An enzyme-linked immunosorbent assay for detection of avian influenza virus subtypes H5 and H7 antibodies

BACKGROUND: Avian influenza virus (AIV) subtypes H5 and H7 attracts particular attention because of the risk of their potential pathogenicity in poultry. The haemagglutination inhibition (HI) test is widely used as subtype specific test for serological diagnostics despite the laborious nature of this method. However, enzyme-linked immunosorbent assays (ELISAs) are being explored as an alternative test method. H5 and H7 specific monoclonal antibodies were experimentally raised and used in the development of inhibition ELISAs for detection of serological response specifically directed against AIV subtypes H5 and H7. The ELISAs were evaluated with polyclonal chicken anti-AIV antibodies against AIV subtypes: H1N2, H5N2, H5N7, H7N1, H7N7, H9N9, H10N4 and H16N3. RESULTS: Both the H5 and H7 ELISA proved to have a high sensitivity and specificity and the ELISAs detected H5 and H7 antibodies earlier during experimental infection than the HI test did. The reproducibility of the ELISA’s performed at different times was high with Pearson correlation coefficients of 0.96-0.98. CONCLUSIONS: The ELISAs are a potential alternative to the HI test for screening of large amounts of avian sera, although only experimental sera were tested in this study

An international parentage and identification panel for the domestic cat (Felis catus)

Seventeen commercial and research laboratories participated in two comparison tests under the auspices of the International Society for Animal Genetics to develop an internationally tested, microsatellite-based parentage and identification panel for the domestic cat (Felis catus). Genetic marker selection was based on the polymorphism information content and allele ranges from seven random-bred populations (n = 261) from the USA, Europe and Brazil and eight breeds (n = 200) from the USA. Nineteen microsatellite markers were included in the comparison test and genotyped across the samples. Based on robustness and efficiency, nine autosomal microsatellite markers were ultimately selected as a single multiplex ‘core’ panel for cat identification and parentage testing. Most markers contained dinucleotide repeats. In addition to the autosomal markers, the panel included two gender-specific markers, amelogenin and zinc-finger XY, which produced genotypes for both the X and Y chromosomes. This international cat parentage and identification panel has a power of exclusion comparable to panels used in other species, ranging from 90.08% to 99.79% across breeds and 99.47% to 99.87% in random-bred cat populations

A single immunization with HA DNA vaccine by electroporation induces early protection against H5N1 avian influenza virus challenge in mice

<p>Abstract</p> <p>Background</p> <p>Developing vaccines for the prevention of human infection by H5N1 influenza viruses is an urgent task. DNA vaccines are a novel alternative to conventional vaccines and should contribute to the prophylaxis of emerging H5N1 virus. In this study, we assessed whether a single immunization with plasmid DNA expressing H5N1 hemagglutinin (HA) could provide early protection against lethal challenge in a mouse model.</p> <p>Methods</p> <p>Mice were immunized once with HA DNA at 3, 5, 7 days before a lethal challenge. The survival rate, virus titer in the lungs and change of body weight were assayed to evaluate the protective abilities of the vaccine. To test the humoral immune response induced by HA DNA, serum samples were collected through the eye canthus of mice on various days after immunization and examined for specific antibodies by ELISA and an HI assay. Splenocytes were isolated after the immunization to determine the antigen-specific T-cell response by the ELISPOT assay.</p> <p>Results</p> <p>Challenge experiments revealed that a single immunization of H5N1 virus HA DNA is effective in early protection against lethal homologous virus. Immunological analysis showed that an antigen-specific antibody and T-cell response could be elicited in mice shortly after the immunization. The protective abilities were correlated with the amount of injected DNA and the length of time after vaccination.</p> <p>Conclusion</p> <p>A single immunization of 100 μg H5 HA DNA vaccine combined with electroporation was able to provide early protection in mice against homologous virus infection.</p

Involvement of Cox-2 in the metastatic potential of chemotherapy-resistant breast cancer cells

