Bat Rabies in the Americas: Is Myotis the Main Ancestral Spreader?

The rabies virus (RABV) is the exclusive lyssavirus affecting both wild and domestic mammalian hosts in the Americas, including humans. Additionally, the Americas stand out as the sole region where bat rabies occurs. While carnivore rabies is being increasingly managed across the region, bats are emerging as significant reservoirs of RABV infection for humans and domestic animals. Knowledge of the bat species maintaining rabies and comprehending cross-species transmission (CST) and host shift processes are pivotal for directing surveillance as well as ecological research involving wildlife reservoir hosts. Prior research indicates that bat RABV CST is influenced by host genetic similarity and geographic overlap, reflecting host adaptation. In this study, we compiled and analyzed a comprehensive nucleoprotein gene dataset representing bat-borne RABV diversity in Argentina and the broader Americas using Bayesian phylogenetics. We examined the association between host genus and geography, finding both factors shaping the global phylogenetic structure. Utilizing a phylogeographic approach, we inferred CST and identified key bat hosts driving transmission. Consistent with CST determinants, we observed monophyletic/paraphyletic clustering of most bat genera in the RABV phylogeny, with stronger CST evidence between host genera of the same family. We further discuss Myotis as a potential ancestral spreader of much of RABV diversity.

Morphological and molecular analyses confirm the presence of the newly described bat species Molossus melini in the Espinal ecoregion from Argentina.

Until now, Molossus melini was known only from its type locality, in the south of Santa Fe province, Argentina. Specimens of this species were collected in 2021 from a roost in a Fraxinus tree of the urban woodland of Paraná city, Entre Ríos province, Argentina. Bat identification was made by comparing external and cranial characters and measurements with those reported in the bibliography and corroborated by a phylogenetic analysis based on the cytochrome b gene. Also, multivariate morphometric analyses showed that cranial measurements, but not external ones, are informative enough to discriminate M. melini from the other Molossus species in Argentina (M. currentium, M. fluminensis, and M. molossus). This new record extends the distribution of M. melini from the south of Santa Fe province by 230 km to the northeast and represents the first record of the species in the Espinal ecoregion. Supplementary information: The online version contains supplementary material available at 10.1007/s13364-023-00679-1.

The Role of Molossidae and Vespertilionidae in Shaping the Diversity of Alphacoronaviruses in the Americas.

Bats are reservoirs of diverse coronaviruses (CoVs), including progenitors of severe acute respiratory syndrome CoV (SARS-CoV) and SARS-CoV-2. In the Americas, there is a contrast between alphacoronaviruses (alphaCoVs) and betaCoVs: while cospeciation prevails in the latter, alphaCoV evolution is dominated by deep and recent host switches. AlphaCoV lineages are maintained by two different bat family groups, Phyllostomidae and Vespertilionidae plus Molossidae. In this study, we used a Bayesian framework to analyze the process of diversification of the lineages maintained by Molossidae and Vespertilionidae, adding novel CoV sequences from Argentina. We provide evidence that the observed CoV diversity in these two bat families is shaped by their geographic distribution and that CoVs exhibit clustering at the level of bat genera. We discuss the causes of the cocirculation of two independent clades in Molossus and Tadarida as well as the role of Myotis as the ancestral host and a major evolutionary reservoir of alphaCoVs across the continent. Although more CoV sampling efforts are needed, these findings contribute to a better knowledge of the diversity of alphaCoVs and the links between bat host species. IMPORTANCE Bats harbor the largest diversity of coronaviruses among mammals. In the Americas, seven alphacoronavirus lineages circulate among bats. Three of these lineages are shared by members of two bat families: Vespertilionidae and Molossidae. Uncovering the relationships between these coronaviruses can help us to understand patterns of cross-species transmission and, ultimately, which hosts are more likely to be involved in spillover events. We found that two different lineages cocirculate among the bat genera Molossus and Tadarida, which share roosts and have common viral variants. The bat genus Myotis functions as a reservoir of coronavirus diversity and, as such, is a key host. Although there were some spillovers recorded, there is a strong host association, showing that once a successful host jump takes place, it is transmitted onward to members of the same bat genus.

Introduction and Differential Diagnosis of Monkeypox in Argentina, 2022.

