RESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects various mammalian species, with farmed minks experiencing the highest number of outbreaks. In Spain, we analyzed 67 whole genome sequences and eight spike sequences from 18 outbreaks, identifying four distinct lineages: B.1, B.1.177, B.1.1.7, and AY.98.1. The potential risk of transmission to humans raises crucial questions about mutation accumulation and its impact on viral fitness. Sequencing revealed numerous not-lineage-defining mutations, suggesting a cumulative mutation process during the outbreaks. We observed that the outbreaks were predominantly associated with different groups of mutations rather than specific lineages. This clustering pattern by the outbreaks could be attributed to the rapid accumulation of mutations, particularly in the ORF1a polyprotein and in the spike protein. Notably, the mutations G37E in NSP9, a potential host marker, and S486L in NSP13 were detected. Spike protein mutations may enhance SARS-CoV-2 adaptability by influencing trimer stability and binding to mink receptors. These findings provide valuable insights into mink coronavirus genetics, highlighting both host markers and viral transmission dynamics within communities.
Assuntos
COVID-19 , Genoma Viral , Vison , Mutação , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , COVID-19/virologia , COVID-19/epidemiologia , COVID-19/transmissão , Animais , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Espanha/epidemiologia , Vison/virologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Adaptação ao Hospedeiro/genética , Humanos , Surtos de Doenças , Pandemias , Filogenia , Sequenciamento Completo do GenomaRESUMO
Nanopore sequencing has emerged as a rapid and cost-efficient tool for diagnostic and epidemiological surveillance of SARS-CoV-2 during the COVID-19 pandemic. This study compared the results from sequencing the SARS-CoV-2 genome using R9 vs R10 flow cells and a Rapid Barcoding Kit (RBK) vs a Ligation Sequencing Kit (LSK). The R9 chemistry provided a lower error rate (3.5%) than R10 chemistry (7%). The SARS-CoV-2 genome includes few homopolymeric regions. Longest homopolymers were composed of 7 (TTTTTTT) and 6 (AAAAAA) nucleotides. The R10 chemistry resulted in a lower rate of deletions in thymine and adenine homopolymeric regions than the R9, at the expenses of a larger rate (~10%) of mismatches in these regions. The LSK had a larger yield than the RBK, and provided longer reads than the RBK. It also resulted in a larger percentage of aligned reads (99 vs 93%) and also in a complete consensus genome. The results from this study suggest that the LSK preparation library provided longer DNA fragments which contributed to a better assembly of the SARS-CoV-2, despite an impaired detection of variants in a R10 flow cell. Nanopore sequencing could be used in epidemiological surveillance of SARS-CoV-2. KEY POINTS: ⢠Sequencing SARS-CoV-2 genome is of great importance for the pandemic surveillance. ⢠Nanopore offers a low cost and accurate method to sequence SARS-CoV-2 genome. ⢠Ligation sequencing is preferred rather than the rapid kit using transposases.
Assuntos
Genoma Viral , Nanoporos , SARS-CoV-2/genética , Análise de Sequência de RNA/métodosRESUMO
Bagaza virus (BAGV; synonymous to Israel turkey meningoencephalomyelitis virus, ITV) is a relevant arthropod-borne epornitic flavivirus. In its first emergence in Europe (southern Spain, 2010) BAGV caused an outbreak, severely affecting red-legged partridges and common pheasants. The effects (pathogenicity, role as reservoir host) of BAGV in other European phasianids are unknown. To fill this gap, grey partridges were experimentally infected with BAGV. The clinical course of the disease was severe, with neurological signs, significant weight loss and 40% mortality. Low viral loads in the blood and the absence of contact transmission suggest a limited-if any-role on BAGV transmission for this European phasianid.
