Haemovigilance survey and screening strategy for arthropod-borne viruses in blood donors from Argentina.

Arthropod-borne viruses (arboviruses) count among emerging infections, which represent a major challenge for transfusion safety worldwide. To assess the risk of arboviruses-transmission by transfusion (ATT), we performed a survey to evaluate the potential threat for transfusion safety. Samples were retrospectively and randomly collected from donors who donated during the peak of dengue incidence in Cordoba (years: 2016 and 2019-2022). A cost-efficient strategy for molecular screening was implemented with a nucleic acid test (NAT) configured with Flavivirus and Alphavirus-universal degenerated primers targeting conserved gene regions. Besides, we evaluated the neutralizing antibody (NAb) prevalence by plaque reduction neutralization test (PRNT). A total of 1438 samples were collected. Among the NAT-screened samples, one resulted positive for Flavivirus detection. Subsequent sequencing of the PCR product revealed Saint Louis Encephalitis Virus (SLEV) infection (GeneBank accession number OR236721). NAb prevalence was 2.95% for anti-Dengue, 9.94% anti-SLEV, 1.09% anti-West Nile Virus, and 0% anti-Chikungunya. One of the NAb-positive samples also resulted positive for IgM against SLEV but negative by ARN detection. This is the first haemovigilance study developed in Argentina that evaluates the potential risk of ATT and the first research to determine the prevalence of NAb against Flavivirus through PNRT to avoid possible cross-reactions between Ab against Flavivirus. Herein, the finding of one SLEV-viremic donor and the detection of anti-SLEV IgM in a different donor demonstrated a potential threat for transfusion safety and emphasized the need for increased vigilance and proactive measures to ensure the safety of blood supplies.

Receptor-binding domain-based SARS-CoV-2 vaccine adjuvanted with cyclic di-adenosine monophosphate enhances humoral and cellular immunity in mice.

Novel adjuvants are highly desired to improve immune responses of SARS-CoV-2 vaccines. This work reports the potential of the stimulator of interferon genes (STING) agonist adjuvant, the cyclic di-adenosine monophosphate (c-di-AMP), in a SARS-CoV-2 vaccine based on the receptor binding domain (RBD). Here, mice immunized with two doses of monomeric RBD adjuvanted with c-di-AMP intramuscularly were found to exhibit stronger immune responses compared to mice vaccinated with RBD adjuvanted with aluminum hydroxide (Al(OH)3 ) or without adjuvant. After two immunizations, consistent enhancements in the magnitude of RBD-specific immunoglobulin G (IgG) antibody response were observed by RBD + c-di-AMP (mean: 15360) compared to RBD + Al(OH)3 (mean: 3280) and RBD alone (n.d.). Analysis of IgG subtypes indicated a predominantly Th1-biased immune response (IgG2c, mean: 14480; IgG2b, mean: 1040, IgG1, mean: 470) in mice vaccinated with RBD + c-di-AMP compared to a Th2-biased response in those vaccinated with RBD + Al(OH)3 (IgG2c, mean: 60; IgG2b: n.d.; IgG1, mean: 16660). In addition, the RBD + c-di-AMP group showed better neutralizing antibody responses as determined by pseudovirus neutralization assay and by plaque reduction neutralization assay with SARS-CoV-2 wild type. Moreover, the RBD + c-di-AMP vaccine promoted interferon-γ secretion of spleen cell cultures after RBD stimulation. Furthermore, evaluation of IgG-antibody titers in aged mice showed that di-AMP was able to improve RBD-immunogenicity at old age after 3 doses (mean: 4000). These data suggest that c-di-AMP improves immune responses of a SARS-CoV-2 vaccine based on RBD, and would be considered a promising option for future COVID-19 vaccines.

Detection of neutralizing antibodies against flaviviruses in free-ranging birds, Paraguay (2016-2018).

