Search

Mosquitoes are vectors of several arboviruses belonging to the genus Alphavirus and Flavivirus. Costa Rica is endemic for several arboviruses. To described and analyze the community structure of vectors in the country, a sampling was performed in two different coastal locations with evidence of arboviral activity during rainy and dry seasons. Encephalitis vector surveillance Traps, CDC Female Gravid Traps and ovitraps were used. Viral detection for several arbovirus was attempted. Blood-meal identification was also performed. A total of 1802 adult mosquitoes belonging to 55 species were captured. Culex quinquefasciatus was the most captured. species The Normalized Difference Vegetation Index (NDVI) was estimated for each area during rainy and dry season. The higher difference between seasonal regional NDVI yield lower values of Chao- Sørensen Similarity Index. Chao2 values and Shannon Index for species diversity were also estimated. There was no viral detection for VEEV, WNV, Madariaga. Dengue virus and Zika virus were detected in two separate Culex quinquefasciatus. The main sources of bloodmeal chickens (60%) and humans (27.5%). Both sampled areas have different seasonal dynamics and population turnover reflected in the Chao2 species richness estimation values and Shannon diversity Index. NDVI influence the mosquito diversity at a regional scale more than at a local scale. However, yearlong continuous sampling is required for a better understanding of local dynamics.

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.

Bats are important natural reservoir hosts of a diverse range of viruses that can be transmitted to humans and have been suggested that could 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 an experimental infection in wild-caught Artibeus bats and sampled several free-living neotropical bats over three countries of the region. Experimental ZIKV infection was made in free-ranging adult bats (4 females and 5 males). The most relevant gross 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 suggestive to be caused by ZIKV, however, since these are wild-caught bats, we can not exclude other agents. Excretion of ZIKV by qPCR was detected (low titles) in only two urine samples in two inoculated animals. All other animals and tissues tested negative. Finally, no virus-neutralizing Abs were found in any animal. To determine ZIKV infection in nature, a total of 2056 bats were blood 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 resulted positive to 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.Author summaryIn previous works made in 2008-2009, we have found the presence of antibodies against Flaviviruses and viral RNA has been detected in Neotropical chiropterans of Mexico, which led us to support the hypothesis that these animals could be reservoirs of Flaviviruses. As controversial opinions have been exposed, and based on a previous (2019) experimental ZIKV infection made in Colorado State University using adult Artibeus males from a captive colony, in this work we also experimentally infected adult Artibeus males complementarily adding females and using free-living animals instead of laboratory bats. We also monitored a diverse range of natural bat populations in Latin America for the presence of viral RNA against ZIKV in blood. A plaque reduction seroneutralization test was used for the detection of antibodies against ZIKV. Similar to the previous work, we found histopathological alteration in male testicles but also in ovaries and oviducts of females, as well as gliosis and multifocal necrosis in pyramidal neurons and Purkinge cells of inoculated animals. Only two urine samples from inoculated animals showed viral RNA. Additionally, leukopenia and lymphoid follicular splenic hyperplasia were evidenced. Differing to what was reported, no neutralizing antibodies against ZIKV were detected in any sample. Viral RNA within the blood was not present in any of the 2056 bat samples collected in French Guyana, Peru and Costa Rica and proceeding from 33 bat genera. These results together suggest that bats are not efficient amplifiers or reservoirs of ZIKV and might not have an important role on ZIKV transmission dynamics.

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.

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.

