The Association of Human Astrovirus with Extracellular Vesicles Facilitates Cell Infection and Protects the Virus from Neutralizing Antibodies.

Viral gastroenteritis has a global distribution and represents a high risk for vulnerable population and children under 5 years due to acute diarrhea, fever and dehydration. Human astroviruses (HAstV) have been identified as the third most important cause of viral gastroenteritis in pediatric and immunocompromised patients. Furthermore, HAstV has been reported in biopsies taken from patients with encephalitis, meningitis and acute respiratory infection, yet it is not clear how the virus reaches these organs. In this work we have tested the possibility that the released astrovirus particles could be associated with extracellular vesicles. Comparison between vesicles purified from HAstV Yuc8 infected and mock-infected cells showed that infection enhances production of vesicles larger than 150 nm. These vesicles contain CD63 and Alix, two markers of vesicular structures. Almost 70% of the extracellular virus present in clarified supernatant at 18 h postinfection was found associated with vesicular membranes, and this association facilitates cell infection in the absence of trypsin activation and protects virions from neutralizing antibodies. Our findings suggest a new pathway for HAstV spread and might represent an explanation for the extra-intestinal presence of some astrovirus strains. IMPORTANCE Astroviruses are an important cause of diarrhea in vulnerable population, particularly children; recently some reports have found these viruses in extra-intestinal organs, including the central nervous system, causing unexpected clinical disease. In this work, we found that human astrovirus strain Yuc8 associates with extracellular vesicles, possibly during or after their cell egress. The association with vesicles doubled astrovirus infectivity in less susceptible cells and rendered virus particles insensitive to neutralization by antibodies. These data suggest that extracellular vesicles could represent a novel pathway for astrovirus to disseminate outside the gastrointestinal tract.

Metagenomic analysis reveals differences in the co-occurrence and abundance of viral species in SARS-CoV-2 patients with different severity of disease.

BACKGROUND: SARS-CoV-2 infections have a wide spectrum of clinical manifestations whose causes are not completely understood. Some human conditions predispose to severe outcome, like old age or the presence of comorbidities, but many other facets, including coinfections with other viruses, remain poorly characterized. METHODS: In this study, the eukaryotic fraction of the respiratory virome of 120 COVID-19 patients was characterized through whole metagenomic sequencing. RESULTS: Genetic material from respiratory viruses was detected in 25% of all samples, whereas human viruses other than SARS-CoV-2 were found in 80% of them. Samples from hospitalized and deceased patients presented a higher prevalence of different viruses when compared to ambulatory individuals. Small circular DNA viruses from the Anneloviridae (Torque teno midi virus 8, TTV-like mini virus 19 and 26) and Cycloviridae families (Human associated cyclovirus 10), Human betaherpesvirus 6, were found to be significantly more abundant in samples from deceased and hospitalized patients compared to samples from ambulatory individuals. Similarly, Rotavirus A, Measles morbillivirus and Alphapapilomavirus 10 were significantly more prevalent in deceased patients compared to hospitalized and ambulatory individuals. CONCLUSIONS: Results show the suitability of using metagenomics to characterize a broader peripheric virological landscape of the eukaryotic virome in SARS-CoV-2 infected patients with distinct disease outcomes. Identified prevalent viruses in hospitalized and deceased patients may prove important for the targeted exploration of coinfections that may impact prognosis.

Genomic Analysis of Early SARS-CoV-2 Variants Introduced in Mexico.

