Genome Analysis of a Tembusu Virus, GX2013H, Isolated from a Cheery Valley Duck in Guangxi, China.

We report here the complete genome sequence of a duck Tembusu virus (DTMUV) strain, GX2013H, isolated from a duck from Cheery Valley in the Guangxi Province of southern China in 2013. We obtained the strain GX2013H from a Cheery Valley duck with severely decreased egg production and neurological signs. The genome of GX2013H is 10,990 nucleotides (nt) in length and contains a single open reading frame encoding a putative polyprotein of 3,425 amino acids (aa). A comparison of the complete sequence and the deduced amino acid sequence of GX2013H with published sequences of 15 other chicken anemia viruses from China showed that the homologies of the nucleotides are approximately 96.5% to 97.5% and the homologies of the deduced amino acid sequences are approximately 98.9% to 99.3%. This report will help to understand the epidemiology and molecular characteristics of TMUV in Guangxi.

Simultaneous typing of nine avian respiratory pathogens using a novel GeXP analyzer-based multiplex PCR assay.

A new, rapid, and high-throughput GenomeLab Gene Expression Profiler (GeXP) analyzer-based multiplex PCR method was developed for simultaneous detection and differentiation of nine avian respiratory pathogens. The respiratory pathogens included in this study were avian influenza subtypes H5, H7, and H9, infectious bronchitis virus (IBV), Newcastle disease virus (NDV), infectious laryngotracheitis virus (ILTV), Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS) and Haemophilus paragallinarum (HPG). Ten pairs of primers were designed using conserved and specific sequence genes of AIV subtypes and respiratory pathogens from GenBank. Single and mixed pathogen cDNA/DNA templates were used to evaluate the specificity of the GeXP-multiplex assay. The corresponding specific DNA products were amplified for each pathogen. The specific DNA product amplification peaks of nine respiratory pathogens were observed on the GeXP analyzer. Non-respiratory avian pathogens, including chicken infectious anemia virus, fowl adenovirus, avian reovirus and infectious bursal disease virus, did not produce DNA products. The detection limit for the GeXP-multiplex assay was determined to be 100 copies/µl using various pre-mixed plasmids/ssRNAs containing known target genes of the respiratory pathogens. Further, GeXP-multiplex PCR assay was 100% specific when 24 clinical samples with respiratory infections were tested in comparison with conventional PCR method. The GeXP-multiplex PCR assay provides a novel tool for simultaneous detection and differentiation of nine avian respiratory pathogens.

Detection of antibodies specific to the non-structural proteins of fowl adenoviruses in infected chickens but not in vaccinated chickens.

Antibodies specific to the non-structural proteins of viruses are detected in virus-infected animals and show promise as a reliable diagnostic marker for virus infections. We examined the potential use of two non-structural proteins of fowl adenovirus (FAdV)-based, 100K and 33K, enzyme-linked immunosorbent assays (ELISAs) in the diagnosis of FAdVs. We cloned and expressed the 100K and 33K non-structural protein genes of the FAdVs in the pGEX-4T-1 plasmid vector. Purified 100K and 33K proteins alone or in combination were used as antigens in ELISAs. Antibodies specific to the 100K and 33K non-structural proteins were detected in chickens experimentally infected with FAdVs, but not in chickens vaccinated with inactivated FAdVs. In contrast, the agar gel precipitation (AGP) test detected FAdV-specific antibodies in 70.3% of the vaccinated chickens, suggesting that the non-structural protein-based ELISA could be used in the differential diagnosis of infected and vaccinated chickens. To further validate the 100K and 33K-based ELISA (100K-33K-ELISA) method, we compared its sensitivity and specificity with that of a whole virus-based ELISA and an AGP test in detecting FAdV-specific antibodies in 350 field samples. The results showed that the 100K-33K-ELISA exhibited a higher sensitivity than the AGP test and a comparable sensitivity and specificity to the whole virus ELISA. Overall, the 100K-33K-ELISA method is sensitive, specific and can be used to distinguish an acute FAdV infection from an inactivated virus-based vaccination response.

Rapid detection of group I avian adenoviruses by a loop-mediated isothermal amplification.

A loop-mediated isothermal amplification (LAMP) assay was optimized for the rapid detection of Group I avian adenoviruses. A set of six primers was designed from the DNA sequences of hexon genes from Group I avian adenovirus. The assay was performed in a water bath for 60 min at 63 C, and the amplification result was visualized by adding a fluorescence dye reagent or by inspecting the white sediment. The results showed that the LAMP assay could detect all 12 serotypes of Group I avian adenovirus and nine Guangxi Group I avian adenovirus isolates. This avian adenovirus Group I-specific LAMP assay could detect 238 copies of avian adenovirus. No cross-reactions were detected using the LAMP assay with avian adenoviruses type II and III or with other avian viruses. The ability of LAMP to detect Group I avian adenovirus isolates was further evaluated with 184 cloacal swab samples from poultry. In total, 72 out of 184 cloacal swab samples from poultry were identified as positive by LAMP, whereas 45 out of 184 were identified as positive by conventional PCR test. The Group I avian adenovirus specific LAMP results were further confirmed by real-time PCR. This specific LAMP method holds promise as a rapid and specific diagnostic assay for detection of samples from birds suspected of adenovirus infection.

Development of a real-time multiplex PCR assay for detection of viral pathogens of penaeid shrimp.

A real-time multiplex polymerase chain reaction (rtm-PCR) assay was developed and optimized to simultaneously detect three viral pathogens of shrimp in one reaction. Three sets of specific oligonucleotide primers for white spot syndrome virus (WSSV), infectious hypodermal and haematopoietic necrosis virus (IHHNV) and Taura syndrome virus (TSV), along with three TaqMan probes specific for each virus were used in the assay. The rtm-PCR results were detected and analyzed using the Light Cycler 2.0 system. Forty-five PCR-positive samples and four negative samples were used to confirm the sensitivity and specificity of the rtm-PCR. The rtm-PCR identified and differentiated the three pathogens. With one viral infection of shrimp, a specific amplified standard curve was displayed. When samples from shrimp infected with two or three pathogens were analyzed, two or three specific standard curves were displayed. The sensitivity of the rtm-PCR assay was 2,000, 20, and 2,000 template copies for WSSV, IHHNV and TSV, respectively. No positive results (standard curves) were displayed when nucleic acid from Vibro spp., and Streptococcus spp. DNA were used as PCR templates. The results indicate that real-time multiplex PCR is able to detect the presence of and differentiate each pathogen in infected shrimp. This real-time multiplex PCR assay is a quick, sensitive, and specific test for detection of WSSV, IHHNV and TSV and will be useful for the control of these viruses in shrimp.

A multiplex RT-PCR for simultaneous differentiation of three viral pathogens of penaeid shrimp.

A multiplex reverse transcription polymerase chain reaction (mRT-PCR) was developed and optimized to simultaneously detect 3 viral pathogens of shrimp. Three sets of specific oligonucleotide primers for Taura syndrome virus (TSV), white spot syndrome virus (WSSV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV) were used in the assay. The mRT-PCR DNA products were visualized by gel electrophoresis and consisted of fragments of 231 bp for TSV, 593 bp for WSSV and 356 bp for IHHNV. No specific bands of the same size were amplified from other penaeid shrimp pathogenic viruses or bacteria. As little as 10 pg of TSV RNA and 100 pg of WSSV DNA and IHHNV DNA could be detected using gel electrophoresis. Studies are in progress to further test the specificity and sensitivity of this mRT-PCR method on viral isolates, as well as on clinical samples.