RESUMEN
The zoonotic parasite Cryptosporidium parvum is a global cause of gastrointestinal disease in humans and ruminants. Sequence analysis of the highly polymorphic gp60 gene enabled the classification of C. parvum isolates into multiple groups (e.g., IIa, IIc, Id) and a large number of subtypes. In Europe, subtype IIaA15G2R1 is largely predominant and has been associated with many water- and food-borne outbreaks. In this study, we generated new whole-genome sequence (WGS) data from 123 human- and ruminant-derived isolates collected in 13 European countries and included other available WGS data from Europe, Egypt, China, and the United States (n = 72) in the largest comparative genomics study to date. We applied rigorous filters to exclude mixed infections and analyzed a data set from 141 isolates from the zoonotic groups IIa (n = 119) and IId (n = 22). Based on 28,047 high-quality, biallelic genomic SNPs, we identified three distinct and strongly supported populations: Isolates from China (IId) and Egypt (IIa and IId) formed population 1; a minority of European isolates (IIa and IId) formed population 2; and the majority of European (IIa, including all IIaA15G2R1 isolates) and all isolates from the United States (IIa) clustered in population 3. Based on analyses of the population structure, population genetics, and recombination, we show that population 3 has recently emerged and expanded throughout Europe to then, possibly from the United Kingdom, reach the United States, where it also expanded. The reason(s) for the successful spread of population 3 remain elusive, although genes under selective pressure uniquely in this population were identified.
Asunto(s)
Criptosporidiosis , Cryptosporidium parvum , Brotes de Enfermedades , Cryptosporidium parvum/genética , Estados Unidos/epidemiología , Europa (Continente)/epidemiología , Humanos , Criptosporidiosis/parasitología , Criptosporidiosis/epidemiología , Animales , Genómica/métodos , Polimorfismo de Nucleótido Simple , Filogenia , Secuenciación Completa del Genoma/métodos , Genoma de Protozoos , China/epidemiología , Egipto/epidemiologíaRESUMEN
Influenza D virus was isolated from pigs on a mixed pig and beef farm in France. Investigation suggested bull-to-pig transmission and spread among pigs. The swine influenza D virus recovered was a reassortant of D/660 and D/OK lineages. Reported mutations in the receptor binding site might be related to swine host adaptation.
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Granjas , Infecciones por Orthomyxoviridae , Filogenia , Virus Reordenados , Enfermedades de los Porcinos , Thogotovirus , Animales , Porcinos , Virus Reordenados/genética , Francia/epidemiología , Infecciones por Orthomyxoviridae/virología , Infecciones por Orthomyxoviridae/veterinaria , Enfermedades de los Porcinos/virología , Bovinos , Thogotovirus/genética , Thogotovirus/clasificación , Thogotovirus/aislamiento & purificación , DeltainfluenzavirusRESUMEN
Cryptosporidium spp. remain a major cause of waterborne diarrhea and illness in developing countries and represent a significant burden to farmers worldwide. Cryptosporidium parvum virus 1 (CSpV1), of the genus Cryspovirus, was first reported to be present in the cytoplasm of C. parvum in 1997. Full-length genome sequences have been obtained from C. parvum from Iowa (Iowa), Kansas (KSU) and China. We aimed at characterizing the genome of CSpV1 from France and used sequence analysis from Cryptosporidium isolates to explore whether CSpV1 genome diversity varies over time, with geographical sampling location, C. parvum genetic diversity, or ruminant host species. A total of 123 fecal samples of cattle, sheep and goats were collected from 17 different French departments (57 diseased animal fecal samples and 66 healthy animal fecal samples). Subtyping analysis of the C. parvum isolates revealed the presence of two zoonotic subtype families IIa and IId. Sequence analysis of CSpV1 revealed that all CSpV1 from France, regardless of the subtype of C. parvum (IIaA15G2R1, IIaA17G2R1 and IIdA18G1R1) are more closely related to CSpV1 from Turkey, and cluster on a distinct branch from CSpV1 collected from C. parvum subtype IIaA15G2R1 from Asia and North America. We also found that samples collected on a given year or successive years in a given location are more likely to host the same subtype of C. parvum and the same CSpV1 strain. Yet, there is no distinct clustering of CSpV1 per French department or ruminants, probably due to trade, and transmission of C. parvum among host species. Our results point towards (i) a close association between CSpV1 movement and C. parvum movement, (ii) recent migrations of C. parvum among distantly located departments and (iii) incidental transmission of C. parvum between ruminants. All together, these results provide insightful information regarding CSpV1 evolution and suggest the virus might be used as an epidemiological tracer for C. parvum. Future studies need to investigate CSpV1's role in C. parvum virulence and on subtype ability to infect different species.