Assuntos
Doenças das Aves/fisiopatologia , Infecções por Flavivirus/veterinária , Flavivirus/fisiologia , Flavivirus/patogenicidade , Galliformes , Animais , Doenças das Aves/mortalidade , Doenças das Aves/virologia , Feminino , Infecções por Flavivirus/mortalidade , Infecções por Flavivirus/fisiopatologia , Infecções por Flavivirus/virologia , Interações Hospedeiro-Patógeno , Masculino , VirulênciaRESUMO
Porcine teschoviruses (PTVs) constitute 1 of the 31 genera within the Picornaviridae family, comprising at least 13 genetic types (PTV-1 to PTV-13), of which only 11 (PTV-1 to PTV-11) have been recognized as serotypes to date. Specific for swine and wild boars, most PTVs are usually non-pathogenic, but some viral variants cause severe disorders in the central nervous system (Teschen disease) or milder signs (Talfan disease), as well as reproductive, digestive and respiratory disorders and skin lesions. Previous studies revealed a high diversity of teschoviruses circulating in Spanish pig populations. Phylogenetic analysis performed with these sequences and others available in GenBank disclosed 13 clusters, 11 of which corresponded to the known PTV serotypes, and 1 of 2 additional groups is represented by isolate CC25, whose full-length genomic sequence has been obtained. This group is new to science, and was putatively named PTV-12. Here, a complete characterization of this isolate is presented, including the experimental infection of minipigs to assess tissue tropism and possible pathogenicity in vivo in the host species. In addition, using this experimental animal model, we investigated whether a pre-existing infection with this PTV-12 isolate could confer cross-protection against infection with a heterotypic PTV-1 virulent strain. Based on phylogenetic analysis and serological data, we propose CC25 as the prototype strain of a new teschovirus serotype, PTV-12.
Assuntos
Proteção Cruzada/imunologia , Infecções por Picornaviridae/imunologia , Doenças dos Suínos/imunologia , Porco Miniatura/virologia , Teschovirus/classificação , Teschovirus/imunologia , Tropismo Viral/fisiologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Infecções por Picornaviridae/virologia , Sorogrupo , Sorotipagem , Espanha , Suínos , Doenças dos Suínos/virologia , Teschovirus/genética , Teschovirus/isolamento & purificação , Viremia/virologiaRESUMO
BACKGROUND: West Nile Virus (WNV) is a zoonotic arbovirus worldwide spread. Seasonal WNV outbreaks occur in the Mediterranean basin since the late 1990's with ever-increasing incidence. In Southern Spain WNV is endemic, as disease foci - caused by WNV lineage 1 (WNV-L1) strains - occur every year. On the contrary, WNV-L2 is the dominant lineage in Europe, so most European WNV sequences available belong to this lineage, WNV-L1 sequences being still scarce. METHODS: To fill this gap, this study reports the genetic characterisation of 27 newly described WNV-L1 strains, involved in outbreaks affecting wild birds and horses during the last decade in South-Western Spain. RESULTS: All strains except one belong to the Western Mediterranean-1 sub-cluster (WMed-1), related phylogenetically to Italian, French, Portuguese, Moroccan and, remarkably, Senegalese strains. This sub-cluster persisted, spread and evolved into three distinguishable WMed-1 phylogenetic groups that co-circulated, notably, in the same province (Cádiz). They displayed different behaviours: from long-term persistence and rapid spread to neighbouring regions within Spain, to long-distance spread to different countries, including transcontinental spread to Africa. Among the different introductions of WNV in Spain revealed in this study, some of them succeeded to get established, some extinguished from the territory shortly afterwards. Furthermore, Spain's southernmost province, Cádiz, constitutes a hotspot for virus incursion. CONCLUSION: Southern Spain seems a likely scenario for emergence of exotic pathogens of African origin. Therefore, circulation of diverse WNV-L1 variants in Spain prompts for an extensive surveillance under a One Health approach.
