Virological evidence of the impact of non-pharmaceutical interventions against COVID-19 in Ecuador, a resource-limited setting.

Ecuador had substantial COVID-19-mortality during 2020 despite early implementation of non-pharmaceutical interventions (NPIs). Resource-limited settings like Ecuador have high proportions of informal labour which entail high human mobility, questioning efficacy of NPIs. We performed a retrospective observational study in Ecuador's national reference laboratory for viral respiratory infections during March 2020-February 2021 using stored respiratory specimens from 1950 patients, corresponding to 2.3% of all samples analysed within the Ecuadorian national surveillance system per week. During 2020, detection of SARS-CoV-2 (Pearson correlation; r = -0.74; p = 0.01) and other respiratory viruses (Pearson correlation; r = -0.68; p = 0.02) by real-time RT-PCR correlated negatively with NPIs stringency. Among respiratory viruses, adenoviruses (Fisher's exact-test; p = 0.026), parainfluenzaviruses (p = 0.04), enteroviruses (p < 0.0001) and metapneumoviruses (p < 0.0001) occurred significantly more frequently during months of absent or non-stringent NPIs (characterized by <55% stringency according to the Oxford stringency index data for Ecuador). Phylogenomic analyses of 632 newly characterized SARS-CoV-2 genomes revealed 100 near-parallel SARS-CoV-2 introductions during early 2020 in the absence of NPIs. NPI stringency correlated negatively with the number of circulating SARS-CoV-2 lineages during 2020 (r = -0.69; p = 0.02). Phylogeographic reconstructions showed differential SARS-CoV-2 dispersion patterns during 2020, with more short-distance transitions potentially associated with recreational activity during non-stringent NPIs. There were also fewer geographic transitions during strict NPIs (n = 450) than during non-stringent or absent NPIs (n = 580). Virological evidence supports that NPIs had an effect on virus spread and distribution in Ecuador, providing a template for future epidemics in resource-limited settings and contributing to a balanced assessment of societal costs entailed by strict NPIs.

Experimental infection of Artibeus lituratus bats and no detection of Zika virus in neotropical bats from French Guiana, Peru, and Costa Rica suggests a limited role of bats in Zika transmission.

Bats are important natural reservoir hosts of a diverse range of viruses that can be transmitted to humans and have been suggested to play an important role in the Zika virus (ZIKV) transmission cycle. However, the exact role of these animals as reservoirs for flaviviruses is still controversial. To further expand our understanding of the role of bats in the ZIKV transmission cycle in Latin America, we carried out an experimental infection in wild-caught Artibeus lituratus bats and sampled several free-living neotropical bats across three countries of the region. Experimental ZIKV infection was performed in wild-caught adult bats (4 females and 5 males). The most relevant findings were hemorrhages in the bladder, stomach and patagium. Significant histological findings included inflammatory infiltrate consisting of a predominance of neutrophils and lymphocytes, in addition to degeneration in the reproductive tract of males and females. This suggests that bat reproduction might be at some level affected by ZIKV. Leukopenia was also observed in some inoculated animals. Hemorrhages, genital alterations, and leukopenia are suggested to be caused by ZIKV; however, since these were wild-caught bats, we cannot exclude other agents. Detection of ZIKV by qPCR was observed at low concentrations in only two urine samples in two inoculated animals. All other animals and tissues tested were negative. Finally, no virus-neutralizing antibodies were found in any animal. To determine ZIKV infection in nature, the blood of a total of 2056 bats was sampled for ZIKV detection by qPCR. Most of the sampled individuals belonged to the genus Pteronotus sp. (23%), followed by the species Carollia sp. (17%), Anoura sp. (14%), and Molossus sp. (13.7%). No sample of any tested species was positive for ZIKV by qPCR. These results together suggest that bats are not efficient amplifiers or reservoirs of ZIKV and may not have an important role in ZIKV transmission dynamics.

Virulent Brucella nosferati infecting Desmodus rotundus has emerging potential due to the broad foraging range of its bat host for humans and wild and domestic animals.

