Fiber Protein Produced in Escherichia coli as a Subunit Vaccine Candidate Against Egg-Drop Syndrome 76.

The egg-drop syndrome '76 (EDS '76) caused by duck atadenovirus A (DAdV-1) infection in laying hens leads to the decrease in egg production, causing heavy economic losses in the poultry industry; thus, vaccines with high safety and immunogenicity are needed. In this study, the DAdV-1 fiber protein expressed in Escherichia coli with codon optimization showed the hemagglutination (HA) titer of 13 log2 after purification (0.6 mg/mL). Compared with inactivated EDS '76 vaccine, the specific pathogen-free chickens immunized with 0.4 mL fiber protein (HA titer of 11 log2) induced an equal level of HA inhibition (HI) titer and neutralizing antibodies. Meanwhile, after immunization with fiber protein, the lowest HI titer that could provide the effect to reduce egg production rate in laying hens after the challenge was 7 log2. Moreover, fiber protein with an HA titer of 7 log2 could induce an HI titer no <7 log2 in laying hens, which was equal to or higher than the lowest HI titer (7 log2) that could reduce egg production against DAdV-1 infection significantly, indicating that it is economically feasible for vaccine development. Importantly, the HI antibodies maintained at a high level up to 180 days postimmunization contribute to the clinical application of the vaccine candidate. Overall, the fiber protein produced in E. coli is an effective subunit vaccine candidate in EDS '76 control for its high immunogenicity and protection in chickens.

Application of phase-conjugate beams in beam correction and underwater optical wireless communication subject to surface wave turbulence.

Water surface wave turbulence is one of the factors affecting the performances of underwater optical wireless communication (UOWC) systems. In our research, a phase-conjugate beam was used to correct the beam distortion and enhance the communication performances when a system is subject to surface wave turbulence. The phase-conjugate beam was generated by a phase-conjugate mirror (PCM), and a turbulence generator was used to generate surface wave turbulence in the experiment. We calculated the beam centroid distribution and the results showed that the phase-conjugate beam had a better propagation performance than the distorted beam at the different water depths. The root mean square (RMS) of the beam centroid for the phase-conjugate beam was 11 times less than that for the distorted beam, which meant that the phase-conjugate beam could effectively correct the beam drift. We further investigated the scintillation index and the signal-to-noise ratio (SNR); the results showed that the phase-conjugate beam was able to reduce the scintillation and an obvious improvement in SNR could be obtained. This research has the potential to be applied in UWC.

Isolation and sequence analysis of the complete H gene of canine distemper virus from domestic dogs in Henan Province, China.

Eighteen canine distemper virus (CDV) isolates were obtained from clinical samples in Henan province, China, between 2012 and 2016. These viruses could not be recognized by 1A4, a monoclonal antibody specific for the H protein of CDV vaccine strains. The complete haemagglutinin (H) genes of all 18 isolates were sequenced, and phylogenetic analysis showed that they segregated into two clusters within the Asia-1 genotype. Moreover, the H genes of four viruses were found to lack a potential N-glycosylation site at position 309, which is the most conserved site within the Asia-1 genotype of CDV, and a novel potential N-glycosylation site (amino acids 517-519) was found in strain HL013, which has not been reported previously. These results will help in achieving a better understanding of the evolution of CDV in China.

Genetic characterization of parvoviruses in domestic cats in Henan province, China.

Feline panleukopenia virus (FPV) and canine parvovirus (CPV) infections are highly contagious and cause serious enteric diseases, with high fatality rates of cats and dogs. Given the importance of cats as a potential source of genetic diversity for parvoviruses, parvovirus strains detected in cats with gastroenteritis signs were analysed, and molecular characterisation, sequence analysis and phylogeny were evaluated on the VP2 gene. The results showed that FPV, new CPV-2a, and CPV-2 are co-circulating in the cat population in Henan province of China. Moreover, CPV-2 strains (F2016020, and F2016021) with Ser297Ala substitution in VP2 protein was for the first time detected in cats with clinical gastroenteritis. This study provided new important findings about the evolutionary of parvovirus infection in domestic cats.

