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We compared cycle thresholds from mpox skin lesions with other specimen sites and over time from onset of clinical signs among 104 patients in Sweden. Cycle thresholds differed by anatomic site. We identified 2 early mpox cases from anorectal swab specimens after skin samples were negative, indicating necessity of sampling multiple sites.
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Mpox , Humanos , Suecia/epidemiología , Reacción en Cadena de la Polimerasa , Monkeypox virusRESUMEN
A previously healthy male patient had detectable monkeypox virus DNA in saliva 76 days after laboratory confirmation of infection. A comprehensive characterization of viral kinetics and a detailed follow-up indicated a declining risk for transmission during the weeks after monkeypox symptoms appeared.
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Mpox , ADN Viral , Brotes de Enfermedades , Estudios de Seguimiento , Humanos , Masculino , Mpox/diagnóstico , Mpox/epidemiología , Monkeypox virus/genética , Suecia/epidemiologíaRESUMEN
BACKGROUND: In order to estimate the prevalence and understand the spread of SARS-CoV-2 in Sweden, the Public Health Agency of Sweden, with support from the Swedish Armed Forces, conducted a series of point prevalence surveys between March and December 2020. METHODS: Sampling material and instructions on how to perform self-sampling of the upper respiratory tract were delivered to the homes of the participants. Samples were analysed by real-time PCR, and the participants completed questionnaires regarding symptoms. FINDINGS: The first survey in the Stockholm region in March 2020 included 707 participants and showed a SARS-CoV-2 prevalence of 2.5%. The following five surveys, performed on a national level, with between 2461 and 2983 participants, showed SARS-CoV-2 prevalences of 0.9% (April), 0.3% (May), 0.0% (August), 0.0% (September), and 0.7% (December). All positive cases who responded to questionnaires reported experiencing symptoms that occurred from 2 weeks before the date of sampling up to and including the date of sampling. INTERPRETATION: None of the individuals shown to be PCR-positive were asymptomatic at the time of sampling or in the 14 days prior to sampling. This is in contrast to many other surveys in which a substantial proportion of positive cases have been reported to be asymptomatic. Our surveys demonstrate a decreasing ratio between notified cases and the observed prevalence throughout the year, in line with increasing testing capacity and the consecutive inclusion of all symptomatic individuals in the case definition for testing.
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COVID-19 , Humanos , COVID-19/epidemiología , Prevalencia , SARS-CoV-2 , Suecia/epidemiología , Salud PúblicaRESUMEN
The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) created an exceptional situation in which numerous laboratories in Europe simultaneously implemented SARS-CoV-2 diagnostics. These laboratories reported in February 2020 that commercial primer and probe batches for SARS-CoV-2 detection were contaminated with synthetic control material, causing delays of regional testing roll-out in various countries.
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Artefactos , Betacoronavirus/genética , Técnicas de Laboratorio Clínico/normas , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/epidemiología , Neumonía Viral/diagnóstico , Neumonía Viral/epidemiología , Juego de Reactivos para Diagnóstico/normas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/normas , Betacoronavirus/patogenicidad , COVID-19 , Prueba de COVID-19 , Técnicas de Laboratorio Clínico/métodos , Proteínas de la Envoltura de Coronavirus , Infecciones por Coronavirus/virología , Cartilla de ADN/análisis , Cartilla de ADN/síntesis química , Sondas de ADN/análisis , Sondas de ADN/síntesis química , Diagnóstico Tardío , Pruebas Diagnósticas de Rutina , Europa (Continente)/epidemiología , Humanos , Laboratorios/organización & administración , Laboratorios/normas , Pandemias , Patología Molecular , Neumonía Viral/virología , ARN Polimerasa Dependiente del ARN/genética , Juego de Reactivos para Diagnóstico/provisión & distribución , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/estadística & datos numéricos , SARS-CoV-2 , Proteínas del Envoltorio Viral/genéticaRESUMEN
Influenza A virus (IAV) has its natural reservoir in wild waterfowl, and new human IAVs often contain gene segments originating from avian IAVs. Treatment options for severe human influenza are principally restricted to neuraminidase inhibitors (NAIs), among which oseltamivir is stockpiled in preparedness for influenza pandemics. There is evolutionary pressure in the environment for resistance development to oseltamivir in avian IAVs, as the active metabolite oseltamivir carboxylate (OC) passes largely undegraded through sewage treatment to river water where waterfowl reside. In an in vivo mallard (Anas platyrhynchos) model, we tested if low-pathogenic avian influenza A(H7N9) virus might become resistant if the host was exposed to low levels of OC. Ducks were experimentally infected, and OC was added to their water, after which infection and transmission were maintained by successive introductions of uninfected birds. Daily fecal samples were tested for IAV excretion, genotype, and phenotype. Following mallard exposure to 2.5 µg/liter OC, the resistance-related neuraminidase (NA) I222T substitution, was detected within 2 days during the first passage and was found in all viruses sequenced from subsequently introduced ducks. The substitution generated 8-fold and 2.4-fold increases in the 50% inhibitory concentration (IC50) for OC (P < 0.001) and zanamivir (P = 0.016), respectively. We conclude that OC exposure of IAV hosts, in the same concentration magnitude as found in the environment, may result in amino acid substitutions, leading to changed antiviral sensitivity in an IAV subtype that can be highly pathogenic to humans. Prudent use of oseltamivir and resistance surveillance of IAVs in wild birds are warranted.