The coronavirus disease 2019 (COVID-19) pandemic has affected most countries in the world. Studying the evolution and transmission patterns in different countries is crucial to enabling implementation of effective strategies for disease control and prevention. In this work, we present the full genome sequence for 17 SARS-CoV-2 isolates corresponding to the earliest sampled cases in Mexico. Global and local phylogenomics, coupled with mutational analysis, consistently revealed that these viral sequences are distributed within 2 known lineages, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineage A/G, containing mostly sequences from North America, and lineage B/S, containing mainly sequences from Europe. Based on the exposure history of the cases and on the phylogenomic analysis, we characterized 14 independent introduction events. Additionally, three cases with no travel history were identified. We found evidence that two of these cases represented local transmission cases occurring in Mexico during mid-March 2020, denoting the earliest events described for the country. Within this local transmission cluster, we also identified an H49Y amino acid change in the Spike protein. This mutation represents a homoplasy occurring independently through time and space and may function as a molecular marker to follow any further spread of these viral variants throughout the country. Our results provide a general picture of the SARS-CoV-2 variants introduced at the beginning of the outbreak in Mexico, setting the foundation for future surveillance efforts.IMPORTANCE Understanding the introduction, spread, and establishment of SARS-CoV-2 within distinct human populations as well as the evolution of the pandemics is crucial to implement effective control strategies. In this work, we report that the initial virus strains introduced in Mexico came from Europe and the United States and that the virus was circulating locally in the country as early as mid-March. We also found evidence for early local transmission of strains with a H49Y mutation in the Spike protein, which could be further used as a molecular marker to follow viral spread within the country and the region.

Emergence and spread of the potential variant of interest (VOI) B.1.1.519 of SARS-CoV-2 predominantly present in Mexico.

SARS-CoV-2 variants emerged in late 2020, and at least three variants of concern (B.1.1.7, B.1.351, and P1) have been reported by WHO. These variants have several substitutions in the spike protein that affect receptor binding; they exhibit increased transmissibility and may be associated with reduced vaccine effectiveness. In the present work, we report the identification of a potential variant of interest, harboring the mutations T478K, P681H, and T732A in the spike protein, within the newly named lineage B.1.1.519, that rapidly outcompeted the preexisting variants in Mexico and has been the dominant virus in the country during the first trimester of 2021.

A cloud-compatible bioinformatics pipeline for ultrarapid pathogen identification from next-generation sequencing of clinical samples.

Unbiased next-generation sequencing (NGS) approaches enable comprehensive pathogen detection in the clinical microbiology laboratory and have numerous applications for public health surveillance, outbreak investigation, and the diagnosis of infectious diseases. However, practical deployment of the technology is hindered by the bioinformatics challenge of analyzing results accurately and in a clinically relevant timeframe. Here we describe SURPI ("sequence-based ultrarapid pathogen identification"), a computational pipeline for pathogen identification from complex metagenomic NGS data generated from clinical samples, and demonstrate use of the pipeline in the analysis of 237 clinical samples comprising more than 1.1 billion sequences. Deployable on both cloud-based and standalone servers, SURPI leverages two state-of-the-art aligners for accelerated analyses, SNAP and RAPSearch, which are as accurate as existing bioinformatics tools but orders of magnitude faster in performance. In fast mode, SURPI detects viruses and bacteria by scanning data sets of 7-500 million reads in 11 min to 5 h, while in comprehensive mode, all known microorganisms are identified, followed by de novo assembly and protein homology searches for divergent viruses in 50 min to 16 h. SURPI has also directly contributed to real-time microbial diagnosis in acutely ill patients, underscoring its potential key role in the development of unbiased NGS-based clinical assays in infectious diseases that demand rapid turnaround times.

DNA microarray for detection of gastrointestinal viruses.

