Sidang Liu

Hydropericardium syndrome outbreak caused by fowl adenovirus serotype 4 in China in 2015.

There have been many outbreaks of hydropericardium syndrome (HPS), which is characterized by pericardial effusion and hepatitis, in Chinese chicken farms since June 2015. HPS was mainly found in miscellaneous meat-type chickens, Ma chickens, layer chicks and Three-yellow chickens, while it was occasionally found in white broilers. To determine the specific causative pathogen and pathogenicity of HPS in chickens, we collected 25 suspected cases and performed clinical pathology and aetiology analyses. The results showed that the 25 cases exhibited multifocal hepatitis with intra-nuclear inclusion bodies and 70 nm-latticed viral particles in the cell nuclei. All samples were positive for fowl adenovirus (FAdV), and sequencing results showed that the hexon gene shared the highest nucleotide similarities with the hexon gene of group 1 serotype 4 (FAdV-4). FAdV-4 was highly pathogenic to embryos and specific pathogen-free chickens, causing 100 and 70 % mortality rates, respectively. Thus, FAdV-4 is associated with HPS outbreaks in China.

The comprehensive diagnosis and prevention of duck plague in northwest Shandong province of China.

Here, we report the first outbreak of duck plague (DP) confirmed in 4 tissue samples that were collected since August 2012 from the northwestern region of Shandong province, China. Among these, 3 were collected from commercial Jin-ding variety layer ducks and one from Cherry Valley meat-breeding ducks. The sick ducks (7 to 49 wk old) were characterized by typical DP symptoms and necroscopic features. The flocks experienced high morbidity and mortality rates, and decreased production performance, which led to tremendous economic losses. The diagnosis of DP infection was confirmed by comprehensive analyses of epidemiological data, clinical signs, necroscopic features, histopathological examinations, and viral isolation and identification. According to the laws of the Peoples Republic of China on Animal Epidemic Prevention, emergency measures were implemented to control the outbreak, which included slaughter of the infected flocks and proper disposal of the bodies, manure, and other wastes, disinfection and thorough cleaning of the duck facilities, fields, tools, utensils, and devices, as well as emergency vaccination of the threatened flocks and the implementations of revised immunization procedures. Possible causes of the DP outbreak and the prevalence of the virus in Shandong province were also analyzed and are discussed herein.

Genetic Diversity and Positive Selection Analysis of Classical Swine Fever Virus Envelope Protein Gene E2 in East China under C-Strain Vaccination.

Classical swine fever virus (CSFV) causes an economically important and highly contagious disease of pigs worldwide. C-strain vaccination is one of the most effective ways to contain this disease. Since 2014, sporadic CSF outbreaks have been occurring in some C-strain vaccinated provinces of China. To decipher the disease etiology, 25 CSFV E2 genes from 169 clinical samples were cloned and sequenced. Phylogenetic analyses revealed that all 25 isolates belonged to subgenotype 2.1. Twenty-three of the 25 isolates were clustered in a newly defined subgenotype, 2.1d, and shared some consistent molecular characteristics. To determine whether the complete E2 gene was under positive selection pressure, we used a site-by-site analysis to identify specific codons that underwent evolutionary selection, and seven positively selected codons were found. Three positively selected sites (amino acids 17, 34, and 72) were identified in antigenicity-relevant domains B/C of the amino-terminal half of the E2 protein. In addition, another positively selected site (amino acid 200) exhibited a polarity change from hydrophilic to hydrophobic, which may change the antigenicity and virulence of CSFV. The results indicate that the circulating CSFV strains in Shandong province were mostly clustered in subgenotype 2.1d. Moreover, the identification of these positively selected sites could help to reveal molecular determinants of virulence or pathogenesis, and to clarify the driving force of CSFV evolution in East China.

