Molecular Characterization of Leptospira Species among Patients with Acute Undifferentiated Febrile Illness from the Municipality of Villeta, Colombia.

Leptospira is a bacterial genus that includes several pathogenic species related to leptospirosis. In Colombia, leptospirosis is a mandatorily reported disease, widely distributed across the country. In the Villeta municipality, leptospirosis has been identified as an important cause of febrile illness; however, to date, no studies have been performed to identify the circulating species. A genus-specific qualitative qPCR was performed on DNA extracted from febrile patients' acute-phase whole-blood samples targeting a fragment of the rrs gene. Positive qPCR samples were further amplified for the adk, icdA, LipL32, LipL41, rrs, and secY genes through conventional PCR for sequencing. All high-quality obtained sequences were further assessed through concatenated phylogenetic analysis. A total of 25% (14/56) of febrile patients' acute blood samples were positive for Leptospira spp. High-quality sequences were obtained for only five genes, and analysis through concatenated phylogeny identified that all sequences clustered within the P1/pathogenic clade; some of them formed a robustly supported clade with Leptospira santarosai, and others were closely related with other Leptospira species but exhibited considerable genetic divergence. We describe the presence of pathogenic Leptospira species among febrile patients from the Villeta municipality and identify L. santarosai and other Leptospira species as causative agents of leptospirosis in the region.

Pathophysiology of chikungunya virus infection associated with fatal outcomes.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes acute, subacute, and chronic human arthritogenic diseases and, in rare instances, can lead to neurological complications and death. Here, we combined epidemiological, virological, histopathological, cytokine, molecular dynamics, metabolomic, proteomic, and genomic analyses to investigate viral and host factors that contribute to chikungunya-associated (CHIK) death. Our results indicate that CHIK deaths are associated with multi-organ infection, central nervous system damage, and elevated serum levels of pro-inflammatory cytokines and chemokines compared with survivors. The histopathologic, metabolite, and proteomic signatures of CHIK deaths reveal hemodynamic disorders and dysregulated immune responses. The CHIKV East-Central-South-African lineage infecting our study population causes both fatal and survival cases. Additionally, CHIKV infection impairs the integrity of the blood-brain barrier, as evidenced by an increase in permeability and altered tight junction protein expression. Overall, our findings improve the understanding of CHIK pathophysiology and the causes of fatal infections.

Clinical and epidemiological characteristics of Madariaga and Venezuelan equine encephalitis virus infections.

Madariaga virus (MADV) and Venezuelan equine encephalitis virus (VEEV) are emerging arboviruses affecting rural and remote areas of Latin America. However, there are limited clinical and epidemiological reports available, and outbreaks are occurring at an increasing frequency. We addressed this gap by analyzing all the available clinical and epidemiological data of MADV and VEEV infections recorded since 1961 in Panama. A total of 168 of human alphavirus encephalitis cases were detected in Panama from 1961 to 2023. Here we describe the clinical signs and symptoms and epidemiological characteristics of these cases, and also explored signs and symptoms as potential predictors of encephalitic alphavirus infection when compared to those of other arbovirus infections occurring in the region. Our results highlight the challenges clinical diagnosis of alphavirus disease in endemic regions with overlapping circulation of multiple arboviruses.

Virome analyses of Amblyomma cajennense and Rhipicephalus microplus ticks collected in Colombia.

Tick-borne viruses (TBV) have gained public health relevance in recent years due to the recognition of human-associated fatal cases and the increase in tick-borne disease and transmission. However, many tick species have not been studied for their potential to transmit pathogenic viruses, especially those found in Latin America. To gain better understanding of the tick virome, we conducted targeted amplification using broadly-reactive consensus-degenerate pan-viral targeting viruses from the genera Flavivirus, Bandavirus, Uukuvirus, and Orthonairovirus genus. Additionally, we conducted unbiased metagenomic analyses to investigate the presence of viral RNA sequences in Amblyomma cajennense, A. patinoi and Rhipicephalus microplus ticks collected from a horse slaughter plant in Medellín, Colombia. While no viral products were detected by PCR, results of the metagenomic analyses revealed the presence of viral genomes belonging to the genera Phlebovirus, Bandavirus, and Uukuvirus, including Lihan Tick Virus (LTV), which was previously reported in Rhipicephalus microplus from Colombia. Overall, the results emphasized the enormous utility of the next-generation sequencing in identifying virus genetic diversity presents in ticks and other species of vectors and reservoirs.