Saint Louis encephalitis virus (SLEV), West Nile virus (WNV) and Ilheus virus (ILHV) are flaviviruses maintained by enzootic transmission networks between mosquitoes and birds. They have been detected in South America, with no records for Paraguay. We detected the presence of neutralizing antibodies for SLEV, WNV and ILHV in free-ranging birds collected in Paraguay (2016-2018). Four positive samples were detected in resident birds: one SLEV (rufous-bellied thrush), one WNV (barred antshrike) and two ILHV (white-tipped dove and shiny cowbird). These results bring new information about enzootic activity of flaviviruses in Paraguay.

Neutralizing response elicited by homologous and heterologous prime booster vaccination against ancestral SARS-CoV-2 B.1, P.1, C.37 and B.1.617.2 variants.

Heterologous Covid-19 vaccination strategies arose due to interruption of vaccination programs plus delay and shortage of vaccine supplies. We analysed neutralizing response against ancestral SARS-CoV-2 B.1 and P.1, C.37 and B.1.67.2 variants elicited by 16 homologous and heterologous protocols combining Gam-COVID-Vac, ChAdOx1-S, Ad5-nCorV, BBIBP-CorV and mRNA-1273 vaccines. Homologous mRNA-1273 and heterologous schemes of a non-replicative viral vector/inactivated virus-based vaccine combined with mRNA-1273 induced significantly broader and greater neutralizing antibody-response. Moreover, serum from participants vaccinated with combinations of ChAdOx1-S/Ad5-nCorV and BBIBP-CorV/non-replicative viral vector-based vaccines showed higher or equivalent neutralizing response compared to homologous protocols, pointing them as good alternative platforms. BBIBP-CorV used as second dose exhibited significantly lower neutralizing response compared to other protocols, demonstrating that it should not be recommended as second dose. The information provided herein is valuable to redesign vaccination strategies, especially for low-income countries that still struggle with low percentages of immunized populations and vaccine supply shortage.

Evaluation of the Gam-COVID-Vac and vaccine-induced neutralizing response against SARS-CoV-2 lineage P.1 variant in an Argentinean cohort.

We evaluated humoral immune-response elicited by Sputnik-V by measuring anti-Spike (S) IgG antibodies (Abs) and neutralizing antibodies (NAb) prior to, 14 and 42 days after-vaccination. The safety and disease rates among vaccinated individuals were also evaluated. Since SARS-CoV-2 lineage P.1 is rapidly spreading in Argentina, virus-neutralizing activity of Sputnik-V-elicited and infection-elicited NAb faced to P.1 were also assessed. A total of 285 participants were recruited; all reported good tolerance, without any severe adverse event. Nine COVID-19 cases were confirmed in fully vaccinated individuals and viable P.1 variant was successfully isolated from one of them. At day 42, 99.65% of the individuals had anti-S IgG; however, 23.15% had not detectable NAbs. Significantly higher neutralization potency against WT compared to P.1 (p < 0·001) was observed. Some samples failed to neutralize P.1, mainly among vaccinated-naїve subjects; however, no significant differences were observed among previously infected-vaccinated individuals. Our results corroborated that Sputnik-V is safe and induces an efficient humoral immune response, although not all immunized subjects develop Nabs. Herein, we show for the first time, evidence of infectious SARS-CoV-2 shedding from Sputnik-V fully vaccinated individuals, by the isolation of viable virus from the nasopharyngeal swab of one participant of our study, 139 days after receiving the second dose. Thereby, we provide evidence indicating that the vaccine might avoid severe forms of COVID-19 but does not prevent infection nor prevents transmission from a fully vaccinated individual.

The extent of infectious SARS-CoV-2 shedding in an Argentinean cohort.