Cell-culture derived fowl adenovirus serotype 4 inactivated vaccine provides complete protection for virus infection on SPF chickens.

Inclusion body hepatitis and hepatitis-hydropericardium syndrome caused by high-pathogenic fowl adenovirus serotype 4 has recently plagued Chinese poultry industry and caused huge economic losses since 2013. So far, there is no commercial vaccine available to control this disease. In this study, we reported the development of both embryo-adapted and cell-culture derived inactivated FAdV-4 vaccines and evaluated their efficacies in chicken. Compared to embryo-adapted vaccine, cell-culture derived vaccine induced significantly earlier and higher serological response measured by AGP and ELISA. After virus challenge, chicken immunized with cell-culture derived vaccine did not showed any gross and histopathological lesions, whereas inclusion body hepatitis was observed in the liver of chicken vaccinated with embryo-adapted vaccine. No mortality was observed in both the vaccinated groups. The above results suggested that cell-culture derived FAdV-4 inactivated vaccine could be a better vaccine candidate than embryo-adapted vaccine to control FADV-4 infections in China.

Positive selection in the hemagglutinin-neuraminidase gene of Newcastle disease virus and its effect on vaccine efficacy.

BACKGROUND: To investigate the relationship between the selective pressure and the sequence variation of the hemagglutinin-neuraminidase (HN) protein, we performed the positive selection analysis by estimating the ratio of non-synonymous to synonymous substitutions with 132 complete HN gene sequences of Newcastle disease viruses (NDVs) isolated in China. RESULTS: The PAML software applying a maximum likelihood method was used for the analysis and three sites (residues 266, 347 and 540) in the HN protein were identified as being under positive selection. Codon 347 was located exactly in a recognized antigenic determinant (residues 345-353) and codon 266 in a predicted linear B-cell epitope. Substitutions at codon 540 contributed to the N-linked glycosylation potential of residue 538. To further evaluate the effect of positively selected sites on the vaccine efficacy, we constructed two recombinant fowlpox viruses rFPV-JS6HN and rFPV-LaSHN, expressing the HN proteins from a genotype VII field isolate Go/JS6/05 (with A266, K347 and A540) and vaccine strain La Sota (with V266, E347 and T540), respectively. Two groups of SPF chickens, 18 each, were vaccinated with the two recombinant fowlpox viruses and challenged by Go/JS6/05 at 3 weeks post-immunization. The results showed that rFPV-JS6HN could elicit more effective immunity against the prevalent virus infection than rFPV-LaSHN in terms of reducing virus shedding. CONCLUSIONS: The analysis of positively selected codons and their effect on the vaccine efficacy indicated that the selective pressure on the HN protein can induce antigenic variation, and new vaccine to control the current ND epidemics should be developed.

[Induction of potent immune responses by recombinant fowlpox virus with deleted ORF73 or ORF214].