West Nile virus (WNV) and Saint Louis encephalitis virus (SLEV) share similar virus transmission cycles that involve birds as amplifiers and mosquitoes as vectors. Mammals, including humans, are dead-end-hosts that may be asymptomatic or develop more severe symptoms. Costa Rica is a hyperendemic country for several flaviviruses such as Dengue (DENV) and Zika (ZIKV) and previous research showed limited and restricted SLEV and WNV circulation in horses, sloths, and monkeys. Nevertheless, actual seroprevalence and high transmission areas are not yet identified. To determine putative WNV and SLEV circulation, we sampled peri domestic and domestic animals, humans, wild birds, and mosquitoes in rural households located in two DENV and ZIKV hyperendemic regions during the rainy and dry seasons of 2017-2018 and conducted PRNT assays for serology and RT PCR for virus detection. At Cuajiniquil, serological evidence of WNV and SLEV was found in equines, humans, chickens, and wild birds. Also, 5 seroconversion events were recorded for WNV (2 equine), SLEV (1 human), and DENV-1 (2 humans). At Talamanca, a lack of WNV circulation was found, but evidence of SLEV circulation was recorded in equines, humans, and wild birds. No evidence of active viral infection was found in any mosquitoes or wild bird samples. This seroconversion evidence supports the active and recent circulation of SLEV and WNV in these two regions. This study provides clear-cut evidence of WNV and SLEV circulation and should be considered by the health and epidemiology authorities for future prevention and differential diagnostics. El virus del Nilo Occidental (VNO) y el virus de la encefalitis de San Luis (SLEV) comparten ciclos de transmisión del virus similares que implican a las aves como amplificadores y a los mosquitos como vectores. Los mamíferos, incluidos los humanos, son huéspedes muertos que pueden ser asintomáticos o desarrollar síntomas más graves. Costa Rica es un país hiperendémico para varios flavivirus como el dengue (DENV) y el zika (ZIKV) y las investigaciones anteriores mostraron una circulación limitada y restringida del SLEV y el WNV en caballos, perezosos y monos. Sin embargo, aún no se ha identificado la seroprevalencia real ni las zonas de alta transmisión. Para determinar la circulación putativa del WNV y el SLEV, tomamos muestras de animales peri domésticos y domésticos, humanos, aves silvestres y mosquitos en hogares rurales ubicados en dos regiones hiperendémicas de DENV y ZIKV durante las estaciones lluviosas y secas de 2017-2018 y realizamos ensayos de PRNT para la serología y RT PCR para la detección del virus. En Cuajiniquil, se encontraron evidencias serológicas de VNO y SLEV en equinos, humanos, pollos y aves silvestres. Además, se registraron 5 eventos de seroconversión para el VNO (2 equinos), el SLEV (1 humano) y el DENV-1 (2 humanos). En Talamanca, no se encontró circulación del VNO, pero se registraron evidencias de circulación del SLEV en equinos, humanos y aves silvestres. No se encontró evidencia de infección viral activa en ningún mosquito ni en muestras de aves silvestres. Estas pruebas de seroconversión apoyan la circulación activa y reciente del SLEV y el VNO en estas dos regiones. Este estudio proporciona pruebas claras de la circulación del VNO y el SLEV y debería ser considerado por las autoridades sanitarias y epidemiológicas para la prevención y el diagnóstico diferencial en el futuro. Universidad Nacional, Costa Rica Escuela de Medicina Veterinaria

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 ��g/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.

Seven government researchers who helped to guide their governments’ responses to the COVID-19 pandemic speak out. Seven government researchers who helped to guide their governments’ responses to the COVID-19 pandemic speak out.

Conventional plaque assays rely on the use of overlays to restrict viral infection allowing the formation of distinct foci that grow in time as the replication cycle continues leading to countable plaques that are visualized with standard techniques such as crystal violet, neutral red, or immuno- labeling. This classical approach takes several days until large enough plaques can be visualized and counted with some variation due to subjectivity in plaque recognition. Since plaques are clonal lesions produced by virus-induced cytopathic effect, we applied DNA fluorescent dyes with differen- tial cell permeability to visualize them by live-cell imaging. We could observe different stages of that cytopathic effect corresponding to an early wave of cells with chromatin-condensation followed by a wave of dead cells with membrane permeabilization within plaques generated by different animal viruses. This approach enables an automated plaque identification using image analysis to increase single plaque resolution compared to crystal violet counterstaining and allows its application to plaque tracking and plaque reduction assays to test compounds for both antiviral and cytotoxic activities. This fluorescent real-time plaque assay sums to those next-generation technologies by combining this robust classical method with modern fluorescence microscopy and image analysis approaches for future applications in virology International Centre for Genetic Engineering and Biotechnology Grant CRP/CRI18-02. UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET) UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiología

Zika virus (ZIKV) is a mosquito-borne flavivirus in which human infection became relevant during recent outbreaks in Latin America due to its unrecognized association with fetal neurological disorders. Currently, there are no approved effective antivirals or vaccines for the treatment or prevention of ZIKV infections. Amantadine and rimantadine are approved antivirals used against susceptible influenza A virus infections that have been shown to have antiviral activity against other viruses, such as dengue virus (DENV). Here, we report the in vitro effectiveness of both amantadine and rimantadine hydrochlorides against ZIKV replication, resulting in a dose-dependent reduction in viral titers of a ZIKV clinical isolate and two different ZIKV reference strains. Additionally, we demonstrate similar in vitro antiviral activity of these drugs against DENV-1 and yellow fever virus (YFV), although at higher drug concentrations for the latter. ZIKV replication was inhibited at drug concentrations well below cytotoxic levels of both compounds, as denoted by the high selectivity indexes obtained with the tested strains. Further work is absolutely needed to determine the potential clinical use of these antivirals against ZIKV infections, but our results suggest the existence of a highly conserved mechanism across flavivirus, susceptible to be blocked by modified more specific adamantane compounds. Centro de Investigación en Enfermedades Tropicales, Facultad de Microbiología, Universidad de Costa Rica UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET) UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiología

In the current global emergency due to SARS-CoV-2 outbreak, passive immunotherapy emerges as a promising treatment for COVID-19. Among animal-derived products, equine formulations are still the cornerstone therapy for treating envenomations due to animal bites and stings. Therefore, drawing upon decades of experience in manufacturing snake antivenom, we developed and preclinically evaluated two anti-SARS-CoV-2 polyclonal equine formulations as potential alternative therapy for COVID-19. We immunized two groups of horses with either S1 (anti-S1) or a mixture of S1, N, and SEM mosaic (anti-Mix) viral recombinant proteins. Horses reached a maximum anti-viral antibody level at 7 weeks following priming, and showed no major adverse acute or chronic clinical alterations. Two whole-IgG formulations were prepared via hyperimmune plasma precipitation with caprylic acid and then formulated for parenteral use. Both preparations had similar physicochemical and microbiological quality and showed ELISA immunoreactivity towards S1 protein and the receptor binding domain (RBD). The anti-Mix formulation also presented immunoreactivity against N protein. Due to high anti-S1 and anti-RBD antibody content, final products exhibited high in vitro neutralizing capacity of SARS-CoV-2 infection, 80 times higher than a pool of human convalescent plasma. Pre-clinical quality profiles were similar among both products, but clinical efficacy and safety must be tested in clinical trials. The technological strategy we describe here can be adapted by other producers, particularly in low- and middle-income countries.

Science diplomacy is a fast-growing field of research, policy, and practice dedicated to understanding and reinforcing the connections between science and international affairs to tackle national, regional, and global issues. By aligning science and diplomacy, countries can attract talent, strengthen their national research ecosystems, provide avenues for participation of scientists in policy, and coordinate integrated solutions to challenges with technical dimensions. While Latin America has a long tradition of bilateral and regional cooperation, science still plays a marginal role in foreign policy, as has become evidenced by the response to the COVID-19 pandemic. With few exceptions, Latin American nations have a relatively immature science, technology, and innovation ecosystem, compounded by low public and private investments in research, coexisting with profound socio-economic inequalities, and large vulnerable populations. Such challenging conditions have created barriers to a fluid relationship between science and diplomacy, fundamentally characterized by inefficient communication between scientists and policymakers, weak collaboration channels, and duplicated roles, which altogether perpetuate siloed mentalities and a lack of trust between the two communities. Over the last decade, a first influential wave of Latin American scientists, diplomats, and other professionals, including five of the co-authors, have undertaken science diplomacy training provided by specialized organizations. Through these experiences, we recognized the need to elevate awareness and build capacities in science diplomacy in our respective countries and overall, across Latin America. Here, we describe emerging efforts and mechanisms to bridge the gap between scientists and policymakers at the national and regional level. Furthermore, we offer recommendations to amplify the impact of those pioneering initiatives toward consolidating a robust science diplomacy practice across the region. The national experiences described from Costa Rica, Mexico, and Panama can serve as a roadmap for other Latin American nations in the early process of developing a science diplomacy strategy, so they can also align themselves to a collective pathway. Most critically, we propose a way forward so that Latin America can leapfrog beyond disjointed training of individuals into integrated institutional strategies that can harness the tools of science diplomacy to enhance science-informed multilateral cooperation and enable more effective science-informed policymaking.

Genome sequencing is a key strategy in the surveillance of SARS-CoV-2, the virus responsible for the COVID-19 pandemic. Latin America is the hardest hit region of the world, accumulating almost 20% of COVID-19 cases worldwide. Costa Rica was first exemplary for the region in its pandemic control, declaring a swift state of emergency on March 16th that led to a low quantity of cases, until measures were lifted in early May. From the first detected case in March 6th to December 31st almost 170 000 cases have been reported in Costa Rica, 99.5% of them from May onwards. We analyzed the genomic variability during the SARS-CoV-2 pandemic in Costa Rica using 185 sequences, 52 from the first months of the pandemic, and 133 from the current wave.Three GISAID clades (G, GH, and GR) and three PANGOLIN lineages (B.1, B.1.1, and B.1.291) are predominant, with phylogenetic relationships that are in line with the results of other Latin American countries, suggesting introduction and multiple re-introductions from other regions of the world. The whole-genome variant calling analysis identified a total of 283 distinct nucleotide variants. These correspond mostly to non-synonymous mutations (51.6%, 146) but 45.6% (129) corresponded to synonymous mutations. The 283 variants showed an expected power-law distribution: 190 single nucleotide mutations were identified in single sequences, only 16 single nucleotide mutations were found in >5% sequences, and only two mutations in >50% genomes. These mutations were distributed through the whole genome. However, 63.6% were present in ORF1ab, 11.7% in Spike gene and 10.6% in the Nucleocapsid gene. Additionally, the prevalence of worldwide-found variant D614G in the Spike (98.9% in Costa Rica), ORF8 L84S (1.1%) is similar to what is found elsewhere. Interestingly, the frequency of mutation T1117I in the Spike has increased during the current pandemic wave beginning in May 2020 in Costa Rica, reaching 29.2% detection in the full genome analyses in November 2020. This variant has been observed in less than 1% of the GISAID reported sequences worldwide in all the 2020. Structural modeling of the Spike protein with the T1117I mutation suggest a potential effect on the viral oligomerization needed for cell infection, but no differences with other genomes on transmissibility, severity nor vaccine effectiveness are predicted. Nevertheless, in-vitro experiments are required to support these in-silico findings. In conclusion, genome analyses of the SARS-CoV-2 sequences over the course of COVID-19 pandemic in Costa Rica suggest introduction of lineages from other countries as travel bans and measures were lifted, similar to results found in other studies, as well as an increase in the Spike-T1117I variant that needs to be monitored and studied in further analyses as part of the surveillance program during the pandemic.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread globally, and the number of cases continues to rise all over the world. Besides humans, the zoonotic origin, as well as intermediate and potential spillback host reservoirs of SARS-CoV-2 are unknown. To circumvent ethical and experimental constraints, and more importantly, to reduce and refine animal experimentation, we employed our airway epithelial cell (AEC) culture repository composed of various domesticated and wildlife animal species to assess their susceptibility to SARS-CoV-2. In this study, we inoculated well-differentiated animal AEC cultures of monkey, cat, ferret, dog, rabbit, pig, cattle, goat, llama, camel, and two neotropical bat species with SARS-CoV-2. We observed that SARS-CoV-2 only replicated efficiently in monkey and cat AEC culture models. Whole-genome sequencing of progeny virus revealed no obvious signs of nucleotide transitions required for SARS-CoV-2 to productively infect monkey and cat epithelial airway cells. Our findings, together with the previously reported human-to-animal spillover events warrants close surveillance to understand the potential role of cats, monkeys, and closely related species as spillback reservoirs for SARS-CoV-2.