BACKGROUND: To analyze the infectious extent of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) in different settings where prevention strategies are critical to limit infection spread, we evaluated SARS-COV-2 viability to guide public health policies regarding isolation criteria and infection control. METHODS: We attempted viral isolation in 82 nasopharyngeal swabs from 72 patients with confirmed SARS-COV-2 infection. Study population was divided into four groups: (i) Patients during the first week of symptoms; (ii) Patients with prolonged positive PCR; (iii) Healthcare workers from a hospital participating of an outbreak investigation, with SARS-COV-2 infection confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) and (iv) Recipients of convalescent immune plasma (CIP).Vero Cl76 cell-line (ATCC CRL-587) was used in assays for virus isolation. Plasma samples of CIP recipients were also tested with plaque-reduction neutralization test. RESULTS: We obtained infectious SARS-COV-2 isolates from 15/84 nasopharyngeal swabs. The virus could not be isolated from upper respiratory tract samples collected 10-day after onset of symptoms (AOS) in patients with mild-moderate disease. CONCLUSION: The knowledge of the extent of SARS-CoV-2 infectivity AOS is relevant for effective prevention measures. This allows to discuss criteria for end isolation despite persistence of positive PCR and improve timing for hospital discharge with consequent availability of critical beds.

Caracterización biológica de cepas del virus Encefalitis San Luis en células de la inmunidad innata / Biological characterization of St. Louis Encephalitis virus strains in cells of innate immunity

Los flavivirus transmitidos por mosquitos son una amenaza actual y emergente en todo el mundo. Dentro de este género, el virus Encefalitis San Luis (VESL) causa una forma severa de enfermedad neuroinvasiva donde la respuesta inmune es un componente crucial de la defensa del huésped. En este trabajo se investigó la interacción entre VESL y células de la inmunidad innata, en un modelo de infección in vitro de monocitos humanos (células U937) con cepas de distinta virulencia y condiciones epidemiológicas de aislamiento (CbaAr-4005 y 78V-6507). Se evaluó la capacidad de infectar y replicar del virus, como también el efecto citopático y la cinética de viabilidad de monocitos durante la infección. Los resultados demostraron la susceptibilidad de los monocitos a la infección, replicación y muerte por ambas cepas virales. Sin embargo, se hallaron diferencias significativas entre ellas. La cepa epidémica y de mayor virulencia CbaAr-4005 registró una tasa de infección y replicación superior a la de la cepa endémica y de menor virulencia 78V-6507. Se comprobó también que el VESL indujo la muerte de monocitos humanos, dependiendo del tiempo post-infección (pi) y de la cepa. Así, CbaAr-4005 provocó a partir del día 3 pi el doble de mortalidad celular que 78V-6507. Además, en los monocitos infectados se observaron alteraciones de parámetros morfológicos que podrían relacionarse con el tipo de mecanismo de muerte celular asociado a la infección por VESL.

Mosquitoes borne Flavivirus infections are an actual and emergent worldwide threat to human health. Within this genus, Saint Louis Encephalitis Virus (SLEV) causes a severe neuroinvasive disease where immune response is crucial for host survival. In this study the interaction between SLEV and innate immune cells was evaluated. An in vitro infection model with human monocytes (U937 cells) and strains with variations in virulence and isolation conditions (CbaAr-4005 and 78V-6507) were used. Infection capacity, replication capacity, cytopathic effect and monocyte viability kinetics were measured. The results showed susceptibility to infection and replication to both strains. However, significant differences were found among them. CbaAr-4005, the epidemic and more virulent strain, showed higher infection and replication ratios compared to 78V-6507. SLEV infection that induces cell death of human monocytes was also found in a post-infection time and in a strain dependent manner. Since day 3 post-infection, twice the mortality in CbaAr-4005 infected cells was observed. Furthermore, infected monocytes showed alterations in morphologic parameters that could be related with apoptosis mechanisms associated to SLEV infections.