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Subtipo H7N9 del Virus de la Influenza A/efectos de los fármacos , Subtipo H7N9 del Virus de la Influenza A/enzimología , Neuraminidasa/metabolismo , Oseltamivir/farmacología , Agua/química , Animales , Patos , Neuraminidasa/genética , Oseltamivir/análogos & derivadosRESUMEN
Influenza A virus (IAV) has its natural reservoir in wild waterfowl, and emerging human IAVs often contain gene segments from avian viruses. The active drug metabolite of oseltamivir (oseltamivir carboxylate [OC]), stockpiled as Tamiflu for influenza pandemic preparedness, is not removed by conventional sewage treatment and has been detected in river water. There, it may exert evolutionary pressure on avian IAV in waterfowl, resulting in the development of resistant viral variants. A resistant avian IAV can circulate among wild birds only if resistance does not restrict viral fitness and if the resistant virus can persist without continuous drug pressure. In this in vivo mallard (Anas platyrhynchos) study, we tested whether an OC-resistant avian IAV (H1N1) strain with an H274Y mutation in the neuraminidase (NA-H274Y) could retain resistance while drug pressure was gradually removed. Successively infected mallards were exposed to decreasing levels of OC, and fecal samples were analyzed for the neuraminidase sequence and phenotypic resistance. No reversion to wild-type virus was observed during the experiment, which included 17 days of viral transmission among 10 ducks exposed to OC concentrations below resistance induction levels. We conclude that resistance in avian IAV that is induced by exposure of the natural host to OC can persist in the absence of the drug. Thus, there is a risk that human-pathogenic IAVs that evolve from IAVs circulating among wild birds may contain resistance mutations. An oseltamivir-resistant pandemic IAV would pose a substantial public health threat. Therefore, our observations underscore the need for prudent oseltamivir use, upgraded sewage treatment, and surveillance for resistant IAVs in wild birds.
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Antivirales/farmacología , Farmacorresistencia Viral , Subtipo H1N1 del Virus de la Influenza A/aislamiento & purificación , Gripe Aviar/virología , Mutación Missense , Neuraminidasa/aislamiento & purificación , Oseltamivir/farmacología , Proteínas Virales/aislamiento & purificación , Sustitución de Aminoácidos , Animales , Patos , Heces/virología , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Proteínas Mutantes/genética , Neuraminidasa/genética , Selección Genética , Proteínas Virales/genéticaRESUMEN
The influenza A virus subtypes H1N1, H1N2 and H3N2 are prevalent in pig populations worldwide. In the present study, two relatively uncommon swine influenza virus (SIV) H1N2 subtypes, isolated in Sweden in 2009 and 2010, were compared regarding their molecular composition and biological characteristics. The differences regarding markers purportedly related to pathogenicity, host adaptation or replication efficiency. They included a truncated PB1-F2 protein in the earlier isolate but a full length version in the more recent one; differences in the number of haemagglutinin glycosylation sites, including a characteristic human one; and a nuclear export protein with altered export signal. Of particular interest, the NS1 amino acid sequence of swine H1N2-2009 and 2010 has a 'unique or very unusual' PDZ binding domain (RPKV) at the C-terminal of the protein, a motif that has been implicated as a virulence marker. Concerning biological properties, these viruses reached lower titre and showed reduced cytopathogenicity in MDCK cells compared with an avian-like H1N1 isolate A/swine/Lidkoping/1193/2002 belonging to the same lineage as the 2009 and 2010 isolates. The findings should contribute to better understanding of factors related to the survival/extinction of this uncommon reassortant variant.