Desmodus rotundus, vampire bats, transmit dangerous infections, and brucellosis is a hazardous zoonotic disease, two adversities that coexist in the subtropical and tropical areas of the American continent. Here, we report a 47.89% Brucella infection prevalence in a colony of vampire bats inhabiting the tropical rainforest of Costa Rica. The bacterium induced placentitis and fetal death in bats. Wide-range phenotypic and genotypic characterization placed the Brucella organisms as a new pathogenic species named Brucella nosferati sp. nov., isolated from bat tissues, including the salivary glands, suggesting feeding behavior might favor transmission to their prey. Overall analyses placed B. nosferati as the etiological agent of a reported canine brucellosis case, demonstrating its potential for infecting other hosts. To assess the putative prey hosts, we analyzed the intestinal contents of 14 infected and 23 non-infected bats by proteomics. A total of 54,508 peptides sorted into 7,203 unique peptides corresponding to 1,521 proteins were identified. Twenty-three wildlife and domestic taxa, including humans, were foraged by B. nosferati-infected D. rotundus, suggesting contact of this bacterium with a broad range of hosts. Our approach is appropriate for detecting, in a single study, the prey preferences of vampire bats in a diverse area, demonstrating its suitability for control strategies where vampire bats thrive. IMPORTANCE The discovery that a high proportion of vampire bats in a tropical area is infected with pathogenic Brucella nosferati and that bats forage on humans and many wild and domestic animals is relevant from the perspective of emerging disease prevention. Indeed, bats harboring B. nosferati in their salivary glands may transmit this pathogenic bacterium to other hosts. This potential is not trivial since, besides the demonstrated pathogenicity, this bacterium possesses all the required virulent arsenal of dangerous Brucella organisms, including those that are zoonotic for humans. Our work has settled the basis for future surveillance actions in brucellosis control programs where these infected bats thrive. Moreover, our strategy to identify the foraging range of bats may be adapted for exploring the feeding habits of diverse animals, including arthropod vectors of infectious diseases, and therefore of interest to a broader audience besides experts on Brucella and bats.

A novel ACE2 decoy for both neutralization of SARS-CoV-2 variants and killing of infected cells.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to millions of infections and deaths worldwide. As this virus evolves rapidly, there is a high need for treatment options that can win the race against new emerging variants of concern. Here, we describe a novel immunotherapeutic drug based on the SARS-CoV-2 entry receptor ACE2 and provide experimental evidence that it cannot only be used for (i) neutralization of SARS-CoV-2 in vitro and in SARS-CoV-2-infected animal models but also for (ii) clearance of virus-infected cells. For the latter purpose, we equipped the ACE2 decoy with an epitope tag. Thereby, we converted it to an adapter molecule, which we successfully applied in the modular platforms UniMAB and UniCAR for retargeting of either unmodified or universal chimeric antigen receptor-modified immune effector cells. Our results pave the way for a clinical application of this novel ACE2 decoy, which will clearly improve COVID-19 treatment.

Detection of Rickettsia spp. in ticks of wildlife fauna from Costa Rica: First report of Rickettsia rhipicephali in Central America.

In the past two decades, new species of Rickettsia have been detected and described worldwide, some of them considered pathogenic for humans. Although Costa Rica is considered a biodiversity hotspot, the knowledge about rickettsiae in sylvatic ecosystems and wild animals is scarce. The aim of this preliminary study was to detect and identify species of Rickettsia in ticks collected from wild animals in Costa Rica. A total 119 ticks were collected from 16 animal host species belonging to diverse vertebrate families (Didelphidae, Procyonidae, Felidae, Choloepodidae, Bradypodidae, Myrmecophagidae, Tayassuidae, Tapiridae, Phyllostomidae, Bufonidae, Geoemydidae, Boidae, Colubridae), and they were grouped into 43 pools to detect the presence of Rickettsia spp. DNA by PCR targeting the gltA gene. In positive pools, amplicons of the ompA, sca5 (ompB), and/or htrA genes were also amplified to identify the species present. The identification of some ticks was also confirmed by molecular methods. Four species of Rickettsia were detected in eight (19%) tick pools: Rickettsia amblyommatis in four pools of Amblyomma geayi (host: Caluromys derbianus) and one pool of Amblyomma cf. parvum (host: Nasua narica), Rickettsia rhipicephali in one pool of Dermacentor latus (host: Tayassu pecari), 'Candidatus Rickettsia colombianensi' in one pool of Amblyomma sp. nymphs (host: Boa constrictor), and Rickettsia sp. genotype IbR/CRC in one pool of Ixodes cf. boliviensis (host: Puma concolor). This is the first molecular detection of R. rhipicephali in Central America, and of 'Candidatus R. colombianensi' in Costa Rica. Results show that diverse wild animals and their ticks are associated with several species of rickettsiae in Costa Rica, which may come in contact with humans and other domestic animals in sylvatic environments.