Os Flavivírus transmitidos por mosquitos são uma ameaça atual e emergente no mundo todo. Nesse gênero, o vírus Encefalite Saint Louis (VESL) causa uma forma grave de doença neuroinvasiva onde a resposta imune é um componente crucial da defesa do hospedeiro. Neste trabalho nos investigamos a interação entre VESL e células de imunidade inata em um modelo de infecção in vitro de monócitos humanos (células U937) com estirpe de diferentes virulências e condições epidemiológicas de isolamento (CbaAr-4005 e 78V-6507). Foi avaliada a capacidade do vírus de infectar e replicar , assim como o efeito citopático e a viabilidade cinética dos monócitos durante a infecção. Os resultados demonstraram a suscetibilidade dos monócitos à infecção, replicação e morte por ambas as estirpes virais. No entanto, foram detectadas diferenças significativas entre eles. A estirpe epidémica e de maior virulenta CbaAr-4005 teve uma maior taxa de infecção e replicação do que a estirpe endémica e menos virulenta 78V-6507. Foi comprovado também que o VESL induziu a morte de monócitos humanos, dependendo do tempo pós-infecção (pi) e da estirpe. Assim, a CbaAr-4005 causou a partir do dia 3 pi o dobro da mortalidade celular o que a 78V- 6507. Além disso, alterações nos parâmetros morfológicos foram observadas nos monócitos infectados que poderiam estar relacionadas ao tipo de mecanismo de morte celular associado à infecção pelo VESL.

Serological survey for Saint Louis encephalitis virus and West Nile virus in domestic mammals in Córdoba, Argentina: are our pets potential sentinels?

We evaluated the seroprevalence of Saint Louis encephalitis virus (SLEV) and West Nile virus (WNV) in dogs and cats in Córdoba, Argentina. Monotypic and heterotypic serological patterns were differentiated by means of a neutralization test. The SLEV seroprevalence in dogs was 14.6% (44/302; 100% monotypic). Two out of 94 (2.1%, 100% monotypic) cats were positive for WNV only. Four dogs (1.3%) exhibited neutralizing antibody titers against SLEV and WNV. During the study, three dogs seroconverted to SLEV. Our study demonstrates that pets were useful for detecting viral activity and could be considered as sentinels in the local surveillance of SLEV and WNV.

Production of recombinant NS1 protein and its possible use in encephalitic flavivirus differential diagnosis.

Saint Louis encephalitis virus (SLEV) and West Nile virus (WNV) are two of the major causes of arboviral encephalitis in the Americas. The co-circulation of related flaviviruses in the Americas and prior vaccination against flaviviruses pose problems to the diagnostic specificity of serological assays due to the development of cross-reactive antibodies. An accurate diagnosis method capable of differentiating these related viruses is needed. NS1 is a glycosylated, nonstructural protein, of about 46 kDa which has a highly conserved structure. Anti-NS1 antibodies can be detected within 4-8 days after the initial exposure and NS1 is the least cross-reactive of the flaviviral antigens. This study was aimed to generate SLEV and WNV NS1 recombinants proteins for the development of a flavivirus diagnostic test. Local Argentinian isolates were used as the source of NS1 gene cloning, expression, and purification. The protein was expressed in Escherichia coli as inclusion bodies and further purified by metal-chelating affinity chromatography (IMAC) under denaturing conditions. Human sera from SLEV and WNV positive cases showed reactivity to the recombinant NS1 proteins by western blot. The unfolded NS1 proteins were also used as immunogens. The polyclonal antibodies elicited in immunized mice recognized the two recombinant proteins with differential reactivity.

Tissue tropism of Saint Louis encephalitis virus: Histopathology triggered by epidemic and non-epidemic strains isolated in Argentina.

Saint Louis encephalitis virus (SLEV) reemerged in South America, and caused encephalitis outbreaks at the beginning of the 21st century. To enhance our knowledge about SLEV virulence, we performed comparative pathogenesis studies in Swiss albino mice inoculated with two different variants, the epidemic strain CbaAr-4005 and the non-epidemic strain CorAn-9275. Only the infection of mice with SLEV strain CbaAr-4005 resulted in high viremia, invasion of peripheral tissues including the lungs, kidney, and spleen, and viral neuroinvasion. This was associated with inflammatory pathology in the lungs, spleen, and brain as well as morbidity and mortality. In contrast, neither signs of desease nor viral replication were observed in mice infected with strain CorAn-9275. Interestingly, important loss of B cells and development of altered germinal centers (GC) were detected in the spleen of mice infected with strain CbaAr-4005, whereas mice infected with SLEV CorAn-9275 developed prominent GC with conserved follicular architecture, and neutralizing antibodies.