We report detection of cases of monkeypox virus infection in Argentina in the context of a marked increase in confounding cases of atypical hand-foot-and-mouth syndrome caused by enterovirus coxsackie A6. We recommend performing an accurate differential virological diagnosis for exanthematous disease in suspected monkeypox cases.

Human Rabies by Secondary Transmission in Argentina, 2021.

Rabies is a zoonotic disease caused by the rabies virus (RABV) that causes fatal encephalitis in mammals. Bats can transmit the disease to urban canines and felines, which rarely infect humans, establishing a secondary link. The last case of human rabies in Argentina was transmitted by a dog in 2008. We present the first case of human rabies originating from an insectivorous bat, Tadarida brasiliensis, transmitted by a feral cat in Buenos Aires province, Argentina.

A Novel Terrestrial Rabies Virus Lineage Occurring in South America: Origin, Diversification, and Evidence of Contact between Wild and Domestic Cycles.

The rabies virus (RABV) is characterized by a history dominated by host shifts within and among bats and carnivores. One of the main outcomes of long-term RABV maintenance in dogs was the establishment of variants in a wide variety of mesocarnivores. In this study, we present the most comprehensive phylogenetic and phylogeographic analysis, contributing to a better understanding of the origins, diversification, and the role of different host species in the evolution and diffusion of a dog-related variant endemic of South America. A total of 237 complete Nucleoprotein gene sequences were studied, corresponding to wild and domestic species, performing selection analyses, ancestral states reconstructions, and recombination analyses. This variant originated in Brazil and disseminated through Argentina and Paraguay, where a previously unknown lineage was found. A single host shift was identified in the phylogeny, from dog to the crab-eating fox (Cerdocyon thous) in the Northeast of Brazil. Although this process occurred in a background of purifying selection, there is evidence of adaptive evolution -or selection of sub-consensus sequences- in internal branches after the host shift. The interaction of domestic and wild cycles persisted after host switching, as revealed by spillover and putative recombination events.

A rapid and simple protocol for concentration of SARS-CoV-2 from sewage.

The aim of this study was to set up a simple protocol to concentrate SARS-CoV-2 from sewage, which can be implemented in laboratories with minimal equipment resources. The method avoids the need for extensive purification steps and reduces the concentration of potential inhibitors of RT-qPCR contained in sewage. The concentration method consists of a single step, in which a small volume (40 mL) of sewage sample is incubated with polyaluminum chloride (PAC)(0.00045 N Al3+ final concentration). Virus particles adsorbed to the precipitate are collected by low-speed centrifugation, after which the recovered pellet is resuspended with a saline buffer. PAC-concentrated samples are stable for at least one week at 4 °C. Therefore, they may be sent refrigerated to a diagnosis center for RNA extraction and RT-qPCR for SARS-CoV-2 RNA detection if the lab does not have such capabilities. The PAC concentration method produced an average shift of 4.5-units in quantification cycle (Cq) values compared to non-concentrated samples, indicating a 25-fold increase in detection sensitivity. The lower detection limit corresponded approximately to 100 viral copies per ml. Kappa index indicated substantial agreement between PAC and polyethylene glycol (PEG) precipitation protocols (k = 0.688, CI 0.457-0.919). This low-cost concentration protocol could be useful to aid in the monitoring of community circulation of SARS-CoV-2, especially in low- and middle-income countries, which do not have massive access to support from specialized labs for sewage surveillance.

Evaluation of RT-qPCR and Loop-Mediated Isothermal Amplification (LAMP) Assays for the Detection of SARS-CoV-2 in Argentina.

Our aim was to evaluate the analytical and clinical performance of the SARS-CoV-2 molecular detection kits used in Argentina. Nine real-time reverse-transcription polymerase chain reaction (RT-qPCR) and three reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assays were evaluated using the World Health Organization (WHO) recommended test as reference method. A secondary standard calibrated for the E, N and RdRp genes against the Pan American Health Organization-World Health Organization-International Standard was used to calculate the limit of detection (LoD). A panel of artificial clinical samples, 32 positive and 30 negative for SARS-CoV-2, were analyzed to estimate the kappa concordance (κ) and the diagnostic performance. Differences among the LoD values for the target genes amplified by each kit were >1 log copies/reaction. The κ for the RT-qPCR kits was greater than 0.9, whereas that for the RT-LAMP assays ranged from 0.75 to 0.93. The clinical performance of RT-qPCR kits showed 100% specificity and high sensitivity, although with variations according to the gene analyzed. The E and N genes provided greater clinical sensitivity, whereas the RdRp gene increased the clinical specificity. The RT-LAMP assays revealed a variable diagnostic performance. The information provided can be useful to choose the most appropriate diagnostic test and may contribute to the establishment of a consensus in the diagnosis of SARS-CoV-2 in Argentina and the region.