Highly Diverse Arenaviruses in Neotropical Bats, Brazil.

We detected arenavirus RNA in 1.6% of 1,047 bats in Brazil that were sampled during 2007-2011. We identified Tacaribe virus in 2 Artibeus sp. bats and a new arenavirus species in Carollia perspicillata bats that we named Tietê mammarenavirus. Our results suggest that bats are an underrecognized arenavirus reservoir.

Effectiveness of Naturally Acquired and Vaccine-Induced Immune Responses to SARS-CoV-2 Mu Variant.

SARS-CoV-2 Mu variant emerged in Colombia in 2021 and spread globally. In 49 serum samples from vaccinees and COVID-19 survivors in Colombia, neutralization was significantly lower (p<0.0001) for Mu than a parental strain and variants of concern. Only the Omicron variant of concern demonstrated higher immune evasion.

Genomic determinants of Furin cleavage in diverse European SARS-related bat coronaviruses.

The furin cleavage site (FCS) in SARS-CoV-2 is unique within the Severe acute respiratory syndrome-related coronavirus (SrC) species. We re-assessed diverse SrC from European horseshoe bats and analyzed the spike-encoding genomic region harboring the FCS in SARS-CoV-2. We reveal molecular features in SrC such as purine richness and RNA secondary structures that resemble those required for FCS acquisition in avian influenza viruses. We discuss the potential acquisition of FCS through molecular mechanisms such as nucleotide substitution, insertion, or recombination, and show that a single nucleotide exchange in two European bat-associated SrC may suffice to enable furin cleavage. Furthermore, we show that FCS occurrence is variable in bat- and rodent-borne counterparts of human coronaviruses. Our results suggest that furin cleavage sites can be acquired in SrC via conserved molecular mechanisms known in other reservoir-bound RNA viruses and thus support a natural origin of SARS-CoV-2.

Frequent Infection of Cats With SARS-CoV-2 Irrespective of Pre-Existing Enzootic Coronavirus Immunity, Brazil 2020.

Carnivores such as cats and minks are highly susceptible to SARS-CoV-2. Brazil is a global COVID-19 hot spot and several cases of human-to-cat transmission have been documented. We investigated the spread of SARS-CoV-2 by testing 547 domestic cats sampled between July-November 2020 from seven states in southern, southeastern, and northeastern Brazil. Moreover, we investigated whether immune responses elicited by enzootic coronaviruses affect SARS-CoV-2 infection in cats. We found infection with significantly higher neutralizing antibody titers against the Gamma variant of concern, endemic in Brazil during 2020, than against an early SARS-CoV-2 B.1 isolate (p<0.0001), validating the use of Gamma for further testing. The overall SARS-CoV-2 seroprevalence in Brazilian cats during late 2020 validated by plaque reduction neutralization test (PRNT90) was 7.3% (95% CI, 5.3-9.8). There was no significant difference in SARS-CoV-2 seroprevalence in cats between Brazilian states, suggesting homogeneous infection levels ranging from 4.6% (95% CI, 2.2-8.4) to 11.4% (95% CI, 6.7-17.4; p=0.4438). Seroprevalence of the prototypic cat coronavirus Feline coronavirus (FCoV) in a PRNT90 was high at 33.3% (95% CI, 24.9-42.5) and seroprevalence of Bovine coronavirus (BCoV) was low at 1.7% (95% CI, 0.2-5.9) in a PRNT90. Neutralizing antibody titers were significantly lower for FCoV than for SARS-CoV-2 (p=0.0001), consistent with relatively more recent infection of cats with SARS-CoV-2. Neither the magnitude of SARS-CoV-2 antibody titers (p=0.6390), nor SARS-CoV-2 infection status were affected by FCoV serostatus (p=0.8863). Our data suggest that pre-existing immunity against enzootic coronaviruses neither prevents, nor enhances SARS-CoV-2 infection in cats. High SARS-CoV-2 seroprevalence already during the first year of the pandemic substantiates frequent infection of domestic cats and raises concerns on potential SARS-CoV-2 mutations escaping human immunity upon spillback.

