A survey of porcine picornaviruses and adenoviruses in fecal samples in Spain.

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.

Real-time fluorogenic reverse transcription polymerase chain reaction assay for detection of African horse sickness virus.

African horse sickness is an arthropod-borne disease of the equine included in the World Organization for Animal Health (OIE) list with important economic consequences for horse trade. The disease is caused by African horse sickness virus (AHSV; family Reoviridae, genus Orbivirus), which is transmitted by Culicoides midges. It is endemic in sub-Saharan Africa, spreading occasionally outside this area where the occurrence of Culicoides vectors allows virus transmission. Currently, only conventional (gel-based) reverse transcription polymerase chain reaction (RT-PCR) protocols are available for its detection; however, these methods are cumbersome and difficult to apply when large numbers of samples are to be tested, as in the case of epizootics. To overcome this problem, a real-time RT-PCR method has been developed, based on a 5'-Taq nuclease-3'-minor groove binder-DNA probe (TaqMan MGB) for detection of a wide range of AHSV serotypes and strains designed to the highly conserved region of the VP7 gene (segment 7). The method was able to detect all prototype strains from the 9 known serotypes of the virus, with a high analytical sensitivity; no cross-reactions were observed with other orbiviruses or with other viruses affecting horses. The diagnostic sensitivity was assessed using a panel of AHSV-positive tissue samples from an epizootic that occurred in Spain between 1987 and 1990. This method, which can be performed in 96-well format, is suitable for large-scale surveillance of AHSV in areas where it can potentially spread.

A fully automated procedure for the high-throughput detection of avian influenza virus by real-time reverse transcription-polymerase chain reaction.

The recent spread of highly pathogenic H5N1 avian influenza (AI) has made it important to develop highly sensitive diagnostic systems for the rapid detection of AI genome and the differentiation of H5N1 variants in a high number of samples. In the present paper, we describe a high-throughput procedure that combines automated extraction, amplification, and detection of AI RNA, by an already described TaqMan real-time reverse transcription-polymerase chain reaction (RRT-PCR) assay targeted at the matrix (M) protein gene of AI virus (AIV). The method was tested in cloacal and tracheal swabs, the most common type of samples used in AI surveillance, as well as in tissue and fecal samples. A robotic system (QIAGEN Biosprint 96) extracted RNA and set up reactions for RRT-PCR in a 96-well format. The recovery of the extracted RNA was as efficient as that of a manual RNA extraction kit, and the sensitivity of the detection system was as high as with previously described nonautomated methods. A system with a basic configuration (one extraction robot plus two real-time 96-well thermocyclers) operated by two persons could account for about 360 samples in 5 hr. Further characterization of AI RNA-positive samples with a TaqMan RRT-PCR specific for H5 (also described here) and/or N1 was possible within 2 hr more. As this work shows, the system can analyze up to 1400 samples per working day by using two nucleic acid extraction robots and a 384-well-format thermocycler.

A real-time TaqMan RT-PCR method for neuraminidase type 1 (N1) gene detection of H5N1 Eurasian strains of avian influenza virus.

This work describes the development of a real-time RT-PCR (RRT-PCR) procedure for detection of the N1 gene from avian influenza virus (AIV), based on the use of specific primers and a TaqMan-MGB (minor groove binder) probe. Nucleotide sequences of the neuraminidase type 1 gene from a collection of H5N1 Eurasian strains of AIV were aligned using ClustalW software. Conserved regions were located and used to design specific primers and a TaqMan-MGB probe using Primer Express software. A one-step RRT-PCR method was optimized using RNA from the Turkey 2005 H5N1 strain of AIV and can be completed in about 2 hr once the RNA is extracted from the sample. The specificity of the assay was assessed with non-N1 AIV strains, another related avian virus, and different avian tissue samples from healthy animals. Sensitivity was determined using 10-fold serial dilutions of the H5N1 Turkey 2005 strain and was compared with the generic RRT-PCR detection method, targeted at the matrix protein gene of AIV, commonly used at the Spanish AIV National Reference Laboratory. The N1 detection method proved to be even more sensitive than the generic (matrix-based) method, allowing a very quick confirmation (or discarding) of any Eurasian N1 strain when a positive result was obtained with the matrix RRT-PCR assay. Combined with RRT-PCR tests for general detection of AIV and H5 typing in use at the NRL, the procedure here described allows characterizing of any H5N1 Eurasian AIV strain in a field sample within a working day.