Gastroenteritis is a clinical illness of humans and other animals that is characterized by vomiting and diarrhea and caused by a variety of pathogens, including viruses. An increasing number of viral species have been associated with gastroenteritis or have been found in stool samples as new molecular tools have been developed. In this work, a DNA microarray capable in theory of parallel detection of more than 100 viral species was developed and tested. Initial validation was done with 10 different virus species, and an additional 5 species were validated using clinical samples. Detection limits of 1 × 10(3) virus particles of Human adenovirus C (HAdV), Human astrovirus (HAstV), and group A Rotavirus (RV-A) were established. Furthermore, when exogenous RNA was added, the limit for RV-A detection decreased by one log. In a small group of clinical samples from children with gastroenteritis (n = 76), the microarray detected at least one viral species in 92% of the samples. Single infection was identified in 63 samples (83%), and coinfection with more than one virus was identified in 7 samples (9%). The most abundant virus species were RV-A (58%), followed by Anellovirus (15.8%), HAstV (6.6%), HAdV (5.3%), Norwalk virus (6.6%), Human enterovirus (HEV) (9.2%), Human parechovirus (1.3%), Sapporo virus (1.3%), and Human bocavirus (1.3%). To further test the specificity and sensitivity of the microarray, the results were verified by reverse transcription-PCR (RT-PCR) detection of 5 gastrointestinal viruses. The RT-PCR assay detected a virus in 59 samples (78%). The microarray showed good performance for detection of RV-A, HAstV, and calicivirus, while the sensitivity for HAdV and HEV was low. Furthermore, some discrepancies in detection of mixed infections were observed and were addressed by reverse transcription-quantitative PCR (RT-qPCR) of the viruses involved. It was observed that differences in the amount of genetic material favored the detection of the most abundant virus. The microarray described in this work should help in understanding the etiology of gastroenteritis in humans and animals.

Gangliosides have a functional role during rotavirus cell entry.

Cell entry of rotaviruses is a complex process, which involves sequential interactions with several cell surface molecules. Among the molecules implicated are gangliosides, glycosphingolipids with one or more sialic acid (SA) residues. The role of gangliosides in rotavirus cell entry was studied by silencing the expression of two key enzymes involved in their biosynthesis--the UDP-glucose:ceramide glucosyltransferase (UGCG), which transfers a glucose molecule to ceramide to produce glucosylceramide GlcCer, and the lactosyl ceramide-α-2,3-sialyl transferase 5 (GM3-s), which adds the first SA to lactoceramide-producing ganglioside GM3. Silencing the expression of both enzymes resulted in decreased ganglioside levels (as judged by GM1a detection). Four rotavirus strains tested (human Wa, simian RRV, porcine TFR-41, and bovine UK) showed a decreased infectivity in cells with impaired ganglioside synthesis; however, their replication after bypassing the entry step was not affected, confirming the importance of gangliosides for cell entry of the viruses. Interestingly, viral binding to the cell surface was not affected in cells with inhibited ganglioside synthesis, but the infectivity of all strains tested was inhibited by preincubation of gangliosides with virus prior to infection. These data suggest that rotaviruses can attach to cell surface in the absence of gangliosides but require them for productive cell entry, confirming their functional role during rotavirus cell entry.

A (H1N1) pdm09 HA D222 variants associated with severity and mortality in patients during a second wave in Mexico.

BACKGROUND: Pandemic type A (H1N1) influenza arose in early 2009, probably in Mexico and the United States, and reappeared in North America in September for seven more months. An amino acid substitution in the hemagglutinin (HA), D222G, has been reported in a significant proportion of patients with a severe and fatal outcome. We studied the prevalence of HA222 substitutions in patients in Mexico during the second wave and its association with clinical outcome and pathogenicity in a mouse model. METHODS: The nucleotide sequences of hemagglutinin (HA) from viruses collected from 77 patients were determined including 50 severe and fatal cases and 27 ambulatory cases. Deep sequencing was done on 5 samples from severe or fatal cases in order to determine the quasispecies proportion. Weight loss and mortality due to infection with cultured influenza viruses were analyzed in a mouse model. RESULTS: Viruses from 14 out of 50 hospitalized patients (28%) had a non aspartic acid residue at the HA 222 position (nD222), while all 27 ambulatory patients had D222 (p=0.0014). G222 was detected as sole species or in coexistence with N222 and D222 in 12 patients with severe disease including 8 who died. N222 in coexistence with D222 was detected in 1 patient who died and co-occurrence of A222 and V222, together with D222, was detected in another patient who died. The patients with a nD222 residue had higher mortality (71.4%), compared to the group with only D222 (22.2%, p=0.0008). Four of the 14 viruses from hospitalized patients were cultured and intranasally infected into mice. Two viruses with G222 were lethal while a third virus, with G222, caused only mild illness in mice similar to the fourth virus that contained D222. CONCLUSIONS: We confirm the elevated incidence of HA222 (H1N1)pdm09 variants in severe disease and mortality. Both clinical and mouse infection data support the idea that nD222 mutations contribute to increased severity of disease but additional determinants in disease outcome may be present.