Asunto(s)
Enfermedades de los Bovinos , Criptosporidiosis , Cryptosporidium parvum , Cryptosporidium , Enfermedades de las Cabras , Enfermedades de las Ovejas , Ovinos , Animales , Bovinos , Cabras , Cryptosporidium parvum/genética , Criptosporidiosis/epidemiología , Francia/epidemiología , Enfermedades de los Bovinos/epidemiología , Enfermedades de las Cabras/epidemiología , Enfermedades de las Ovejas/epidemiologíaRESUMEN
Currently, turkey coronaviruses (TCoV) are isolated from homogenized intestines of experimentally infected embryos to ensure a maximum recovery of viral particles from all components of the intestines. However, the process of homogenization also ensures release of a significant amount of cellular RNAs into the sample that hinders downstream viral genome sequencing. This is especially the case for next generation sequencing (NGS) which sequences molecules at random. This characteristic means that the heavily abundant cellular RNA in the sample drowns out the minority viral RNA during the sequencing process and, consequently, very little to no viral genome data are obtained. To address this problem, a method was developed, in which 10 descendent isolates of the European strain of TCoV were recovered uniquely from the intestinal lumen without homogenization of the tissue. For nine out of 10 samples, NGS produced viral RNA reads with good coverage depth over the entire TCoV genomes. This is a much-needed new, simple and cost effective method of isolating TCoV that facilitates downstream NGS of viral RNA and should be considered as an alternative method for isolating other avian enteric coronaviruses in the interest of obtaining full-length genome sequences.
Asunto(s)
Coronavirus del Pavo , Enfermedades de las Aves de Corral , Animales , Coronavirus del Pavo/genética , Genoma Viral , Secuenciación de Nucleótidos de Alto Rendimiento/veterinaria , Intestinos , ARN Viral/genética , PavosRESUMEN
Gilthead seabream represents a species of importance in Mediterranean aquaculture. The larval stage is particularly sensitive and frequently impacted in suboptimal environmental or sanitary conditions. In the present study, investigations were carried out in a seabream hatchery following an unusual mortality reaching 70% among 50-day post-hatching. Anorexia, loss of appetite and abnormal swimming behaviour were observed in absence of parasites or pathogenic bacteria. Proliferation of rod-shaped bacteria in the gut lumen was associated with focal degeneration in the intestinal mucosa. Cytopathic effects on an EK-1 cell line after 21 days of culture at 14°C and 20°C in contact with homogenized affected larvae revealed the presence of a viral agent. Molecular characterization by high-throughput sequencing showed a typical picornavirus genome organization with a polyprotein precursor of 2276 amino acids sharing 46.3% identity with that of the Eel Picornavirus-1. A specific real-time PCR confirmed the presence of the viral genome in affected larval homogenate and corresponding cell culture supernatant. We propose the name Potamipivirus daurada for this novel species within the genus Potamipivirus. The etiological role of this virus remains uncertain at this time, and future studies will be necessary to investigate its prevalence in natural and aquaculture-reared populations as well as its ability to cause diseases in gilthead seabream.
Asunto(s)
Enfermedades de los Peces , Dorada , Animales , Virus ADN/genética , Larva , Reacción en Cadena en Tiempo Real de la Polimerasa , Dorada/genéticaRESUMEN
We detected 3 genotypes of highly pathogenic avian influenza A(H5N8) virus in France during winter 2016-17. Genotype A viruses caused dramatic economic losses in the domestic duck farm industry in southwestern France. Our phylogenetic analysis suggests that genotype A viruses formed 5 distinct geographic clusters in southwestern France. In some clusters, local secondary transmission might have been started by a single introduction. The intensity of the viral spread seems to correspond to the density of duck holdings in each production area. To avoid the introduction of disease into an unaffected area, it is crucial that authorities limit the movements of potentially infected birds.