A new fluorogenic real-time RT-PCR assay for detection of lineage 1 and lineage 2 West Nile viruses.

West Nile virus represents an emerging threat for animal and human health worldwide. This virus exhibits a marked genetic variation, with at least 2 distinct evolutionary lineages. Lineage 1 has been recognized in Africa, Asia, Europe, Oceania, and more recently in the Americas, whereas lineage 2 is restricted to Africa. Perhaps for this reason, the available real-time RT-PCR methods for detecting West Nile virus genome have mainly focused on lineage 1. However, both viruses may potentially be spread beyond their endemic areas by migratory birds. This report describes a new real-time reverse transcription-PCR (RT-PCR) method based on a 5'-Taq nuclease-3' minor groove binder DNA probe (TaqMan MGB) that allows the detection of a wide range of West Nile virus isolates, including both lineages 1 and 2. This method was able to detect West Nile viruses from different origins (North and Central Africa, Middle East, Europe, and North America), whereas other flaviviruses (Usutu, Dengue, Yellow fever) analyzed in parallel remained negative. The sensitivity achieved by this assay was 10(-2)-10(-3) pfu/tube. This method, which can be performed in 96-well format, could be suitable for the large-scale surveillance of West Nile virus in areas where both lineages can potentially spread.

High throughput detection of bluetongue virus by a new real-time fluorogenic reverse transcription-polymerase chain reaction: application on clinical samples from current Mediterranean outbreaks.

A real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for the detection of bluetongue virus (BTV) in blood samples. A combination of primers specific for a highly conserved region in RNA segment 5 (based on Mediterranean BTV sequences) and a DNA probe bound to 5'-Taq nuclease-3' minor groove binder (TaqMan MGB) was used to detect a range of isolates. This real-time RT-PCR assay could detect 5.4 x 10(-3) tissue culture infectious doses (TCID50) of virus per milliliter of sample, which was comparable to our current BTV diagnostic nested RT-PCR assay. The assay detected all recent Mediterranean isolates (including serotypes 2, 4, and 16), BTV vaccine strains for serotypes 2 and 4, and 15 out of the 24 BTV reference strains available (all serotypes), but did not detect the related orbiviruses epizootic hemorrhagic disease and African horse sickness viruses. Following assay evaluation, the ability of this assay to identify BTV in recent isolates (2003, 2004) from ovine and bovine samples from an epizootic outbreak in Spain was also tested. Minor nucleotide changes (detected by sequencing viral genomes) within the probe-binding region were found to have a profound effect on virus detection. This assay has the benefits of being fast and simple, and the 96-well format enables large-scale epidemiological screening for BTV, especially when combined with a high-throughput nucleic acid extraction method.

Analyzing the genetic diversity of teschoviruses in Spanish pig populations using complete VP1 sequences.

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.

Prevalence of West Nile virus neutralizing antibodies in colonial aquatic birds in southern Spain.

The rapid expansion of West Nile virus (WNV) throughout the New World has raised interest in understanding the population dynamics and patterns of dispersal of emerging infectious diseases by wildlife. WNV affects humans, although its main reservoirs are various species of birds. Here we analyse the prevalence of WNV-neutralizing antibodies in nearly full-grown chicks belonging to seven different species of colonial waterbirds at three localities in southern Spain. Chicks with neutralizing antibodies against WNV were detected in three species and at all three localities. However, the low antibody titres suggest the presence of antibodies is probably due to maternal transfer of antibody, presumably from exposure of the adult birds to WNV or a similar flavivirus at some stage of their lives. The analyses of the movements of tagged birds confirmed that all species with antibody visit regions that have had reports of WNV infection over the past decade.

Seroconversion in wild birds and local circulation of West Nile virus, Spain.

A serosurvey for neutralizing antibodies against West Nile virus (WNV) in common coots (Fulica atra) was conducted in Doñana, Spain. Antibody prevalence was highest in 2003, intermediate in 2004, and lowest in 2005. Some birds seroreverted <1 year after first capture. Seroconversion of birds suggests local circulation of the virus.