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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.

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.

The emergence and dissemination of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the resulting COVID-19 pandemic triggered a global public health crisis. Although several SARS-CoV-2 vaccines have been developed, demand far exceeds supply, access to them is inequitable, and thus, populations in low- and middle-income countries are unlikely to be protected soon (1). Furthermore, there are no specific therapies available, which is a challenge for COVID-19 patient care (2). Thus, the appearance of SARS-CoV-2 variants and reports of reinfections associated with immune escape (3, 4) highlight the urgent need for effective and broad coverage COVID-19 therapeutics. Intravenous administration of human or heterologous antibodies is a therapy successfully used in patients with viral respiratory diseases (5). Accordingly, formulations containing SARS-CoV-2 specific antibodies are an attractive therapeutic option for COVID-19 patients (6). SARS-CoV-2 specific antibodies could limit infection by direct virion neutralization and/or by targeting infected cells for elimination via complement or antibody-mediated cytotoxicity (6). Specific SARS-CoV-2 antibody-based therapeutics include convalescent plasma (CP), monoclonal antibodies (mAbs), human polyclonal IgG formulations purified from CP or transgenic animals, and heterologous hyperimmune polyclonal antibodies (pAbs) (6). Although the window for using antibody-based therapeutics varies, clinical data show that they are mainly effective if administered early after symptoms onset (6). Universidad de Costa Rica/[741-C0-198]/UCR/Costa Rica Caja Costarricense del Seguro Social/[]/CCSS/Costa Rica Banco Centroamericano de Integración Económica/[]/BCIE/Costa Rica German academic exchange services/[57592642]/DAAD/Alemania UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP) UCR::Vicerrectoría de Docencia::Salud::Facultad de Medicina::Escuela de Medicina UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET)

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.

Acknowledgments: We are grateful to all contributors who assisted with sampling endeavors. We would also like to thank the National System of Conservation Areas (SINAC) and the Ministry of Environment and Energy (MINAE) of Costa Rica. We thank the Arbovirus Reference Collection (ARC) from the Centers for Disease Control and Prevention, National Center for Emerging and Zoonotic Infectious Diseases, Division of Vector-Borne Diseases, Arboviral Diseases Branch, Diagnostic and Reference Team, Reference and Reagent Laboratory, and Sanofi Pasteur for supplying the chimeric viruses 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. Los virus transmitidos por artrópodos pertenecientes al género flavivirus tienen una enorme relevancia en la salud pública. Se ha propuesto que los primates neotropicales no humanos (PN) sean susceptibles a flavivirus debido a sus hábitos arborícolas y diurnos, a su similitud genética con los seres humanos y a su relativa cercanía con ellos. su relativa cercanía a los humanos. Sin embargo, el único flavivirus conocido en el continente americano de la fiebre amarilla es el único flavivirus conocido en el continente americano que se mantiene mediante ciclos silvícolas en los que participan los PN, y el papel de los PN como posibles de otros flavivirus es aún desconocido. Aquí examinamos la exposición a flavivirus en 86 muestras de suero suero, incluyendo un 83,7% de muestras de PN en libertad y un 16,3% de PN en cautividad que viven en regiones endémicas de flavivirus en Costa Rica. de flavivirus en regiones endémicas de Costa Rica. Las muestras de suero se recogieron de forma oportunista en toda Costa Rica en 2000-2015. Se utilizó una prueba de neutralización por reducción de microplaca altamente específica (micro-PRNT) para determinar la presencia de anticuerpos contra el YFV, el virus del dengue 1-4 (DENV), el virus del Zika, el virus del Nilo Occidental (WNV) y el virus de la encefalitis de San Luis (SLEV). Encontramos pruebas de PN seropositivas con reactividad homotípica al SLEV 11,6% (10/86), al DENV 10,5% (9/86) y al WNV 2,3% (2/86). La reactividad heterotípica heterotípica se determinó en el 3,5% (3/86) de los individuos contra el DENV, el 1,2% (1/86) contra el SLEV y el 1,2% (1/86) contra el VN. El 13,9% (12/86) de los PN fueron positivos para una especie indeterminada de especie indeterminada de flavivirus. No se encontraron anticuerpos contra el DENV-3, el DENV-4, el YFV o el ZIKV. Este trabajo proporciona pruebas serológicas convincentes de la exposición a flavivirus en los PN de Costa Rica, en particular a DENV, SLEV y WNV. El rango de años de muestreo y la región de donde se detectaron los positivos fueron detectados coinciden con aquellos en los que se registraron picos de DENV en poblaciones humanas, lo que sugiere una exposición bidireccional debida al contacto entre humanos y animales salvajes o a vectores puente. Nuestro trabajo Nuestro trabajo sugiere la exposición continua de las poblaciones silvestres a diversos flavivirus de importancia para la salud pública y subraya la necesidad de de salud pública y subraya la necesidad de una mayor vigilancia de los flavivirus en la interfaz hombre-vida silvestre en Centroamérica. en la interfaz hombre-vida silvestre en Centroamérica. Escuela de Medicina Veterinaria

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.

