Full genome analysis of swine genotype 3 hepatitis E virus isolated from eastern China.

Hepatitis E is believed to occur in both endemic and sporadic forms in developing countries, which causes a major public health problem in Asia and Africa (Meng, 2010; Wang et al., 2016a). Recent studies have documented that the disease is also endemic in many industrialized countries (Wenzel et al., 2011). The causative agent, hepatitis E virus (HEV), belonging to the genus Orthohepevirus, is a non-enveloped RNA virus with a single-stranded, positive-sense genome of approximately 7.2 kb (Smith et al., 2014). The genome consists of a short 5' un-translated region (UTR), three open reading frames (ORFs), and a 3' UTR containing a poly(A) tail (Meng, 2011). Four recognized major genotypes of HEV are identified: genotype 1 (Asian and African strains), genotype 2 (a Mexican strain), genotype 3 (primarily from America and Europe, and some Asian countries), and genotype 4 (mainly Asian strains) (Smith et al., 2016). Previous study revealed that HEV genotype 4 is the dominant zoonotic HEV genotype in China (Wang et al., 2016a). However, infections with HEV 3 have been found more commonly in recent years in China (Liu et al., 2012; Zhang et al., 2013). To date, only one full genome of Chinese swine genotype 3 HEV strain from Shanghai has been documented (Si et al., 2009). We report here the first full genome sequence of a genotype 3 swine HEV strain from Zhejiang, China.

Temporal genetic variability and host sources of Escherichia coli associated with fecal pollution from domesticated animals in the shellfish culture environment of Xiangshan Bay, East China Sea.

This study was conducted to analyze the genetic variability of Escherichia coli from domesticated animal wastes for microbial source tracking (MST) application in fecal contaminated shellfish growing waters of Xiangshan Bay, East China Sea. (GTG)(5) primer was used to generate 1363 fingerprints from E. coli isolated from feces of known 9 domesticated animal sources around this shellfish culture area. Jackknife analysis of the complete (GTG)(5)-PCR DNA fingerprint library indicated that isolates were assigned to the correct source groups with an 84.28% average rate of correct classification. Based on one-year source tracking data, the dominant sources of E. coli were swine, chickens, ducks and cows in this water area. Moreover, annual and spatial changes of E. coli concentrations and host sources may affect the level and distribution of zoonotic pathogen species in waters. Our findings will further contribute to preventing fecal pollution in aquatic environments and quality control of shellfish.

Immune responses of Fenneropenaeus chinensis against white spot syndrome virus after oral delivery of VP28 using Bacillus subtilis as vehicles.

The protective efficacy of oral administration of VP28 using Bacillus subtilis as vehicles (rVP28-bs) in shrimp, Fenneropenaeus chinensis, upon challenge with white spot syndrome virus (WSSV) was investigated. The calculated relative percent survival (RPS) value of rVP28-bs fed shrimp was 83.3% when challenged on the 14th day post-administration, which is significantly higher (p < 0.001) than that of the group administered recombinant Escherichia coli over-expressing rVP28 (rVP28-e21). After immunization, activities of phenoloxidase (PO), superoxide dismutase (SOD) and inducible nitric oxide synthase (iNOS) in hemolymph were analyzed. It was found that the supplementation of rVP28-bs into shrimp food pellets resulted in the most pronounced increase of iNOS activity (p < 0.001), but had the least influence on activities of PO and SOD. Besides, in the shrimp orally administered with rVP28-bs, the caspase-3 activity was one-fifth that of the control, though the signs of apoptosis (chromatin margination, nuclear fragmentation and apoptotic bodies) could not be observed by transmission electron microscope (TEM). These results suggest that by oral delivery of rVP28-bs, shrimp showed significant resistance to WSSV and an effect on the innate immune system of shrimp. The remarkably enhanced level of iNOS after rVP28-bs administration might be responsible for antiviral defense in shrimp.

A survey on porcine circovirus type 2 infection and phylogenetic analysis of its ORF2 gene in Hangzhou, Zhejiang Province, China.

Porcine circovirus type 2 (PCV2) is closely related to the postweaning multisystemic wasting syndrome (PMWS). In this study, the pig serum and tissue samples collected from different regions of Hangzhou District in Zhejiang Province of China between 2003 and 2005 were analyzed by enzyme-linked immunosorbent assay (ELISA) for PCV2 antibody and by polymerase chain reaction (PCR) for ORF2 gene. The results show that out of 1250 randomly collected serum samples, 500 sera (40%) were seropositive for PCV2. PCR results demonstrate that Hangzhou PCV2 with more than 50% Chinese PCV2 strains and French PCV2 formed Cluster A. Only one PCV2 from Hangzhou belonged to Cluster B with some other Chinese PCV2 and Netherlands's isolates. Cluster C consisted of PCV2 isolates from China, US, Canada, UK and Germany. The results indicate that the PCV2 infection was widespread in Hangzhou.

