Presence of pandemic H1N1 influenza virus by qRT-PCR in samples collected at 3, 5, and 7 days post infection (dpi).^*

*<p>15 pigs were infected with either the A/CA/04/2009 (CA/09) or A/Mexico/4108/2009 (MX/09) pandemic H1N1 virus isolates. Number of pigs positive out of 5 is reported from each group euthanized on 3, 5, or 7 dpi; LN = inguinal lymph node tissue sample.</p

Table_1_Development of a Digital RT-PCR Method for Absolute Quantification of Bluetongue Virus in Field Samples.xlsx

Bluetongue (BT) is a major Office International des Epizooties (OIE)-listed disease of wild and domestic ruminants caused by several serotypes of Bluetongue virus (BTV), a virus with a segmented dsRNA genome belonging to the family Reoviridae, genus Orbivirus. BTV is transmitted through the bites of Culicoides midges. The aim of this study was to develop a new method for quantification of BTV Seg-10 by droplet digital RT-PCR (RTdd-PCR), using nucleic acids purified from complex matrices such as blood, tissues, and midges, that notoriously contain strong PCR inhibitors. First, RTdd-PCR was optimized by using RNAs purified from serially 10-fold dilutions of a BTV-1 isolate (105.43TCID50/ml up to 10−0.57 TCID50/ml) and from the same dilutions spiked into fresh ovine EDTA-blood and spleen homogenate. The method showed a good degree of linearity (R2 ≥ 0.995). The limit of detection (LoD) and the limit of quantification (LoQ) established were 10−0.67TCID50/ml (0.72 copies/μl) and 100.03TCID50/ml (3.05 copies/μl) of BTV-1, respectively. Second, the newly developed test was compared, using the same set of biological samples, to the quantitative RT-PCR (RT-qPCR) detecting Seg-10 assay widely used for the molecular diagnosis of BTV from field samples. Results showed a difference mean of 0.30 log between the two assays with these samples (p < 0.05). Anyway, the analysis of correlation demonstrated that both assays provided similar measurements with a very close agreement between the systems.</p

Presence of pandemic H1N1 influenza virus by virus isolation in samples collected at 3, 5, and 7 days post infection (dpi).^*

*<p>15 pigs were infected with either the A/CA/04/2009 (CA/09) or A/Mexico/4108/2009 (MX/09) pandemic H1N1 virus isolates. Number of pigs positive out of 5 is reported from each group euthanized on 3, 5, or 7 dpi; LN = inguinal lymph node tissue sample.</p

Neighbour-joining tree inferred from multiple nucleotide sequence alignment of the H encoding gene of selected CDV strains retrieved from GenBank.

<p>Sequences obtained in this study are marked with a circle.</p

Data on wild animals infected with CDV and detected by RT-PCR.

<p>CDV, canine distemper virus; CPV-2, canine parvovirus type 2. IHC, immunohystochemistry.</p

Map illustrating the distribution of CDV positive animals in Abruzzi region.

<p>Map was generated using Quantum GIS (QGIS) software, version 1.8.0.</p

Immunohistochemistry (IHC) for CDV on lung tissue of wolf Wa. Bronchiolar epithelial cells and inflammatory cells filling the airways show a strong and specific CDV immuno-reactivity.

<p>Scale bar = 100 µm.</p

Arctic Lineage-Canine Distemper Virus as a Cause of Death in Apennine Wolves (<i>Canis lupus</i>) in Italy

<div><p>Canine distemper virus (CDV) infection is a primary threat affecting a wide number of carnivore species, including wild animals. In January 2013, two carcasses of Apennine wolves (<i>Canis lupus</i>) were collected in Ortona dei Marsi (L'Aquila province, Italy) by the local Veterinary Services. CDV was immediately identified either by RT-PCR or immunohistochemistry in lung and central nervous tissue samples. At the same time, severe clinical signs consistent with CDV infection were identified and taped (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082356#pone.0082356.s001" target="_blank">Videos S1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082356#pone.0082356.s003" target="_blank">S3</a></b>) from three wolves rescued in the areas surrounding the National Parks of the Abruzzi region by the Veterinary Services. The samples collected from these symptomatic animals also turned out CDV positive by RT-PCR. So far, 30 carcasses of wolves were screened and CDV was detected in 20 of them. The sequencing of the haemagglutinin gene and subsequent phylogenetic analysis demonstrated that the identified virus belonged to the CDV Arctic lineage. Strains belonging to this lineage are known to circulate in Italy and in Eastern Europe amongst domestic dogs. To the best of our knowledge this is the first report of CDV Arctic lineage epidemics in the wild population in Europe.</p></div

Indirect immunofluorescence (IIF) for detection IgM antibodies performed on the serum sample of wolf W1.

<p>Scale bar = 100 µm.</p

Immunohistochemistry (IHC) for CDV on cerebral cortex tissue of wolf Wa. xNeurons from the cerebral cortex show a strong and specific CDV immuno-reactivity.

<p>Scale bar = 100 µm.</p