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

Several studies have shown Dengue Virus (DENV) nucleic acids and/or antibodies present in Neotropical wildlife including bats, suggesting that some bat species may be susceptible to DENV infection. Here we aim to elucidate the role of house-roosting bats in the DENV transmission cycle. Bats were sampled in households located in high and low dengue incidence regions during rainy and dry seasons in Costa Rica. We captured 318 bats from 12 different species in 29 households. Necropsies were performed in 205 bats to analyze virus presence in heart, lung, spleen, liver, intestine, kidney, and brain tissue. Histopathology studies from all organs showed no significant findings of disease or infection. Sera were analyzed by PRNT90 for a seroprevalence of 21.2% (51/241), and by PCR for 8.8% (28/318) positive bats for DENV RNA. From these 28 bats, 11 intestine samples were analyzed by RT-PCR. Two intestines were DENV RNA positive for the same dengue serotype detected in blood. Viral isolation from all positive organs or blood was unsuccessful. Additionally, viral load analyses in positive blood samples by qRT-PCR showed virus concentrations under the minimal dose required for mosquito infection. Simultaneously, 651 mosquitoes were collected using EVS-CO2 traps and analyzed for DENV and feeding preferences (bat cytochrome b). Only three mosquitoes were found DENV positive and none was positive for bat cytochrome b. Our results suggest an accidental presence of DENV in bats probably caused from oral ingestion of infected mosquitoes. Phylogenetic analyses suggest also a spillover event from humans to bats. Therefore, we conclude that bats in these urban environments do not sustain DENV amplification, they do not have a role as reservoirs, but function as epidemiological dead end hosts for this virus., Author summary Dengue is the most important human vector-borne disease. Several studies have shown DENV presence in mammalian wildlife such as bats, thus considering them putative reservoirs or hosts. We aimed to elucidate if bats that cohabit in houses in close proximity with humans may be involved in a dengue transmission cycle. We sampled bats in low and high dengue incidence areas during the dry (low mosquito abundance) and wet (high mosquito abundance) seasons. We analyzed blood and several organs. As previously reported, we found DENV nucleic acid and neutralizing antibodies in a small percentage of blood samples, but virus detection in all organs was negative. We were able to show that dengue found in all positive samples was in low concentration and thus virus isolation was unsuccessful. We found positive intestine samples which may suggest infection through DENV-positive mosquito ingestion. Furthermore, mosquitoes sampled in close vicinity of bats’ roosting place were not feeding on these mammals. Virus sequence analysis from bats and humans show a spillover effect from humans to bats. Taken together, our results indicate that bats do not sustain sufficient virus amplification in order to function as reservoirs and exclude them as players in the dengue virus transmission cycle.

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)