Asunto(s)
Subtipo H5N8 del Virus de la Influenza A , Gripe Aviar , Animales , Animales Salvajes , Aves , Brotes de Enfermedades , Francia/epidemiología , Subtipo H5N8 del Virus de la Influenza A/genética , Gripe Aviar/epidemiología , FilogeniaRESUMEN
Guinea fowl fulminating enteritis has been reported in France since the 1970s. In 2014, a coronavirus was identified and appeared as a possible viral pathogen involved in the disease. In the present study, intestinal content from a guinea fowl involved in a new case of the disease in 2017 was analysed by deep sequencing, revealing the presence of a guinea fowl coronavirus (GfCoV) and a picornavirus (GfPic). Serial passage assays into the intra-amniotic cavity of 13-day-old specific pathogen-free chicken eggs and 20-day-old conventional guinea fowl eggs were attempted. In chicken eggs, isolation assays failed, but in guinea fowl eggs, both viruses were successfully obtained. Furthermore, two GfCoV and two GfPic isolates were obtained from the same bird but from different sections of its intestines. This shows that using eggs of the same species, in which the virus has been detected, can be the key for successful isolation. The consensus sequence of the full-length genomes of both GfCoV isolates was highly similar, and correlated to those previously described in terms of genome organization, ORF length and phylogenetic clustering. According to full-length genome analysis and the structure of the Internal Ribosome Entry Site, both GfPic isolates belong to the Anativirus genus and specifically the species Anativirus B. The availability of the first isolates of GfCoV and GfPic will now provide a means of assessing their pathogenicity in guinea fowl in controlled experimental conditions and to assess whether they are primary viral pathogens of the disease "guinea fowl fulminating enteritis".RESEARCH HIGHLIGHTSFirst isolation of guinea fowl coronaviruses and picornaviruses.Eggs homologous to the infected species are key for isolation.Isolates available to precisely evaluate the virus roles in fulminating enteritis.First full-length genome sequences of guinea fowl picornaviruses.
Asunto(s)
Coronavirus/clasificación , Enteritis/virología , Galliformes/virología , Picornaviridae/clasificación , Animales , Coronavirus/aislamiento & purificación , Infecciones por Coronavirus/veterinaria , Infecciones por Coronavirus/virología , Enteritis/veterinaria , Genoma Viral , Filogenia , Picornaviridae/aislamiento & purificación , Infecciones por Picornaviridae/veterinaria , Infecciones por Picornaviridae/virología , Enfermedades de las Aves de Corral/virologíaRESUMEN
BACKGROUND: Rabbit haemorrhagic disease virus Lagovirus europaeus/GI.1d variant (GI.1d/RHDV) was identified in 1990 in France, and until the emergence of the new genotype GI.2, it was the main variant circulating in the country. The early stages of RHDV infection have been described in a few studies of rabbits experimentally infected with earlier strains, but no information was given on the minimum infective dose. We report the genomic and phenotypic characterisation of a GI.1d/RHDV strain collected in 2000 in France (GI.1d/00-21). RESULTS: We performed in vivo assays in rabbits to study virus replication kinetics in several tissues at the early stage of infection, and to estimate the minimum infective dose. Four tested doses, negligible (10- 1 viral genome copies), low (104), high (107) and very high (1011) were quantified using a method combining density gradient centrifugation of the viral particles and an RT-qPCR technique developed to quantify genomic RNA (gRNA). The GI.1d/00-21 genome showed the same genomic organisation as other lagoviruses; however, a substitution in the 5' untranslated region and a change in the potential p23/2C-like helicase cleavage site were observed. We showed that the liver of one of the two rabbits inoculated via the oral route was infected at 16 h post-infection and all tissues at 39 h post-infection. GI.1d/00-21 induced classical RHD signs (depression) and lesions (haemorrhage and splenomegaly). Although infective dose estimation should be interpreted with caution, the minimum infective dose that infected an inoculated rabbit was lower or equal to 104 gRNA copies, whereas between 104 and 107 gRNA copies were required to also induce mortality. CONCLUSIONS: These results provide a better understanding of GI.1d/RHDV infection in rabbits. The genome analysis showed a newly observed mutation in the 5' untranslated region of a lagovirus, whose role remains unknown. The phenotypic analysis showed that the pathogenicity of GI.1d/00-21 and the replication kinetics in infected organs were close to those reported for the original GI.1 strains, and could not alone explain the observed selective advantage of the GI.1d strains. Determining the minimum dose of viral particles required to cause mortality in rabbits is an important input for in vivo studies.
