Huabin Shao

Genome Sequence of a Fowl Adenovirus Serotype 4 Strain Lethal to Chickens, Isolated from China

ABSTRACT We report here the complete genome sequence of virulent fowl adenovirus serotype 4 strain HB1510, isolated from a diseased chicken with hydropericardium-hepatitis syndrome in Hubei, China, in 2015. The viral genome is 43,721 bp long, and sequence analysis showed an 11-amino-acid deletion in open reading frame 29 (ORF29).

Complete genome sequence and molecular characterization of thermostable Newcastle disease virus strain TS09-C

A thermostable avirulent Newcastle disease virus (NDV) strain TS09-C was developed by serial passage of V4 strain in BHK-21 cells. The complete genome sequence of strain TS09-C was determined and compared with the sequences of other NDV isolates representing different thermostable phenotypes. The TS09-C genome was 15,186 nucleotides long and consisted of eight genes in the order of 3′-NP-P/V/W-M-F-HN-L-5′. High levels of nucleotide and amino acid sequence identity existed among the thermostable NDV isolates. Thermostable strains TS09-C, V4, I-2, and Ulster all belonged to genotype I. The F protein cleavage site of strain TS09-C was 112G-K-Q-R–R-L117, with an isolated basic amino acid and a pair of contiguous basic amino acids, differing from that of its parental V4 strain (112G-K-Q-G-R-L117). Our characterization of the complete genome of thermostable avirulent NDV strain TS09-C may facilitate the development of a thermostable NDV reverse genetic system and further study of the mechanism of thermostability of NDV.

A one-step real-time reverse transcription-polymerase chain reaction detection of classical swine fever virus using a minor groove binding probe

The aim of this study was to develop a one-step real-time reverse transcription-polymerase chain reaction assay using the minor groove binding probe (MGB rRT-PCR) for rapid and quantitative detection of classical swine fever virus (CSFV). The method, which targets the 5′-nontranslated region (5′NTR) of the viral genome, detected all CSFV isolate tested, but not heterologous pathogens. Using an in vitro transcript of the 5′NTR as a quantitative standard for the CSFV genome copy number, the assay had a detection limit of 10 copies/reaction, and the standard curve had a linear range from 10 to 107 copies/reaction, with good reproducibility. As determined by an end-point dilution comparison, in most case, the sensitivity of the MGB rRT-PCR was approximately 10-fold higher than that of virus isolation and the rRT-PCR using the standard Taqman probe (standard rRT-PCR). The agreement between the MGB rRT-PCR and standard rRT-PCR, or virus isolation was 93.3% and 76.7%, respectively, when detecting 261 field samples. Due to its rapidity, high specificity and sensitivity, the MGB rRT-PCR assay provides a valuable tool for diagnosis and molecular studies of CSFV biology.

Correlation between gyrA and CmeR Box Polymorphism and Fluoroquinolone Resistance in Campylobacter jejuni Isolates in China

ABSTRACT Sequence analysis of 79 ciprofloxacin-resistant Campylobacter jejuni isolates collected in China showed resistance-related sequence variations in gyrA and CmeR-Box. All the isolates contain an identical Thr-86-Ile substitution in GyrA. Several novel CmeR-Box variations, including point substitutions, deletion, and insertion, were identified. The point insertion or deletion led to dramatically reduced binding of CmeR to the cmeABC promoter, which significantly increases the expression of cmeABC and contributes to the high fluoroquinolone resistance.

Molecular basis for the thermostability of Newcastle disease virus.

Thermostable Newcastle disease virus (NDV) vaccines have been used widely to protect village chickens against Newcastle disease, due to their decreased dependence on cold chain for transport and storage. However, the genetic basis underlying the NDV thermostability is poorly understood. In this study, we generated chimeric viruses by exchanging viral genes between the thermostable TS09-C strain and thermolabile LaSota strain using reverse genetics technology. Evaluations of these chimeric NDVs demonstrated that the thermostability of NDV was dependent on the origin of HN protein. Chimeras bearing the HN protein derived from thermostable virus exhibited a thermostable phenotype, and vice versa. Both hemagglutinin and neuraminidase activities of viruses bearing the TS09-C HN protein were more thermostable than those containing LaSota HN protein. Furthermore, the newly developed thermostable virus rLS-T-HN, encoding the TS09-C HN protein in LaSota backbone, induced significantly higher antibody response than the TS09-C virus, and conferred complete protection against virulent NDV challenge. Taken together, the data suggest that the HN protein of NDV is a crucial determinant of thermostability, and the HN gene from a thermostable NDV could be engineered into a thermolabile NDV vaccine strain for developing novel thermostable NDV vaccine.