Heterogeneous taxonomic resolution of cytochrome b gene identification of bats from Argentina: Implications for field studies.

Bats are among the most diverse, widespread, and abundant mammals. In Argentina, 67 species of bats have been recorded, belonging to 5 families and 29 genera. These high levels of biodiversity are likely to complicate identification at fieldwork, especially between closely related species, where external morphology-based approaches are the only immediate means for a priori species assignment. The use of molecular markers can enhance species identification, and acquires particular relevance in capture-release studies. In this study, we discuss the extent of the use of the mitochondrial cytochrome b gene for species identification, comparing external morphology identification with a molecular phylogenetic classification based on this marker, under the light of current bat systematics. We analyzed 33 samples collected in an eco-epidemiological survey in the province of Santa Fe (Argentina). We further sequenced 27 museum vouchers to test the accuracy of cytochrome b -based phylogenies in taxonomic identification of bats occurring in the Pampean/Chacoan regions of Argentina. The cytochrome b gene was successfully amplified in all Molossid and Vespertilionid species except for Eptesicus, for which we designed a new reverse primer. The resulting Bayesian phylogeny was congruent with current systematics. Cytochrome b proved useful for species-level delimitation in non-conflicting genera (Eumops, Dasypterus, Molossops) and has infrageneric resolution in more complex lineages (Eptesicus, Myotis, Molossus). We discuss four sources of incongruence that may act separately or in combination: 1) molecular processes, 2) biology, 3) limitations in identification, and 4) errors in the current taxonomy. The present study confirms the general applicability of cytochrome b -based phylogenies in eco-epidemiological studies, but its resolution and reliability depend mainly, but not solely, on the level of genetic differentiation within each bat genus.

Evaluation of Two Real-Time, TaqMan Reverse Transcription-PCR Assays for Detection of Rabies Virus in Circulating Variants from Argentina: Influence of Sequence Variation.

In rabies diagnosis, it is essential to count on a rapid test to give a quick response. The combined sensitivity and robustness of the TaqMan RT-PCR assays (qRT-PCR) have made these methods a valuable alternative for rabies virus (RABV) detection. We conducted a study to compare the applicability of two widely used qRT-PCR assays targeting the nucleoprotein gene (LysGT1 assay) and leader sequences (LN34 qRT-PCR assay) of RABV genomes, in all variants circulating in Argentina. A total of 44 samples obtained from bats, dogs, cattle, and horses, that were previously tested for rabies by FAT and conventional RT-PCR, were used in the study. All variants were successfully detected by the pan-lyssavirus LN34 qRT-PCR assay. The LysGT1 assay failed to detect three bat-related variants. We further sequenced the region targeted by LysGT1 and demonstrated that the presence of three or more mismatches with respect to the primers and probe sequences precludes viral detection. We conclude that the LysGT1 assay is prone to yield variant-dependent false-negative test results, and in consequence, the LN34 assay would ensure more effective detection of RABV in Argentina.

Enfermedad de mano-pie-boca atípica causada por virus Coxsackie A6 en Argentina, 2015 / Atypical hand, foot, and mouth disease caused by Coxsackievirus A6 in Argentina in 2015

We present two groups of cases of atypical hand, foot, and mouth disease (HFMD) caused by Coxsackievirus A6 (CV-A6) detected in Argentina in 2015. The first group involved 14 patients from Chubut province and the second group affected 12 patients from San Luis province. Molecular analysis of the complete VP1 protein gene revealed the circulation of E2 sublineage, the most predominant worldwide. To our knowledge, this is the first report of CV-A6 infections associated with atypical HFMD in Argentina and South America.

