oryctolagus cuniculus) and the Hyalomma lusitanicum (Acari: Ixodidae) in a Meso-Mediterranean Ecosystem

(1) Background: Q fever is a worldwide zoonosis caused by Coxiella burnetii that have cases reported in humans and animals almost everywhere. The aim of this study was to describe the seasonality of Coxiella burnetii in the wild rabbit (Oryctolagus cuniculus) and the tick Hyalomma lusitanicum in a meso-Mediterranean ecosystem. (2) Methods: two populations of wild rabbits that differ in whether or not they share habitat with ungulates, mainly red deer (Cervus elaphus) were sampled for a year to collect ticks, blood and vaginal or anal swabs. Presence of C. burnetii DNA in swabs and the tick H. lusitanicum was determined by PCR and serum antibodies by ELISA. (3) Results: C. burnetii DNA was detected in 47.2% of 583 rabbits, in 65.5% of sera, and in more than half of the H. lusitanicum. There were small variations according to sex and age of the rabbits but significant according to the habitat (4) Conclusions: The results indicate that C. burnetii circulates freely between wild rabbits and H. lusitanicum and the sylvatic cycle in meso-Mediterranean environments relies in the presence of wild rabbits and H. lusitanicum above all if sharing habitat with red deer.

Phylogenetic relationships of a rabies virus isolate in Costa Rica

Introduction: The sylvatic cycle of rabies is a significant sanitary burden in Central America. The Costa Rican government monitors cases since 1985 and infections from bats are still reported for wild animals, livestock, and humans, generating a need of further pathogen characterization in the region. Objective: To compare rabies phylogenetic analyses from complete genomes with nucleoprotein gene studies. Methods: For the phylogenetic analyses we used four rabies tissue samples collected in 2018, and generated complete genomes by Next-Generation sequencing (NGS). We also extracted RNA from tissues of confirmed cases and generated ssDNA using several primers. Double-stranded DNA was generated and used to generate genomic libraries. Results: We describe, for the first-time, the complete genome of four sequences of the rabies virus isolated in Costa Rica in 2018. Complete genome trees resembled the topology of nucleoprotein gene trees. All isolates were related to Desmodus rotundus. One sample group into Lineage (L)2, and the remaining samples group in L1, matched previous reports from regional rabies viruses. Conclusion: Our method produces valid viral assemblies from clinical specimens without target enrichment or viral isolation.

Fast surveillance response and genome sequencing reveals the circulation of a new Yellow Fever Virus sublineage in 2021, in Minas Gerais, Brazil

Yellow fever virus (YFV) exhibits a sylvatic cycle of transmission involving wild mosquitoes and non-human primates (NHP). In Brazil, YFV is endemic in the Amazon region, from where waves of epidemic expansion towards other Brazilian states eventually occur. During such waves, the virus usually follows the route from North to the Central-West and Southeast Brazilian regions. Amidst these journeys, outbreaks of Yellow Fever (YF) in NHPs, with spillovers to humans have been observed. In the present work, we describe a surveillance effort encompassing the technology of smartphone applications and the coordinated action of several research institutions and health services that succeeded in the first confirmation of YFV in NHPs in the state of Minas Gerais (MG), Southeast region, in 2021, followed by genome sequencing in an interval of only ten days. Samples from two NHPs (one of the species Alouatta caraya in the municipality of Icarai de Minas and other of the species Callithrix penicillata in the municipality of Ubai) were collected and the presence of YFV was confirmed by RT-qPCR. We generated three near-complete and one partial-genome by Nanopore sequencer MinION. Phylogenetic analysis revealed that all viral genomes recovered are equal and related to lineage South America 1, clustering with a genome detected in the Amazon region (Para state) in 2017. These findings reveal the occurrence of a new wave of viral expansion in MG, six years after detection in the state of Para in 2015, with no human outbreaks to date reported. Coordinated work between local surveillance and support laboratories has been of major importance to ensure a quick response and implementation of contingency measures towards avoiding the occurrence of YF cases in humans.

Serological Positivity against Selected Flavivirus and Alphavirus in Free-ranging Bats and Birds from Costa Rica Evidence Exposure to Arboviruses Seldom Reported Locally in Humans

Arboviruses have two ecological transmission cycles, sylvatic and urban. For some, the sylvatic cycle has not been thoroughly described in America. To study the role of wildlife in a putative sylvatic cycle, we sampled free-ranging bats and birds in two arbovirus endemic locations and analyzed them using molecular, serological, and histological methods. No current infection was detected, and no significant arbovirus-associated histological changes were observed. Neutralizing antibodies were detected against selected arboviruses. In bats, positivity in 34.95% for DENV-1, 16.26% for DENV-2, 5.69% for DENV-3, 4.87% for DENV-4, 2.43% for WNV, 4.87% for SLEV, 0,81% for YFV, 7.31% for EEEV, and 0.81% for VEEV was found. Antibodies against ZIKV were not detected. In birds, PRNT results were positive against WNV in 0.80%, SLEV in 5.64%, EEEV in 8.4%, and VEEV in 5.63%. An additional retrospective PRNT analysis was performed using bat samples from three additional DENV endemic sites resulting in a 3.27% prevalence for WNV and 1.63% for SLEV. Interestingly one sample resulted unequivocally WNV positive confirmed by serum titration. These results suggest that free-ranging bats and birds are exposed to not currently reported hyperendemic-human infecting Flavivirus and Alphavirus, however, their role as reservoirs or hosts is still undetermined.