Differential virome composition and richness between children's diarrheagenic stools kept at ultra-low temperatures for long-term.

INTRODUCTION: Diarrhoeal illness is the second cause of morbidity/mortality among children from less-developed regions worldwide. Nonetheless, there is scarce information regarding their gut microbiome. AIM: Microbiome characterization, with an emphasis on the virome, of children's stools with diarrhoea, by a commercial microbiome array. METHODOLOGY: Nucleic acids extraction, optimised for viral identification, of stool samples from 20 Mexican children with diarrhoea (10 children < 2 and 10 ≥ 2-years-old), collected 16 years ago and kept at -70 °C, were analysed for the presence of viruses, bacteria, archaea, protozoa, and fungi species sequences. RESULTS: Only viral and bacterial species sequences were identified among children's stools. Most stool samples harboured species belonging to the bacteriophages (95%), anellovirus (60%), diarrhoeagenic viruses (40%), and non-human pathogens viruses (45% avian virus and 40% plant viruses) groups. Among the children's stools, virome inter-individual species composition was observed, even in presence of illness. The < 2-years-old children group has significantly higher viral richness (p = 0.01), conferred mainly by bacteriophages and diarrheagenic-viruses (p = 0.01) species, in comparison with the ≥ 2-years-old group. CONCLUSIONS: The virome of stools of children with diarrhoea revealed inter-individual viral species composition. Similarly, to the few virome studies in healthy young children, the bacteriophages group was the most abundant. A significantly higher viral richness, conferred by bacteriophages and diarrheagenic-viral species, was observed among < 2-years-old children in comparison with older children. Stools preserved at -70 °C for long term can successfully be used for microbiome studies.

The fecal and oropharyngeal eukaryotic viromes of healthy infants during the first year of life are personal.

Using a metagenomic sequencing approach, we described and compared the diversity and dynamics of the oropharyngeal and fecal eukaryotic virome of nine asymptomatic children in a semi-rural community setting located in the State of Morelos, Mexico. Ninety oropharyngeal swabs and 97 fecal samples were collected starting 2 weeks after birth and monthly thereafter until 12 months of age. In both niches, more than 95% of the total sequence reads were represented by viruses that replicate either in humans or in plants. Regarding human viruses, three families were most abundant and frequent in the oropharynx: Herpesviridae, Picornaviridae, and Reoviridae; in fecal samples, four virus families predominated: Caliciviridae, Picornaviridae, Reoviridae, and Anelloviridae. Both niches showed a high abundance of plant viruses of the family Virgaviridae. Differences in the frequency and abundance of sequence reads and diversity of virus species were observed in both niches and throughout the year of study, with some viruses already present in the first months of life. Our results suggest that the children's virome is dynamic and likely shaped by the environment, feeding, and age. Moreover, composition analysis suggests that the virome composition is mostly individual. Whether this constant exposition to different viruses has a long-term impact on children's health or development remains to be studied.

Monitoring SARS-CoV-2 in the Wastewater and Rivers of Tapachula, a Migratory Hub in Southern Mexico.