Se describen dos grupos de casos de enfermedad de mano-pie-boca (HFMD) atípica causada por el virus Coxsackie A6 (Coxsackievirus A6, CV-A6) detectados en Argentina en el año 2015. El primero de los grupos involucró a 14 pacientes de Chubut y el segundo a 12 pacientes de San Luis. El análisis molecular del gen de la proteína VP1 completa reveló la circulación del sublinaje E2, el predominante a nivel global. Hasta donde sabemos, este es el primer reporte de infecciones CV-A6 asociadas con HFMD atípica en Argentina y Sudamérica.

Acute Flaccid Myelitis Associated with Enterovirus D68 in Children, Argentina, 2016.

After a 2014 outbreak of severe respiratory illness caused by enterovirus D68 in the United States, sporadic cases of acute flaccid myelitis have been reported worldwide. We describe a cluster of acute flaccid myelitis cases in Argentina in 2016, adding data to the evidence of association between enterovirus D68 and this polio-like illness.

Atypical hand, foot, and mouth disease caused by Coxsackievirus A6 in Argentina in 2015.

We present two groups of cases of atypical hand, foot, and mouth disease (HFMD) caused by Coxsackievirus A6 (CV-A6) detected in Argentina in 2015. The first group involved 14 patients from Chubut province and the second group affected 12 patients from San Luis province. Molecular analysis of the complete VP1 protein gene revealed the circulation of E2 sublineage, the most predominant worldwide. To our knowledge, this is the first report of CV-A6 infections associated with atypical HFMD in Argentina and South America.

Diagnosis and molecular typing of rabies virus in samples stored in inadequate conditions.

INTRODUCTION: The exposure of nervous tissue samples to high temperatures affects the sensitivity of rabies virus diagnostic tests, causing degradation of the viral structure. This study evaluated reverse transcriptase polymerase chain reaction (RT-PCR) for the diagnosis and molecular characterization of brain tissue samples in an advanced state of decomposition and poorly conserved viral isolates by comparing it with routine diagnostic tests. METHODOLOGY: A panel of three canine brain samples exposed to controlled decomposition for 7, 15, 30, and 120 days were evaluated using fluorescence antibody test (FAT), mouse inoculation test (MIT), and RT-PCR. In addition, 14 isolates of rabies variants, representing the largest circulation in Argentina, preserved in inadequate cooling for six to eight years were analyzed. Molecular typing of strains was performed using a 159-nucleotide region corresponding to the nucleoprotein gene. RESULTS: The three samples analyzed were positive by RT-PCR at all the decomposition times evaluated, in contrast to results observed with FAT and MIT, which rapidly became negative. In addition, 100% of the inadequately preserved samples were characterized molecularly. The limit of detection of RT-PCR was 0.5 MICDL50/0.03 mL. CONCLUSION: RT-PCR can be useful for rabies diagnosis and typing of putrefying samples or rabies isolates stored in inadequate conditions.

Environmental surveillance of norovirus in Argentina revealed distinct viral diversity patterns, seasonality and spatio-temporal diffusion processes.

Norovirus (NoV) contamination was evaluated in five rivers of Argentina between 2005 and 2011. NoV was present in all sampled rivers, with distinct NoV patterns in waters impacted by different-sized communities. In rivers affected by medium-sized populations (Salta and Córdoba cities) only one or two genotypes were present, GII.4 being the main one, with winter seasonality. In contrast, in the much more heavily populated area of Buenos Aires city the prevalent GII.4 was accompanied by several additional genotypes (GII.4, GII.b, GII.2, GII.7, GII.17, GII.e and GII.g) and one ungenotyped GII NoV, with no clear seasonality. GII.4 2006b was the main variant detected (60.9%). Phylogeographic and phylodynamic analyses performed in region D of the VP1 gene showed a most recent common ancestor in 2002 and a substitution rate of 3.7×10(-3) substitutions per site per year (HPD95%=2.3×10(-3)-5.2×10(-3)) for this variant still involving a significant population size with a slight decrease since 2008. The spatio-temporal diffusion analysis proposed Europe as an intermediate path between the American Continent and the rest of the World for NoV dissemination. Given the importance of NoV as a cause of epidemic gastroenteritis and the likelihood of its environmental transmission, the results of this work should increase public and institutional awareness of the health risk involved in sewage discharges into the environment. Environmental surveillance of enteric viruses could be a very useful tool not only to prevent waterborne outbreaks, but also to describe the epidemiology of the viruses. The detailed analysis of the viral genomes disposed into the environment contributed to the characterization of the dissemination, diversity and seasonality of NoV in its natural host population. In future studies, environmental surveillance and molecular analysis should be complemented with a quantitative viral risk assessment for estimating the disease burden from viruses in the environment.

