A novel linear B cell epitope of the canine coronavirus nucleocapsid protein identified by a monoclonal antibody.

The infection of canine coronavirus (CCoV) causes a highly contagious disease in dogs with acute gastroenteritis. The efficient serological diagnostics is critical for controlling the disease caused by CCoV. Nucleocapsid (N) protein of CCoV is an important target for developing serological approaches. However, little is known about the antigenic sites in the N protein of CCoV. In this study, we generated a monoclonal antibody (mAb) against the N protein of CCoV, designated as 13E8, through the fusion of the sp2/0 cells with the spleen cells from a mouse immunized with the purified recombinant GST-N protein. Epitope mapping revealed that mAb 13E8 recognized a novel linear B cell epitope in N protein at 294-314aa (named as EP-13E8) by using a serial of truncated N protein through Western blot and ELISA. Sequence analysis showed that the sequence of EP-13E8 was highly conserved (100 %) among different CCoV strains analyzed, but exhibited a low similarity (31.8-63.6 %) with the responding sequence in other coronaviruses of the same genus such as FCoV, PEDV and HCoV except for TGEV (95.5 % identity). Structural assay suggested that the epitope of EP-13E8 were located in the close proximity on the surface of the N protein. Overall, the mAb 13E8 against N protein generated and its epitope EP-13E8 identified here paid the way for further developing epitope-based serological diagnostics for CCoV.

Identification and phylogenetic analysis of two canine coronavirus strains.

Canine coronavirus (CCoV), a member of the genus Alphacoronavirus, is an enveloped, single-stranded positive-sense RNA virus that responsible for gastroenteritis in dogs. In this study, two CCoV isolates were successfully propagated from 53 CCoV-positive clinical specimens by serial passaging in A-72 cells. These two strains, CCoV JS1706 and CCoV JS1712, caused cytopathic effects in A-72 cells. The sizes of virus plaque formed by them differed in early passages. Electron microscopy revealed a large quantity of typical coronavirus particles with 80-120 nm in diameter in cell culture media and cytoplasm of infected cells, in which they appeared as inclusion bodies. RT-PCR analysis of S gene indicated that these two isolates were belonged to CCoV IIa subtype. Homology of RdRp, S, M and N proteins between the two strains were 100, 99.6, 99.2 and 100.0%, respectively, whereas they were 99.4-100%, 83.1-95.2%, 88.5-99.2% and 91.9-99.7% identity compared to CCoV II reference strains. Phylogenetic analysis of RdRp, S, M and N protein showed that they were closely related to CCoV II strains. These two subtype IIa isolates will be useful for evaluating the pathogenesis and evolution of CCoV and for developing diagnostic reagents and vaccines.

Single Immunization with Newcastle Disease Virus-Vectored H7N9 Vaccine Confers a Complete Protection Against Challenge with Highly Pathogenic Avian Influenza H7N9 Virus.

In the fifth wave of the H7N9 avian influenza epidemic, highly pathogenic avian influenza (HPAI) A (H7N9) viruses have emerged and pose a great challenge to public health and the poultry industry. In addition, there are apparent genetic and antigenic variations between the classical H7N9 avian influenza virus and the newly-emerged H7N9 virus. Therefore, an antigenic-match vaccine is required for the prevention and control of H7N9 avian influenza in poultry in China. In this study, a recombinant Newcastle disease virus (NDV)-vectored vaccine expressing the HA derived from a prevailing HPAI H7N9 virus (GD15) was generated using reverse genetics. The recombinant virus (rAI4HA) showed virus yield and growth capacity in chicken embryos comparable to the parental virus (rAI4). Expression of the HA protein was detected in chicken embryo fibroblasts inoculated with rAI4HA. A chicken immunization study demonstrated that both rAI4HA and rAI4 induced similar anti-NDV hemagglutination inhibition (HI) antibody titers at weeks 2, 3, and 4 after a single immunization. However, rAI4HA-immunized chickens had a low seroconversion rate (20%) and negative HI titers against H7N9. Additionally, rAI4HA elicited high levels of H7N9-specifc IgY antibody as measured by ELISA. More importantly, the recombinant vaccine provided a complete protection against a lethal challenge with HPAI H7N9 virus and significantly inhibited virus shedding after a single immunization. Our results suggest that the recombinant NDV-vectored H7N9 vaccine expressing the antigenic-match HA can confer a complete protection against HPAI H7N9 challenge after a single immunization.