Bats are hosts of diverse coronaviruses (CoVs) known to potentially cross the host–species barrier. For analysing coronavirus diversity in a bat species-rich country, a total of 421 anal swabs/faecal samples from Costa Rican bats were screened for CoV RNA-dependent RNA polymerase (RdRp) gene sequences by a pancoronavirus PCR. Six families, 24 genera and 41 species of bats were analysed. The detection rate for CoV was 1%. Individuals (n = 4) from four different species of frugivorous (Artibeus jamaicensis, Carollia perspicillata and Carollia castanea) and nectivorous (Glossophaga soricina) bats were positive for coronavi- rus-derived nucleic acids. Analysis of 440 nt. RdRp sequences allocated all Costa Rican bat CoVs to the a-CoV group. Several CoVs sequences clustered near previously described CoVs from the same species of bat, but were phylogeneti- cally distant from the human CoV sequences identified to date, suggesting no recent spillover events. The Glossophaga soricina CoV sequence is sufficiently dis- similar (26% homology to the closest known bat CoVs) to represent a unique coronavirus not clustering near other CoVs found in the same bat species so far, implying an even higher CoV diversity than previously suspected. Los murciélagos son huéspedes de diversos coronavirus (CoV) que se sabe que pueden cruzar la la barrera de la especie huésped. Para analizar la diversidad de coronavirus en un país rico en especies de murciélagos de murciélagos, se analizaron 421 muestras de hisopos anales y heces de murciélagos costarricenses de la RNA polimerasa dependiente del CoV (RdRp) mediante una PCR de pancoronavirus. PCR de pancoronavirus. Se analizaron seis familias, 24 géneros y 41 especies de murciélagos. La tasa de detección del CoV fue del 1%. Se analizaron individuos (n = 4) de cuatro especies diferentes de murciélagos frugívoros (n = 4). especies de murciélagos frugívoros (Artibeus jamaicensis, Carollia perspicillata y Carollia castanea) y nectivoros (Glossophaga soricina) resultaron positivos a los ácidos nucleicos derivados del coronavi rus. El análisis de las secuencias de 440 nt. RdRp asignó todos los CoV de los murciélagos de Costa de Costa Rica al grupo a-CoV. Varias secuencias de CoVs se agruparon cerca de CoVs previamente descritos de la misma especie de murciélago, pero eran filogenéticamente distantes del CoV humano. de los CoV humanos identificados hasta la fecha, lo que sugiere que no se han producido de la contaminación reciente. La secuencia del CoV de Glossophaga soricina es lo suficientemente dis (26% de homología con los CoVs de murciélagos conocidos más cercanos) como para representar un único coronavirus que no se agrupa cerca de otros CoVs encontrados en la misma especie de murciélago hasta ahora, lo que implica una diversidad de CoV aún mayor de lo que se sospechaba. Universidad Nacional, Costa Rica Escuela de Medicina Veterinaria

Human cytomegalovirus (HCMV) establishes lifelong infection with recurrent episodes of virus production and shedding despite the presence of adaptive immunological memory responses including HCMV immune immunoglobulin G (IgG). Very little is known how HCMV evades from humoral and cellular IgG-dependent immune responses, the latter being executed by cells expressing surface receptors for the Fc domain of IgG (FcγRs). Remarkably, HCMV expresses the RL11-encoded gp34 and UL119-118-encoded gp68 type I transmembrane glycoproteins which bind Fcγ with nanomolar affinity. Using a newly developed FcγR activation assay, we tested if the HCMV-encoded Fcγ binding proteins (HCMV FcγRs) interfere with individual host FcγRs. In absence of gp34 or/and gp68, HCMV elicited a much stronger activation of FcγRIIIA/CD16, FcγRIIA/CD32A and FcγRI/CD64 by polyclonal HCMV-immune IgG as compared to wildtype HCMV. gp34 and gp68 co-expression culminates in the late phase of HCMV replication coinciding with the emergence of surface HCMV antigens triggering FcγRIII/CD16 responses by polyclonal HCMV-immune IgG. The gp34- and gp68-dependent inhibition of HCMV immune IgG was fully reproduced when testing the activation of primary human NK cells. Their broad antagonistic function towards FcγRIIIA, FcγRIIA and FcγRI activation was also recapitulated in a gain-of-function approach based on humanized monoclonal antibodies (trastuzumab, rituximab) and isotypes of different IgG subclasses. Surface immune-precipitation showed that both HCMV-encoded Fcγ binding proteins have the capacity to bind trastuzumab antibody-HER2 antigen complexes demonstrating simultaneous linkage of immune IgG with antigen and the HCMV inhibitors on the plasma membrane. Our studies reveal a novel strategy by which viral FcγRs can compete for immune complexes against various Fc receptors on immune cells, dampening their activation and antiviral immunity., Author Summary Herpes viruses persist lifelong continuously alternating between latency and virus production and transmission. The latter events occur despite the presence of immune IgG antibodies. IgG acts by neutralization of virions and activation of immune cells bearing one or more surface receptors, called FcγRs, recognizing the constant Fc domain of IgG. Activating FcγRs induce a wide range of immune responses, including antibody dependent cellular cytotoxicity (ADCC) of virus-infected cells by natural killer (NK) cells, cytokine secretion and the uptake of immune complexes to enhance antigen presentation to T cells. We demonstrate that the HCMV glycoproteins RL11/gp34 and UL119-118/gp68 block IgG-mediated activation of FcγRs. A novel reporter cell-based assay was used to test FcγRs individually and assess their relative susceptibility to each antagonist. This approach revealed that gp34 and gp68 block triggering of activating FcγRs, i.e. FcγRI (CD64), FcγRII (CD32A) and FcγRIII (CD16). Co-immunoprecipitation showed the formation of ternary complexes containing IgG, IgG-bound antigen and the viral antagonists on the cell surface. Assigning the redundant abilities of HCMV to hinder IgG effector responses to the viral Fc binding proteins, we discuss gp34 and gp68 as potential culprits which might contribute to the limited efficacy of therapeutic IgG against HCMV.