<p>Abstract</p> <p>Background</p> <p>A major problem with the use of current chemotherapy regimens for several cancers, including breast cancer, is development of intrinsic or acquired drug resistance, which results in disease recurrence and metastasis. However, the mechanisms underlying this drug resistance are unknown. To study the molecular mechanisms underlying the invasive and metastatic activities of drug-resistant cancer cells, we generated a doxorubicin-resistant MCF-7 breast cancer cell line (MCF-7/DOX).</p> <p>Methods</p> <p>We used MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays, flow cytometry assays, DNA fragmentation assays, Western blot analysis, cell invasion assays, small interfering RNA (siRNA) transfection, reverse transcription-polymerase chain reaction, experimental lung metastasis models, and gelatin and fibrinogen/plasminogen zymography to study the molecular mechanism of metastatic activities in MCF-7/DOX cells.</p> <p>Results</p> <p>We found that MCF-7/DOX acquired invasive activities. In addition, Western blot analysis showed increased expression of epidermal growth factor receptor (EGFR) and Cox-2 in MCF-7/DOX cells. Inhibition of Cox-2, phosphoinositide 3-kinase (PI3K)/Akt, or mitogen-activated protein kinase (MAPK) pathways effectively inhibited the invasive activities of MCF-7/DOX cells. Gelatin and fibrinogen/plasminogen zymography analysis showed that the enzymatic activities of matrix metalloproteinase-2 (MMP-2), MMP-9, and urokinase-type plasminogen activator were markedly higher in MCF-7/DOX cells than in the MCF-7 cells. <it>In vitro </it>invasion assays and mouse models of lung metastasis demonstrated that MCF-7/DOX cells acquired invasive abilities. Using siRNAs and agonists specific for prostaglandin E (EP) receptors, we found that EP1 and EP3 played important roles in the invasiveness of MCF-7/DOX cells.</p> <p>Conclusions</p> <p>We found that the invasive activity of MCF-7/DOX cells is mediated by Cox-2, which is induced by the EGFR-activated PI3K/Akt and MAPK pathways. In addition, EP1 and EP3 are important in the Cox-2-induced invasion of MCF-7/DOX cells. Therefore, not only Cox-2 but also EP1 and EP3 could be important targets for chemosensitization and inhibition of metastasis in breast cancers that are resistant to chemotherapy.</p

Different Domains of the RNA Polymerase of Infectious Bursal Disease Virus Contribute to Virulence

BACKGROUND: Infectious bursal disease virus (IBDV) is a pathogen of worldwide significance to the poultry industry. IBDV has a bi-segmented double-stranded RNA genome. Segments A and B encode the capsid, ribonucleoprotein and non-structural proteins, or the virus polymerase (RdRp), respectively. Since the late eighties, very virulent (vv) IBDV strains have emerged in Europe inducing up to 60% mortality. Although some progress has been made in understanding the molecular biology of IBDV, the molecular basis for the pathogenicity of vvIBDV is still not fully understood. METHODOLOGY, PRINCIPAL FINDINGS: Strain 88180 belongs to a lineage of pathogenic IBDV phylogenetically related to vvIBDV. By reverse genetics, we rescued a molecular clone (mc88180), as pathogenic as its parent strain. To study the molecular basis for 88180 pathogenicity, we constructed and characterized in vivo reassortant or mosaic recombinant viruses derived from the 88180 and the attenuated Cu-1 IBDV strains. The reassortant virus rescued from segments A of 88180 (A88) and B of Cu-1 (BCU1) was milder than mc88180 showing that segment B is involved in 88180 pathogenicity. Next, the exchange of different regions of BCU1 with their counterparts in B88 in association with A88 did not fully restore a virulence equivalent to mc88180. This demonstrated that several regions if not the whole B88 are essential for the in vivo pathogenicity of 88180. CONCLUSION, SIGNIFICANCE: The present results show that different domains of the RdRp, are essential for the in vivo pathogenicity of IBDV, independently of the replication efficiency of the mosaic viruses

A microsatellite baseline for genetic stock identification of European Atlantic salmon (Salmo salar L.)

Atlantic salmon (Salmo salar L.) populations from different river origins mix in the North Atlantic during the marine life stage. To facilitate marine stock identification, we developed a genetic baseline covering the European component of the species’ range excluding the Baltic Sea, from the Russian River Megra in the north-east, the Icelandic Ellidaar in the west, and the Spanish Ulla in the south, spanning 3737 km North to South and 2717 km East to West. The baseline encompasses data for 14 microsatellites for 26 822 individual fish from 13 countries, 282 rivers, and 467 sampling sites. A hierarchy of regional genetic assignment units was defined using a combination of distance-based and Bayesian clustering. At the top level, three assignment units were identified comprising northern, southern, and Icelandic regions. A second assignment level was also defined, comprising eighteen and twenty-nine regional units for accurate individual assignment and mixed stock estimates respectively. The baseline provides the most comprehensive geographical coverage for an Atlantic salmon genetic data-set, and a unique resource for the conservation and management of the species in Europe. It is freely available to researchers to facilitate identification of the natal origin of European salmon

Isolation and characterization of microsatellite loci from Iguana delicatissima (Reptilia: Iguanidae), new perspectives for investigation of hybridization events with Iguana iguana

International audienceA (GA)n and (GT)n microsatellite-enriched library was constructed and 25 nuclear simple sequence repeat (SSR) loci were characterized in the Lesser Antillean Iguana (Iguana delicatissima). All SSR loci were found to be polymorphic after screening for diversity in different cultivars, and a cross-taxa amplification tests showed the potential transferability of most SSR markers in Iguana iguana. First to be published for I. delicatissima, this new SSR resource will be a powerful tool for intraspecific genetic studies and for investigation of hybridization events with Iguana iguana

Compared pathogenicity of mc88180, mcCu-1, A88BCU1 and ACU1B88 in seven-week old SPF chickens (experiment <i>ii</i>).

