Myxoma virus jumps species to the Iberian hare.

The study of myxoma virus (MYXV) infections in the European rabbit (Oryctolagus cuniculus) has produced one of the most accepted host-pathogen evolutionary models. To date, myxomatosis has been limited to the European rabbit with sporadic reports in hares. However, reports of widespread mortalities in the Iberian hare (Lepus granatensis) with myxomatosis-like clinical signs indicate a potential species jump has occurred. The presence of MYXV DNA was confirmed by PCR in 244 samples received from regional veterinary services, animal health laboratories, hunters or rangers over a 5-month period. PCR analysis of 4 MYXV positive hare samples revealed a 2.8 kb insertion located within the M009 gene with respect to MYXV. The presence of this insertion was subsequently confirmed in 20 samples from 18 Spanish provinces. Sanger sequencing and subsequent analysis show that the insert contained 4 ORFs which are phylogenetically related to MYXV genes M060, M061, M064 and M065. The complete MYXV genome from hare tissue was sequenced using Ion torrent next-generation technology and a summary of the data presented here. With the exception of the inserted region, the virus genome had no large scale modifications and 110 mutations with respect to the MYXV reference strain Lausanne were observed. The next phase in the evolution of MYXV has taken place as a host species jump from the European rabbit to the Iberian hare an occurrence which could have important effects on this naïve population.

Rapid molecular haemagglutinin subtyping of avian influenza isolates by specific real-time RT-PCR tests.

Sixteen haemagglutinin (HA) subtypes of avian influenza viruses (AIV) have been described to date. Rapid subtype identification of any AIV is of major interest because of the possible serious consequences for the poultry industry and even public health. Molecular techniques currently allow immediate accurate subtype characterisation prior to virus isolation. In this study, a set of fourteen specific real-time RT-PCR methods were developed and evaluated for AIV HA subtyping (H1-H4, H6-H8, H10-H16), H5 and H9 being excluded on the basis of the current validity of the European Union (EU) recommended specific assays. Specific primers and probes sets for each HA-subtype were designed to hybridise the largest isolates range within each single subtype, considering the Eurasian lineage as a major target. The robustness and general application of the 14 HA-subtype methods were verified by the analysis of 110 AIV isolates belonging to all 16 HA-subtypes, performed in different laboratories. The developed real-time RT-PCR assays proved to be highly specific and revealed suitable sensitivity, allowing direct HA-subtyping of clinical material. In summary, this study provides for the first time a panel of molecular tests using specific hydrolysis probes for rapid and complete AIV HA-subtype identification.

Real-time fluorogenic reverse transcription polymerase chain reaction assay for the specific detection of Bagaza virus.

In September 2010, an outbreak of disease in 2 wild bird species (red-legged partridge, Alectoris rufa; ring-necked pheasant, Phasianus colchicus) occurred in southern Spain. Bagaza virus (BAGV) was identified as the etiological agent of the outbreak. BAGV had only been reported before in Western Africa (Central African Republic, Senegal) and in India. The first occurrence of BAGV in Spain stimulated a demand for rapid, reliable, and efficacious diagnostic methods to facilitate the surveillance of this disease in the field. This report describes a real-time reverse transcription polymerase chain reaction (RT-PCR) method based on a commercial 5'-Taq nuclease-3' minor groove binder DNA probe and primers targeting the Bagaza NS5 gene. The method allowed the detection of BAGV with a high sensitivity, whereas other closely related flaviviruses (Usutu virus, West Nile virus, and Japanese encephalitis virus) were not detected. The assay was evaluated using field samples of red-legged partridges dead during the outbreak (n = 11), as well as samples collected from partridges during surveillance programs (n = 81). The results were compared to those obtained with a pan-flaviviral hemi-nested RT-PCR followed by nucleotide sequencing, which was employed originally to identify the virus involved in the outbreak. The results obtained with both techniques were 100% matching, indicating that the newly developed real-time RT-PCR is a valid technique for BAGV genome detection, useful in both diagnosis and surveillance studies.

Bagaza virus in partridges and pheasants, Spain, 2010.

In September 2010, an unusually high number of wild birds (partridges and pheasants) died in Cádiz in southwestern Spain. Reverse transcription PCR and virus isolation detected flavivirus infections. Complete nucleotide sequence analysis identified Bagaza virus, a flavivirus with a known distribution that includes sub-Saharan Africa and India, as the causative agent.

Evaluation of a fluorogenic real-time reverse transcription-polymerase chain reaction method for the specific detection of all known serotypes of porcine teschoviruses.

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.

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.