[Serological evidence of St. Louis encephalitis virus circulation in birds from Buenos Aires City, Argentina]. / Evidencia serológica de circulación del virus de la encefalitis de San Luis en aves de la Ciudad Autónoma de Buenos Aires, Argentina.

Our goal was to determine the presence of neutralizing antibodies against St. Louis encephalitis virus (SLEV) and West Nile virus (WNV) in sera of wild and domestic birds from Buenos Aires City, Argentina. From October 2012 to April 2013, 180 samples were collected and processed by the microneutralization technique. A 7.2% of the sampled birds were seropositive for SLEV, while no seropositive birds for WNV were detected.

Evidencia serológica de circulación del virus de la encefalitis de San Luis en aves de la Ciudad Autónoma de Buenos Aires, Argentina / Serological evidence of St. Louis encephalitis virus circulation in birds from Buenos Aires City, Argentina

Nuestro objetivo fue conocer la presencia de anticuerpos neutralizantes contra el virus de la encefalitis de San Luis (St. Louis encephalitis virus [SLEV]) y el virus del Nilo Occidental (West Nile virus [WNV]) en sueros de aves silvestres y domésticas de la Ciudad Autónoma de Buenos Aires, Argentina. Desde octubre del 2012 hasta abril del 2013 se colectaron 180 muestras que fueron procesadas por la técnica de microneutralización. El 7,2% de las aves muestreadas resultaron seropositivas para SLEV, mientras que no se detectaron aves seropositivas para WNV

Our goal was to determine the presence of neutralizing antibodies against St. Louis encephalitis virus (SLEV) and West Nile virus (WNV) in sera of wild and domestic birds from Buenos Aires City, Argentina. From October 2012 to April 2013, 180 samples were collected and processed by the microneutralization technique. A 7.2 % of the sampled birds were seropositive for SLEV, while no seropositive birds for WNV were detected

Evidence of hepatitis A virus circulation in central Argentina: seroprevalence and environmental surveillance.

BACKGROUND: Hepatitis A virus (HAV) has shown intermediate endemicity in Argentina, but notification of clinical cases has decreased since the introduction of the vaccine in 2005. OBJECTIVES: In order to get insight into the local circulation of this virus after four years of the official introduction of the vaccine, the aims of this study were to provide information on HAV immune status of the adult population of Córdoba city and to conduct environmental surveillance of HAV in sewage and river samples in the same region. STUDY DESIGN: The prevalence of anti-HAV was determined by EIA in 416 samples of people (without prior vaccination) from Córdoba city (2009-2010). Spline regression models were estimated under generalized additive models. Environmental surveillance was conducted in river and sewage samples collected in the same period. Viral detection was performed by RT-Nested PCR of the 5'UTR. RESULTS: In Córdoba, the global prevalence of anti-HAV was 73.5%. It increased with age (p<0.0001) and it was associated with the low-income population (OR: 1.14; 95% CI 1.05-1.25). This prevalence decreased in younger age groups, especially in the high-income population. Environmental monitoring revealed the presence of HAV (IA) in 20.8% and 16.1% of wastewater and river samples, respectively. CONCLUSIONS: As a consequence of a decrease in HAV circulation due to improvements in immunization, socio-economic and hygienic conditions, young adults are becoming increasingly susceptible to HAV infections. Environmental monitoring demonstrated that HAV circulates in the local population; therefore, health care systems should consider the implementation of preventive measures for susceptible adults in order to reduce the risk of HAV infection.

Seroprevalence of St. Louis encephalitis virus and West Nile virus (Flavivirus, Flaviviridae) in horses, Uruguay.

St. Louis encephalitis virus (SLEV) and West Nile virus (WNV) belong to the Japanese encephalitis antigenic complex (Flavivirus genus, Flaviviridae family). They show antigenic close relationships and share many similarities in their ecology. Both are responsible for serious human diseases. The aim of this study was to investigate the presence of neutralizing antibodies to these viruses in horses from Uruguay. To do this, 425 horse sera were collected in 2007 and analyzed by plaque reduction neutralization tests. As a result, 205 sera (48.2%) were found positive for SLEV, with titers ranging between 10 and 80. Two sera remained inconclusive, since they showed low titers to WNV and SLEV (10 and 20), not allowing us to demonstrate activity of WNV in our territory. This is the first report of circulation of SLEV in horses in Uruguay.

