RESUMEN

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.

Asunto(s)

Herpesviridae , Picornaviridae , Niño , Humanos , Lactante , Eucariontes , Viroma , Heces , Orofaringe , Metagenómica/métodos

RESUMEN

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.

Asunto(s)

COVID-19 , Coinfección , Virus , Humanos , SARS-CoV-2/genética , Coinfección/epidemiología , Virus/genética , ADN Circular , Índice de Severidad de la Enfermedad

RESUMEN

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.

Asunto(s)

COVID-19 , SARS-CoV-2 , COVID-19/epidemiología , Humanos , México/epidemiología , Pandemias , Filogenia , SARS-CoV-2/genética

RESUMEN

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.

Asunto(s)

Infecciones por Astroviridae , Vesículas Extracelulares , Gastroenteritis , Mamastrovirus , Anticuerpos Neutralizantes , Infecciones por Astroviridae/inmunología , Infecciones por Astroviridae/virología , Vesículas Extracelulares/virología , Gastroenteritis/virología , Humanos , Mamastrovirus/inmunología

RESUMEN

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.

Asunto(s)

COVID-19 , SARS-CoV-2 , COVID-19/epidemiología , Humanos , México/epidemiología , Pandemias , ARN Viral/genética , Ríos , SARS-CoV-2/genética , Aguas del Alcantarillado , Aguas Residuales , Agua

RESUMEN

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.

Asunto(s)

COVID-19 , SARS-CoV-2 , COVID-19/epidemiología , Genoma Viral , Humanos , México/epidemiología , Filogenia , SARS-CoV-2/genética

RESUMEN

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.

Asunto(s)

Infecciones por Caliciviridae/epidemiología , Caliciviridae/genética , Caliciviridae/metabolismo , Metagenómica , Caliciviridae/clasificación , Heces , Femenino , Gastroenteritis , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Incidencia , Lactante , Estudios Longitudinales , Masculino , Metagenoma , Epidemiología Molecular , Norovirus/genética , Prevalencia , Sapovirus/genética

RESUMEN

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.

Asunto(s)

COVID-19/epidemiología , Genoma Viral , SARS-CoV-2/genética , Adulto , Factores de Edad , Atención Ambulatoria/estadística & datos numéricos , COVID-19/complicaciones , COVID-19/mortalidad , Análisis por Conglomerados , Femenino , Genotipo , Hospitalización/estadística & datos numéricos , Humanos , Masculino , México/epidemiología , Persona de Mediana Edad , Mutación , Fenotipo , Filogenia , Cobertura de Afecciones Preexistentes/estadística & datos numéricos , SARS-CoV-2/clasificación , SARS-CoV-2/aislamiento & purificación , Adulto Joven

RESUMEN

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.

Asunto(s)

COVID-19 , Diabetes Mellitus , Hipertensión , Masculino , Humanos , Anciano , Recién Nacido , Lactante , Preescolar , Niño , Adolescente , COVID-19/epidemiología , SARS-CoV-2 , Pandemias , Estudios Longitudinales , México/epidemiología , Estudios de Seguimiento , ARN Viral , Diabetes Mellitus/epidemiología , Hipertensión/epidemiología

RESUMEN

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.

Asunto(s)

COVID-19/virología , Variación Genética , SARS-CoV-2/genética , COVID-19/epidemiología , Evolución Molecular , Pruebas Genéticas , Genoma Viral , Humanos , México/epidemiología , Filogenia , SARS-CoV-2/clasificación , Secuenciación Completa del Genoma

RESUMEN

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.

Asunto(s)

COVID-19/epidemiología , COVID-19/virología , SARS-CoV-2/genética , COVID-19/transmisión , Genoma Viral/genética , Humanos , México/epidemiología , Mutación , Filogenia , Prevalencia , SARS-CoV-2/clasificación , SARS-CoV-2/aislamiento & purificación , Glicoproteína de la Espiga del Coronavirus/genética

RESUMEN

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.