Effect of age on the pathogenesis of duck tembusu virus in Cherry Valley ducks

The effect of host age on the outcome of duck tembusu virus (DTMUV) infection was studied in ducks. Three groups of Cherry Valley ducks at 1, 3, and 7 weeks of age were intramuscularly infected with DTMUV to systematically observe the clinical symptoms, pathological changes, tissue viral loads, and immune responses. Severe clinical symptoms and neurological dysfunction were observed in 1-week-old ducks as early as 2 days post infection (dpi) and some died at 5–7 dpi. Three weeks-old ducks showed similar but milder symptoms and no deaths. However, 7-weeks-old ducks showed only transient loss of appetite. Gross lesions gradually reduced in severity as ducks matured. One-week-old ducks showed endocardial hemorrhage, splenomegaly, swelling in the lymph follicles of the ileum, liver, and kidney swelling with degeneration, and meningeal hyperemia. Three-weeks-old ducks showed only mild pathological lesions. No visible lesions were observed in 7-weeks-old ducks. However, pathological histology analysis demonstrated all infected ducks displayed viral encephalitis. DTMUV could be detected in the brains of 1-week-old ducks as early as 1 dpi and virus titers of most organs in 1-week-old ducks were significantly higher than that of 3- and 7-weeks-old ducks at 3–5 dpi. The patterns of IFN-γ, IL-2, and serum neutralizing antibodies were similar, and there were significant difference between the youngest ducks and the older ducks at early infection stage (P < 0.05). More important is that although the antibody titers of all infected ducks were similar from 9 to 17 dpi, reduced clearance of virus was observed in the youngest groups comparing with the other two groups, indicating that immune system maturity was more important than the presence of neutralizing antibody. In summary, this study demonstrates that viral pathogenesis is strongest in 1-week-old ducks and the age-related immune response plays an important role in the pathogenesis of DTMUV in ducks.

Distribution of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) in different stages of gestation sows: HP-PRRSV distribution in gestation sows

Highly pathogenic PRRSV (HP-PRRSV) emerged in China in 2006 and caused severe reproductive losses, particularly in late-term sows. To determine whether these reproductive failures were related to the susceptibility of late-term sows to HP-PRRV, 60- and 90-days of gestation sows were infected with HP-PRRSV isolate TA-12 (GenBank accession HQ417620). A monoclonal antibody specific to the C-terminal of the nucleocapsid protein was used to evaluate viral distribution by IHC. This showed that HP-PRRSV had a similar distribution in both sets of sows. However, HP-PRRSV infection led to dramatically decreased serum levels of luteinizing hormone (LH) and 17-β-estradiol (E2) in late-term sows, while only E2 was decreased in the 60-day sows. These results indicate that HP-PRRSV-induced reproductive failure is more likely due to reproductive hormone level imbalances rather than tissue tropism differences.

Comparison of genomes of Brucella melitensis M28 and the B. melitensis M5-90 derivative vaccine strain highlights the translation elongation factor Tu gene tuf2 as an attenuation-related gene.

ABSTRACT Brucella melitensis causes brucellosis, a disease affecting sheep, cattle, and sometimes humans. Attenuated B. melitensis strain M5-90, derived from virulent strain M28, is widely used as a live vaccine in ruminants in China. Genetic differences between the strains may cast light on the mechanism of attenuation. We recently reported the complete genomic sequences of M28 and M5-90. Genome organization is highly conserved between these isolates, and also with virulent strains 16 M and ATCC 23457. Analysis revealed 23 open reading frames (ORFs) with consistent differences between M5-90 and the virulent strains. Notably, the tuf2 gene encoding translation elongation factor EF-Tu from M5-90 contained 50 single nucleotide polymorphisms (SNPs) and 9 gaps (indels) compared to tuf2 of M28 or of the other virulent strains. There were no changes in tuf1. To evaluate the potential role of EF-Tu in pathogenesis, tuf1 and tuf2 mutants of M28 and an M5-90 strain harboring wild-type tuf2 were constructed, and their virulence/attenuation was evaluated in vivo. We report that the tuf2 gene plays an important role in the attenuation of M5-90 virulence.