Landscape determinants of Saint Louis encephalitis human infections in Córdoba city, Argentina during 2010.

Saint Louis encephalitis virus (SLEV) is endemic in Argentina. During 2005 an outbreak occurred in Córdoba. From January to April of 2010 a new outbreak occurred in Córdoba city with a lower magnitude than the one reported in 2005. Understanding the association of different landscape elements related to SLEV hosts and vectors in urban environments is important for identifying high risk areas for human infections, which was here evaluated. The current study uses a case-control approach at a household geographical location, considering symptomatic and asymptomatic human infections produced by SLEV during 2010 in Córdoba city. Geographical information systems and logistic regression analysis were used to study the distribution of infected human cases and their proximity to water bodies, vegetation abundance, agricultural fields and housing density classified as high/low density urban constructions. Population density at a neighborhood level was also analyzed as a demographic variable. Logistic regression analysis revealed vegetation abundance was significantly (p<0.01) associated with the presence of human infections by SLEV. A map of probability of human infections in Córdoba city was derived from the logistic model. The model highlights areas that are more likely to experience SLEV infections. Landscape variables contributing to the outbreak were the proximity to places with vegetation abundance (parks, squares, riversides) and the presence of low density urban constructions, like residential areas. The population density analysis shows that SLEV infections are more likely to occur when population density by neighborhood is lower. These findings and the predictive map developed could be useful for public health surveillance and to improve prevention of vector-borne diseases.

Serological detection of St. Louis encephalitis virus and West Nile virus in equines from Santa Fe, Argentina.

St. Louis encephalitis virus (SLEV) and West Nile virus (WNV) present ecological and antigenic similarities and are responsible for serious human diseases. In addition, WNV is a significant pathogen in terms of equine health. The purpose of our study was to analyse the seroprevalence of SLEV and WNV in equine sera collected in Santa Fe Province, Argentina. The seroprevalence determined using the plaque reduction neutralisation test was 12.2% for SLEV, 16.2% for WNV and 48.6% for a combination of both viruses. These results provide evidence of the co-circulation of SLEV and WNV in equines in Santa Fe.

Serological detection of St. Louis encephalitis virus and West Nile virus in equines from Santa Fe, Argentina

St. Louis encephalitis virus (SLEV) and West Nile virus (WNV) present ecological and antigenic similarities and are responsible for serious human diseases. In addition, WNV is a significant pathogen in terms of equine health. The purpose of our study was to analyse the seroprevalence of SLEV and WNV in equine sera collected in Santa Fe Province, Argentina. The seroprevalence determined using the plaque reduction neutralisation test was 12.2% for SLEV, 16.2% for WNV and 48.6% for a combination of both viruses. These results provide evidence of the co-circulation of SLEV and WNV in equines in Santa Fe.

First detection of Rio Negro virus (Venezuelan equine encephalitis complex subtype VI) in Córdoba, Argentina.

Rio Negro virus (RNV) (Venezuelan equine encephalitis subtype VI) circulates only in Argentina; in northern provinces, isolates have been obtained from mosquitoes and rodents since 1980 and have been associated with acute febrile illness in humans. However, no studies of RNV have been performed in the central area of the country. We carried out molecular and serological detection of RNV in Córdoba, a province of the central part of the country, in mosquitoes and humans, respectively. One mosquito pool tested positive for alphavirus RNA by reverse transcriptase-nested polymerase chain reaction (RT-nested PCR). Subsequent sequencing determined that this alphavirus grouped with RNV. Serological studies detected antibodies to RNV in one human serum sample, which was obtained during the same period that RNV was detected using the aforementioned molecular methods. This is the first report of RNV circulation in the central area of Argentina, indicating an expansion of its original distribution. These results highlight the importance of strengthening surveillance procedures in endemic areas, as well as in new regions where RNV may emerge.