Serological Evidence That SARS-CoV-2 Has Not Emerged in Deer in Germany or Austria during the COVID-19 Pandemic.

Spillover of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) to North American white-tailed deer (Odocoileus virginianus) has been documented. However, it is unclear if this is a phenomenon specific to North American deer or is a broader problem. We evaluated pre and pandemic exposure of German and Austrian deer species using a SARS-CoV-2 pseudoneutralization assay. In stark contrast to North American white-tailed deer, we found no evidence of SARS-CoV-2 exposure.

Emergence of SARS-CoV-2 Delta Variant, Benin, May-July 2021.

Severe acute respiratory syndrome coronavirus 2 Delta variant epidemiology in Africa is unknown. We found Delta variant was introduced in Benin during April-May 2021 and became predominant within 2 months, after which a steep increase in reported coronavirus disease incidence occurred. Benin might require increased nonpharmaceutical interventions and vaccination coverage.

High SARS-CoV-2 Seroprevalence in Rural Peru, 2021: a Cross-Sectional Population-Based Study.

Latin America has been severely affected by the COVID-19 pandemic. The COVID-19 burden in rural settings in Latin America is unclear. We performed a cross-sectional, population-based, random-selection SARS-CoV-2 serologic study during March 2021 in the rural population of San Martin region, northern Peru. In total, 563 persons from 288 houses across 10 provinces were enrolled, reaching 0.2% of the total rural population of San Martin. Screening for SARS-CoV-2 IgG antibodies was done using a chemiluminescence immunoassay (CLIA), and reactive sera were confirmed using a SARS-CoV-2 surrogate virus neutralization test (sVNT). Validation of the testing algorithm using prepandemic sera from two regions of Peru showed false-positive results in the CLIA (23/84 sera; 27%) but not in the sVNT, highlighting the pitfalls of SARS-CoV-2 antibody testing in tropical regions and the high specificity of the two-step algorithm used in this study. An overall 59.0% seroprevalence (95% confidence interval [CI], 55 to 63%) corroborated intense SARS-CoV-2 spread in San Martin. Seroprevalence rates between the 10 provinces varied from 41.3 to 74.0% (95% CI, 30 to 84%). Higher seroprevalence was not associated with population size, population density, surface area, mean altitude, or poverty index in Spearman correlations. Seroprevalence and reported incidence diverged substantially between provinces, suggesting regional biases of COVID-19 surveillance data. Potentially, limited health care access due to environmental, economic, and cultural factors might lead to undetected infections in rural populations. Additionally, test avoidance to evade mandatory quarantine might affect rural regions more than urban regions. Serologic diagnostics should be pursued in resource-limited settings to inform country-level surveillance and vaccination strategies and to support control measures for COVID-19. IMPORTANCE Latin America is a global hot spot of the COVID-19 pandemic. Serologic studies in Latin America have been mostly performed in urban settings. Rural populations comprise 20% of the total Latin American population. Nevertheless, information on COVID-19 spread in rural settings is scarce. Using a representative population-based seroprevalence study, we detected a high seroprevalence in rural populations in San Martin, northern Peru, in 2021, reaching 41 to 74%. However, seroprevalence and reported incidence diverged substantially between regions, potentially due to limited health care access or test avoidance due to mandatory quarantine. Our results suggest that rural populations are highly affected by SARS-CoV-2 even though they are sociodemographically distinct from urban populations and that highly specific serological diagnostics should be performed in resource-limited settings to support public health strategies of COVID-19 control.

High Efficacy of Therapeutic Equine Hyperimmune Antibodies Against SARS-CoV-2 Variants of Concern.