Etiological characterization of acute undifferentiated febrile illness in Apartadó and Villeta municipalities, Colombia, during COVID-19 pandemic.

Background: Acute undifferentiated febrile illness (AUFI) is one of the leading causes of illness in tropical regions. Although malaria is the most important cause, other pathogens such as Dengue (DENV), Leptospira and recently, Coronavirus Disease 2019 (COVID-19) have gained importance. In Colombia, few studies aimed to identify the etiology of AUFI. Most of them performed in Apartadó and Villeta municipalities, identifying the active circulation of several pathogens. Thus, we conducted a cross-sectional study in these municipalities to characterize the etiologies of AUFI during COVID-19 pandemic. Methods: An active surveillance was conducted between September and December 2021 in local hospitals of Apartadó and Villeta municipalities. Febrile patients were enrolled after voluntarily agreeing to participate in the study. Ten different etiologies were evaluated through direct, serological, molecular and rapid diagnostic methods. Results: In Apartadó a confirmed etiology was found in 60% of subjects, DENV (25%) being the most frequent, followed by leptospirosis (16.7%), malaria (10%), COVID-19 (8.3%), spotted fever group (SFG) rickettsiosis (6.7%) and Chikungunya (1.7%). In Villeta, a specific etiology was confirmed in 55.4% of patients, of which SFG rickettsiosis (39.3%) was the most frequent, followed by leptospirosis (21.4%), DENV (3.6%) and malaria (1.8%). No cases due to Mayaro, Yellow Fever, Oropouche and Venezuelan Equine Encephalitis viruses were detected. Conclusion: We confirm the relevance of dengue fever, leptospirosis, SFG rickettsiosis, COVID-19 and malaria as causes of AUFI in the municipality of Apartadó, and highlight the great importance of SFG rickettsiosis as the main cause of AUFI in the municipality of Villeta.

Differential Susceptibility of Aedes aegypti and Aedes albopictus Mosquitoes to Infection by Mayaro Virus Strains.

Mayaro virus (MAYV) is an arthropod-borne virus (arbovirus) belonging to the family Togaviridae, genus Alphavirus. In recent years, the geographic distribution of MAYV may have expanded north from South and Central America into the Caribbean Islands. Although Haemagogus janthinomys is considered the main vector for MAYV, the virus has also been isolated from other mosquitoes, including Aedes aegypti, a widespread species that serves as the main vector for highly epidemic viruses. Given the possible expansion and outbreaks of MAYV in Latin America, it is possible that MAYV might be adapting to be efficiently transmitted by urban vectors. Therefore, to investigate this possibility, we evaluated the vector competence of Ae. aegypti and Ae. albopictus mosquitoes to transmit MAYV isolated during a year of low or high MAYV transmission. Adult Ae. aegypti and Ae. albopictus were orally infected with the MAYV strains, and the infection, dissemination, and transmission rates were calculated to evaluate their vector competence. Overall, we found higher infection, dissemination, and transmission rates in both Ae. aegypti and Ae. albopictus mosquitoes infected with the strain isolated during a MAYV outbreak, whereas low/no transmission was detected with the strain isolated during a year of low MAYV activity. Our results confirmed that both Ae. aegypti and Ae. albopictus are competent vectors for the emergent MAYV. Our data suggest that strains isolated during MAYV outbreaks might be better fit to infect and be transmitted by urban vectors, raising serious concern about the epidemic potential of MAYV.

Arbovirus Antibody Seroprevalence in the Human Population from Cauca, Colombia.

Several arboviruses have emerged or reemerged into the New World during the past several decades, causing outbreaks of significant proportion. In particular, the outbreaks of Dengue virus (DENV), Zika virus, and Chikungunya virus (CHIKV) have been explosive and unpredictable, and have led to significant adverse health effects. These viruses are considered the leading cause of acute undifferentiated febrile illnesses in Colombia. However, Venezuelan equine encephalitis virus (VEEV) is endemic in Colombia, and arboviruses such as the Mayaro virus (MAYV) and the Oropouche virus (OROV) cause febrile illnesses in neighboring countries. Yet, evidence of human exposure to MAYV and OROV in Colombia is scarce. In this study, we conducted a serosurvey study in healthy individuals from the Cauca Department in Colombia. We assessed the seroprevalence of antibodies against multiple arboviruses, including DENV serotype 2, CHIKV, VEEV, MAYV, and OROV. Based on serological analyses, we found that the overall seroprevalence for DENV serotype 2 was 30%, 1% for MAYV, 2.6% for CHIKV, 4.4% for VEEV, and 2% for OROV. This study provides evidence about the circulation of MAYV and OROV in Colombia, and suggests that they-along with VEEV and CHIKV-might be responsible for cases of acute undifferentiated febrile illnesses that remain undiagnosed in the region. The study results also highlight the need to strengthen surveillance programs to identify outbreaks caused by these and other vector-borne pathogens.