High diversity of rabies viruses associated with insectivorous bats in Argentina: presence of several independent enzootics.

BACKGROUND: Rabies is a fatal infection of the central nervous system primarily transmitted by rabid animal bites. Rabies virus (RABV) circulates through two different epidemiological cycles: terrestrial and aerial, where dogs, foxes or skunks and bats, respectively, act as the most relevant reservoirs and/or vectors. It is widely accepted that insectivorous bats are not important vectors of RABV in Argentina despite the great diversity of bat species and the extensive Argentinean territory. METHODS: We studied the positivity rate of RABV detection in different areas of the country, and the antigenic and genetic diversity of 99 rabies virus (RABV) strains obtained from 14 species of insectivorous bats collected in Argentina between 1991 and 2008. RESULTS: Based on the analysis of bats received for RABV analysis by the National Rabies system of surveillance, the positivity rate of RABV in insectivorous bats ranged from 3.1 to 5.4%, depending on the geographic location. The findings were distributed among an extensive area of the Argentinean territory. The 99 strains of insectivorous bat-related sequences were divided into six distinct lineages associated with Tadarida brasiliensis, Myotis spp, Eptesicus spp, Histiotus montanus, Lasiurus blosseviilli and Lasiurus cinereus. Comparison with RABV sequences obtained from insectivorous bats of the Americas revealed co-circulation of similar genetic variants in several countries. Finally, inter-species transmission, mostly related with Lasiurus species, was demonstrated in 11.8% of the samples. CONCLUSIONS: This study demonstrates the presence of several independent enzootics of rabies in insectivorous bats of Argentina. This information is relevant to identify potential areas at risk for human and animal infection.

Diagnóstico de un caso de rabia humana en Jujuy, 2008 / Diagnosis of a human rabies case in Jujuy, 2008

El 22 de Julio de 2008, un niño de 8 años de edad, residente en la provincia de Jujuy, Argentina, falleció por una encefalitis producida por el virus de la rabia. El diagnóstico se realizó mediante la detección de anticuerpos en suero y se confirmó por inmunofluorescencia en el cerebro. La tipificación antigénica correspondió a la variante 1 trasmitida por perros. El análisis molecular estableció que el virus detectado es de la misma variante genética que circula en Jujuy desde 2003. Este trabajo resume la evolución clínica del paciente y la posterior investigación epidemiológica que reveló el antecedente de mordedura por un perro 60 días antes de la iniciación de la enfermedad y la ausencia de un tratamiento antirrábico post-exposición.

On July 22, 2008, a previously healthy 8 years old boy from Jujuy, Argentina, died of encephalitis later confirmed as rabies. Diagnosis was made on the basis rabies-specific antibodies presence in a serum sample and it was confirmed by detection of the viral antigens in brain necropsy using the inmunofluorescent test. Antigenic characterization identified dog as source of infection. Molecular analysis recognized the same genetic variant circulating in Jujuy since 2003. This report presents the patient's clinical course and the epidemiologic investigation that revealed a dog bite 60 days before the illness onset and the lack of rabies treatment.

[Diagnosis of a human rabies case in Jujuy, 2008]. / Diagnóstico de un caso de rabia humana en Jujuy, 2008.

On July 22, 2008, a previously healthy 8 years old boy from Jujuy, Argentina, died of encephalitis later confirmed as rabies. Diagnosis was made on the basis rabies-specific antibodies presence in a serum sample and it was confirmed by detection of the viral antigens in brain necropsy using the immunofluorescent test. Antigenic characterization identified dog as source of infection. Molecular analysis recognized the same genetic variant circulating in Jujuy since 2003. This report presents the patient's clinical course and the epidemiologic investigation that revealed a dog bite 60 days before the illness onset and the lack of rabies treatment.