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Brotes de Enfermedades/veterinaria , Subtipo H1N2 del Virus de la Influenza A/genética , Infecciones por Orthomyxoviridae/veterinaria , Enfermedades de los Porcinos/virología , Porcinos/virología , Animales , Línea Celular , Perros , Genoma Viral , Glicosilación , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Subtipo H1N2 del Virus de la Influenza A/crecimiento & desarrollo , Subtipo H1N2 del Virus de la Influenza A/aislamiento & purificación , Neuraminidasa/genética , Proteínas de la Nucleocápside , Infecciones por Orthomyxoviridae/epidemiología , Filogenia , ARN Viral/genética , Proteínas de Unión al ARN/genética , Análisis de Secuencia de ADN , Suecia/epidemiología , Proteínas del Núcleo Viral/genética , Proteínas no Estructurales Virales/genética , Proteínas Virales/genéticaRESUMEN
Traditionally the focus for control of food-borne disease has been bacteria. During the last decade viruses have emerged as important sources of food borne human disease. Since the traditional bacteriological indicators, are not reliable for viral contamination, new methods are needed. PCR has enhanced the detection of virus in food. A challenge for developing detection reliable methods for viruses in food is that food matrices vary in composition, high sequence variability and inhibitors may be present. Therefore it is necessary to develop assays that have high diagnostic sensitivity, are broad and robust, and combine sample concentration and removal of inhibitors.
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Rapid and adaptable diagnostic capabilities are of great importance in the face of emerging infectious diseases. In an outbreak, timely establishment of diagnostic routines is crucial to identifying cases and preventing the spread of the disease, especially when faced with high-consequence pathogens. In this article, we describe a multiagency exercise including the rapid deployment and diagnostic adaptation of the Swedish Armed Forces mobile laboratory (biological field analysis laboratory) in the context of COVID-19. This deployment was initiated as a high-readiness exercise at the end of January 2020, when the global development of the outbreak was still uncertain. Through collaboration with the Public Health Agency of Sweden and a civilian hospital, a real-time reverse transcriptase polymerase chain reaction method specific to SARS-CoV-2 was made available and adapted to the mobile laboratory, and the team established and evaluated a functional and efficient diagnostic asset along with a logistical support chain. We also organized and evaluated mobile testing teams, and the method was later used in large-scale, national, cross-sectional COVID-19 surveys in several regions of Sweden. In this article, we focus on the challenges of overbridging the civil-military interface in this context and identifying lessons learned and added values to the response during the early pandemic. We propose that the experiences from this exercise and governmental agency collaboration are valuable in preparation for future outbreaks.
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COVID-19 , Personal Militar , Estudios Transversales , Humanos , Laboratorios , SARS-CoV-2RESUMEN
We evaluated the performance of 11 SARS-CoV-2 antibody tests using a reference set of heat-inactivated samples from 278 unexposed persons and 258 COVID-19 patients, some of whom contributed serial samples. The reference set included samples with a variation in SARS-CoV-2 IgG antibody titers, as determined by an in-house immunofluorescence assay (IFA). The five evaluated rapid diagnostic tests had a specificity of 99.0% and a sensitivity that ranged from 56.3 to 81.6% and decreased with low IFA IgG titers. The specificity was > 99% for five out of six platform-based tests, and when assessed using samples collected ≥ 22 days after symptom onset, two assays had a sensitivity of > 96%. These two assays also detected samples with low IFA titers more frequently than the other assays. In conclusion, the evaluated antibody tests showed a heterogeneity in their performances and only a few tests performed well with samples having low IFA IgG titers, an important aspect for diagnostics and epidemiological investigations.