No evidence of SARS-CoV-2 infection in Neotropical Primates sampled during COVID-19 pandemic in Minas Gerais and Rio Grande do Sul, Brazil.

In 2019, a new coronavirus disease (COVID-19) was detected in China. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was capable to infect domestic and captive mammals like cats, tigers and minks. Due to genetic similarities, concern about the infection of Non-Human Primates (NHPs) and the establishment of a sylvatic cycle has grown in the Americas. In this study, neotropical primates (NP) were sampled in different areas from Brazil to investigate whether they were infected by SARS-CoV-2. A total of 89 samples from 51 NP of four species were examined. No positive samples were detected via RT-qPCR, regardless of the NHP species, tissue or habitat tested. This work provides the first report on the lack of evidence of circulation of SARS-CoV-2 in NP. The expand of wild animals sampling is necessary to understand their role in the epidemiology of SARS-CoV-2.

Long-Term Determinants of the Seroprevalence of the Hepatitis E Virus in Wild Boar (Sus scrofa)

The hepatitis E virus (HEV) is an emerging zoonotic pathogen whose main reservoir is suids. Most of the ecological and epidemiological aspects of its sylvatic cycle remain unknown. Thus, in this work, we study the drivers of HEV exposure in the wild boar population of Doñana National Park (DNP, southwest Spain) operating in the medium and long-term (2005–2018). Anti-HEV antibodies are widely distributed throughout the wild boar (46.7 ± 3.8%, 327 out of 700 sampled), showing a statistically significant age-increasing pattern. The temporal pattern displayed important interannual fluctuations. This could be mediated by marked variations in the population control of the wild boar, and subsequent changes in abundance rates, and its interplay with climatic conditions; as wet years together with a low abundance of wild boar led to the lowest seroprevalence. The fact that seroprevalence is high during conditions of high abundance, and not affected by rainfall level, is probably due to the increased interactions among the animals, and possibly, the subsequent higher environmental contamination with HEV particles. The proximity to the marshland (the main water body of the study area) is associated with a higher risk of testing positive, which is probably mediated by the preferential use of this area during the dry season and the favourable environmental conditions for the survival of HEV particles. A deeper understanding of the epidemiology of HEV in host communities deserves future research concerning other susceptible species. Most importantly, wild boar population control remains a challenge at the international level, and an increase of shared pathogen-related conflicts associated with this species is expected, as exemplified by HEV. Therefore, surveillance of wild boar diseases, including integrated population monitoring and sustainable population control programmes, will be essential to control the associated risks.

Database of Sabethes and Haemagogus (Diptera: Culicidae) in Argentina: Sylvatic Vectors of the Yellow Fever Virus

Yellow fever is an endemic disease in America caused by an arbovirus that circulates in the sylvatic cycle between nonhuman primates and mosquitoes of the genera Sabethes Robineau-Desvoidy and Haemagogus Williston. The main goal of this work is to report the distribution patterns of these genera in Argentina through an updated database built from published records as well as from own sample collections. These genera are represented in Argentina by a total of 18 species distributed in 14 provinces and 10 ecoregions. The ecoregions with greatest biodiversity were Paranense Forest, Yungas, Campos and Malezales. This database will also allow generating distribution maps for these mosquito genera, and their respective species in Argentina, to establish areas with high probability of viral circulation that are an essential input for vector surveillance, as a tool for public health decision-makers.

American Mammals Susceptibility to Dengue According to Geographical, Environmental, and Phylogenetic Distances

Many human emergent and re-emergent diseases have a sylvatic cycle. Yet, little effort has been put into discovering and modeling the wild mammal reservoirs of dengue (DENV), particularly in the Americas. Here, we show a species-level susceptibility prediction to dengue of wild mammals in the Americas as a function of the three most important biodiversity dimensions (ecological, geographical, and phylogenetic spaces), using machine learning protocols. Model predictions showed that different species of bats would be highly susceptible to DENV infections, where susceptibility mostly depended on phylogenetic relationships among hosts and their environmental requirement. Mammal species predicted as highly susceptible coincide with sets of species that have been reported infected in field studies, but it also suggests other species that have not been previously considered or that have been captured in low numbers. Also, the environment (i.e., the distance between the species' optima in bioclimatic dimensions) in combination with geographic and phylogenetic distance is highly relevant in predicting susceptibility to DENV in wild mammals. Our results agree with previous modeling efforts indicating that temperature is an important factor determining DENV transmission, and provide novel insights regarding other relevant factors and the importance of considering wild reservoirs. This modeling framework will aid in the identification of potential DENV reservoirs for future surveillance efforts.