[Epidemiology of enterovirus associated with neurologic diseases]. / Epidemiologia de los enterovirus asociados a enfermedades neurologicas.

This report describes the results of human enterovirus (HEV) detection and characterization using cell culture, polymerase chain reaction and molecular typing in 2167 samples obtained from acute flaccid paralysis, aseptic meningitis and acute encephalitis patients, from 1991 to 1998 in Argentina. HEV were isolated in 130 out of 666 cases (19.5%) and 36 out of 666 (5.4%). HEV RNA was detected in 28.8% (231/801) and 3.0% (21/700) of the patients with meningitis and encephalitis, respectively. Children with ages ranging from 1 to 9 years accounted for 75.3% of the meningitis cases and from 1 to 4 years for 58% of acute flaccid paralysis patients. Echovirus 4 (E4), E9, E30 and E17 were identified from meningitis outbreaks. Coxsackievirus A2 (CAV2), CBV2, CBV5, E7, E11, E19, E24, E29 and enterovirus 71 were recovered only from sporadic cases. Three different serotypes were identified in encephalitis patients: E4, E7 and E24. A total of 28 different serotypes of non-polio enteroviruses were detected from acute flaccid paralysis cases. The information here presented contributes to improving our knowledge about enteroviruses epidemiology in Argentina and their relationship with different neurological diseases. This study provides valuable data that could be useful to further research.

Epidemiología de los enterovirus asociados a enfermedades neurológicas / Epidemiology of enterovirus associated with neurologic diseases

El presente estudio describe los resultados de la investigación de los enterovirus humanos (HEV) mediante cultivo celular y reacción en cadena de la polimerasa y su tipificación molecular en 2167 casos de parálisis fláccida aguda, meningitis aséptica y encefalitis aguda, obtenidos entre 1991 y 1998 en la Argentina. La frecuencia de detección de HEV en parálisis fláccida aguda fue 19.5% (130/666) y de poliovirus Sabin 5.4% (36/666). La tasa de detección de HEV en los casos de meningitis fue 28.8% (231/801) y en encefalitis 3.0% (21/700). El grupo etario más afectado por las meningitis fue entre 1 y 9 años (75.3%) y en los casos de parálisis fláccida aguda, de 1 a 4 años (58%). En muestras de brotes de meningitis se identificó echovirus (E) 4, E9, E30 y E17, y en casos esporádicos virus coxsackie A (CAV) 2, B (CBV) 2 y CBV5, E7, E11, E19, E24 y E29, y enterovirus (EV) 71. Finalmente, en casos de encefalitis se detectó E4, E7 y E24. En casos de parálisis fláccida aguda se identificaron 28 serotipos distintos de enterovirus no polio. En la Argentina y en otros países latinoamericanos existe escasa información acerca de la circulación de los HEV y su relación con diversas enfermedades neurológicas. Este estudio proporciona información que puede servir como base para posteriores investigaciones.(AU)

This report describes the results of human enterovirus (HEV) detection and characterization using cell culture, polymerase chain reaction and molecular typing in 2167 samples obtained from acute flaccid paralysis, aseptic meningitis and acute encephalitis patients, from 1991 to 1998 in Argentina. HEV were isolated in 130 out of 666 cases (19.5%) and 36 out of 666 (5.4%). HEV RNA was detected in 28.8% (231/801) and 3.0% (21/700) of the patients with meningitis and encephalitis, respectively. Children with ages ranging from 1 to 9 years accounted for 75.3% of the meningitis cases and from 1 to 4 years for 58% of acute flaccid paralysis patients. Echovirus 4 (E4), E9, E30 and E17 were identified from meningitis outbreaks. Coxsackievirus A2 (CAV2), CBV2, CBV5, E7, E11, E19, E24, E29 and enterovirus 71 were recovered only from sporadic cases. Three different serotypes were identified in encephalitis patients: E4, E7 and E24. A total of 28 different serotypes of non-polio enteroviruses were detected from acute flaccid paralysis cases. The information here presented contributes to improving our knowledge about enteroviruses epidemiology in Argentina and their relationship with different neurological diseases. This study provides valuable data that could be useful to further research.(AU)