High-level secretion of a chimeric thermostable lichenase from Bacillus subtilis by screening of site-mutated signal peptides with structural alterations.

A chimeric gene mHG (669 bp) was constructed by substitution of Clostridium thermocellum ZJL4 lichenase (CG) N-terminal fragment (except its signal sequence) for the counterpart of Bacillus sp. A3 lichenase (BG). To acquire high-level secretion of the chimeric lichenase (mHG) in Bacillus subtilis, a series of site-mutated signal peptides were designed. The activity of mHG, which was directed by an artificial hydrophobic signal peptide H1 (MMARKIAGMATSLLVIFSSSAVA) from cytoplasm into growth medium, reached 80.56 U/ml after 22-h incubation, indicating that signal peptide hydrophobicity appears to be critical for early stages in mHG export. By purification of the mHG (approximately 25.3 kDa) from cultures of B. subtilis (pBSG-H1), enzymatic property assays showed that the common optima for mHG were 70 degrees C and pH 5.0. The residual activity of mHG at 90 degrees C for 10 min was 83.45% of its maximum activity, which was almost similar to that of CG (90 degrees C, 10 min, 84.33%). This constructed shuttle expression vector with a novel signal peptide exhibited its applicability for high-level production of heterologous proteins from B. subtilis. Moreover, the high-level secreted mHG with relatively high thermostability could be a potential candidate for feed industrial applications.

Detection of PCV2 DNA by SYBR Green I-based quantitative PCR.

We developed an assay for the detection and quantitation of porcine circovirus type 2 (PCV2) with the SYBR Green I-based real-time PCR. The real-time PCR provides a broad dynamic range, detecting from 10(3) to 10(11) copies of DNA per reaction. No cross-reactions were found in specimens containing PCV1. Because of the high sensitivity and specificity of the assay with a relatively rapid and simple procedure, real-time PCR can be used as a routine assay for the clinical diagnosis of PCV2 infection. In this study we applied real-time PCR assay to 80 clinical samples, collected from 40 pigs with postweaning multisystemic wasting syndrome (PMWS) and 40 healthy pigs in comparison with conventional PCR assay. In 56 of 80 samples, PCV2 DNA was detected by conventional PCR assay. All samples positive for PCV2 DNA in conventional PCR assay were also positive in real-time assay, and 12 of 24 samples that tested negative for PCV2 DNA in the conventional assay were tested positive in real-time PCR assay. Real-time PCR assay increased the number of samples in which PCV2 was detected by 15%. It is, therefore, considered to be a useful tool for the detection of PCV2.

Protection of Carassius auratus Gibelio against infection by Aeromonas hydrophila using specific immunoglobulins from hen egg yolk.

Specific immunoglobulin (IgY) from egg yolk against Aeromonas hydrophila was produced by immunization of White Leghorn hens with formalin-killed whole cells of A. hydrophila. ELISA test using A. hydrophila as the coating antigen revealed that the specific antibody titer started to increase in the egg yolk at the 13th day post-immunization (P/N=2.18), reached the peak at the 56th day (P/N=13.82), and remained at high level until day 133 (P/N=7.03). The antibody was purified by saturated ammonium sulphate with a recovery rate of 63.5%. The specific IgY inhibited the growth of A. hydrophila at a concentration of 1.0 mg/ml during the 18 h incubation. Pre-treatment of polyploid gibel carps Carassius auratus Gibelio with specific IgY had a protection rate of 60% (6/10) against challenge with A. hydrophila, while none of the fishes in the control groups receiving sterile phosphate buffered saline (PBS) or non-specific IgY survived the challenge. Treatment of fishes with the specific IgY 4 h after the challenge also had lower mortality (70%, 7/10), a 30% reduction against the control PBS or non-specific IgY groups (10/10). These results indicate that specific IgY antibodies could be obtained easily from hens immunized with an inactivated A. hydrophila and could provide a novel alternative approach to control of diseases in fishes caused by this organism.

[Characterization of a recombinant Listeria monocytogenes strain containing the fusion protein gene of Newcastle disease virus].