Assuntos
Aves , Filogenia , Febre do Nilo Ocidental , Vírus do Nilo Ocidental , Vírus do Nilo Ocidental/genética , Vírus do Nilo Ocidental/classificação , Vírus do Nilo Ocidental/isolamento & purificação , Febre do Nilo Ocidental/epidemiologia , Febre do Nilo Ocidental/virologia , Febre do Nilo Ocidental/transmissão , Animais , Espanha/epidemiologia , Aves/virologia , Doenças das Aves/virologia , Doenças das Aves/epidemiologia , Cavalos/virologia , Europa (Continente)/epidemiologia , Surtos de Doenças , África/epidemiologia , Doenças dos Cavalos/virologia , Doenças dos Cavalos/epidemiologia , Humanos , Animais Selvagens/virologiaRESUMO
The African horse sickness virus (AHSV) belongs to the Genus Orbivirus, family Sedoreoviridae, and nine serotypes of the virus have been described to date. The AHSV genome is composed of ten linear segments of double-stranded (ds) RNA, numbered in decreasing size order (Seg-1 to Seg-10). Genome segment 2 (Seg-2) encodes outer-capsid protein VP2, the most variable AHSV protein and the primary target for neutralizing antibodies. Consequently, Seg-2 determines the identity of the virus serotype. An African horse sickness (AHS) outbreak in an AHS-free status country requires identifying the serotype as soon as possible to implement a serotype-specific vaccination program. Considering that nowadays 'polyvalent live attenuated' is the only commercially available vaccination strategy to control the disease, field and vaccine strains of different serotypes could co-circulate. Additionally, in AHS-endemic countries, more than one serotype is often circulating at the same time. Therefore, a strategy to rapidly determine the virus serotype in an AHS-positive sample is strongly recommended in both epidemiological situations. The main objective of this study is to describe the development and validation of three triplex real-time RT-PCR (rRT-PCR) methods for rapid AHSV serotype detection. Samples from recent AHS outbreaks in Kenia (2015-2017), Thailand (2020), and Nigeria (2023), and from the AHS outbreak in Spain (1987-1990), were included in the study for the validation of these methods.
Assuntos
Vírus da Doença Equina Africana , Doença Equina Africana , Orbivirus , Vacinas Virais , Animais , Cavalos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Doença Equina Africana/diagnóstico , Doença Equina Africana/epidemiologia , Doença Equina Africana/prevenção & controle , Orbivirus/genética , Anticorpos NeutralizantesRESUMO
In September 2022, more than 50 years after its eradication from Spain, Sheep pox virus was confirmed by laboratory analysis in sheep showing characteristic lesions. This was the start of an outbreak that lasted 9 months and infected 30 farms dispersed over two different areas, Andalusia and Castilla-La Mancha. Early after the initial confirmation, an active surveillance based on clinical inspection with laboratory confirmation of sheep with clinical signs was started in restricted areas. This allowed the confirmation of Sheep pox in 22 out of 28 suspected farms, where limited numbers of sheep with mainly erythema and papules were found, indicative of early detection. Nevertheless, to improve active surveillance and stop the outbreak, clinical inspection was reinforced by laboratory analysis in all inspected farms, even when no clinically diseased sheep were detected. Although more than 35,000 oral swabs from 335 farms were analysed by real-time PCR in pools of five, only two out of six reported outbreaks in this period were detected by laboratory analysis before clinical signs were observed. Furthermore, additional insights were gained from the extensive laboratory surveillance performed on samples collected under field conditions. No evidence of Sheep pox virus infection was found in goats. Oral swabs proved to be the sample of choice for early detection in the absence of scabs and could be tested in pools of five without extensive loss in sensitivity; serology by ELISA was not useful in outbreak detection. Finally, a non-infectious genome of the virus could be detected months after cleaning and disinfection; thus, real-time PCR results should be interpreted with caution in sentinel animals during repopulation. In conclusion, the outbreak of Sheep pox virus in Spain showed that active clinical inspection with laboratory confirmation of clinically diseased sheep via oral swab testing proved a sensitive method for detection of infected farms, providing insights in laboratory surveillance that will be helpful for other countries confronted with Sheep pox outbreaks.