A modified porous silicon microparticle potentiates protective systemic and mucosal immunity for SARS-CoV-2 subunit vaccine.

Development of optimal SARS-CoV-2 vaccines to induce potent, long-lasting immunity and provide cross-reactive protection against emerging variants remains a high priority. Here, we report that a modified porous silicon microparticle (mPSM) adjuvant to SARS-CoV-2 receptor-binding domain (RBD) vaccine activated dendritic cells and generated more potent and durable systemic humoral and type 1 helper T (Th) cell- mediated immune responses than alum-formulated RBD following parenteral vaccination, and protected mice from SARS-CoV-2 and Beta variant challenge. Notably, mPSM facilitated the uptake of SARS-CoV-2 RBD antigens by nasal and airway epithelial cells. Parenteral and intranasal prime and boost vaccinations with mPSM-RBD elicited stronger lung resident T and B cells and IgA responses compared to parenteral vaccination alone, which led to markedly diminished viral loads and inflammation in the lung following SARS-CoV-2 Delta variant challenge. Overall, our results suggest that mPSM is effective adjuvant for SARS-CoV-2 subunit vaccine in both systemic and mucosal vaccinations.

Analytical characterization of the SARS-CoV-2 EURM-017 reference material.

BACKGROUND: Current serological methods for SARS-CoV-2 lack adequate standardization to a universal standard reference material. Standardization will allow comparison of results across various lab-developed and commercial assays and publications. SARS-CoV-2 EURM-017 is human sera reference material containing antibodies directed against SARS-CoV-2 proteins, S1/S2 (full-length spike [S]), S1 receptor-binding domain (S1 RBD), S1, S2, and nucleocapsid (N) protein. The goal of this study was to characterize five antigen-specific serum fractions in EURM-017 for standardization of serology assays. METHODS: Five antigen-specific serum fractions were affinity purified, quantified, and PRNT50 titers compared. Standardization methods were established for two anti-S1 RBD (IgG and Total Ig) and one N protein assay. For the anti-S1 RBD assays, standardization involved determining assay index values for serial dilutions of S1-RBD anti-sera. Index values for the anti-S1 RBD IgG assay and PRNT50 titers were determined for 44 symptomatic COVID-19 patient sera. The index values were converted to EURM-017 ug/mL. RESULTS: Anti-sera protein content was as follows: S1 (17.7 µg/mL), S1 RBD (17.4 µg/mL), S1/S2 (full-length S) (34.1 µg/mL), S2 (29.7 µg/mL), and N protein (72.5 µg/mL). S1 anti-serum had the highest neutralization activity. A standardization method for S1 RBD anti-serum and an anti-S1 RBD IgG assay yielded the linear equation (y = 0.75x-0.10; y = index, x=µg/mL anti-serum). Patient sample index values for the S1-RBD IgG assay correlated well with PRNT50 titers (Pearson r = 0.84). Using the equation above, patient index values were converted to standardized µg/mL. CONCLUSIONS: Standardization of different lab-developed and commercial assays to EURM-017 antigen-specific anti-sera will allow comparison of results across studies globally due to traceability to a single standard reference material.

Serological Responses in Patients Infected with Mayaro Virus and Evaluation of Cross-Protective Responses against Chikungunya Virus.

Mayaro virus (MAYV) is an alphavirus endemic to both Latin America and the Caribbean. Recent reports have questioned the ability of MAYV and its close relative, Chikungunya virus (CHIKV), to generate cross-reactive, neutralizing antibodies to one another. Since CHIKV was introduced to South America in 2013, discerning whether individuals have cross-reactive antibodies or whether they have had exposures to both viruses previously has been difficult. Using samples obtained from people infected with MAYV prior to the introduction of CHIKV in the Americas, we performed neutralizing assays and observed no discernable neutralization of CHIKV by sera from patients previously infected with MAYV. These data suggest that a positive CHIKV neutralization test cannot be attributed to prior exposure to MAYV and that previous exposure to MAYV may not be protective against a subsequent CHIKV infection.