In the current global emergency due to SARS-CoV-2 outbreak, passive immunotherapy emerges as a promising treatment for COVID-19. Among animal-derived products, equine formulations are still the cornerstone therapy for treating envenomations due to animal bites and stings. Therefore, drawing upon decades of experience in manufacturing snake antivenom, we developed and preclinically evaluated two anti-SARS-CoV-2 polyclonal equine formulations as potential alternative therapy for COVID-19. We immunized two groups of horses with either S1 (anti-S1) or a mixture of S1, N, and SEM mosaic (anti-Mix) viral recombinant proteins. Horses reached a maximum anti-viral antibody level at 7 weeks following priming, and showed no major adverse acute or chronic clinical alterations. Two whole-IgG formulations were prepared via hyperimmune plasma precipitation with caprylic acid and then formulated for parenteral use. Both preparations had similar physicochemical and microbiological quality and showed ELISA immunoreactivity towards S1 protein and the receptor binding domain (RBD). The anti-Mix formulation also presented immunoreactivity against N protein. Due to high anti-S1 and anti-RBD antibody content, final products exhibited highin vitroneutralizing capacity of SARS-CoV-2 infection, 80 times higher than a pool of human convalescent plasma. Pre-clinical quality profiles were similar among both products, but clinical efficacy and safety must be tested in clinical trials. The technological strategy we describe here can be adapted by other producers, particularly in low- and middle-income countries.

In this review, we attempt to give the reader an update on clinical diagnosis, management, and treatment of Chikungunya virus (CHIKV) infection regarding the course of the disease and the role of clinical laboratory. CHIKV infection evolves through three phases: acute, sub-acute, and chronic. Protocol for patient assessment and management should be in agreement with each phase requirements. Despite its benignity, CHIKV infection has shown an increase in severe cases during recent outbreaks. Among the most affected group are neonates, elderly population, and patients with co-morbidities. Furthermore, since CHIKV geographical distribution and clinical spectrum overlaps with other arboviruses, differential diagnosis becomes crucial for physicians to ensure good management and treatment for patients. In this review, we describe and compare the main symptoms, complications, and risk groups for CHIKV, Dengue (DENV), and Zika (ZIKV) infections, regarding key features will guide to an accurate diagnosis. CHIKV is an emergent and reemergent arbovirus that arrived in the Americas in 2014, causing thousands of infections. The acute febrile phase is similar to that of dengue but with a higher proportion of debilitating arthralgia that can persist for months or even years after infection. Severe forms of CHIKV infection can lead to chronic polyarthralgia, neurologic symptoms, or multiorgan failure. Treatment of CHIKV infection is mainly focused on supportive care, which includes the use of analgesic and anti-inflammatory medication, rehydration, and rest.

Dengue is the most widespread arboviral disease affecting humans. Bats are recognized carriers of emerging viral zoonoses and have been proposed as dengue reservoirs, since RNA/NS1 and/or antiviral antibodies have been detected. Yet, experimental inoculation of Artibeus bats failed to show virus replication. This conflicting results prevent drawing further conclusions of whether bats sustain dengue infection. To test bat cellular permissivity to dengue infection, we established primary bat embryonic cells from diverse organs and tissues of Artibeus jamaicensis, Molossus sinaloae, and Desmodus rotundus. We observed a limited serotype-, organ-, and bat species- specific dengue susceptibility. Only some Molossusderived primary cells sustained poorly initial Dengue serotype-1 replication, though it was latter absent. To elucidate if Molossus bats may play a role in dengue replication, ecological or in vivo experiments must be performed. Taken together our results show that Dengue did not replicate efficiently in cell lines derived from Neotropical bat species. UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiología UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET)