Asunto(s)
Infecciones por Caliciviridae/veterinaria , Variación Genética , Genoma Viral , Virus de la Enfermedad Hemorrágica del Conejo/genética , Conejos/virología , Replicación Viral , Animales , Infecciones por Caliciviridae/epidemiología , Infecciones por Caliciviridae/virología , Francia/epidemiología , Filogenia , ARN Viral/genéticaRESUMEN
In 2018, a veterinarian became sick shortly after swabbing sows exhibiting respiratory syndrome on a farm in France. Epidemiologic data and genetic analyses revealed consecutive human-to-swine and swine-to-human influenza A(H1N1)pdm09 virus transmission, which occurred despite some biosecurity measures. Providing pig industry workers the annual influenza vaccine might reduce transmission risk.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A , Gripe Humana/transmisión , Infecciones por Orthomyxoviridae/transmisión , Enfermedades de los Porcinos/transmisión , Zoonosis/transmisión , Animales , Brotes de Enfermedades/estadística & datos numéricos , Brotes de Enfermedades/veterinaria , Femenino , Francia/epidemiología , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Gripe Humana/epidemiología , Infecciones por Orthomyxoviridae/epidemiología , Filogenia , Porcinos , Enfermedades de los Porcinos/epidemiología , Zoonosis/epidemiología , Zoonosis/virologíaRESUMEN
Quality management and independent assessment of high-throughput sequencing-based virus diagnostics have not yet been established as a mandatory approach for ensuring comparable results. The sensitivity and specificity of viral high-throughput sequence data analysis are highly affected by bioinformatics processing using publicly available and custom tools and databases and thus differ widely between individuals and institutions. Here we present the results of the COMPARE [Collaborative Management Platform for Detection and Analyses of (Re-)emerging and Foodborne Outbreaks in Europe] in silico virus proficiency test. An artificial, simulated in silico data set of Illumina HiSeq sequences was provided to 13 different European institutes for bioinformatics analysis to identify viral pathogens in high-throughput sequence data. Comparison of the participants' analyses shows that the use of different tools, programs, and databases for bioinformatics analyses can impact the correct identification of viral sequences from a simple data set. The identification of slightly mutated and highly divergent virus genomes has been shown to be most challenging. Furthermore, the interpretation of the results, together with a fictitious case report, by the participants showed that in addition to the bioinformatics analysis, the virological evaluation of the results can be important in clinical settings. External quality assessment and proficiency testing should become an important part of validating high-throughput sequencing-based virus diagnostics and could improve the harmonization, comparability, and reproducibility of results. There is a need for the establishment of international proficiency testing, like that established for conventional laboratory tests such as PCR, for bioinformatics pipelines and the interpretation of such results.