A modified porous silicon microparticle promotes mucosal delivery of SARS-CoV-2 antigen and induction of potent and durable systemic and mucosal T helper 1 skewed protective immunity.

Development of optimal SARS-CoV-2 vaccines to induce potent, long-lasting immunity and provide cross-reactive protection against emerging variants remains a high priority. Here, we report that a modified porous silicon microparticle (mPSM)-adjuvanted SARS-CoV-2 receptor-binding domain (RBD) vaccine activated dendritic cells and generated more potent and durable SARS-CoV-2-specific systemic humoral and type 1 helper T (Th) cell-mediated immune responses than alum-formulated RBD following parenteral vaccination, and protected mice from SARS-CoV-2 and Beta variant infection. mPSM facilitated the uptake of SARS-CoV-2 RBD antigens by nasal and airway epithelial cells. Parenteral and intranasal prime and boost vaccinations with mPSM-RBD elicited potent systemic and lung resident memory T and B cells and SARS-CoV-2 specific IgA responses, and markedly diminished viral loads and inflammation in the lung following SARS-CoV-2 Delta variant infection. Our results suggest that mPSM can serve as potent adjuvant for SARS-CoV-2 subunit vaccine which is effective for systemic and mucosal vaccination.

Closing the Rift: Discovery of a novel virus receptor.

In this issue of Cell, Ganaie et al. reports the identification of LRP1 as a receptor of the highly pathogenic Rift Valley fever virus. By using genome-wide CRISPR-Cas9 screening and functional studies, Ganaie et al. identified LRP1 and several co-factors as essential elements for virus infection.

Antiviral activity of oleandrin and a defined extract of Nerium oleander against SARS-CoV-2.

With continued expansion of the coronavirus disease (COVID-19) pandemic, caused by severe acute respiratory syndrome 2 (SARS-CoV-2), both antiviral drugs as well as effective vaccines are desperately needed to treat patients at high risk of life-threatening disease. Here, we present in vitro evidence for significant inhibition of SARS-CoV-2 by oleandrin and a defined extract of N. oleander (designated as PBI-06150). Using Vero cells, we found that prophylactic (pre-infection) oleandrin (as either the pure compound or as the active principal ingredient in PBI-06150) administration at concentrations as low as 0.05 µg/ml exhibited potent antiviral activity against SARS-CoV-2, with an 800-fold reduction in virus production, and a 0.1 µg/ml concentration resulted in a greater than 3000-fold reduction in infectious virus production. The half maximal effective concentration (EC50) values were 11.98 ng/ml when virus output was measured at 24 h post-infection, and 7.07 ng/ml measured at 48 h post-infection. Therapeutic (post-infection) treatment up to 24 h after SARS-CoV-2 infection of Vero cells also reduced viral titers, with 0.1 µg/ml and 0.05 µg/ml concentrations causing greater than 100-fold reduction as measured at 48 h, and the 0.05 µg/ml concentration resulting in a 78-fold reduction. Concentrations of oleandrin up to 10 µg/ml were well tolerated in Vero cells. We also present in vivo evidence of the safety and efficacy of defined N. oleander extract (PBI-06150), which was administered to golden Syrian hamsters in a preparation containing as high as 130 µg/ml of oleandrin. In comparison to administration of control vehicle, PBI-06150 provided a statistically significant reduction of the viral titer in the nasal turbinates (nasal conchae). The potent prophylactic and therapeutic antiviral activities demonstrated here, together with initial evidence of its safety and efficacy in a relevant hamster model of COVID-19, support the further development of oleandrin and/or defined extracts containing this molecule for the treatment of SARS-CoV-2 and associated COVID-19 disease and potentially also for reduction of virus spread by persons diagnosed early after infection.

A trans-complementation system for SARS-CoV-2 recapitulates authentic viral replication without virulence.