The roles of RelA/(p)ppGpp in glucose-starvation induced adaptive response in the zoonotic Streptococcus suis

The (p)ppGpp signal molecules play a central role in the stringent response (SR) to adapt to nutrient starvation in bacteria, yet the carbohydrate starvation induced adaptive response and the roles of SR in this response is not well characterized, especially in Gram-positives. Here, two (p)ppGpp synthetases RelA and RelQ are identified in Streptococcus suis, an important emerging zoonotic Gram-positive bacterium, while only RelA is functional under glucose starvation. To characterize the roles of RelA/(p)ppGpp in glucose starvation response in S. suis, the growth curves and transcriptional profiles were compared between the mutant strain ΔrelA [a (p)ppGpp0 strain under glucose starvation] and its parental strain SC-19 [(p)ppGpp+]. The results showed great difference between SC-19 and ΔrelA on adaptive responses when suffering glucose starvation, and demonstrated that RelA/(p)ppGpp plays important roles in adaptation to glucose starvation. Besides the classic SR including inhibition of growth and related macromolecular synthesis, the extended adaptive response also includes inhibited glycolysis, and carbon catabolite repression (CCR)-mediated carbohydrate-dependent metabolic switches. Collectively, the pheno- and genotypic characterization of the glucose starvation induced adaptive response in S. suis makes a great contribution to understanding better the mechanism of SR.

Genome Sequence of a Virulent Genotype III Newcastle Disease Virus Isolated from Laying Ducks in China

ABSTRACT Here, we report the complete genome sequence of a virulent Newcastle disease virus (NDV) strain HN1007, isolated from diseased duck flocks in Henan, China, in 2010. The isolate has a genome length of 15,186 nucleotides, and was classified as a member of genotype III of class II.

Evaluation of a real-time RT-PCR assay using minor groove binding probe for specific detection of Chinese wild-type classical swine fever virus

A one-step real-time RT-PCR assay using a minor groove binding probe was developed for the specific detection of Chinese wild-type classical swine fever virus (CSFV). The assay detected wild-type CSFV strains representing different genotypes, but did not amplify viral RNA from the Hog Cholera Lipinized Virus (HCLV) vaccine-strain and other porcine viruses. The assay had a detection limit of 10 copies/reaction or 3.0 median tissue culture infective dose/reaction. In comparison to the sequencing nested RT-PCR assay, the sensitivity and specificity of the assay were 98.3% and 94.3%, respectively, when testing 515 veterinary samples. Wild-type CSFV RNA was detected in nasal swabs 2-4 days before detection in serum samples from pigs exposed to infection by contact, and 2-4 days prior to the onset of clinical disease. HCLV RNA remained undetectable in nasal swabs and serum samples from vaccinated pigs. In conclusion, the novel assay described in this study provides a rapid and sensitive method for differentiating between wild-type and the HCLV-strain of CSFV. It could be used for monitoring in CSF outbreak areas or as a screening method for CSFV eradication strategies.

Evaluation of a thermostable Newcastle disease virus strain TS09-C as an in-ovo vaccine for chickens

In-ovo vaccination is an attractive immunization approach for poultry industry. However, most of the Newcastle disease virus (NDV) vaccine strains used after hatch are unsafe, as in-ovo vaccines, due to their high pathogenicity for chicken embryos. In this study, we evaluated the safety and immunogenicity of a thermostable NDV strain TS09-C, derived from V4 strain, as in-ovo vaccine. Chickens in-ovo vaccinated with the parental V4 strain displayed greatly reduced hatchability and severe histopathological lesions in both trachea and intestine tissues, while the hatchability was not affected by in-ovo vaccination withTS09-C strain. The safe dose that infected all chicken embryos without obviously histopathological lesions was 103.0 EID50 per bird. In-ovo vaccination of chickens with TS09-C virus conferred complete protection against virulent NDV challenge. Results suggest that the thermostable NDV strain TS09-C is a safe and immunogenic in-ovo vaccine candidate that can be delivered quickly and uniformly, and induce earlier immune response.

Protein Expressions and Their Immunogenicity from Riemerella anatipestifer Cultured in Iron Restriction Medium

Riemerella anatipestifer was cultured in both iron restriction media and normal media. Two-dimensional gel electrophoresis identified 23 proteins that significantly increased in the iron restriction media. Of them 12 proteins were analyzed with mass spectrography. Nine of 12 proteins belong to 6 different protein families: fibronectin type iii domain protein, secreted subtilase family protein, phosphoglycerate kinase, translation elongation factor, leucine-rich repeat-containing protein, and Galactose-binding domain-like protein. Other 3 proteins were novel with unknown function. Two novel proteins (Riean_1750 and Riean_1752) were expressed in prokaryotic expression systems. The specificities of these 2 novel proteins to R. anatipestifer were confirmed by western-blotting analysis. The ducks immunized with either protein had low mortality challenged by R. anatipestifer, 33.3% and 16.7%, respectively. The ducks developed 100% immunity when immunized with combined Riean_1750 and Riean_1752 proteins. The data suggested 2 novel proteins play important roles in the bacterial survival in the iron restricted environment. They could be used as subunit vaccines of R. anatipestifer.