Sloths are genetically and physiologically divergent mammals. Phleboviruses are major arthropod‐borne viruses (arboviruses) causing disease in humans and other animals globally. Sloths host arboviruses, but virus detections are scarce. A phlebovirus termed Anhanga virus (ANHV) was isolated from a Brazilian Linnaeus's two‐toed sloth (Choloepus didactylus) in 1962. Here, we investigated the presence of phleboviruses in sera sampled in 2014 from 74 Hoffmann's two‐toed (Choloepus hoffmanni, n = 65) and three‐toed (Bradypus variegatus, n = 9) sloths in Costa Rica by broadly reactive RT‐PCR. A clinically healthy adult Hoffmann's two‐toed sloth was infected with a phlebovirus. Viral load in this animal was high at 8.5 × 107 RNA copies/ml. The full coding sequence of the virus was determined by deep sequencing. Phylogenetic analyses and sequence distance comparisons revealed that the new sloth virus, likely representing a new phlebovirus species, provisionally named Penshurt virus (PEHV), was most closely related to ANHV, with amino acid identities of 93.1%, 84.6%, 94.7% and 89.0% in the translated L, M, N and NSs genes, respectively. Significantly more non‐synonymous mutations relative to ANHV occurred in the M gene encoding the viral glycoproteins and in the NSs gene encoding a putative interferon antagonist compared to L and N genes. This was compatible with viral adaptation to different sloth species and with micro‐evolutionary processes associated with immune evasion during the genealogy of sloth‐associated phleboviruses. However, gene‐wide mean dN/dS ratios were low at 0.02–0.15 and no sites showed significant evidence for positive selection, pointing to comparable selection pressures within sloth‐associated viruses and genetically related phleboviruses infecting hosts other than sloths. The detection of a new phlebovirus closely‐related to ANHV, in sloths from Costa Rica fifty years after and more than 3,000 km away from the isolation of ANHV confirmed the host associations of ANHV‐related phleboviruses with the two extant species of two‐toed sloths.

Dengue virus (DENV) infection of humans is presently the most important arthropod-borne viral global threat, for which no suitable or reliable animal model exists. Reports addressing the effect of DENV on vascular components other than endothelial cells are lacking. Dengue virus infection of vascular smooth muscle cells, which play a physiological compensatory response to hypotension in arteries and arterioles, has not been characterized, thus precluding our understanding of the role of these vascular components in dengue pathogenesis. Therefore, we studied the permissiveness of primary human umbilical artery smooth muscle cells (HUASMC) to DENV 1-4 infection and compared with the infection in the previously reported primary human umbilical vein endothelial cells (HUVEC) and the classically used, non-transformed, and highly permissive Lilly Laboratories Cell-Monkey Kidney 2 cells. Our results show that HUASMC are susceptible and productive to infection with the four DENV serotypes, although to a lesser extent when compared with the other cell lines. This is the first report of DENV permissiveness in human smooth muscle cells, which might represent an unexplored pathophysiological contributor to the vascular collapse observed in severe human dengue infection. Universidad de Costa Rica/[803-A5-025]/UCR/Costa Rica UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiología UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET)

Genome sequencing is a key strategy in the surveillance of SARS-CoV-2, the virus responsible for the COVID-19 pandemic. Latin America is the hardest-hit region of the world, accumulating almost 20% of COVID-19 cases worldwide. In Costa Rica, from the first detected case on March 6th to December 31st almost 170,000 cases have been reported. We analyzed the genomic variability during the SARS-CoV-2 pandemic in Costa Rica using 185 sequences, 52 from the first months of the pandemic, and 133 from the current wave. Three GISAID clades (G, GH, and GR) and three PANGOLIN lineages (B.1, B.1.1, and B.1.291) were predominant, suggesting multiple re-introductions from other regions. The whole-genome variant calling analysis identified a total of 283 distinct nucleotide variants, following a power-law distribution with 190 single nucleotide mutations in a single sequence, and only 16 mutations were found in >5% sequences. These mutations were distributed through the whole genome. The prevalence of worldwide-found variant D614G in the Spike (98.9% in Costa Rica), ORF8 L84S (1.1%) is similar to what is found elsewhere. Interestingly, the frequency of mutation T1117I in the Spike has increased during the current pandemic wave beginning in May 2020 in Costa Rica, reaching 29.2% detection in the full genome analyses in November 2020. This variant has been observed in less than 1% of the GISAID reported sequences worldwide in 2020. Structural modeling of the Spike protein with the T1117I mutation suggests a potential effect on the viral oligomerization needed for cell infection, but no differences with other genomes on transmissibility, severity nor vaccine effectiveness are predicted. In conclusion, genome analyses of the SARS-CoV-2 sequences over the course of the COVID-19 pandemic in Costa Rica suggest the introduction of lineages from other countries and the detection of mutations in line with other studies, but pointing out the local increase in the detection of Spike-T1117I variant. The genomic features of this virus need to be monitored and studied in further analyses as part of the surveillance program during the pandemic., Graphical abstract Unlabelled Image