Homologous recombination was utilized for construction of a recombinant strain of L. monocytogenes carrying a gene from the Newcastle diseases virus by insertional mutation targeting its listeriolysin O gene (hly). The gene encoding fusion protein of the Newcastle disease virus (NDV-F) was used as the model heterologous gene. The F gene was inserted into hly downstream to its promoter and signal sequence by overlapping extension polymerase chain reaction, which was then subcloned into the shuttle plasmid pKSV7 for allelic exchange with L. monocytogenes chromosome. PCR amplification of the target genes indicated insertion of the F gene into the chromosome DNA of L. monocytogenes. RT-PCR showed transcription of F gene from the recombinant L. monocytogenes strain. Comparisons were then made between the recombinant strain and its wild parent strain in terms of the hemolytic activity, adhesion and invasiveness to cultured HeLa cells, virulence to mice and chicken embryos, and growth kinetics in broth medium as well as its stability upon repeated subculturing and serial passages in mice. The recombinant L. monocytogenes lost its hemolytic activity on the blood agar and had no hemolytic titer from its culture supernatants as compared with the titer of 24 in the supernatant from the wild parent strain. The recombinant strain also had lower adhesiveness (P > 0.05) and significantly lower relative invasiveness to the HeLa cells than its wild type strain (P < 0.05). Such insertional mutation resulted in reduced virulence, about 3.7 logs and 6.5 logs less than its parent strain L. monocytogenes 10403S as shown by the 50% lethal dose assays in the mouse and chicken embryonated egg models respectively. The recombinant strain was relatively stable as shown by amplification of the target gene NDV-F from its genomic DNA after subculturing in BHI broth or in mice for 5 times.

Immunogenicity of formaldehyde and binary ethylenimine inactivated infectious bursal disease virus in broiler chicks.

Infectious bursal disease virus (IBDV) was inactivated by two different chemicals--formaldehyde and binary ethylenimine (BEI). Formaldehyde was used at 0.1% and 0.2%, while BEI was used at concentrations of 0.001 and 0.002 mol/L. These four vaccines were tested for their efficiency in generating humoral immune response in different groups of broiler chicks. Both BEI-inactivated vaccines gave relatively higher antibody titers and were almost twice as efficient as formaldehyde-inactivated ones.

Virulence phenotyping and molecular characterization of a low-pathogenicity isolate of Listeria monocytogenes from cow's milk.

A low-pathogenicity isolate of Listeria monocytogenes from cow's milk, as screened in mouse and chicken embryonated egg models, was examined for virulence-related phenotypic traits. Corresponding virulence genes (iap, prfA, plcA, hly, mpl, actA, plcB, InlA and InlB) were compared with L. monocytogenes reference strains 10403S and EGD to elucidate the possible molecular mechanisms of low virulence. Although L. monocytogenes H4 exhibited similar patterns to strain 10403S in terms of hemolytic activity, in vitro growth and invasiveness and even had higher adhesiveness, faster intracellular growth and higher phospholipase activity in vitro, it was substantially less virulent than the strain 10403S in mouse and chicken embryo models (50% lethal dose: 10(8.14) vs. 10(5.49) and 10(6.73) vs. 10(1.9), respectively). The genes prfA, plcA and mpl were homologous among L. monocytogenes strains H4, 10403S and EGD (>98%). Genes iap, hly, plcB, InlA and InlB of L. monocytogenes 10403S had higher homology to those of strain EGD (>98%) than isolate H4. The homology of the gene hly between strain 10403S and isolate H4 was 96.9% at the nucleotide level, but 98.7% at the amino acid level. The actA gene of isolate H4 had deletions of 105 nucleotides corresponding to 35 amino acid deletions falling within the proline-rich region. Taken together, this study presents some clues as to reduced virulence to mice and chicken embryos of the isolate H4 probably as a result of deletion mutations of actA.

[Stability of recombinant plasmids in attenuated Salmonella typhimurium and their effects on bacterial invasiveness and multiplication].

Effects of plasmid type and insertion sequence on in vitro and in vivo multiplication and invasiveness of attenuated Salmonella typhimurium were examined following transformation of the bacteria with eukaryotic expression plasmids pcDNA3 and pCI with or without heterologous gene (Newcastle disease virus F gene). Exogenous plasmids had negative impacts on the replication or invasiveness of the attenuated S. typhimurium in LB broth and/or HeLa cell monolayers as well as on its survival in live chicks. The plasmid pCI had more significant effects than pcDNA3. Introduction of heterologous gene into the plasmids not only exhibited additional negative influence on the host strain but also on their own stability therein. All these results suggest that full consideration should be given to the types of plasmids and their stability within the host strain as well as to their effects on replication and invasiveness of attenuated bacteria as the DNA vaccine delivery vector for improved immune protection.