The biosafety level 3 (BSL-3) requirement to culture severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a bottleneck for research. Here, we report a trans-complementation system that produces single-round infectious SARS-CoV-2 that recapitulates authentic viral replication. We demonstrate that the single-round infectious SARS-CoV-2 can be used at BSL-2 laboratories for high-throughput neutralization and antiviral testing. The trans-complementation system consists of two components: a genomic viral RNA containing ORF3 and envelope gene deletions, as well as mutated transcriptional regulator sequences, and a producer cell line expressing the two deleted genes. Trans-complementation of the two components generates virions that can infect naive cells for only one round but does not produce wild-type SARS-CoV-2. Hamsters and K18-hACE2 transgenic mice inoculated with the complementation-derived virions exhibited no detectable disease, even after intracranial inoculation with the highest possible dose. Thus, the trans-complementation platform can be safely used at BSL-2 laboratories for research and countermeasure development.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Isothermal Recombinase Polymerase Amplification-Lateral Flow Point-of-Care Diagnostic Test for Heartland Virus.

The detection of novel or re-emergent pathogens necessitates the development of rapid, easy-to-use diagnostic tests that can be readily adapted and utilized in both clinical laboratories and field settings. Heartland virus (HRTV) is the first pathogenic Phlebovirus responsible for serious and fatal cases in the United States. We developed a qualitative test based on recombinase-polymerase-amplification coupled with lateral flow reading (RPA-LF) for rapid detection of HRTV. The RPA-LF detected HRTV with a limit of detection of 1.19-1.54 plaque-forming unit equivalents/reaction. In addition, the RPA-LF was able to detect 0.6075 copies/µL of HRTV nucleoprotein gene-containing plasmid. We evaluated six clinical samples that were previously found to be real-time PCR positive for HRTV and found five out of six samples to be positive by RPA-LF, yielding 83.3% concordance with real-time PCR. All six samples had Ct values between 29 and 39 by real-time PCR. We also determined that the HRTV primers and probe do not cross-react with other tick-transmitted viruses such as Bourbon and Powassan, or other related viruses, including Lonestar tick virus and Sunday canyon virus (100% specificity). This is the first isothermal amplification test developed for a tick-borne virus, which will allow for rapid differentiation between HRTV and other pathogens producing similar clinical manifestations.

Prophylactic and Therapeutic Inhibition of In Vitro SARS-CoV-2 Replication by Oleandrin.

With continued expansion of the COVID-19 pandemic, antiviral drugs are desperately needed to treat patients at high risk of life-threatening disease and even to limit spread if administered early during infection. Typically, the fastest route to identifying and licensing a safe and effective antiviral drug is to test those already shown safe in early clinical trials for other infections or diseases. Here, we tested in vitro oleandrin, derived from the Nerium oleander plant and shown previously to have inhibitory activity against several viruses. Using Vero cells, we found that prophylactic oleandrin administration at concentrations down to 0.05 µg/ml exhibited potent antiviral activity against SARS-CoV-2, with an 800-fold reduction in virus production, and a 0.1 µg/ml dose resulted in a greater than 3,000-fold reduction in infectious virus production. The EC 50 values were 11.98ng/ml when virus output was measured at 24 hours post-infection, and 7.07ng/ml measured at 48 hours post-infection. Therapeutic (post-infection) treatment up to 24 hours after infection of Vero cells also reduced viral titers, with the 0.1 µg/ml dose causing greater than 100-fold reductions as measured at 48 hours, and the 0.05 µg/ml dose resulting in a 78-fold reduction. The potent prophylactic and therapeutic antiviral activities demonstrated here strongly support the further development of oleandrin to reduce the severity of COVID-19 and potentially also to reduce spread by persons diagnosed early after infection. IMPORTANCE: COVID-19, a pandemic disease caused by infection with SARS-CoV-2, has swept around the world to cause millions of infections and hundreds-of-thousands of deaths due to the lack of vaccines and effective therapeutics. We tested oleandrin, derived from the Nerium oleander plant and shown previously to reduce the replication of several viruses, against SARS-CoV-2 infection of Vero cells. When administered both before and after virus infection, nanogram doses of oleandrin significantly inhibited replication by up to 3,000-fold, indicating the potential to prevent disease and virus spread in persons recently exposed to SARS-CoV-2, as well as to prevent severe disease in persons at high risk. These results indicate that oleandrin should be tested in animal models and in humans exposed to infection to determine its medical usefulness in controlling the pandemic.

Persistence of Severe Acute Respiratory Syndrome Coronavirus 2 in Aerosol Suspensions.

We aerosolized severe acute respiratory syndrome coronavirus 2 and determined that its dynamic aerosol efficiency surpassed those of severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome. Although we performed experiment only once across several laboratories, our findings suggest retained infectivity and virion integrity for up to 16 hours in respirable-sized aerosols.