Pathogenicity of duck plague and innate immune responses of the Cherry Valley ducks to duck plague virus

Duck plague caused by duck plague virus (DPV) is an acute and contagious disease. To better understand the pathogenic mechanism of duck plague virus in ducklings, an infection experiment was performed. Our results showed that typical symptoms were observed in the infected ducklings. DPV could replicate quickly in many tissues, leading to pathological lesions, especially on the spleen. Real-time quantitative PCR demonstrated that expression of many innate immune-related genes was mostly up-regulated in the brain, and the antiviral innate immune response was established, but not sufficient to restrict viral replication. In contrast, although the expression of many major pattern recognition receptors (PRRs) increased in the spleen, the expression of most cytokines was declined. Our study indicates that DPV is a pantropic virus that can replicate rapidly in tissues, causing serious pathological lesions but the immune responses are different in the spleen and brain. To our knowledge, this is the first report to systematically explore the expression profiles of the immune genes in the DPV-infected ducks. Our data provide a foundation for further study of the pathogenicity of duck plague.

Florfenicol-induced Mitochondrial Dysfunction Suppresses Cell Proliferation and Autophagy in Fibroblasts

Florfenicol (FLO) is one of the most popular antibiotics used in veterinary clinic and aquaculture. FLO can inhibit both bacterial and mitochondrial protein synthesis. However, the effects of FLO on mitochondrial function and cellular homeostasis remain unclear. Here we show that FLO inhibits expression of mitochondrial DNA-encoded proteins, decreases mitochondrial membrane potential, and promotes generation of reactive oxygen species (ROS) in vitro. As a result, activities of mitochondrial respiratory chain complex I and IV and the cellular ATP level are decreased and mitochondrial morphology is damaged. FLO represses cell growth and proliferation by suppression of phosphorylation of p70S6K through AMPK/mTOR/p70S6K pathway. Furthermore, FLO also induces G0/G1 cell cycle arrest via increase of p21 levels through activating ROS/p53/p21 pathway. Moreover, the clearance of damaged mitochondria by autophagy is impaired, leading to cell proliferation inhibition and promotes cell senescence. In addition, FLO-induced upregulation of cytosolic p53 may contribute to mitophagy deficiency via regulation of Parkin recruitment. In summary, our data suggest that florfenicol is an inhibitor of mitochondrial protein synthesis that can induce noticeable cytotoxicity. Thus, these findings can be useful for guiding the proper use of FLO and the development of safe drugs.

Emergence of H3N8 equine influenza virus in donkeys in China in 2017

Equine influenza virus is a major respiratory pathogen in horses. Although both horses and donkeys belong to the genus Equus, donkey infection with influenza viruses is rare. In March 2017, an influenza outbreak occurred in donkeys in Shandong province, China. The causative virus, A/donkey/Shandong/1/2017(H3N8), was isolated from a dead donkey. Genetic analysis indicated that the virus originated from influenza A (H3N8) clade 2 of the Florida sub-lineage that has been circulating in Asian equine populations. Comparison of the deduced amino acid sequence of the HA gene of this causative virus with that of the A/equine/Richmond/1/2007 vaccine strain showed that substitutions had occurred in the antigenic regions A, B, and C. This study provides insight into the currently circulating and newly emerging H3N8 strains in donkeys in China.

Epidemiological investigation of outbreaks of fowl adenovirus infections in commercial chickens in China

One hundred and five fowl adenovirus (FAdV) strains were isolated in China from 2015 to 2016 from poultry with inclusion body hepatitis (IBH) and hydropericardium syndrome (HPS). Polymerase chain reactions determined that 68 were FAdV species C, five were FAdV species D, two were FAdV species E, and 30 contained two or more different FAdV strains. A phylogenetic analysis showed that the isolated FAdV strains clustered into three major groups: FAdV-C, FAdV-D and FAdV-E. Based on a hexon gene sequencing analysis, these viruses were genetically related to FAdV-4, FAdV-7, FAdV-8b and FAdV-11, of which FAdV-4 was dominant (93% of the strains). An epidemiological analysis showed that FAdVs had been circulating in broilers, domestic chickens, and layers, and co-infections with other immunosuppressive pathogens, such as chicken infectious anaemia virus, Mareks disease virus and reticuloendotheliosis virus, were identified. To control FAdVs, strict biosecurity protection measures are necessary, and a continued surveillance of FAdVs is needed to increase our understanding of the epidemiology of the viruses that are associated with IBH and HPS.