OBJECTIVE: To determine if the fowlpox virus (FPV) ORF73 or ORF214 gene encoding protein has the function of IL-18 binding protein, and to assess the role that ORF73 or ORF214 gene in regulating the immune response. METHODS: We constructed recombinant fowlpox virus (rFPV) vector-based rFPV(LP)-delta73LRH5A or rFPV(LP)-delta 214LRH5A, expressing avian influenza haemagglutinin gene (H5A) with ORF73 or ORF214 gene deletion, respectively. The parental recombinant virus expressing avian influenza haemagglutinin (rFPV(LP)-12LSH5A) was used as the control virus. The production of interferon (IFN) in vitro by splenocytes and peripheral blood mononuclear leukocytes (PBML) of SPF chickens stimulated with rFPVs was detected. The immune efficacy, antibody responses, ratio of CD4+/ CD8+ T lymphocyte and multiplication capacity of PBML induced by the rFPVs vector-based rFPV(LP)-delta73LRH5A, rFPV(LP)-delta214LRH5A and rFPV(LP)-12LSH5A vaccine were evaluated in SPF chickens. RESULTS: The level of IFN production from splenocytes stimulated with rFPV(LP)-delta73LRH5A and rFPV(LP)-delta214LRH5A was significantly higher than that with rFPV(LP)-12LSH5A in vitro, whereas the ratio of CD4+/CD8+ T lymphocyte in rFPV(LP)-delta73LRH5A and rFPV(LP)-delta214LRH5A groups was significantly lower than that in rFPV(LP)-12LSH5A groups after 10 days post immunization (dpi). These rFPVs stimulated the proliferation of PBML without significant difference. All chickens immunized with rFPV(LP)-delta73LRH5A, rFPV(LP)-delta214LRH5A or rFPV(LP)-12LSH5A produced HI antibodies against H5 AIV HA antigen, and rFPV(LP)-delta214LRH5A induced significantly lower HI titer than rFPV(LP)-12LSH5A after 14 dpi. All immunized chickens were fully protected against lethal challenge of H5N1 AIV. CONCLUSION: ORF73 and ORF214 encoding proteins blocked induction of IFN, suggesting that they are provided with IL-18BP functionality. Despite of decreasing humoral response and cell-mediated immune response, rFPV with deleted FPV73 or FPV214 gene could induce the effective efficacy in SPF chickens.

[Purification and identification of avian influenza viruses causing mixed multi-infection].

OBJECTIVE: To purify and identify avian influenza viruses causing multi-subtype mixed infection. METHODS: By using chicken embryo end-point dilution or combining with specific antiserum neutralizing tests, the mix-infection samples of 2 or 3 subtyping avian influenza viruses were purified, and the results of purification were identified by RT-PCR and hemagglutination inhibition tests. These methods were used to purify and identify 214 positive samples of avian influenza viruses. RESULTS: The method of chicken embryo end-point dilution was able to purify the viruses from samples by dilution and subculturing six or seven passages. However, combining this method with the specific antiserum neutralizing tests, the samples could be purified after four or five passages. The accuracy of identification could be obviously enhanced by using RT-PCR and hemagglutination inhibition tests simultaneously. Based on these methods, 233 of avian influenza viruses, which constituted 13 subtypes, were purified from 214 samples. CONCLUSION: Both chicken embryo end-point dilution method and its combination with specific antiserum neutralizing tests could be used to purify avian influenza viruses of mix-infection, however, the latter may be more focused and valid. In addition, the results of purification should also be evaluated by multiple means.

[Phylogenetic analysis of the neuraminidase genes of subtype N1 viruses in domestic ducks in eastern China].

To examine the phylogenetic information regarding the gene pool of AIV in domestic ducks in eastern China, the NA genes of twenty-six viruses isolated during 2002-2006, including two H1N1 strains, tenH3N1 strains and fourteen HSN1 strains, which reflected the predominant N1 subtype viruses were subjected to phylogenetic analysis. The results indicated that AIVs of N1 subtype circulating in domestic ducks in eastern China were undergoing a gradual evolution. Analysis of the deduced amino acid sequences revealed that NAs from all isolated H5N1 viruses had a 20-aa deletion in the stalk region (residues 49-68), whereas no deletion was seen in the NAs from other HA subtype viruses. The viruses of H3N1 and H1N1 might have a propensity for reassortment of NA genes, whereas no direct evidence of reassortment of NA gene was obtained in H5N1 viruses.

A reverse transcription-PCR for subtyping of the neuraminidase of avian influenza viruses.

To date, nine neuraminidase (NA) subtypes of avian influenza viruses have been identified. In order to differentiate the NA of avian influenza viruses rapidly, a reverse transcription PCR (RT-PCR) was developed. Nine pairs of NA-specific primers for the RT-PCR were designed based on the analysis of 509 complete NA sequences in GenBank. The primers were designed to amplify partial NA genes and each pair is unique to a single NA subtype (N1-N9). By nine RT-PCRs simultaneously in a set of separate tubes, the subtype of NA was determined by subsequent agarose gel electrophoresis and ethidium bromide staining, since only one of the nine RT-PCRs would give a product of expected size for each virus strain. In comparison with the established method of sequence analysis of 101 reference strains or isolates of avian influenza viruses, the RT-PCR method had a sensitivity of 97.3% and a specificity of 91.1% in subtyping avian influenza viruses. These results indicate that the RT-PCR method described below provides a specific and sensitive alternative to conventional NA-subtyping methods.

