Presentation_1_A Novel Diagnostic Method to Detect Duck Tembusu Virus: A Colloidal Gold-Based Immunochromatographic Assay.pdf

<p>Duck Tembusu virus (DTMUV) is an emerging pathogenic flavivirus that has resulted in large economic losses to the duck-rearing industry in China since 2010. Therefore, an effective diagnostic approach to monitor the spread of DTMUV is necessary. Here, a novel diagnostic immunochromatographic strip (ICS) assay was developed to detect DTMUV. The assay was carried out using colloidal gold coated with purified monoclonal antibody A12D3 against envelope E protein. Purified polyclonal C12D1 antibodies from BALB/c mice against the envelope E protein were used as the capture antibody. Goat anti-mouse IgG was used to detect DTMUV, which was also assembled on the ICS. Results showed that the ICS could specifically detect DTMUV within 10 min. It also could be stored 25 and 4°C for 4 and 6 months, respectively. The sensitivity of the ICS indicated that the dilution multiples of positive allantoic fluid of DTMUV (LD50: 10^4.33/0.2 ml) was up to 200. Its specificity and sensibility showed no significant change under the above storage situations. Fifty clinical samples were simultaneously detected by ICS and reverse-transcription polymerase chain reaction with a 93.9% coincidence rate between them. It proved that the ICS in the present study was highly specific, sensitive, repeatable, and more convenient to rapidly detect DTMUV in clinical samples.</p

Primary screening of epitope with mAb 3G2.

<p>One 16-AA polypeptide of TMUV NS1 protein (NS1-27) was screened with mAb 3G2 by indirect ELISA. Mouse serum against TMUV NS1 protein and normal mouse serum were used as positive and negative controls, respectively. Each sample was detected in triplicate. Error bars were expressed as standard deviation of the means (n = 3). The mean value was statistically significant, calculated by the two-tailed Student’s unpaired t-test (*P < 0.05).</p

Western-blot identification of epitope.

<p>The 16-AA polypeptide of NS1-27 reacted with mAb 3G2 in Western-blot assay. Lane M, PageRuler Prestained Protein Ladder (Fermentas, Canada); Lane 1, GST-tag didn’t react with mAb 3G2; Lane 2, The band of NS1-27-GST fusion protein was visualized with mAb 3G2.</p

Sequences of the overlapping polypeptides from TMUV NS1 (SDSG strain, Accession number: KJ740747.1).

<p>Sequences of the overlapping polypeptides from TMUV NS1 (SDSG strain, Accession number: KJ740747.1).</p

The accurate mapping of one B cell epitope with mAb.

<p>NS1-27 polypeptide was truncated from the carboxy and amino terminals. After the truncated peptides were expressed as a GST fusion protein, they were probed with mAb 3G2 by indirect ELISA respectively. The minimal unit of the peptide was the sequence of 8 AA and 3 AA truncated from the carboxy and amino terminals of NS1-27.</p

IFA and western-blot identification of mAb 3G2.

<p>A: Western-blot identification Lane 1, Control, GST-tag didn’t react with mAb 3G2; Lane 2, The band of NS1-GST fusion protein was reacted with mAb 3G2; Lane M, Blue plusIIprotein Marker (14-120kda, Transgen Biotech). B: IFA identification Monoclonal antibody against TMUV NS1 protein was used to perform IFA on TMUV-infected BHK-21 cells. BHK-21 cells infected with TMUV yielded significant fluorescence with six MAbs in the cytoplasm; Control BHK-21 cells didn’t yield any fluorescence.</p

Sequences of the truncated NS1-27 polypepide.

<p>Sequences of the truncated NS1-27 polypepide.</p

Additional file 1: of Preparation and evaluation of goose reovirus inactivated vaccine

Alignment data of the sequence among JS-01 strain and other reovirus strains. (TXT 61 kb

Epidemiological investigation of fowl adenovirus (FAdV) infections in ducks and geese in Shandong Province, China

Outbreaks of fowl adenoviruses (FAdVs) infection with the main clinical signs of hepatitis-hydropericardium-syndrome or inclusion body hepatitis have been frequently reported in ducks and geese in recent years, causing economic losses for the Chinese waterfowl industry. This study investigated 792 samples (391 fattening ducks, 192 breeder ducks and 209 fattening geese) of suspected FAdV-infected waterfowl from 38 farms (21 fattening duck farms, nine breeder duck farms and eight fattening geese farms) in Shandong Province between 2019 and 2022. The results showed a 60.23% infection rate for FAdVs (477/792), while the infection rate for breeder ducks was almost the same as that for fattening geese (55.73% vs. 54.55%). Notably, co-infection with avian influenza virus H9N2 (H9N2), Tembusu virus (TMUV), duck hepatitis virus (DHV), duck circovirus (DuCV), goose astrovirus (GAstV) and duck parvovirus (DPV)/goose parvovirus (GPV) was common among the 477 FAdVs positive cases. Phylogenetic analysis of complete hexon genes of 22 FAdV strains in Shandong Province showed the presence of four species (FAdV-A, C, D, E) and five serotypes (FAdV-1, 4, 8a, 8b, 11). These findings provide the first data on the prevalence and co-infection status of FAdVs in Shandong Province and can be used as a basis for FAdV prevention in the field. Samples of suspected FAdV-infected waterfowl from farms in Shandong Province were collected from 2019 to 2022.Single infections with FAdV were less frequent than mixed infections.477 out of 792 samples (60.23%) tested positive for FAdV nucleic acids.Detection rate of FAdV was 65.47% in fattening duck farms, 55.73% in breeder duck farms and 54.55% in fattening geese farms. Samples of suspected FAdV-infected waterfowl from farms in Shandong Province were collected from 2019 to 2022. Single infections with FAdV were less frequent than mixed infections. 477 out of 792 samples (60.23%) tested positive for FAdV nucleic acids. Detection rate of FAdV was 65.47% in fattening duck farms, 55.73% in breeder duck farms and 54.55% in fattening geese farms.</p

Sequence alignments of Parvovirus PLA₂ Motifs and sPLA₂ representatives between SDLC01 strain and Adeno-associated virus -2.

<p>Sequence alignments of Parvovirus PLA₂ Motifs and sPLA₂ representatives between SDLC01 strain and Adeno-associated virus -2.</p