SARS-CoV-2 variants of concern show reduced neutralization by vaccine-induced and therapeutic monoclonal antibodies; therefore, treatment alternatives are needed. We tested therapeutic equine polyclonal antibodies (pAbs) that are being assessed in clinical trials in Costa Rica against five globally circulating variants of concern: alpha, beta, epsilon, gamma and delta, using plaque reduction neutralization assays. We show that equine pAbs efficiently neutralize the variants of concern, with inhibitory concentrations in the range of 0.146-1.078 µg/mL, which correspond to extremely low concentrations when compared to pAbs doses used in clinical trials. Equine pAbs are an effective, broad coverage, low-cost and a scalable COVID-19 treatment.

Genomic epidemiology of SARS-CoV-2 transmission lineages in Ecuador.

Characterisation of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genetic diversity through space and time can reveal trends in virus importation and domestic circulation and permit the exploration of questions regarding the early transmission dynamics. Here, we present a detailed description of SARS-CoV-2 genomic epidemiology in Ecuador, one of the hardest hit countries during the early stages of the coronavirus-19 pandemic. We generated and analysed 160 whole genome sequences sampled from all provinces of Ecuador in 2020. Molecular clock and phylogeographic analysis of these sequences in the context of global SARS-CoV-2 diversity enable us to identify and characterise individual transmission lineages within Ecuador, explore their spatiotemporal distributions, and consider their introduction and domestic circulation. Our results reveal a pattern of multiple international importations across the country, with apparent differences between key provinces. Transmission lineages were mostly introduced before the implementation of non-pharmaceutical interventions, with differential degrees of persistence and national dissemination.

Self-prescribed Ivermectin use is associated with a lower rate of seroconversion in health care workers diagnosed with COVID, in a dose-dependent response.

BACKGROUND: Over-the-counter use of ivermectin amongst other drugs as SARS-CoV-2 treatment has been increasingly common, despite the lack of evidence on its clinical efficacy. OBJECTIVE: To evaluate the effect of ivermectin use on production of antibodies against SARS-CoV-2 in health care workers (HCW) diagnosed with COVID-19 and of Th1/Th2 cytokines by stimulated peripheral blood mononuclear cells of the same cohort (PBMCs). METHODS: This cross-sectional study evaluated seroconversion and neutralizing antibodies production in HCW at Complexo Hospitalar Universitário Professor Edgard Santos (Salvador, Brazil), diagnosed with COVID-19 from May to July, 2020, as well as in vitro production of antibody against SARS-CoV-2 and Th1/Th2 cytokines. Analyses were performed between December 2020 and February 2021. Participants were stratified according to the use of ivermectin (≤ 1 dose vs. multiple doses) for treatment of COVID-19. RESULTS: 45 HCW were included (62% women). Mean age was 39 years, and disease severity was similar across groups. Neutralizing antibodies were detected less frequently in multiple doses (70%) vs. ≤ 1 dose (97%) groups, p = 0.02). PBMCs of patients in multiple doses group also were less likely to produce antibodies against SARS-CoV-2 following in vitro stimulation with purified spike protein in comparison with patients in ≤ 1 dose group (p < 0.001). PBMC´s production of Th1/Th2 cytokines levels was similar across groups. Abdominal pain (15% vs 46%, p = 0.04), diarrhea (21% vs. 55%, p = 0.05) and taste perversion (0% vs. 18%, p = 0.05) were more frequently reported by participants that used multiple doses of ivermectin. CONCLUSIONS: Although there was no evidence for differential disease severity upon ivermectin use for treatment of COVID-19 it was associated with more gastro-intestinal side-effects and impairment of anti-SARS-CoV2 antibodies production, in a dose dependent manner. This potentially impacts the effectiveness of immune response and the risk of reinfection and warrants additional studies for clarifying the mechanisms and consequences of such immunomodulatory effects.

Mutations Associated with SARS-CoV-2 Variants of Concern, Benin, Early 2021.

Intense transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Africa might promote emergence of variants. We describe 10 SARS-CoV-2 lineages in Benin during early 2021 that harbored mutations associated with variants of concern. Benin-derived SARS-CoV-2 strains were more efficiently neutralized by antibodies derived from vaccinees than patients, warranting accelerated vaccination in Africa.

Rabies in Costa Rica - Next Steps Towards Controlling Bat-Borne Rabies After its Elimination in Dogs.