[Distribution of avian influenza virus subtypes among domestic ducks in eastern China].

OBJECTIVE: To identify the distribution of avian influenza virus subtypes among domestic ducks in eastern China. METHODS: One hundred and eighty avian influenza viruses isolated from domestic ducks between 2002 and 2006 were tested for their Hemagglutinin subtypes, and 88 of them were followed by monitoring for Neuraminidase (NA) subtypes. RESULTS: At least 9 HA subtypes and 6 NA subtypes, which constituted 13 subtypes of avian influenza viruses in domestic ducks, including H1N1, H3N1, H3N2, H3N8, H4N6, H5N1, H5N2, H6N2, H6N8, H8N4, H9N2, H10N3 and H11N2, were circulating in eastern China in recent years. CONCLUSION: Multiple subtypes of avian influenza viruses were distributed among domestic ducks in eastern China in recent years. The surveillance and prevention of avian influenza viruses in domestic ducks must be strengthened.

[Recombinant fowlpox virus coexpressing HA and NA gene from subtype H5N1 of avian influenza virus and its protective efficacy].

The hemagglutinin (HA) and neuraminidase(NA)gene from subtype H5N1 avian influenza virus were directly inserted into the transferring vector pllS, resulting in the recombinant transferring vector p11SH5ANA. Then p11SH5ANA was transfected into the chicken embryo fibroblasts (CEF), which was pre-infected with wild type fowlpox virus, to generate the recombinant fowlpox virus coexpressing H5A and NA (rFPV-11SH5ANA). By selection of blue plaques on the CEF, rFPV-11SH5ANA was obtained and purified. Experiments on SPF chickens demonstrated that the HI antibody titers in chickens vaccinated with HA-NA coexpressed vaccine was higher than those with HA expressed monovalent vaccines, and all chickens receiving either rFPV-11SH5ANA or rFPV-11SH5A were completely protected from the virulent AIV(H5N1) challenge, while those receiving wt-FPV experienced 100% mortality. The results showed that the rFPV-11SH5ANA was a safe and highly efficient gene engineering vaccine candidate for preventing HPAI.

[Influence of nonessential region on protective efficacy of recombinant fowlpox viruses].

Because of the interference of maternal antibodies, the recombinant fowlpox virus (rFPV) vaccine has not been used widely. The selection of a well-defined FPV nonessential region might be a desirable way to solve this problem. Two pairs of primers were designed according to the genome of a pathogenic FPV to amplify two flanking regions (FPV1 and FPV2) of the supposed nonessential region by PCR, and then a series of plasmid vectors were constructed to generate the expression vector pP12LS, which containing FPV1, FPV2, the expression cassette of P11-LacZ reporter gene and the promoter Ps. To abtain the vector pP12LSF, the F gene of ZJ1 strain Newcastle Disease Virus (NDV) was inserted into pP12LS, in which the F gene was located downstream of the promoter Ps. pP12LSF was transfected into chicken embryo fibroblast (CEF) pre-nfected with 282E4 strain FPV. The recombinant FPV, rFPV-FSC, was purified by blue plaque selection. The LacZ and F genes could be expressed by rFPV-FSC after 20 successive passages in CEF. The FPV nonessential region was the only difference between rFPV-FSC and rFPV-FSB. These two rFPVs could induce completely protection in SPF chickens against lethal challenge with F48E8 strain NDV. However, the protective efficacy showed a significant difference in commercial chickens with maternal antibodies. The protective efficacy of rFPV-FSC was 100% and rFPV-FSB was 61.54%. The results indicate that the selection of a well-defined FPV nonessencial region is an effective way to increase the protective efficacy of rFPVs, especially in chickens with maternal antibodies.