t IgG responses are fundamental to adaptive immunity and document immunological memory of previous pathogen encounter. While specific antigen recognition is mediated by the variable F(ab′)2 domain of IgG, various effector functions become activated via the constant Fcγ part bridging IgG-opsonized targets to FcγR-expressing immune effector cells. Traditionally, neutralizing IgG is considered the most appropriate correlate of protective humoral immunity to viruses. However, evidence is increasing that antiviral IgG mediates protection to viruses via activation of FcγRs. Using a test system allowing quantitative detection of virus-immune IgG able to activate FcγRs, sera of healthy individuals and vaccinees were assessed with regard to two prototypical human pathogenic viruses: measles and human cytomegalovirus. Marked differences in the capacity of individuals to generate FcγRI-, FcγRIIand FcγRIII-activating responses were noted. Comparison of FcγR-activating IgG with neutralizing and ELISA IgG concentrations did not correlate for HCMV and only very poorly for MV. Since neither neutralizing IgG nor overall IgG responses faithfully predict the activation of FcγRs, only the simultaneous quantification of IgGs activating defined FcγRs will aid to delineate individual “immunograms” of virus IgG immunity. Such new multiparametric assessment of antiviral IgG qualities could be instrumental in defining correlates of protection and disease progression. Deutsche Forschungsgemeinschaft/[He 2526/6-2, GK1045]//Alemania Helmholtz Association/[VISTRIE VH-VI-242]// European Commission/[QLRT-2001-01112]// European Commission/[MRTN-CT-2005-019248]// UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET)

Recently, two new influenza A-like viruses have been discovered in bats, A/little yellow-shouldered bat/Guatemala/060/2010 (HL17NL10) and A/flat-faced bat/Peru/033/2010 (HL18NL11). The hemagglutinin (HA)-like (HL) and neuraminidase (NA)-like (NL) proteins of these viruses lack hemagglutination and neuraminidase activities, despite their sequence and structural homologies with the HA and NA proteins of conventional influenza A viruses. We have now investigated whether the NS1 proteins of the HL17NL10 and HL18NL11 viruses can functionally replace the NS1 protein of a conventional influenza A virus. For this purpose, we generated recombinant influenza A/Puerto Rico/8/1934 (PR8) H1N1 viruses containing the NS1 protein of the PR8 wild-type, HL17NL10, and HL18NL11 viruses. These viruses (r/NS1PR8, r/NS1HL17, and r/NS1HL18, respectively) were tested for replication in bat and nonbat mammalian cells and in mice. Our results demonstrate that the r/NS1HL17 and r/NS1HL18 viruses are attenuated in vitro and in vivo . However, the bat NS1 recombinant viruses showed a phenotype similar to that of the r/NS1PR8 virus in STAT1 −/− human A549 cells and mice, both in vitro and in vivo systems being unable to respond to interferon (IFN). Interestingly, multiple mouse passages of the r/NS1HL17 and r/NS1HL18 viruses resulted in selection of mutant viruses containing single amino acid mutations in the viral PB2 protein. In contrast to the parental viruses, virulence and IFN antagonism were restored in the selected PB2 mutants. Our results indicate that the NS1 protein of bat influenza A-like viruses is less efficient than the NS1 protein of its conventional influenza A virus NS1 counterpart in antagonizing the IFN response and that this deficiency can be overcome by the influenza virus PB2 protein. IMPORTANCE Significant gaps in our understanding of the basic features of the recently discovered bat influenza A-like viruses HL17NL10 and HL18NL11 remain. The basic biology of these unique viruses displays both similarities to and differences from the basic biology of conventional influenza A viruses. Here, we show that recombinant influenza A viruses containing the NS1 protein from HL17NL10 and HL18NL11 are attenuated. This attenuation was mediated by their inability to antagonize the type I IFN response. However, this deficiency could be compensated for by single amino acid replacements in the PB2 gene. Our results unravel a functional divergence between the NS1 proteins of bat influenza A-like and conventional influenza A viruses and demonstrate an interplay between the viral PB2 and NS1 proteins to antagonize IFN.