The COVID-19 pandemic has been monitored by applying different strategies, including SARS-CoV-2 detection with clinical testing or through wastewater-based epidemiology (WBE). We used the latter approach to follow SARS-CoV-2 dispersion in Tapachula city, located in Mexico's tropical southern border region. Tapachula is a dynamic entry point for people seeking asylum in Mexico or traveling to the USA. Clinical testing facilities for SARS-CoV-2 monitoring are limited in the city. A total of eighty water samples were collected from urban and suburban rivers and sewage and a wastewater treatment plant over 4 months in Tapachula. We concentrated viral particles with a PEG-8000-based method, performed RNA extraction, and detected SARS-CoV-2 particles through RT-PCR. We considered the pepper mild mottle virus as a fecal water pollution biomarker and analytical control. SARS-CoV-2 viral loads (N1 and N2 markers) were quantified and correlated with official regional statistics of COVID-19 bed occupancy and confirmed cases (r > 91%). Our results concluded that WBE proved a valuable tool for tracing and tracking the COVID-19 pandemic in tropical countries with similar water temperatures (21-29 °C). Monitoring SARS-CoV-2 through urban and suburban river water sampling would be helpful in places lacking a wastewater treatment plant or water bodies with sewage discharges.

High Prevalence and Diversity of Caliciviruses in a Community Setting Determined by a Metagenomic Approach.

We recently carried out a metagenomic study to determine the fecal virome of infants during their first year of life in a semirural community in Mexico. A total of 97 stool samples from nine children were collected starting 2 weeks after birth and monthly thereafter until 12 months of age. In this work, we describe the prevalence and incidence of caliciviruses in this birth cohort. We found that 54 (56%) and 24 (25%) of the samples were positive for norovirus and sapovirus sequence reads detected by next-generation sequencing, respectively. Potential infections were arbitrarily considered when at least 20% of the complete virus genome was determined. Considering only these samples, there were 3 cases per child/year for norovirus and 0.33 cases per child/year for sapovirus. All nine children had sequence reads related to norovirus in at least 2 and up to 10 samples, and 8 children excreted sapovirus sequence reads in 1 and up to 5 samples during the study. The virus in 35 samples could be genotyped. The results showed a high diversity of both norovirus (GI.3[P13], GI.5, GII.4, GII.4[P16], GII.7[P7], and GII.17[P17]) and sapovirus (GI.1, GI.7, and GII.4) in the community. Of interest, despite the frequent detection of caliciviruses in the stools, all children remained asymptomatic during the study. Our results clearly show that metagenomic studies in stools may reveal a detailed picture of the prevalence and diversity of gastrointestinal viruses in the human gut during the first year of life. IMPORTANCE Human caliciviruses are important etiological agents of acute gastroenteritis in children under 5 years of age. Several studies have characterized their association with childhood diarrhea and their presence in nondiarrheal stool samples. In this work, we used a next-generation sequencing approach to determine, in a longitudinal study, the fecal virome of infants during their first year of life. Using this method, we found that caliciviruses can be detected significantly more frequently than previously reported, providing a more detailed picture of the prevalence and genetic diversity of these viruses in the human gut during early life.

Genomic Characterization of SARS-CoV-2 Isolated from Patients with Distinct Disease Outcomes in Mexico.

The coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has shown a wide spectrum of clinical manifestations ranging from asymptomatic infections to severe disease and death. Pre-existing medical conditions and age have been mainly linked to the development of severe disease; however, the potential association of viral genetic characteristics with different clinical conditions remains unclear. SARS-CoV-2 variants with increased transmissibility were detected early in the pandemics, and several variants with potential relevance for public health are currently circulating around the world. In this study, we characterized 57 complete SARS-CoV-2 genomes during the exponential growth phase of the early epidemiological curve in Mexico, in April 2020. Patients were categorized under distinct disease severity outcomes: mild disease or ambulatory care, severe disease or hospitalized, and deceased. To reduce bias related to risk factors, the patients were less than 60 years old and with no diagnosed comorbidities A trait-association phylogenomic approach was used to explore genotype-phenotype associations, represented by the co-occurrence of mutations, disease severity outcome categories, and clusters of Mexican sequences. Phylogenetic results revealed a higher genomic diversity compared to the initial viruses detected during the early stage of the local epidemic. We identified a total of 90 single nucleotide variants compared to the Wuhan-Hu-1 genome, including 54 nonsynonymous mutations. We did not find evidence for the co-occurrence of mutations associated with specific disease outcomes. Therefore, in the group of patients studied, disease severity was likely mainly driven by the host genetic background and other demographic factors. IMPORTANCE The genetic association of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) with different clinical conditions remains unclear and needs further investigation. In this study, we characterized 57 complete SARS-CoV-2 genomes from patients in Mexico with distinct disease severity outcomes: mild disease or ambulatory care, severe disease or hospitalized, and deceased. To reduce bias related to risk factors the patients were less than 60 years old and with no diagnosed comorbidities. We did not find evidence for the co-occurrence of mutations associated with specific disease outcomes. Therefore, in the group of patients studied, disease severity was likely mainly driven by the host genetic background and other demographic factors.

Two-year follow-up of the COVID-19 pandemic in Mexico.

Background: After the initial outbreak in China (December 2019), the World Health Organization declared COVID-19 a pandemic on March 11th, 2020. This paper aims to describe the first 2 years of the pandemic in Mexico. Design and methods: This is a population-based longitudinal study. We analyzed data from the national COVID-19 registry to describe the evolution of the pandemic in terms of the number of confirmed cases, hospitalizations, deaths and reported symptoms in relation to health policies and circulating variants. We also carried out logistic regression to investigate the major risk factors for disease severity. Results: From March 2020 to March 2022, the coronavirus disease 2019 (COVID-19) pandemic in Mexico underwent four epidemic waves. Out of 5,702,143 confirmed cases, 680,063 were hospitalized (11.9%), and 324,436 (5.7%) died. Even if there was no difference in susceptibility by gender, males had a higher risk of death (CFP: 7.3 vs. 4.2%) and hospital admission risk (HP: 14.4 vs. 9.5%). Severity increased with age. With respect to younger ages (0-17 years), the 60+ years or older group reached adjusted odds ratios of 9.63 in the case of admission and 53.05 (95% CI: 27.94-118.62) in the case of death. The presence of any comorbidity more than doubled the odds ratio, with hypertension-diabetes as the riskiest combination. While the wave peaks increased over time, the odds ratios for developing severe disease (waves 2, 3, and 4 to wave 1) decreased to 0.15 (95% CI: 0.12-0.18) in the fourth wave. Conclusion: The health policy promoted by the Mexican government decreased hospitalizations and deaths, particularly among older adults with the highest risk of admission and death. Comorbidities augment the risk of developing severe illness, which is shown to rise by double in the Mexican population, particularly for those reported with hypertension-diabetes. Factors such as the decrease in the severity of the SARS-CoV2 variants, changes in symptomatology, and advances in the management of patients, vaccination, and treatments influenced the decrease in mortality and hospitalizations.

Dominance of Three Sublineages of the SARS-CoV-2 Delta Variant in Mexico.

In this study, we analyzed the sequences of SARS-CoV-2 isolates of the Delta variant in Mexico, which has completely replaced other previously circulating variants in the country due to its transmission advantage. Among all the Delta sublineages that were detected, 81.5 % were classified as AY.20, AY.26, and AY.100. According to publicly available data, these only reached a world prevalence of less than 1%, suggesting a possible Mexican origin. The signature mutations of these sublineages are described herein, and phylogenetic analyses and haplotype networks are used to track their spread across the country. Other frequently detected sublineages include AY.3, AY.62, AY.103, and AY.113. Over time, the main sublineages showed different geographical distributions, with AY.20 predominant in Central Mexico, AY.26 in the North, and AY.100 in the Northwest and South/Southeast. This work describes the circulation, from May to November 2021, of the primary sublineages of the Delta variant associated with the third wave of the COVID-19 pandemic in Mexico and highlights the importance of SARS-CoV-2 genomic surveillance for the timely identification of emerging variants that may impact public health.