Assuntos
Capripoxvirus , Surtos de Doenças , Infecções por Poxviridae , Doenças dos Ovinos , Animais , Espanha/epidemiologia , Surtos de Doenças/veterinária , Ovinos , Infecções por Poxviridae/veterinária , Infecções por Poxviridae/epidemiologia , Infecções por Poxviridae/diagnóstico , Infecções por Poxviridae/virologia , Doenças dos Ovinos/epidemiologia , Doenças dos Ovinos/virologia , Doenças dos Ovinos/diagnóstico , Capripoxvirus/genética , Capripoxvirus/isolamento & purificação , Cabras , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Fazendas , Monitoramento Epidemiológico/veterináriaRESUMO
West Nile Virus (WNV) is a mosquito vector-borne zoonosis with an increasing incidence in Europe that has become a public health concern. In Spain, although local circulation has been known for decades, until 2020, when a large outbreak occurred, West Nile Virus cases were scarce and mostly occurred in southern Spain. Since then, there have been new cases every year and the pathogen has spread to new regions. Thus, monitoring of circulating variants and lineages plays a fundamental role in understanding WNV evolution, spread and dynamics. In this study, we sequenced WNV consensus genomes from mosquito pools captured in 2022 as part of a newly implemented surveillance program in southern Spain and compared it to other European, African and Spanish sequences. Characterization of WNV genomes in mosquitoes captured in 2022 reveals the co-circulation of two WNV lineage 1 variants, the one that caused the outbreak in 2020 and another variant that is closely related to variants reported in Spain in 2012, France in 2015, Italy in 2021-2022 and Senegal in 2012-2018. The geographic distribution of these variants indicates that WNV L1 dynamics in southern Europe include an alternating dominance of variants in some territories.
Assuntos
Culicidae , Febre do Nilo Ocidental , Vírus do Nilo Ocidental , Animais , Humanos , Vírus do Nilo Ocidental/genética , Febre do Nilo Ocidental/epidemiologia , Espanha/epidemiologia , Europa (Continente)/epidemiologiaRESUMO
This study aimed to compare the infection dynamics of three genotype II African swine fever viruses (ASFV) circulating in Europe. Eighteen domestic pigs divided into three groups were infected intramuscularly or by direct contact with two haemadsorbent ASFVs (HAD) from Poland (Pol16/DP/ OUT21) and Estonia (Est16/WB/Viru8), and with the Latvian non-HAD ASFV (Lv17/WB/Rie1). Parameters, such as symptoms, pathogenicity, and distribution of the virus in tissues, humoral immune response, and dissemination of the virus by blood, oropharyngeal and rectal routes, were investigated. The Polish ASFV caused a case of rapidly developing fatal acute disease, while the Estonian ASFV caused acute to sub-acute infections and two animals survived. In contrast, animals infected with the ASFV from Latvia developed a more subtle, mild, or even subclinical disease. Oral excretion was sporadic or even absent in the attenuated group, whereas in animals that developed an acute or sub-acute form of ASF, oral excretion began at the same time the ASFV was detected in the blood, or even 3 days earlier, and persisted up to 22 days. Regardless of virulence, blood was the main route of transmission of ASFV and infectious virus was isolated from persistently infected animals for at least 19 days in the attenuated group and up to 44 days in the group of moderate virulence. Rectal excretion was limited to the acute phase of infection. In terms of diagnostics, the ASFV genome was detected in contact pigs from oropharyngeal samples earlier than in blood, independently of virulence. Together with blood, both samples could allow to detect ASFV infection for a longer period. The results presented here provide quantitative data on the spread and excretion of ASFV strains of different virulence among domestic pigs that can help to better focus surveillance activities and, thus, increase the ability to detect ASF introductions earlier.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Doenças dos Suínos , Febre Suína Africana/epidemiologia , Vírus da Febre Suína Africana/genética , Animais , Genótipo , Sus scrofa , Suínos , VirulênciaRESUMO
In the course of an epidemiologic surveillance program for swine diseases carried out in Spain, 206 cytopathic viruses were isolated from 600 porcine fecal samples between 2004 and 2005. The virus isolates were examined using reverse transcription polymerase chain reaction (RT-PCR) methods specific for different types of porcine picornaviruses, including members of the Teschovirus, Enterovirus, and Sapelovirus genera, and PCR for porcine adenoviruses. Of the 206 isolates, 97 (47%) were identified as teschoviruses, 18 (9%) as sapeloviruses, and 7 (3%) as porcine adenoviruses. Neither Porcine enterovirus B nor Swine vesicular disease virus was found among the isolates. The present study confirms that teschoviruses are highly prevalent in porcine fecal samples, at least in Spain. It also reveals that these viruses commonly circulate among apparently healthy pigs.