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Prueba Serológica para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/aislamiento & purificación , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , COVID-19/sangre , COVID-19/inmunología , Prueba Serológica para COVID-19/economía , Niño , Preescolar , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Masculino , Persona de Mediana Edad , SARS-CoV-2/inmunología , Sensibilidad y Especificidad , Factores de Tiempo , Adulto JovenRESUMEN
Torque teno virus (TTV) is an unenveloped, circular, single stranded DNA virus with a genome size of approximately 3.8 kb. Previous studies have demonstrated varying grades of association between TTV DNA levels and immune deficiencies related to age, chronic infections and cancer. Alzheimer's disease (AD) has been related to persistent viral infections such as HSV-1 and CMV, but it is not known whether TTV viral load could serve as a functional biomarker of cellular immunity in this setting. Therefore, the objective of this study was to investigate whether TTV infection and viral load is related to AD status, CMV immunity, systemic inflammation or HLA types connected to anti-viral immunity. A total of 50 AD subjects and 51 non-demented controls were included in the study. AD subjects were diagnosed according to NINCDS-ADRDA and DSM-IV criteria and neuroradiologic findings were consistent with the diagnosis. TTV viral load was analyzed in plasma samples using a quantitative real-time PCR. Using a cut-off for TTV status at 200 copies/ml, 88% (89/101) of the study subjects were classified as TTV positive. TTV viral load significantly increased with age (beta 0.049 per year, p<0.001) but significantly decreased in relation to CMV IgG levels (beta -0.022 per 1000 units, p = 0.005) and HLA-B27 positivity (beta -0.53, p = 0.023). In conclusion, TTV immune control is not significantly affected by AD status, but appears related to age, CMV humoral immune response and HLA type.
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Enfermedad de Alzheimer/inmunología , Enfermedad de Alzheimer/virología , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/virología , Infecciones por Virus ADN/inmunología , Infecciones por Virus ADN/virología , Torque teno virus/patogenicidad , Carga Viral , Factores de Edad , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/complicaciones , Estudios de Casos y Controles , Infecciones por Citomegalovirus/complicaciones , Infecciones por Virus ADN/complicaciones , Femenino , Prueba de Histocompatibilidad , Humanos , Inmunidad Celular , Masculino , Torque teno virus/genética , Torque teno virus/inmunología , Carga Viral/inmunologíaRESUMEN
Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is commonly diagnosed by reverse transcription polymerase chain reaction (RT-PCR) to detect viral RNA in patient samples, but RNA extraction constitutes a major bottleneck in current testing. Methodological simplification could increase diagnostic availability and efficiency, benefitting patient care and infection control. Here, we describe methods circumventing RNA extraction in COVID-19 testing by performing RT-PCR directly on heat-inactivated or lysed samples. Our data, including benchmarking using 597 clinical patient samples and a standardised diagnostic system, demonstrate that direct RT-PCR is viable option to extraction-based tests. Using controlled amounts of active SARS-CoV-2, we confirm effectiveness of heat inactivation by plaque assay and evaluate various generic buffers as transport medium for direct RT-PCR. Significant savings in time and cost are achieved through RNA-extraction-free protocols that are directly compatible with established PCR-based testing pipelines. This could aid expansion of COVID-19 testing.
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Betacoronavirus/genética , Betacoronavirus/aislamiento & purificación , Técnicas de Laboratorio Clínico/métodos , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/virología , Neumonía Viral/diagnóstico , Neumonía Viral/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Benchmarking , COVID-19 , Prueba de COVID-19 , Técnicas de Laboratorio Clínico/normas , Técnicas de Laboratorio Clínico/estadística & datos numéricos , Infecciones por Coronavirus/epidemiología , Cartilla de ADN/genética , Calor , Humanos , Pandemias , Neumonía Viral/epidemiología , ARN Viral/genética , ARN Viral/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/normas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/estadística & datos numéricos , SARS-CoV-2 , Sensibilidad y Especificidad , Suecia/epidemiología , Ensayo de Placa Viral/métodosRESUMEN
Surveillance of wild birds is critical in monitoring for highly pathogenic avian influenza A viruses (AIVs). However, a successful surveillance regime requires proper treatment of samples in the field - rapid placement of samples in -80°C and subsequent maintenance of cold-chain. Given the logistical difficulties of this, many avian taxa and/or geographic locations are not sampled, or, when sampled may result in false negatives due to poor sample treatment in the field. Here, we assessed the utility of RNAlater® as a stabilization agent for AIV sampling. We found no difference in real time PCR performance between virus transport media at optimal conditions and RNAlater® at -80°C, -20°C, 4°C or room temperature up to two weeks, at either low or high virus load. Not only was RNAlater® useful in comparison of spiked samples or those from duck experiments, it was employed successfully in a field study of backyard birds in China. We detected AIV in cloacal and oropharyngeal samples from chickens and a sample with a low Cq was successfully subtyped as H9, although sample storage conditions were suboptimal. Thus, despite limitations in downstream characterization such virus isolation and typing, RNAlater® is a viable option for AIV sampling under logistically challenging circumstances.