Asunto(s)

Virus ADN/aislamiento & purificación , Tracto Gastrointestinal/virología , Viroma/genética , Bacteriófagos/genética , Bacteriófagos/aislamiento & purificación , Virus ADN/genética , Heces/virología , Enfermedades Gastrointestinales/virología , Humanos , Lactante , Recién Nacido , México

RESUMEN

Plant viruses have been reported to be common in the gut of human adults, presumably as result of food ingestion. In this work, we report that plant viruses can also be found frequently in the gut and oropharynx of children during their first year of life, even when they are exclusively breast-fed. Fecal and oropharynx samples were collected monthly, from birth to 1 year of age, from three apparently healthy children in a semi-rural community and analyzed by next generation sequencing. In 100% of the fecal samples and 65% of the oropharynx samples at least one plant virus was identified. Tobamoviruses in the Virgaviridae family were by far the most frequently detected, with tropical soda apple mosaic virus, pepper mild mottle virus, and opuntia tobamovirus 2 being the most common species. Seventeen complete virus genomes could be assembled, and phylogenetic analyses showed a large diversity of virus strains circulating in the population. These results suggest that children are continuously exposed to an extensive and highly diverse collection of tobamoviruses. Whether the common presence of plant viruses at an early age influences the infant's immune system, either directly or through interaction with other members of the microbiota, remains to be investigated.

Asunto(s)

Tracto Gastrointestinal/virología , Orofaringe/virología , Virus de Plantas/aislamiento & purificación , Tobamovirus/aislamiento & purificación , Heces/virología , Femenino , Variación Genética , Genoma Viral/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Lactante , Recién Nacido , Masculino , Filogenia , Virus de Plantas/clasificación , Virus de Plantas/genética , Tobamovirus/clasificación , Tobamovirus/genética

RESUMEN

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.

Asunto(s)

Betacoronavirus/genética , Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/virología , Variación Genética , Genoma Viral , Genómica , Neumonía Viral/epidemiología , Neumonía Viral/virología , Sustitución de Aminoácidos , Betacoronavirus/clasificación , COVID-19 , Biología Computacional/métodos , Infecciones por Coronavirus/transmisión , Genómica/métodos , Humanos , México/epidemiología , Mutación , Pandemias , Filogenia , Neumonía Viral/transmisión , SARS-CoV-2

RESUMEN

Rotaviruses are the leading cause of viral gastroenteritis among children under five years of age. Rotavirus cell entry has been extensively studied; however, rotavirus cell release is still poorly understood. Specifically, the mechanism by which rotaviruses leave the cell before cell lysis is not known. Previous works have found rotavirus proteins and viral particles associated with extracellular vesicles secreted by cells. These vesicles have been shown to contain markers of exosomes; however, in a recent work they presented characteristics more typical of microparticles, and they were associated with an increase in the infectivity of the virus. In this work, we purified different types of vesicles from rotavirus-infected cells. We analyzed the association of virus with these vesicles and their possible role in promotion of rotavirus infection. We confirmed a non-lytic rotavirus release from the two cell lines tested, and observed a notable stimulation of vesicle secretion following rotavirus infection. A fraction of the secreted viral particles present in the cell supernatant was protected from protease treatment, possibly through its association with membranous vesicles; the more pronounced association of the virus was with fractions corresponding to cell membrane generated microvesicles. Using electron microscopy, we found different size vesicles with particles resembling rotaviruses associated from both- the outside and the inside. The viral particles inside the vesicles were refractory to neutralization with a potent rotavirus neutralizing monoclonal antibody, and were able to infect cells even without trypsin activation. The association of rotavirus particles with extracellular vesicles suggests these might have a role in virus spread.

Asunto(s)

Vesículas Extracelulares/virología , Infecciones por Rotavirus/metabolismo , Rotavirus/metabolismo , Células CACO-2/virología , Vesículas Extracelulares/ultraestructura , Humanos , Microscopía Electrónica de Transmisión , Rotavirus/ultraestructura , Virión/metabolismo , Liberación del Virus

RESUMEN

We report two full-genome sequences of vesicular stomatitis New Jersey virus (VSNJV) obtained by Illumina next-generation sequencing of RNA isolated from epithelial suspensions of cattle naturally infected in Mexico. These genomes represent the first full-genome sequences of vesicular stomatitis New Jersey viruses circulating in Mexico deposited in the GenBank database.

RESUMEN

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.