Asunto(s)
Biología Computacional/métodos , Simulación por Computador , Secuenciación de Nucleótidos de Alto Rendimiento/normas , Ensayos de Aptitud de Laboratorios/estadística & datos numéricos , Análisis de Secuencia de ADN/normas , Virus/genética , Análisis de Datos , Europa (Continente) , Genoma Viral , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Colaboración Intersectorial , Ensayos de Aptitud de Laboratorios/organización & administración , Reproducibilidad de los Resultados , Análisis de Secuencia de ADN/estadística & datos numéricos , Virus/patogenicidadRESUMEN
The H1N1 influenza virus responsible for the most recent pandemic in 2009 (H1N1pdm) has spread to swine populations worldwide while it replaced the previous seasonal H1N1 virus in humans. In France, surveillance of swine influenza A viruses in pig herds with respiratory outbreaks led to the detection of 44 H1N1pdm strains between 2009 and 2017, regardless of the season, and findings were not correlated with pig density. From these isolates, 17 whole-genome sequences were obtained, as were 6 additional hemagglutinin (HA)/neuraminidase (NA) sequences, in order to perform spatial and temporal analyses of genetic diversity and to compare evolutionary patterns of H1N1pdm in pigs to patterns for human strains. Following mutation accumulation and fixation over time, phylogenetic analyses revealed for the first time the divergence of a swine-specific genogroup within the H1N1pdm lineage. The divergence is thought to have occurred around 2011, although this was demonstrated only through strains isolated in 2015 to 2016 in the southern half of France. To date, these H1N1pdm swine strains have not been related to any increased virulence in swine herds and have not exhibited any antigenic drift compared to seasonal human strains. However, further monitoring is encouraged, as diverging evolutionary patterns in these two species, i.e., swine and humans, may lead to the emergence of viruses with a potentially higher risk to both animal and human health.IMPORTANCE Pigs are a "mixing vessel" for influenza A viruses (IAVs) because of their ability to be infected by avian and human IAVs and their propensity to facilitate viral genomic reassortment events. Also, as IAVs may evolve differently in swine and humans, pigs can become a reservoir for old human strains against which the human population has become immunologically naive. Thus, viruses from the novel swine-specific H1N1pdm genogroup may continue to diverge from seasonal H1N1pdm strains and/or from other H1N1pdm viruses infecting pigs and lead to the emergence of viruses that would not be covered by human vaccines and/or swine vaccines based on antigens closely related to the original H1N1pdm virus. This discovery confirms the importance of encouraging swine IAV monitoring because H1N1pdm swine viruses could carry an increased risk to both human and swine health in the future as a whole H1N1pdm virus or gene provider in subsequent reassortant viruses.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/clasificación , Infecciones por Orthomyxoviridae/epidemiología , Enfermedades de los Porcinos/virología , Secuenciación Completa del Genoma/métodos , Animales , Evolución Molecular , Francia/epidemiología , Hemaglutininas/genética , Subtipo H1N1 del Virus de la Influenza A/genética , Neuraminidasa/genética , Infecciones por Orthomyxoviridae/virología , Pandemias , Filogenia , Vigilancia de la Población , Análisis Espacio-Temporal , Porcinos , Enfermedades de los Porcinos/epidemiología , Proteínas Virales/genética , Secuenciación Completa del Genoma/veterinariaRESUMEN
BACKGROUND: Next generation sequencing (NGS) is becoming widely used among diagnostics and research laboratories, and nowadays it is applied to a variety of disciplines, including veterinary virology. The NGS workflow comprises several steps, namely sample processing, library preparation, sequencing and primary/secondary/tertiary bioinformatics (BI) analyses. The latter is constituted by a complex process extremely difficult to standardize, due to the variety of tools and metrics available. Thus, it is of the utmost importance to assess the comparability of results obtained through different methods and in different laboratories. To achieve this goal, we have organized a proficiency test focused on the bioinformatics components for the generation of complete genome sequences of salmonid rhabdoviruses. METHODS: Three partners, that performed virus sequencing using different commercial library preparation kits and NGS platforms, gathered together and shared with each other 75 raw datasets which were analyzed separately by the participants to produce a consensus sequence according to their own bioinformatics pipeline. Results were then compared to highlight discrepancies, and a subset of inconsistencies were investigated more in detail. RESULTS: In total, we observed 526 discrepancies, of which 39.5% were located at genome termini, 14.1% at intergenic regions and 46.4% at coding regions. Among these, 10 SNPs and 99 indels caused changes in the protein products. Overall reproducibility was 99.94%. Based on the analysis of a subset of inconsistencies investigated more in-depth, manual curation appeared the most critical step affecting sequence comparability, suggesting that the harmonization of this phase is crucial to obtain comparable results. The analysis of a calibrator sample allowed assessing BI accuracy, being 99.983%. CONCLUSIONS: We demonstrated the applicability and the usefulness of BI proficiency testing to assure the quality of NGS data, and recommend a wider implementation of such exercises to guarantee sequence data uniformity among different virology laboratories.