Rabies is an acute, progressive encephalitis caused by a lyssavirus, with the highest case fatality of any conventional infectious disease. More than 17 different lyssaviruses have been described, but rabies virus is the most widely distributed and important member of the genus. Globally, tens of thousands of human fatalities still occur each year. Although all mammals are susceptible, most human fatalities are caused by the bites of rabid dogs, within lesser developed countries. A global plan envisions the elimination of human rabies cases caused via dogs by the year 2030. The combination of prophylaxis of exposed humans and mass vaccination of dogs is an essential strategy for such success. Regionally, the Americas are well on the way to meet this goal. As one example of achievement, Costa Rica, a small country within Central America, reported the last autochthonous case of human rabies transmitted by a dog at the end of the 1970s. Today, rabies virus transmitted by the common vampire bat, Desmodus rotundus, as well as other wildlife, remains a major concern for humans, livestock, and other animals throughout the region. This review summarizes the historical occurrence of dog rabies and its elimination in Costa Rica, describes the current occurrence of the disease with a particular focus upon affected livestock, discusses the ecology of the vampire bat as the primary reservoir relevant to management, details the clinical characteristics of recent human rabies cases, and provides suggestions for resolution of global challenges posed by this zoonosis within a One Health context.

Serosurvey of Nonhuman Primates in Costa Rica at the Human-Wildlife Interface Reveals High Exposure to Flaviviruses.

Arthropod-borne viruses belonging to the flavivirus genus possess an enormous relevance in public health. Neotropical non-human primates (NPs) have been proposed to be susceptible to flavivirus infections due to their arboreal and diurnal habits, their genetic similarity to humans, and their relative closeness to humans. However, the only known flavivirus in the American continent maintained by sylvatic cycles involving NPs is yellow fever virus (YFV), and NPs' role as potential hosts of other flaviviruses is still unknown. Here, we examined flavivirus exposure in 86 serum samples including 83.7% samples from free-range and 16.3% from captive NPs living in flavivirus-endemic regions of Costa Rica. Serum samples were opportunistically collected throughout Costa Rica in 2000-2015. We used a highly specific micro-plaque reduction neutralization test (micro-PRNT) to determine the presence of antibodies against YFV, dengue virus 1-4 (DENV), Zika virus, West Nile virus (WNV), and Saint Louis encephalitis virus (SLEV). We found evidence of seropositive NPs with homotypic reactivity to SLEV 11.6% (10/86), DENV 10.5% (9/86), and WNV 2.3% (2/86). Heterotypic reactivity was determined in 3.5% (3/86) of individuals against DENV, 1.2% (1/86) against SLEV, and 1.2% (1/86) against WNV. We found that 13.9% (12/86) of NPs were positive for an undetermined flavivirus species. No antibodies against DENV-3, DENV-4, YFV, or ZIKV were found. This work provides compelling serological evidence of flavivirus exposure in Costa Rican NPs, in particular to DENV, SLEV, and WNV. The range of years of sampling and the region from where positives were detected coincide with those in which peaks of DENV in human populations were registered, suggesting bidirectional exposure due to human-wildlife contact or bridging vectors. Our work suggests the continuous exposure of wildlife populations to various flaviviruses of public health importance and underscores the necessity of further surveillance of flaviviruses at the human-wildlife interface in Central America.

Susceptibility of Well-Differentiated Airway Epithelial Cell Cultures from Domestic and Wild Animals to Severe Acute Respiratory Syndrome Coronavirus 2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally, and the number of worldwide cases continues to rise. The zoonotic origins of SARS-CoV-2 and its intermediate and potential spillback host reservoirs, besides humans, remain largely unknown. Because of ethical and experimental constraints and more important, to reduce and refine animal experimentation, we used our repository of well-differentiated airway epithelial cell (AEC) cultures from various domesticated and wildlife animal species to assess their susceptibility to SARS-CoV-2. We observed that SARS-CoV-2 replicated efficiently only in monkey and cat AEC culture models. Whole-genome sequencing of progeny viruses revealed no obvious signs of nucleotide transitions required for SARS-CoV-2 to productively infect monkey and cat AEC cultures. Our findings, together with previous reports of human-to-animal spillover events, warrant close surveillance to determine the potential role of cats, monkeys, and closely related species as spillback reservoirs for SARS-CoV-2.