IgG responses are crucial in antiviral defence and instrumental for the serodiagnosis of infections. Fcγ receptors (FcγRs), which recognize the Fc-part of IgG, differ regarding their IgG binding affinity, IgG subclass preference, cellular expression profile and pathogen elimination mechanisms elicited upon activation. Assessing their activation in vitro is of fundamental importance, but technically difficult. Therefore, a novel assay for measuring antiviral IgG antibodies triggering activation of individual host Fcγ receptors was established. The assay comprises the co-cultivation of virus-infected target cells with immune IgG antibodies and mouse BW5147 hybridoma cells stably expressing chimeric FcγR–CD3ζ chain molecules consisting of the extracellular domain of human FcγRIIIA, FcγRIIA or FcγRI, respectively, fused to the transmembrane and intracellular domains of the mouse CD3ζ chain. Triggering of the chimeric FcγR receptors by immune complexes formed on the surface of IgG-opsonized virus-infected target cells resulted in FcγR activation leading to IL-2 secretion by BW5147 cells, which was quantified as a surrogate marker in an ELISA. Target cells infected with various human pathogenic viruses including herpes simplex virus type 1 (HSV-1), Epstein–Barr virus (EBV), human cytomegalovirus (HCMV), measles virus (MV), and respiratory syncytial virus (RSV) or displaying human immunodeficiency virus-1 (HIV-1) gp120 evoke dose-dependent IgG responses demonstrating the universal applicability of the assay. Taken together, a new reliable and simple tool for measuring antibodies triggering activation of Fcγ receptors was established. This assay will be instrumental for defining novel correlates of IgG immunity and the design of new therapeutic IgGs. UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiología

Objetivo. Evaluar, bajo una perspectiva ecológica, la presencia de Aedes albopictus y su infección natural por virus dengue (DENV) en una zona de actividad piñera de Costa Rica. Método: Se colectaron mosquitos adultos en galerías forestales colindantes con piñeras, viviendas en proximidad a cultivos (‹1 km) y viviendas en lejanía (1- 10 km). Se empleó el índice de Shannon-Wiener para estimar biodiversidad. La infestación larvaria se evaluó en plantas de piña y viviendas y se calcularon índices aédicos de viviendas (IV) y de contenedores (IC). La detección de DENV en adultos (cuerpos y cabezas) y en larvas de Ae. albopictus se efectuó mediante RT-PCR y secuenciación. Resultados. Se colectaron 1376 adultos en total: Ae. albopictus (5,81%), Anopheles apicimacula (5,01%), Culex coronator (11,55%), Cx. inflictus (6,1%), Cx. nigripalpus (48,11%), Cx. quinquefasciatus (23,34%) y Limatus durhamii (0,07%). El índice de biodiversidad fue mayor en galerías forestales. Ae. albopictus adultos fueron colectados principalmente en el área de piñeras (73/80), aunque sólo dos larvas en las plantas de piña. Los índices aédicos en proximidad (IV: 40,7%, IC: 26,9%) y en lejanía (IV: 51,7%, IC: 29,6%) no mostraron diferencias significativas (IV Z=0,56, p=0,58; IC Z=0,16, p=0,87). Se detectó DENV-2 y DENV-3 en 2/20 grupos de cabezas y DENV-1 en 2/74 grupos de larvas de Ae. albopictus. Discusión. Las galerías forestales próximas a cultivos de piña podrían considerarse “islas ecológicas” adecuadas para el refugio de Ae. albopictus. La presencia de DENV en adultos y larvas sugiere un papel activo de Ae. albopictus en la transmisión de virus en este ecosistema. Objective. To evaluate, under an ecological perspective, the presence of Aedes albopictus and the wild infection by dengue viruses (DENV) in an area of pineapple activity in Costa Rica.Materials and methods. Adult mosquitoes were collected in forest galleries limiting pineapple plantations, houses adjacent to plantations (