Asunto(s)
Biología Computacional/métodos , Biología Computacional/normas , Secuenciación de Nucleótidos de Alto Rendimiento/normas , Virus de la Necrosis Hematopoyética Infecciosa/genética , Novirhabdovirus/genética , Análisis de Secuencia de ADN/normas , Animales , Peces , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Control de Calidad , Reproducibilidad de los Resultados , Análisis de Secuencia de ADN/métodosRESUMEN
This report describes the detection of a triple reassortant swine influenza A virus of H1avN2 subtype. It evolved from an avian-like swine H1avN1 that first acquired the N2 segment from a seasonal H3N2, then the M segment from a 2009 pandemic H1N1, in two reassortments estimated to have occurred 10 years apart. This study illustrates how recurrent influenza infections increase the co-infection risk and facilitate evolutionary jumps by successive gene exchanges. It recalls the importance of appropriate biosecurity measures inside holdings to limit virus persistence and interspecies transmissions, which both contribute to the emergence of new potentially zoonotic viruses.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/fisiología , Subtipo H1N2 del Virus de la Influenza A/fisiología , Subtipo H3N2 del Virus de la Influenza A/fisiología , Virus Reordenados/fisiología , Enfermedades de los Porcinos/virología , Animales , Francia , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N2 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/genética , Sus scrofa , PorcinosRESUMEN
Novirhabdoviruses like the Viral hemorrhagic septicemia virus (VHSV) are rhabdoviruses infecting fish. In the current study, RNA genomes of different VHSV field isolates classified as high, medium or low virulent phenotypes have been sequenced by next-generation sequencing and compared. Various amino acid changes, depending on the VHSV phenotype, have been identified in all the VHSV proteins. As a starting point, we focused our study on the non-virion (NV) non-structural protein in which an arginine residue (R116) is present in all the virulent isolates and replaced by a serine/asparagine residue S/N116 in the attenuated isolates. A recombinant virus derived from a virulent VHSV strain in which the NV R116 residue has been replaced by a serine, rVHSVNVR116S, was generated by reverse genetics and used to infect juvenile trout. We showed that rVHSVNVR116S was highly attenuated and that surviving fish were almost completely protected from a challenge with the wild-type VHSV.
Asunto(s)
Sustitución de Aminoácidos , Enfermedades de los Peces/patología , Enfermedades de los Peces/virología , Novirhabdovirus/patogenicidad , Infecciones por Rhabdoviridae/veterinaria , Proteínas Virales/genética , Factores de Virulencia/genética , Animales , Genoma Viral , Novirhabdovirus/genética , Novirhabdovirus/aislamiento & purificación , Fenotipo , Genética Inversa , Infecciones por Rhabdoviridae/patología , Infecciones por Rhabdoviridae/virología , Análisis de Secuencia de ADN , Trucha , VirulenciaRESUMEN
Several new highly pathogenic (HP) H5 avian influenza virus (AIV) have been detected in poultry farms from south-western France since November 2015, among which an HP H5N1. The zoonotic potential and origin of these AIVs immediately became matters of concern. One virus of each subtype H5N1 (150169a), H5N2 (150233) and H5N9 (150236) was characterised. All proved highly pathogenic for poultry as demonstrated molecularly by the presence of a polybasic cleavage site in their HA protein - with a sequence (HQRRKR/GLF) previously unknown among avian H5 HPAI viruses - or experimentally by the in vivo demonstration of an intravenous pathogenicity index of 2.9 for the H5N1 HP isolate. Phylogenetic analyses based on the full genomes obtained by NGS confirmed that the eight viral segments of the three isolates were all part of avian Eurasian phylogenetic lineage but differed from the Gs/Gd/1/96-like lineage. The study of the genetic characteristics at specific amino acid positions relevant for modulating the adaptation to and the virulence for mammals showed that presently, these viruses possess most molecular features characteristic of AIV and lack some major characteristics required for efficient respiratory transmission to or between humans. The three isolates are therefore predicted to have no significant pandemic potential.