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Fijadores/farmacología , Virus de la Influenza A/aislamiento & purificación , ARN Viral/aislamiento & purificación , Manejo de Especímenes/métodos , Animales , Animales Salvajes , Aves/virología , Pollos/virología , China , Cloaca/virología , Patos/virología , Gripe Aviar/virología , Orofaringe/virología , Manejo de Especímenes/instrumentaciónRESUMEN
Urbanization is intensifying worldwide, and affects the epidemiology of infectious diseases. However, the effect of urbanization on natural host-pathogen systems remains poorly understood. Urban ducks occupy an interesting niche in that they directly interact with both humans and wild migratory birds, and either directly or indirectly with food production birds. Here we have collected samples from Mallards (Anas platyrhynchos) residing in a pond in central Uppsala, Sweden, from January 2013 to January 2014. This artificial pond is kept ice-free during the winter months, and is a popular location where the ducks are fed, resulting in a resident population of ducks year-round. Nine hundred and seventy seven (977) fecal samples were screened for RNA viruses including: influenza A virus (IAV), avian paramyxovirus 1, avian coronavirus (CoV), and avian astrovirus (AstroV). This intra-annual dataset illustrates that these RNA viruses exhibit similar annual patterns to IAV, suggesting similar ecological factors are at play. Furthermore, in comparison to wild ducks, autumnal prevalence of IAV and CoV are lower in this urban population. We also demonstrate that AstroV might be a larger burden to urban ducks than IAV, and should be better assessed to demonstrate the degree to which wild birds contribute to the epidemiology of these viruses. The presence of economically relevant viruses in urban Mallards highlights the importance of elucidating the ecology of wildlife pathogens in urban environments, which will become increasingly important for managing disease risks to wildlife, food production animals, and humans.
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Infecciones por Astroviridae/veterinaria , Astroviridae/genética , Infecciones por Coronavirus/veterinaria , Gammacoronavirus/genética , Virus de la Influenza A/genética , Gripe Aviar/epidemiología , Enfermedad de Newcastle/epidemiología , Virus de la Enfermedad de Newcastle/genética , Migración Animal/fisiología , Animales , Animales Salvajes , Anseriformes , Astroviridae/clasificación , Astroviridae/aislamiento & purificación , Infecciones por Astroviridae/epidemiología , Infecciones por Astroviridae/virología , Ciudades , Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/virología , Heces/virología , Gammacoronavirus/clasificación , Gammacoronavirus/aislamiento & purificación , Virus de la Influenza A/clasificación , Virus de la Influenza A/aislamiento & purificación , Gripe Aviar/virología , Enfermedad de Newcastle/virología , Virus de la Enfermedad de Newcastle/clasificación , Virus de la Enfermedad de Newcastle/aislamiento & purificación , Filogenia , Estanques , ARN Viral/genética , Suecia/epidemiología , UrbanizaciónRESUMEN
The human genome contains around 1000 betaretrovirus-like copies, human mouse mammary tumour virus (MMTV)-like (HML) groups 1-10, also referred to as human endogenous retrovirus "HERV-K". Despite many efforts, it is not established whether betaretroviruses, exo- or endogenous, are involved in the etiology of breast cancer, or other cancer diseases, in humans. Quantitative real-time PCR (QPCR) TaqMan-based assays for HML groups 1-7, targeting the conserved reverse transcriptase (RT) and integrase (IN) domains of the pol gene were designed. Plasmids containing the entire pol gene of HML1-7 were used as standards. The RT and IN based QPCRs could detect 10(0)-10(3) copies per PCR reaction of the plasmids. However, not all plasmids gave a signal in both RT and IN QPCRs, probably due to mismatches. Furthermore, RT and IN based HML6 specific QPCRs were developed. They were specific for amplification of transcripts for the whole HML6 group. The methods allow the monitoring in body fluids and tissues of expression of a wide range of betaretrovirus-like sequences. Betaretrovirus-like RNA was studied in normal human tissues and of HML6 in brains of multiple sclerosis (MS) patients. Brain, adrenal gland and testis had a high betaretrovirus-like expression. Multiple sclerosis plaques contained the same HML6 RNA concentration as control tissue. These assays are expected to enhance studies on involvement of betaretroviruses in physiology and disease.