Assuntos
Adenoviridae/isolamento & purificação , Fezes/virologia , Picornaviridae/isolamento & purificação , Doenças dos Suínos/virologia , Suínos/virologia , Adenoviridae/classificação , Animais , Linhagem Celular , Enterovirus/genética , Enterovirus/isolamento & purificação , Humanos , Rim/virologia , Picornaviridae/classificação , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sorotipagem , Espanha , Doenças dos Suínos/classificação , Teschovirus/classificação , Teschovirus/isolamento & purificaçãoRESUMO
High impact, mosquito-borne flaviviruses such as West Nile virus (WNV), Usutu virus (USUV), Japanese encephalitis virus (JEV), Tembusu virus (TMUV), and Bagaza/Israel turkey meningoencephalomyelitis virus (BAGV/ITV) are emerging in different areas of the world. These viruses belong to the Japanese encephalitis (JE) serocomplex (JEV, WNV, and USUV) and the Ntaya serocomplex (TMUV and BAGV/ITV). Notably, they share transmission route (mosquito bite) and reservoir host type (wild birds), and some of them co-circulate in the same areas, infecting overlapping mosquito and avian population. This may simplify epidemiological surveillance, since it allows the detection of different infections targeting the same population, but also represents a challenge, as the diagnostic tools applied need to detect the whole range of flaviviruses surveyed, and correctly differentiate between these closely related pathogens. To this aim, a duplex real-time RT-PCR (dRRT-PCR) method has been developed for the simultaneous and differential detection of JE and Ntaya flavivirus serocomplexes. The method has been standardized and evaluated by analyzing a panel of 49 flaviviral and non-flaviviral isolates, and clinical samples of different bird species obtained from experimental infections or from the field, proving its value for virus detection in apparently healthy or suspicious animals. This new dRRT-PCR technique is a reliable, specific and highly sensitive tool for rapid detection and differentiation of JE and Ntaya flavivirus groups in either domestic or wild animals. This novel method can be implemented in animal virology diagnostic laboratories as screening tool in routine surveillance and in the event of bird encephalitis emergence.
RESUMO
The increasing incidence of West Nile virus (WNV) in the Euro-Mediterranean area warrants the implementation of effective surveillance programs in animals. A crucial step in the fight against the disease is the evaluation of the capacity of the veterinary labs to accurately detect the infection in animal populations. In this context, the animal virology network of the MediLabSecure project organized an external quality assessment (EQA) to evaluate the WNV molecular and serological diagnostic capacities of beneficiary veterinary labs. Laboratories from 17 Mediterranean and Black Sea countries participated. The results of the triplex real time RT-PCR for simultaneous detection and differentiation of WNV lineage 1 (L1), lineage 2 (L2) and Usutu virus (USUV) were highly satisfactory, especially for L1 and L2, with detection rates of 97.9% and 100%, respectively. For USUV, 75% of the labs reported correct results. More limitations were observed for the generic detection of flaviviruses using conventional reverse-transcription polymerase chain reaction (RT-PCR), since only 46.1% reported correct results in the whole panel. As regards the serological panel, the results were excellent for the generic detection of WNV antibodies. More variability was observed for the specific detection of IgM antibodies with a higher percentage of incorrect results mainly in samples with low titers. This EQA provides a good overview of the WNV (and USUV) diagnostic performance of the involved veterinary labs and demonstrates that the implemented training program was successful in upgrading their diagnostic capacities.
RESUMO
Rift Valley fever (RVF) is an arboviral zoonosis that primarily affects ruminants but can also cause illness in humans. The increasing impact of RVF in Africa and Middle East and the risk of expansion to other areas such as Europe, where competent mosquitos are already established, require the implementation of efficient surveillance programs in animal populations. For that, it is pivotal to regularly assess the performance of existing diagnostic tests and to evaluate the capacity of veterinary labs of endemic and non-endemic countries to detect the infection in an accurate and timely manner. In this context, the animal virology network of the MediLabSecure project organized between October 2016 and March 2017 an external quality assessment (EQA) to evaluate the RVF diagnostic capacities of beneficiary veterinary labs. This EQA was conceived as the last step of a training curriculum that included 2 diagnostic workshops that were organized by INIA-CISA (Spain) in 2015 and 2016. Seventeen veterinary diagnostic labs from 17 countries in the Mediterranean and Black Sea regions participated in this EQA. The exercise consisted of two panels of samples for molecular and serological detection of the virus. The laboratories were also provided with positive controls and all the kits and reagents necessary to perform the recommended diagnostic techniques. All the labs were able to apply the different protocols and to provide the results on time. The performance was good in the molecular panel with 70.6% of participants reporting 100% correct results, and excellent in the serological panel with 100% correct results reported by 94.1% of the labs. This EQA provided a good overview of the RVFV diagnostic capacities of the involved labs and demonstrated that most of them were able to correctly identify the virus genome and antibodies in different animal samples.