The Alpha Variant (B.1.1.7) of SARS-CoV-2 Failed to Become Dominant in Mexico.

During the coronavirus disease 2019 (COVID-19) pandemic, the emergence and rapid increase of the B.1.1.7 (Alpha) lineage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), first identified in the United Kingdom in September 2020, was well documented in different areas of the world and became a global public health concern because of its increased transmissibility. The B.1.1.7 lineage was first detected in Mexico during December 2020, showing a slow progressive increase in its circulation frequency, which reached its maximum in May 2021 but never became predominant. In this work, we analyzed the patterns of diversity and distribution of this lineage in Mexico using phylogenetic and haplotype network analyses. Despite the reported increase in transmissibility of the B.1.1.7 lineage, in most Mexican states, it did not displace cocirculating lineages, such as B.1.1.519, which dominated the country from February to May 2021. Our results show that the states with the highest prevalence of B.1.1.7 were those at the Mexico-U.S. border. An apparent pattern of dispersion of this lineage from the northern states of Mexico toward the center or the southeast was observed in the largest transmission chains, indicating possible independent introduction events from the United States. However, other entry points cannot be excluded, as shown by multiple introduction events. Local transmission led to a few successful haplotypes with a localized distribution and specific mutations indicating sustained community transmission. IMPORTANCE The emergence and rapid increase of the B.1.1.7 (Alpha) lineage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) throughout the world were due to its increased transmissibility. However, it did not displace cocirculating lineages in most of Mexico, particularly B.1.1.519, which dominated the country from February to May 2021. In this work, we analyzed the distribution of B.1.1.7 in Mexico using phylogenetic and haplotype network analyses. Our results show that the states with the highest prevalence of B.1.1.7 (around 30%) were those at the Mexico-U.S. border, which also exhibited the highest lineage diversity, indicating possible introduction events from the United States. Also, several haplotypes were identified with a localized distribution and specific mutations, indicating that sustained community transmission occurred in the country.

Different rotavirus strains enter MA104 cells through different endocytic pathways: the role of clathrin-mediated endocytosis.

Rotaviruses, the single most important agents of acute severe gastroenteritis in children, are nonenveloped viruses formed by a three-layered capsid that encloses a genome formed by 11 segments of double-stranded RNA. The mechanism of entry of these viruses into the host cell is not well understood. The best-studied strain, RRV, which is sensitive to neuraminidase (NA) treatment of the cells, uses integrins alpha2 beta1 and alphav beta3 and the heat shock protein hsc70 as receptors and enters MA104 cells through a non-clathrin-, non-caveolin-mediated pathway that depends on a functional dynamin and on the presence of cholesterol on the cell surface. In this work, using a combination of pharmacological, biochemical, and genetic approaches, we compared the entry characteristics of four rotavirus strains known to have different receptor requirements. We chose four rotavirus strains that represent all phenotypic combinations of NA resistance or sensitivity and integrin dependence or independence. We found that even though all the strains share their requirements for hsc70, dynamin, and cholesterol, three of them differ from the simian strain RRV in the endocytic pathway used. The human strain Wa, porcine strain TFR-41, and bovine strain UK seem to enter the cell through clathrin-mediated endocytosis, since treatments that inhibit this pathway block their infectivity; consistent with this entry route, these strains were sensitive to changes in the endosomal pH. The inhibition of other endocytic mechanisms, such as macropinocytosis or caveola-mediated uptake, had no effect on the internalization of the rotavirus strains tested here.

The gut virome of healthy children during the first year of life is diverse and dynamic.