Recently, two influenza A virus (FLUAV) genomes were identified in Central and South American bats. These sequences exhibit notable divergence from classical FLUAV counterparts, and functionally, bat FLUAV glycoproteins lack canonical receptor binding and destroying activity. Nevertheless, other features that distinguish these viruses from classical FLUAVs have yet to be explored. Here, we studied the viral nonstructural protein NS1, a virulence factor that modulates host signaling to promote efficient propagation. Like all FLUAV NS1 proteins, bat FLUAV NS1s bind double-stranded RNA and act as interferon antagonists. Unexpectedly, we found that bat FLUAV NS1s are unique in being unable to bind host p85β, a regulatory subunit of the cellular metabolism-regulating enzyme, phosphoinositide 3-kinase (PI3K). Furthermore, neither bat FLUAV NS1 alone nor infection with a chimeric bat FLUAV efficiently activates Akt, a PI3K effector. Structure-guided mutagenesis revealed that the bat FLUAV NS1-p85β interaction can be reengineered (in a strain-specific manner) by changing two to four NS1 residues (96L, 99M, 100I, and 145T), thereby creating a hydrophobic patch. Notably, ameliorated p85β-binding is insufficient for bat FLUAV NS1 to activate PI3K, and a chimeric bat FLUAV expressing NS1 with engineered hydrophobic patch mutations exhibits cell-type-dependent, but species-independent, propagation phenotypes. We hypothesize that bat FLUAV hijacking of PI3K in the natural bat host has been selected against, perhaps because genes in this metabolic pathway were differentially shaped by evolution to suit the unique energy use strategies of this flying mammal. These data expand our understanding of the enigmatic functional divergence between bat FLUAVs and classical mammalian and avian FLUAVs. IMPORTANCE The potential for novel influenza A viruses to establish infections in humans from animals is a source of continuous concern due to possible severe outbreaks or pandemics. The recent discovery of influenza A-like viruses in bats has raised questions over whether these entities could be a threat to humans. Understanding unique properties of the newly described bat influenza A-like viruses, such as their mechanisms to infect cells or how they manipulate host functions, is critical to assess their likelihood of causing disease. Here, we characterized the bat influenza A-like virus NS1 protein, a key virulence factor, and found unexpected functional divergence of this protein from counterparts in other influenza A viruses. Our study dissects the molecular changes required by bat influenza A-like virus NS1 to adopt classical influenza A virus properties and suggests consequences of bat influenza A-like virus infection, potential future evolutionary trajectories, and intriguing virus-host biology in bat species.

Candidatus Rickettsia amblyommii' is a spotted fever group rickettsia that is not considered pathogenic, although there is serologic evidence of possible infection in animals and humans. The aim of this study was to evaluate the pathogenic potential of a Costa Rican strain of ' Candidatus R. amblyommii' in guinea pigs and determine its capacity to generate protective immunity against a subsequent infection with a local strain of Rickettsia rickettsii isolated from a human case. Six guinea pigs were inoculated with ' Candidatus R. amblyommii' strain 9-CC-3-1 and two controls with cell culture medium. Health status was evaluated, and necropsies were executed at days 2, 4, and 13. Blood and tissues were processed by PCR to detect the gltA gene, and end titers of anti-' Candidatus R. amblyommii' IgG were determined by indirect immunofluorescence. To evaluate protective immunity, another 5 guinea pigs were infected with ' Candidatus R. amblyommii' (IGPs). After 4 weeks, these 5 IGPs and 3 controls (CGPs) were inoculated with pathogenic R. rickettsii. Clinical signs and titers of anti-. Rickettsia IgG were determined. IgG titers reached 1:512 at day 13 post-infection with ' Candidatus R. amblyommii' On day 2 after inoculation, two guinea pigs had enlarged testicles and ' Candidatus R. amblyommii' DNA was detected in testicles. Histopathology confirmed piogranulomatous orchitis with perivascular inflammatory infiltrate in the epididymis. In the protective immunity assay, anti-. Rickettsia IgG end titers after R. rickettsii infection were lower in IGPs than in CGPs. IGPs exhibited only transient fever, while CGP showed signs of severe disease and mortality. R. rickettsii was detected in testicles and blood of CGPs. Results show that the strain 9-CC-3-1 of ' Candidatus R. amblyommii' was able to generate pathology and an antibody response in guinea pigs. Moreover, its capacity to generate protective immunity against R. rickettsii may modulate the epidemiology and severity of Rocky Mountain spotted fever in areas where both species circulate. Candidatus Rickettsia amblyommii' es un grupo de fiebre maculosa rickettsia que no se considera patógena, aunque existen pruebas serológicas de una posible infección en animales y seres humanos. El objetivo de este estudio fue evaluar el potencial patógeno de una cepa costarricense de ' Candidatus R. amblyommii' en conejillos de indias y determinar su capacidad para generar inmunidad protectora contra una infección posterior con una cepa local de Rickettsia rickettsii aislada de un caso humano. Se inocularon seis cobayas con la cepa 9-CC-3-1 de ' Candidatus R. amblyommii' y dos controles con medio de cultivo celular. Se evaluó el estado de salud y se realizaron necropsias en los días 2, 4 y 13. La sangre y los tejidos fueron procesados por PCR para detectar el gen gltA, y los títulos finales de la IgG de ' Candidatus R. amblyommii' fueron determinados por inmunofluorescencia indirecta. Para evaluar la inmunidad protectora, otros 5 conejillos de indias fueron infectados con ' Candidatus R. amblyommii' (IGP). Después de 4 semanas, estos 5 IGP y 3 controles (CGP) fueron inoculados con R. rickettsii patógeno. Los signos clínicos y los títulos de anti. Rickettsia IgG fueron determinados. Los títulos de IgG alcanzaron 1:512 en el día 13 después de la infección con ' Candidatus R. amblyommii' El día 2 después de la inoculación, dos conejillos de indias tenían los testículos agrandados y el ADN de ' Candidatus R. amblyommii' fue detectado en los testículos. La histopatología confirmó la orquitis piogranulomatosa con infiltrado inflamatorio perivascular en el epidídimo. En el ensayo de inmunidad protectora, el anti. Los títulos finales de IgG de Rickettsia después de la infección de R. rickettsii fueron menores en los IGP que en los CGP. Los IGP sólo mostraron fiebre transitoria, mientras que los CGP mostraron signos de enfermedad grave y mortalidad. R. rickettsii se detectó en los testículos y en la sangre de los CGP. Los resultados muestran que la cepa 9-CC-3-1 de ' Candidatus R. amblyommii' fue capaz de generar una patología y una respuesta de anticuerpos en los conejillos de indias. Además, su capacidad de generar inmunidad protectora contra R. rickettsii puede modular la epidemiología y la gravedad de la fiebre maculosa de las Montañas Rocosas en las zonas donde circulan ambas especies. Universidad Nacional, Costa Rica Escuela de Medicina Veterinaria