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Retrovirus Endógenos/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , ARN Viral/análisis , Glándulas Suprarrenales/virología , Secuencia de Bases , Líquidos Corporales/virología , Encéfalo/virología , Retrovirus Endógenos/genética , Expresión Génica , Genes pol , Humanos , Integrasas/genética , Masculino , Datos de Secuencia Molecular , Plásmidos/genética , ARN Viral/genética , ADN Polimerasa Dirigida por ARN/genética , Estándares de Referencia , Sensibilidad y Especificidad , Testículo/virología , Proteínas Virales/genéticaRESUMEN
Coronaviruses (CoVs) are found in a wide variety of wild and domestic animals, and constitute a risk for zoonotic and emerging infectious disease. In poultry, the genetic diversity, evolution, distribution and taxonomy of some coronaviruses have been well described, but little is known about the features of CoVs in wild birds. In this study we screened 764 samples from 22 avian species of the orders Anseriformes and Charadriiformes in Sweden collected in 2006/2007 for CoV, with an overall CoV prevalence of 18.7%, which is higher than many other wild bird surveys. The highest prevalence was found in the diving ducks--mainly Greater Scaup (Aythya marila; 51.5%)--and the dabbling duck Mallard (Anas platyrhynchos; 19.2%). Sequences from two of the Greater Scaup CoV fell into an infrequently detected lineage, shared only with a Tufted Duck (Aythya fuligula) CoV. Coronavirus sequences from Mallards in this study were highly similar to CoV sequences from the sample species and location in 2011, suggesting long-term maintenance in this population. A single Black-headed Gull represented the only positive sample from the order Charadriiformes. Globally, Anas species represent the largest fraction of avian CoV sequences, and there seems to be no host species, geographical or temporal structure. To better understand the eitiology, epidemiology and ecology of these viruses more systematic surveillance of wild birds and subsequent sequencing of detected CoV is imperative.
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Aves/virología , Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/virología , Coronavirus/clasificación , Algoritmos , Animales , Biodiversidad , Charadriiformes/virología , Coronavirus/genética , ARN Polimerasas Dirigidas por ADN/genética , Bases de Datos Factuales , Patos/virología , Gansos/virología , Variación Genética , Epidemiología Molecular , Filogenia , Prevalencia , ARN Viral/genética , SueciaRESUMEN
This study identified and characterized Brucella species in the informal milk chain in Uganda. A total of 324 cattle bulk milk samples were screened for the genus Brucella by real-time PCR with primers targeting the bcsp31 gene and further characterized by the omp25 gene. Of the samples tested, 6.5% were positive for Brucella species. In the omp25 phylogeny, the study sequences were found to form a separate clade within the branch containing B. abortus sequences. The study shows that informally marketed cattle milk in Uganda is a likely risk factor for human brucellosis and confirms that B. abortus is present in the cattle population. This information is important for potential future control measures, such as vaccination of cattle.
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Brucellosis is endemic in livestock and humans in Uganda and its transmission involves a multitude of risk factors like consumption of milk from infected cattle. To shed new light on the epidemiology of brucellosis in Uganda the present study used phenotypic and molecular approaches to delineate the Brucella species, biovars, and genotypes shed in cattle milk. Brucella abortus without a biovar designation was isolated from eleven out of 207 milk samples from cattle in Uganda. These isolates had a genomic monomorphism at 16 variable number tandem repeat (VNTR) loci and showed in turn high levels of genetic variation when compared with other African strains or other B. abortus biovars from other parts of the world. This study further highlights the usefulness of MLVA as an epidemiological tool for investigation of Brucella infections.