Assuntos
Febre do Vale de Rift/diagnóstico , Animais , Mar Negro , Culicidae , Europa (Continente) , Genoma Viral , Humanos , Laboratórios , Mar Mediterrâneo , Febre do Vale de Rift/virologia , Vírus da Febre do Vale do Rift/genética , RuminantesRESUMO
A non-haemadsorbing (non-HAD) ASF virus (ASFV) genotype II, namely Lv17/WB/Rie1, was isolated from a hunted wild boar in Latvia in 2017. Domestic pigs experimentally infected with the non-HAD ASFV developed a nonspecific or subclinical form of the disease. Two months later, these animals were fully protected when exposed to other domestic pigs infected with a related virulent HAD genotype II ASFV.
Assuntos
Vírus da Febre Suína Africana/imunologia , Febre Suína Africana/virologia , Febre Suína Africana/epidemiologia , Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/isolamento & purificação , Vírus da Febre Suína Africana/patogenicidade , Animais , Infecções Assintomáticas , Proteção Cruzada , Europa (Continente)/epidemiologia , Genótipo , Letônia/epidemiologia , Sus scrofa , SuínosRESUMO
This study aims at assessing the serological cross-reactions existing between three mosquito-borne flaviviruses with avian reservoirs co-circulating in Europe: West Nile (WNV), Usutu (USUV) and Bagaza (BAGV). The study is useful for a better interpretation of serological results in diagnostics and surveillance. Serum samples obtained from a natural host, the red-legged partridge (Alectoris rufa), experimentally infected with WNV, USUV or BAGV were analysed using two commercially available WNV competition ELISAs suitable for serological surveillance, and by the confirmatory virus neutralization test (VNT). The ELISAs examined showed different levels of specificity for WNV, as judged by cross-reaction observed with the other flaviviruses. By VNT, virus-specific antibodies were confirmed in 80%, 50% or 0% of sera from WNV-, BAGV-, or USUV-inoculated birds, respectively. The results indicate how the co-circulation of cross-reacting flaviviruses may affect the outcomes of WNV serological surveillance when applying currently available serological tools. On the one hand, the choice of the ELISA test for antibody screening should consider the differences found in specificity, since one test is more specific for WNV while the other one is more suitable for detection of a broader range of flavivirus antibodies. On the other hand, besides corroborating that cross-neutralization occurs between flaviviruses from different serocomplexes (WNV/USUV and BAGV), this study points out that cross-neutralization between WNV and USUV is not symmetric, and reveals the difficulty to identify USUV infections serologically. This finding indicates that actual USUV infections might be underestimated in the current diagnostic schemes.