The constant region of IgG antibodies mediates antiviral activities upon engaging host Fcγ receptors (FcγRs) expressed by a variety of immune cells, such as antibody-dependent cellullar cytotoxcity (ADCC) executed by natural killer (NK)cells. Human cytomegalovirus (HCMV) is unique among viruses by encoding also an array of several Fcγ-binding glycoproteins with cell surface disposition and concomitant incorporation into the virion. Evidence is increasing that the virus-encoded Fcγ receptors differ in their Fcγ binding mode but effectively operate as adversaries of host FcγRs since they are able to prevent IgG-mediated triggering of activating host FcγRs, i.e., FcγRI, FcγRIIA, and FcγRIIIA. Here we discuss virus-encoded FcγRs as the first known HCMV inhibitors of IgG-mediated immunity which could account for the limited efficacy of HCMV hyperimmune globulin in clinical settings. A better understanding of their molecular mode of action opens up new perspectives for improving IgG therapies against HCMV disease. DFG through He 2,526/6–2 European Commission through QLRT-2001–01112 European Commission through MRTN-CT-2005–019248 Helmholtz Association through VISTRIE VH-VI-242 UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiología

Dengue virus (DENV) (Flavivirus, Flaviviridae) is a reemerging arthropod-borne virus with a worldwide circulation, transmitted mainly by Aedes aegypti and Aedes albopictus mosquitoes. Since the first detection of its main transmitting vector in 1992 and the invasion of DENV-1 in 1993, Costa Rica has faced dengue outbreaks yearly. In 2007 and 2013, Costa Rica experienced two of the largest outbreaks in terms of total and severe cases. To provide genetic information about the etiologic agents producing these outbreaks, we conducted phylogenetic analysis of viruses isolated from human samples. A total of 23 DENV-1 and DENV-2 sequences were characterized. These analyses signaled that DENV-1 genotype V and DENV-2 American/Asian genotype were circulating in those outbreaks. Our results suggest that the 2007 and 2013 outbreak viral strains of DENV-1 and DENV-2 originated from nearby countries and underwent in situ microevolution. UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET) UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiología

Rickettsiae are intracellular bacteria commonly associated with hematophagous arthropods. Most of them have been described in hard ticks, but some have been found in soft ticks. Here we report the detection and isolation of a new Rickettsia from Ornithodoros knoxjonesi larvae collected from Balantiopteryx plicata (Emballonuridae) in Nicoya, Costa Rica. Two ticks were processed to detect Rickettsia spp. genes gltA, ompA, ompB, and htrA by PCR. Part of the macerate was also inoculated into Vero E6 and C6/36 cell lines, and cells were evaluated by Giménez stain, indirect immunofluorescence assay (IFA), and PCR. Both ticks were positive by PCR and rickettsial growth was successful in Vero E6 cells. Amplification and sequencing of near full length rrs, gltA, sca4 genes, and fragments of ompA and ompB showed that the Rickettsia sp. was different from described species. The highest homologies were with ‘Candidatus Rickettsia wissemanii’ and Rickettsia peacockii: 99.70% (1321/1325) with both sequences for rrs, 99.58% (1172/1177) and 99.76% (1246/1249) for gltA, 99.26% with both sequences (2948/2970 and 2957/2979) for sca4, 98.78% (485/491) and 98.39% (2069/2115) for ompA, and 98.58 (1453/1474) and 98.92% (1459/1475) for ompB; respectively. Bat blood, spleen, liver, and lung samples analyzed for Rickettsia detection were negative. Results demonstrate that the Rickettsia isolated from O. knoxjonesi is probably an undescribed species that belongs to the spotted fever group, for which ‘Candidatus Rickettsia nicoyana’ is proposed. Considering that B. plicata inhabits areas where contact with humans may occur and that human parasitism by Ornithodoros has been reported in the country, it will be important to continue with the characterization of this species and its pathogenic potential. Universidad de Costa Rica/[803-B4-055 ]/UCR/Costa Rica Universidad de Costa Rica/[803-B4-656]/UCR/Costa Rica UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiología UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET) UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Estructuras Microscópicas (CIEMIC)