Asunto(s)
Subtipo H5N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/patogenicidad , Subtipo H5N2 del Virus de la Influenza A/genética , Subtipo H5N2 del Virus de la Influenza A/patogenicidad , Gripe Aviar/virología , Animales , Aves , Pollos , Brotes de Enfermedades , Patos , Francia/epidemiología , Genes Virales/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Humanos , Subtipo H5N1 del Virus de la Influenza A/clasificación , Subtipo H5N1 del Virus de la Influenza A/aislamiento & purificación , Subtipo H5N2 del Virus de la Influenza A/clasificación , Subtipo H5N2 del Virus de la Influenza A/aislamiento & purificación , Gripe Aviar/epidemiología , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Aves de Corral/virología , Enfermedades de las Aves de Corral/virología , ARN Viral/genética , Análisis de Secuencia de ADNRESUMEN
Porcine endogenous retroviruses (PERVs) are present in the genomes of pig cells. The PERV-A/C recombinant virus can infect human cells and is a major risk of zoonotic disease in the case of xenotransplantation of pig organs to humans. Raltegravir (RAL) is a viral integrase (IN) inhibitor used in highly active antiretroviral treatment. In the present study, we explored the potential use of RAL against PERV-A/C. We report (i) a three-dimensional model of the PERV-A/C intasome complexed with RAL, (ii) the sensitivity of PERV-A/C IN to RAL in vitro and (iii) the sensitivity of a PERV-A/C-IRES-GFP recombinant virus to RAL in cellulo. We demonstrated that RAL is a potent inhibitor against PERV-A/C IN and PERV-A/C replication with IC50s in the nanomolar range. To date, the use of retroviral inhibitors remains the only way to control the risk of zoonotic PERV infection during pig-to-human xenotransplantation.
Asunto(s)
Antivirales/farmacología , Retrovirus Endógenos/enzimología , Retrovirus Endógenos/fisiología , Integrasas/análisis , Raltegravir Potásico/farmacología , Integración Viral/efectos de los fármacos , Animales , Antivirales/química , Cristalografía por Rayos X , Retrovirus Endógenos/efectos de los fármacos , Concentración 50 Inhibidora , Integrasas/química , Unión Proteica , Conformación Proteica , Raltegravir Potásico/química , PorcinosRESUMEN
Metagenomic shotgun sequencing (mNGS) can serve as a generic molecular diagnostic tool. An mNGS proficiency test (PT) was performed in six European veterinary and public health laboratories to detect porcine astroviruses in fecal material and the extracted RNA. While different mNGS workflows for the generation of mNGS data were used in the different laboratories, the bioinformatic analysis was standardized using a metagenomic read classifier as well as read mapping to selected astroviral reference genomes to assess the semiquantitative representation of astrovirus species mixtures. All participants successfully identified and classified most of the viral reads to the two dominant species. The normalized read counts obtained by aligning reads to astrovirus reference genomes by Bowtie2 were in line with Kraken read classification counts. Moreover, participants performed well in terms of repeatability when the fecal sample was tested in duplicate. However, the normalized read counts per detected astrovirus species differed substantially between participants, which was related to the different laboratory methods used for data generation. Further modeling of the mNGS data indicated the importance of selecting appropriate reference data for mNGS read classification. As virus- or sample-specific biases may apply, caution is needed when extrapolating this swine feces-based PT for the detection of other RNA viruses or using different sample types. The suitability of experimental design to a given pathogen/sample matrix combination, quality assurance, interpretation, and follow-up investigation remain critical factors for the diagnostic interpretation of mNGS results. IMPORTANCE: Metagenomic shotgun sequencing (mNGS) is a generic molecular diagnostic method, involving laboratory preparation of samples, sequencing, bioinformatic analysis of millions of short sequences, and interpretation of the results. In this paper, we investigated the performance of mNGS on the detection of porcine astroviruses, a model for RNA viruses in a pig fecal material, among six European veterinary and public health laboratories. We showed that different methods for data generation affect mNGS performance among participants and that the selection of reference genomes is crucial for read classification. Follow-up investigation remains a critical factor for the diagnostic interpretation of mNGS results. The paper contributes to potential improvements of mNGS as a diagnostic tool in clinical settings.