In this work, we determined the diversity and dynamics of the gut virome of infants during the first year of life. Fecal samples were collected monthly, from birth to one year of age, from three healthy children living in a semi-rural village in Mexico. Most of the viral reads were classified into six families of bacteriophages including five dsDNA virus families of the order Caudovirales, with Siphoviridae and Podoviridae being the most abundant. Eukaryotic viruses were detected as early as two weeks after birth and remained present all along the first year of life. Thirty-four different eukaryotic virus families were found, where eight of these families accounted for 98% of all eukaryotic viral reads: Anelloviridae, Astroviridae, Caliciviridae, Genomoviridae, Parvoviridae, Picornaviridae, Reoviridae and the plant-infecting viruses of the Virgaviridae family. Some viruses in these families are known human pathogens, and it is surprising that they were found during the first year of life in infants without gastrointestinal symptoms. The eukaryotic virus species richness found in this work was higher than that observed in previous studies; on average between 7 and 24 virus species were identified per sample. The richness and abundance of the eukaryotic virome significantly increased during the second semester of life, probably because of an increased environmental exposure of infants with age. Our findings suggest an early and permanent contact of infants with a diverse array of bacteriophages and eukaryotic viruses, whose composition changes over time. The bacteriophages and eukaryotic viruses found in these children could represent a metastable virome, whose potential influence on the development of the infant's immune system or on the health of the infants later in life, remains to be investigated.

Genetic Analysis of SARS-CoV-2 Variants in Mexico during the First Year of the COVID-19 Pandemic.

During the first year of the SARS-CoV-2 pandemic in Mexico, more than two million people were infected. In this study, we analyzed full genome sequences from 27 February 2020 to 28 February 2021 to characterize the geographical and temporal distribution of SARS-CoV-2 lineages and identify the most common circulating lineages during this period. We defined six different geographical regions with particular dynamics of lineage circulation. The Northeast and Northwest regions were the ones that exhibited the highest lineage diversity, while the Central south and South/Southeast regions presented less diversity with predominance of a certain lineage. Additionally, by late February 2021, lineage B.1.1.519 represented more than 89% of all circulating lineages in the country.

Characterization of viroplasm formation during the early stages of rotavirus infection.

BACKGROUND: During rotavirus replication cycle, electron-dense cytoplasmic inclusions named viroplasms are formed, and two non-structural proteins, NSP2 and NSP5, have been shown to localize in these membrane-free structures. In these inclusions, replication of dsRNA and packaging of pre-virion particles occur. Despite the importance of viroplasms in the replication cycle of rotavirus, the information regarding their formation, and the possible sites of their nucleation during the early stages of infection is scarce. Here, we analyzed the formation of viroplasms after infection of MA104 cells with the rotavirus strain RRV, using different multiplicities of infection (MOI), and different times post-infection. The possibility that viroplasms formation is nucleated by the entering viral particles was investigated using fluorescently labeled purified rotavirus particles. RESULTS: The immunofluorescent detection of viroplasms, using antibodies specific to NSP2 showed that both the number and size of viroplasms increased during infection, and depend on the MOI used. Small-size viroplasms predominated independently of the MOI or time post-infection, although at MOI's of 2.5 and 10 the proportion of larger viroplasms increased. Purified RRV particles were successfully labeled with the Cy5 mono reactive dye, without decrease in virus infectivity, and the labeled viruses were clearly observed by confocal microscope. PAGE gel analysis showed that most viral proteins were labeled; including the intermediate capsid protein VP6. Only 2 out of 117 Cy5-labeled virus particles colocalized with newly formed viroplasms at 4 hours post-infection. CONCLUSIONS: The results presented in this work suggest that during rotavirus infection the number and size of viroplasm increases in an MOI-dependent manner. The Cy5 in vitro labeled virus particles were not found to colocalize with newly formed viroplasms, suggesting that they are not involved in viroplasm nucleation.