Asunto(s)

Gastroenteritis/diagnóstico , Gastroenteritis/virología , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Virosis/diagnóstico , Virosis/virología , Virus/clasificación , Virus/genética , Preescolar , Gastroenteritis/epidemiología , Humanos , Lactante , Recién Nacido , Análisis de Secuencia por Matrices de Oligonucleótidos/normas , Prevalencia , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Virosis/epidemiología

RESUMEN

Viruses are the most frequent cause of respiratory disease in children. However, despite the advanced diagnostic methods currently in use, in 20 to 50% of respiratory samples a specific pathogen cannot be detected. In this work, we used a metagenomic approach and deep sequencing to examine respiratory samples from children with lower and upper respiratory tract infections that had been previously found negative for 6 bacteria and 15 respiratory viruses by PCR. Nasal washings from 25 children (out of 250) hospitalized with a diagnosis of pneumonia and nasopharyngeal swabs from 46 outpatient children (out of 526) were studied. DNA reads for at least one virus commonly associated to respiratory infections was found in 20 of 25 hospitalized patients, while reads for pathogenic respiratory bacteria were detected in the remaining 5 children. For outpatients, all the samples were pooled into 25 DNA libraries for sequencing. In this case, in 22 of the 25 sequenced libraries at least one respiratory virus was identified, while in all other, but one, pathogenic bacteria were detected. In both patient groups reads for respiratory syncytial virus, coronavirus-OC43, and rhinovirus were identified. In addition, viruses less frequently associated to respiratory infections were also found. Saffold virus was detected in outpatient but not in hospitalized children. Anellovirus, rotavirus, and astrovirus, as well as several animal and plant viruses were detected in both groups. No novel viruses were identified. Adding up the deep sequencing results to the PCR data, 79.2% of 250 hospitalized and 76.6% of 526 ambulatory patients were positive for viruses, and all other children, but one, had pathogenic respiratory bacteria identified. These results suggest that at least in the type of populations studied and with the sampling methods used the odds of finding novel, clinically relevant viruses, in pediatric respiratory infections are low.

Asunto(s)

Virus ADN/fisiología , Virus ARN/fisiología , Infecciones del Sistema Respiratorio/virología , Niño , Niño Hospitalizado , Preescolar , Coronavirus/genética , Coronavirus/fisiología , Virus ADN/clasificación , Virus ADN/genética , ADN Viral/análisis , Femenino , Humanos , Lactante , Masculino , Nasofaringe/virología , Filogenia , Neumonía/diagnóstico , Neumonía/virología , Virus ARN/clasificación , Virus ARN/genética , ARN Viral/análisis , Virus Sincitiales Respiratorios/genética , Virus Sincitiales Respiratorios/fisiología , Infecciones del Sistema Respiratorio/patología , Rhinovirus/genética , Rhinovirus/fisiología , Análisis de Secuencia de ADN , Análisis de Secuencia de ARN

RESUMEN

In this work, nineteen influenza A/H3N2 viruses isolated in Mexico between 2003 and 2012 were studied. Our findings show that different human A/H3N2 viral lineages co-circulate within a same season and can also persist locally in between different influenza seasons, increasing the chance for genetic reassortment events. A novel minor cluster was also identified, named here as Korea, that circulated worldwide during 2003. Frequently, phylogenetic characterization did not correlate with the determined antigenic identity, supporting the need for the use of molecular evolutionary tools additionally to antigenic data for the surveillance and characterization of viral diversity during each flu season. This work represents the first long-term molecular epidemiology study of influenza A/H3N2 viruses in Mexico based on the complete genomic sequences and contributes to the monitoring of evolutionary trends of A/H3N2 influenza viruses within North and Central America.

Asunto(s)

Brotes de Enfermedades , Subtipo H3N2 del Virus de la Influenza A/genética , Gripe Humana/epidemiología , Polimorfismo Genético , Genoma Viral , Humanos , Subtipo H3N2 del Virus de la Influenza A/aislamiento & purificación , México , Filogenia

RESUMEN

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.

Asunto(s)

Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Gripe Humana/mortalidad , Gripe Humana/patología , Índice de Severidad de la Enfermedad , Factores de Virulencia/genética , Adulto , Animales , Secuencia de Bases , Peso Corporal , Modelos Animales de Enfermedad , Femenino , Histocitoquímica , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/aislamiento & purificación , Gripe Humana/epidemiología , Gripe Humana/virología , Pulmón/patología , Masculino , México/epidemiología , Ratones , Persona de Mediana Edad , Datos de Secuencia Molecular , Mutación Missense , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/virología , ARN Viral/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Análisis de Supervivencia