RESUMEN
BACKGROUND: Avian influenza A (AI) viruses of subtypes H5 can cause serious disease outbreaks in poultry including panzootic due to H5N1 highly pathogenic (HP) viruses. These viruses are a threat not only for animal health but also public health due to their zoonotic potential. The domestic duck plays a major role in the epidemiological cycle of influenza virus subtypes H5 but little is known concerning host/pathogen interactions during influenza infection in duck species. In this study, a subtracted library from duck trachea (a primary site of influenza virus infection) was constructed to analyse and compare the host response after a highly or low pathogenic (LP) H5N1-infection. RESULTS: Here, we show that more than 200 different genes were differentially expressed in infected duck trachea to a significant degree. In addition, significant differentially expressed genes between LPAI- and HPAI-infected tracheas were observed. Gene ontology annotation was used and specific signalling pathways were identified. These pathways were different for LPAI and HPAI-infected tracheas, except for the CXCR4 signalling pathway which is implicated in immune response. A different modulation of genes in the CXCR4 signalling pathway and TRIM33 was induced in duck tracheas infected with a HPAI- or a LPAI-H5N1. CONCLUSION: First, this study indicates that Suppressive Subtractive Hybridization (SSH) is an alternative approach to gain insights into the pathogenesis of influenza infection in ducks. Secondly, the results indicate that cellular gene expression in the duck trachea was differently modulated after infection with a LPAI-H5N1 or after infection with a HPAI-H5N1 virus. Such difference found in infected trachea, a primary infection site, could precede continuation of infection and could explain appearance of respiratory symptoms or not.
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Perfilación de la Expresión Génica , Interacciones Huésped-Patógeno , Subtipo H5N1 del Virus de la Influenza A/patogenicidad , Gripe Aviar/patología , Gripe Aviar/virología , Tráquea/patología , Tráquea/virología , Animales , Patos , Hibridación de Ácido Nucleico/métodos , Transducción de Señal/genéticaRESUMEN
The European sea bass Dicentrarchus labrax is the main species reared in Mediterranean aquaculture. Its larval stage, which is very sensitive and highly affected by sanitary and environmental conditions, is particularly scrutinized in hatcheries. Recently, a Mediterranean sea bass farm had to deal with an abnormal increase in mortality, especially between 20 and 35 days post-hatching (dph). Biological investigations led to the observation of cytopathic effects on three different fish cell lines after almost 3 weeks of culture at 14 °C in contact with homogenized affected larvae, suggesting the presence of a viral agent. High-throughput sequencing revealed a 6818-nucleotide-long RNA genome with six putative ORFs, corresponding to the organization of viruses belonging to the Totiviridae family. This genome clustered with the newly described and suggested Pistolvirus genus, sharing 45.5% to 37.2% nucleotide identity with other piscine toti-like viruses such as Cyclopterus lumpus toti-like virus (CLuTLV) or piscine myocarditis virus (PMCV), respectively. Therefore, we propose to name this new viral agent sea bass toti-like virus (SBTLV). Specific real-time RT-PCR confirmed the presence of the viral genome in the affected larval homogenate from different production batches and the corresponding cell culture supernatant. Experimental infections performed on sea bass fingerlings did not induce mortality, although the virus could be detected in various organs and a specific immune response was developed. Additional studies are needed to understand the exact involvement of this virus in the mortality observed in hatcheries and the potential associated cofactors.
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Lubina , Enfermedades de los Peces , Virus , Animales , Lubina/genética , Genoma , Acuicultura , Virus/genética , NucleótidosRESUMEN
The summer temperatures recorded in Poland in 2022 were among the highest in over 30 years and, combined with higher-than-expected rainfall, gave the impression of an almost tropical climate. Such climatic conditions were ideal for the transmission of vector-borne zoonotic diseases such as West Nile fever. In northeastern Poland, in the Mazowieckie region, the Polish event-based surveillance network reported increased fatalities of free-living hooded crows (Corvus corone cornix). West Nile virus (WNV) lineage 2 was identified for the first time as the etiological agent responsible for the death of the birds. WNV was detected in 17 out of the 99 (17.17%) free-living birds tested in this study. All the WNV-infected dead birds were collected in the same area and were diagnosed in September by the NVRI and confirmed by the EURL for equine diseases, ANSES, in October 2022. Unnaturally high temperatures recorded in Poland in 2022 likely favored the infection and spread of the virus in the avian population. A nationwide alert and awareness raising of blood transfusion centers and hospitals was carried out to prevent human infections by WNV.