<p>On the first day of the experiment, SPF chickens were housed in five groups of 20 chickens of comparable sex and weight. On the same day, each bird was inoculated by the intranasal route with a standardized dose of the relevant virus (10⁵ EID₅₀ by bird, except for the ACU1 B88 virus which was inoculated with 10^3.6 EID₅₀ by bird). A group was kept as a mock-inoculated control receiving PBS only. See in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028064#pone.0028064.s003" target="_blank">Table S3</a> the summarized protocol of the animal experiment. <b>A</b>) % cumulated mortality at 5 days post infection, <b>B</b>) BF to body weights ratio (‰) at day 20 (n = 7 to 10 chickens depending on the group). The box plots show the median (horizontal line) flanked by the 2^nd and 3^rd quartile. The outer bars show the range of values. <b>C</b>) and <b>D</b>) Histological lesion scoring at day 4 and 20, respectively (n = 5 chickens per group). The outer bars show the range of values. <b>E</b>) VN titer at day 20 (in log₂, n = 10 chickens per group). <b>F</b>) Virus titer at day 4 (in log₁₀ EID_50/gr, n = 5 chickens per group). Treatments sharing the same lowercase letter do not differ significantly according to the kruskal-Wallis test (BF to body weights ratio, lesion score, VN titer and virus titer) or according to the Chi-squared test (% mortality), at the p≤0.05 confidence level.</p

Compared pathogenicity of recombinant or wild type IBDV strains mc88180, 88180, F52/70 and 89163 in six-week old SPF chickens (experiment <i>i</i>).

<p>On the first day of the experiment, SPF chickens were housed in five groups of 30 chickens of comparable sex and weight. On the same day, each bird was inoculated by the intranasal route with a standardized dose of the relevant virus (10⁵ EID₅₀ by bird). A group was kept as a mock-inoculated control receiving PBS only. See in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028064#pone.0028064.s003" target="_blank">Table S3</a> the summarized protocol of the animal experiment. <b>A</b>) % cumulated mortality at 5 days post infection, <b>B</b>) BF to body weights ratio (‰) at day 17 (n = 21 to 25 chickens depending on the group). The box plots show the median (horizontal line) flanked by the 2^nd and 3^rd quartile. The outer bars show the range of values. <b>C</b>) and <b>D</b>) Histological lesion scoring at day 4 and 17, respectively (n = 5 to 6 chickens per group). The outer bars show the range of values. <b>E</b>) VN titer at day 17 (in log₂, n = 21 to 25 chickens depending on the group). <b>F</b>) virus titer at day 4 post infection (in log₁₀ EID_50/gr, n = 5 chickens per group). Treatments sharing the same lowercase letter do not differ significantly according to the kruskal-Wallis test (BF to body weights ratio, lesion score, VN titer and virus titer) or according to the Chi-squared test (% mortality), at the p≤0.05 confidence level.</p

Compared pathogenicity of mc88180, A88BCU1 and five recombinant viruses with mosaic segment B in five-week-old SPF chickens (experiment <i>iii</i>).

<p>See in Table S1 and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028064#pone-0028064-g001" target="_blank">Figure 1</a> the genetic make up of the viruses with mosaic segment B and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028064#pone.0028064.s003" target="_blank">Table S3</a> the summarized protocol of the animal experiment. On the first day of the experiment, SPF chickens were housed in eight groups of 20 chickens of comparable sex and weight. On the same day, each bird was inoculated by the intranasal route with a standardized dose of the relevant virus (10⁵ EID₅₀ by bird). A group was kept as a mock-inoculated control receiving PBS only. <b>A</b>) % cumulated mortality at 5 days post infection, <b>B</b>) BF to body weights ratio (‰) at day 21 (n = 10 chickens per group). The box plots show the median (horizontal line) flanked by the 2^nd and 3^rd quartile. The outer bars show the range of values. <b>C</b>) and <b>D</b>) Histological lesion scoring at day 4 and 21, respectively (n = 5 chickens per group). The outer bars show the range of values. <b>E</b>) VN titer at day 21 (in log₂, n = 8 to 10 chickens depending on the group). <b>F</b>) Virus titer at day 4 (in log₁₀ EID_50/gr, n = 5 chickens per group). Treatments sharing the same lowercase letter do not differ significantly according to the kruskal-Wallis test (BF to body weights ratio, lesion score, VN titer and virus titer) or according to the Chi-squared test (% mortality), at the p≤0.05 confidence level.</p