Assuntos
Infecções por Flavivirus/veterinária , Flavivirus/isolamento & purificação , Febre do Nilo Ocidental/epidemiologia , Vírus do Nilo Ocidental/isolamento & purificação , Animais , Anticorpos Antivirais/sangue , Aves/virologia , Reações Cruzadas , Culicidae/virologia , Ensaio de Imunoadsorção Enzimática/veterinária , Monitoramento Epidemiológico , Europa (Continente)RESUMO
Teschovirus and Sapelovirus are two genera of the Picornaviridae family, comprising highly variable and heterogeneous enteric viruses, commonly found in faecal samples from domestic pigs. Although both of them are also known to infect wild boar, studies on their presence in these wild suids are scarce. The present study aimed at determining the presence of porcine teschovirus (PTV) and sapelovirus (PSV) in free-living wild boar populations, as well as to study their relationships with similar viruses present in pigs. Fresh faecal samples (n=63) from wild boar were collected in Doñana Biological Reserve (SW Spain) during 2007 and 2011, and analysed using multiplex RT-PCR for the simultaneous detection and differentiation of PTV and PSV. A total of 32 samples (50.8%) presented positive PTV bands, while PSV amplicons were detected in 4 samples (6.4%). All PSV-positive samples were also positive for PTV, which indicated co-infection with both viruses. Virus isolation was successful from 6 samples, 4 of which were identified as PTV by RT-PCR, and three of these were further characterized by sequencing of the VP1 capsid protein. The remaining two isolates were negative for PTV or PSV. Genetic characterization of PSV-positive faecal samples, using the VP4 protein coding gene, was successful in 4 stool samples. Close phylogenetic relationship was found among wild boar and domestic pig strains in both PTV and PSV. More studies are needed to ascertain the epizootiological significance of these findings.
Assuntos
Coinfecção/veterinária , Fezes/virologia , Infecções por Picornaviridae/veterinária , Picornaviridae/isolamento & purificação , Sus scrofa/virologia , Doenças dos Suínos/virologia , Teschovirus/isolamento & purificação , Animais , Proteínas do Capsídeo/genética , Coinfecção/virologia , Filogenia , Picornaviridae/classificação , Picornaviridae/genética , Picornaviridae/fisiologia , Infecções por Picornaviridae/virologia , Espanha , Suínos , Doenças dos Suínos/genética , Teschovirus/genética , Teschovirus/fisiologiaRESUMO
Performance of a real-time reverse-transcription polymerase chain reaction method for the rapid, simple and reliable detection of porcine teschovirus (PTV) was assessed. The method was based on the use of a set of oligonucleotides consisting of two specific primers and a fluorogenic TaqMan-MGB probe. Reverse transcription and PCR reactions were performed sequentially in one step. As a result the whole procedure was simple and rapid, taking less than 3h for completion. The method reacted in a dose-dependent manner with prototype strains for the eleven known PTV serotypes (PTV1-11), with higher analytical sensitivity than other gel-based RT-PCR methods described, which were performed in parallel to allow for a comparison. The assay did not cross-react with other related viruses or porcine viruses tested. The diagnostic performance of the method was analyzed using a panel of field samples consisting of pig fecal and pig slurry samples. As a conclusion, this technique is adequate and convenient for porcine teschovirus detection, both for diagnosis as well as in environmental investigations.
Assuntos
Infecções por Picornaviridae/veterinária , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Doenças dos Suínos/virologia , Teschovirus/isolamento & purificação , Microbiologia da Água , Poluição da Água , Animais , Primers do DNA , Infecções por Picornaviridae/epidemiologia , Infecções por Picornaviridae/virologia , RNA Viral/análise , RNA Viral/genética , Sensibilidade e Especificidade , Sorotipagem , Suínos , Doenças dos Suínos/epidemiologia , Teschovirus/classificação , Teschovirus/genéticaRESUMO
Porcine teschoviruses (PTVs) have been previously shown to be the most abundant cytopathic viruses found in swine feces. In the present study, the diversity of PTVs was studied, using PTV isolates collected between 2004 and 2009 in a wide territory in Spain. In order to characterize genetically the isolates, phylogeny reconstructions were made using maximum likelihood and Bayesian inference methods, based on the 1D (VP1) gene, and including sequences available in public databases. The phylogenetic trees obtained indicated that PTVs present 12 main lineages, 11 corresponding to the PTV serotypes described to date, and one lineage distinct from the rest. The geographic distribution of the different lineages does not seem to be strongly associated to particular territories, and co-circulation of multiple lineages was found in the same geographic areas. Nevertheless, some spatial structuring of the viral populations studied is indicated by the differences found between Spanish samples with respect to other European countries. A coalescent-based approach indicated that mutation may have been the main factor in originating the genetic diversity observed in the VP1 gene region. This study revealed a high diversity of teschoviruses circulating in the pig populations studied, and showed that molecular analysis of the complete VP1 protein is a suitable method for the identification of members of the porcine teschovirus group. However, further analyses are needed to clarify the geographical structuring of the different PTV populations.