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Brucella abortus/aislamiento & purificación , Productos Lácteos/microbiología , Leche/microbiología , Animales , Técnicas de Tipificación Bacteriana/métodos , Brucella abortus/genética , Brucelosis/microbiología , Bovinos , Variación Genética/genética , Genotipo , Repeticiones de Minisatélite/genética , UgandaRESUMEN
BACKGROUND: Wild waterfowl is the natural reservoir of influenza A virus (IAV); hosted viruses are very variable and provide a source for genetic segments which can reassort with poultry or mammalian adapted IAVs to generate novel species crossing viruses. Additionally, wild waterfowl act as a reservoir for highly pathogenic IAVs. Exposure of wild birds to the antiviral drug oseltamivir may occur in the environment as its active metabolite can be released from sewage treatment plants to river water. Resistance to oseltamivir, or to other neuraminidase inhibitors (NAIs), in IAVs of wild waterfowl has not been extensively studied. AIM AND METHODS: In a previous in vivo Mallard experiment, an influenza A(H6N2) virus developed oseltamivir resistance by the R292K substitution in the neuraminidase (NA), when the birds were exposed to oseltamivir. In this study we tested if the resistance could be maintained in Mallards without drug exposure. Three variants of resistant H6N2/R292K virus were each propagated during 17 days in five successive pairs of naïve Mallards, while oseltamivir exposure was decreased and removed. Daily fecal samples were analyzed for viral presence, genotype and phenotype. RESULTS AND CONCLUSION: Within three days without drug exposure no resistant viruses could be detected by NA sequencing, which was confirmed by functional NAI sensitivity testing. We conclude that this resistant N2 virus could not compete in fitness with wild type subpopulations without oseltamivir drug pressure, and thus has no potential to circulate among wild birds. The results of this study contrast to previous observations of drug induced resistance in an avian H1N1 virus, which was maintained also without drug exposure in Mallards. Experimental observations on persistence of NAI resistance in avian IAVs resemble NAI resistance seen in human IAVs, in which resistant N2 subtypes do not circulate, while N1 subtypes with permissive mutations can circulate without drug pressure. We speculate that the phylogenetic group N1 NAs may easier compensate for NAI resistance than group N2 NAs, though further studies are needed to confirm such conclusions.
Asunto(s)
Farmacorresistencia Viral , Patos , Inhibidores Enzimáticos/farmacología , Virus de la Influenza A/genética , Gripe Aviar/virología , Mutación , Neuraminidasa/genética , Oseltamivir/farmacología , Animales , Patos/virologíaRESUMEN
Low-pathogenic avian influenza (LPAI) viruses in wild birds are important as they can constitute the basis for the development of highly pathogenic avian influenza viruses or form part of human-adapted strains with pandemic potential. However, the pathogenesis of LPAI viruses is not well characterized in dabbling ducks, one of the natural reservoirs of LPAI viruses. Between 21 September 2009 and 21 December 2009, we used real-time reverse transcriptase polymerase chain reaction (q-PCR), histopathology, and immunohistochemistry (IHC) to study Mallards (Anas platyrhynchos) infected with an influenza A/H1N1 virus isolated from a wild Mallard in Sweden. The ducks were either inoculated intraesophageally ("artificial infection") or infected by virus shed by other ducks in the experiment ("contact infection"). The ducks were subjected to three low concentrations (80 ng/L, 1 µg/L, and 80 µg/L) of the active metabolite of oseltamivir (Tamiflu(®)), oseltamivir carboxylate (OC), which resulted in the development of the viral resistance mutation H274Y at 1 and 80 µg/L. The LPAI virus infection was localized to the intestinal tract and cloacal bursa except in one Mallard. The exception was a duck euthanized 1 day postinoculation, whose infection was located solely in the lung, possibly due to intratracheal deposition of virus. The intestinal infection was characterized by occasional degenerating cells in the lamina propria and presence of viral antigen as detected by IHC, as well as positive q-PCR performed on samples from feces and intestinal contents. Histopathologic changes, IHC positivity, and viral shedding all indicated that the infection peaked early, around 2 days postinfection. Furthermore, more viral antigen and viral RNA were detected with IHC and q-PCR in the proximal parts early in the infection. There was no obvious difference in the course of the infection in artificial versus contact infection, when the level of OC was increased from 80 ng/L to 1 µg/L (based on IHC and q-PCR), when the level of OC was increased to 80 µg/L, or when the resistance mutation H274Y developed (based on q-PCR).