La inmunización única con una vacuna con un virus de la enfermedad de Newcastle recombinante contra el subtipo H7N9 del virus de la influenza aviar confiere una protección completa contra el desafío con el virus H7N9 de la influenza aviar altamente patógena. Durante la quinta ola de la epidemia de influenza aviar H7N9, surgieron virus altamente patógenos de influenza aviar A (H7N9) que representan un gran desafío para la salud pública y para la industria avícola. Además, existen variaciones genéticas y antigénicas aparentes entre el virus de la influenza aviar H7N9 clásica y el virus H7N9 de reciente aparición. Por lo tanto, se requiere una vacuna antigénica para la prevención y el control de la influenza aviar H7N9 en la avicultura en China. En este estudio, se generó una vacuna vectorizada con un virus recombinante de la enfermedad de Newcastle recombinante que expresaba el gene HA derivado de un virus de influenza aviar de alta patogenicidad H7N9 prevalente (GD15) utilizando genética inversa. El virus recombinante (rAI4HA) mostró el rendimiento y la capacidad de crecimiento viral en embriones de pollo comparables con el virus paterno (rAI4). La expresión de la proteína HA se detectó en fibroblastos de embrión de pollo inoculados con el virus rAI4HA. Un estudio de inmunización en pollos demostró que tanto el virus rAI4HA como el virus rAI4 indujeron títulos de anticuerpos de inhibición de hemaglutinación (HI) contra el virus de Newcastle similares en las semanas dos, tres y cuatro después de una inmunización única. Sin embargo, los pollos inmunizados con el virus rAI4HA tuvieron una tasa de seroconversión baja (20%) y títulos de HI negativos contra H7N9. Además, el virus rAI4HA provocó altos niveles de anticuerpos IgY específicos contra H7N9 medidos por ELISA. Más importante aún, la vacuna recombinante proporcionó una protección completa contra un desafío letal con un virus de alta patogenicidad H7N9 e inhibió significativamente la propagación de virus después de una sola inmunización. Estos resultados sugieren que la vacuna H7N9 vectorizada con un virus de la enfermedad de Newcastle recombinante que expresa al subtipo HA antigénicamente adecuado puede conferir una protección completa contra el desafío con un virus de alta patogenicidad H7N9 después de una inmunización única.

Signature-tagged mutagenesis screening revealed a novel smooth-to-rough transition determinant of Salmonella enterica serovar Enteritidis.

BACKGROUND: Salmonella enterica serovar Enteritidis (S. Enteritidis) has emerged as one of the most important food-borne pathogens for humans. Lipopolysaccharide (LPS), as a component of the outer membrane, is responsible for the virulence and smooth-to-rough transition in S. Enteritidis. In this study, we screened S. Enteritidis signature-tagged transposon mutant library using monoclonal antibody against somatic O9 antigen (O9 MAb) and O9 factor rabbit antiserum to identify novel genes that are involved in smooth-to-rough transition. RESULTS: A total of 480 mutants were screened and one mutant with transposon insertion in rfbG gene had smooth-to-rough transition phenotype. In order to verify the role of rfbG gene, an rfbG insertion or deletion mutant was constructed using λ-Red recombination system. Phenotypic and biological analysis revealed that rfbG insertion or deletion mutants were similar to the wild-type strain in growth rate and biochemical properties, but the swimming motility was reduced. SE Slide Agglutination test and ELISA test showed that rfbG mutants do not stimulate animals to produce agglutinating antibody. In addition, the half-lethal dose (LD50) of the rfbG deletion mutant strain was 106.6 -fold higher than that of the parent strain in a mouse model when injected intraperitoneally. CONCLUSIONS: These data indicate that the rfbG gene is involved in smooth-to-rough transition, swimming motility and virulence of S. Enteritidis. Furthermore, somatic O-antigen antibody-based approach to screen signature-tagged transposon mutants is feasible to clarify LPS biosynthesis and to find suitable markers in DIVA-vaccine research.

The immune response of a recombinant fowlpox virus coexpressing the HA gene of the H5N1 highly pathogenic avian influenza virus and chicken interleukin 6 gene in ducks.

Ducks have played an important role in the emergence of H5N1 subtype of highly pathogenic avian influenza (HPAI), and the development of an effective vaccine against HPAI in ducks is a top priority. It has been shown that a recombinant fowlpox virus (FPV)-vectored vaccine can provide protection against HPAI in ducks. In this study, a recombinant fowlpox virus (rFPV-AIH5AIL6) coexpressing the haemagglutinin (HA) gene of the H5N1 subtype of the avian influenza virus (AIV) and chicken interleukin 6 gene was constructed and tested in Gaoyou and cherry valley ducks to evaluate the immune response in ducks. These animal studies demonstrated that rFPV-AIH5AIL6 induced a higher anti-AIV HI antibody response, an enhanced lymphocyte proliferation response, an elevated immune protection, and a reduction in virus shedding compared to a recombinant fowlpox virus expressing the HA gene alone (rFPV-SYHA). These data indicate that rFPV-AIH5AIL6 may be a potential vaccine against the H5 subtype of avian influenza in ducks and chicken interleukin 6 may be an effective adjuvant for increasing the immunogenicity of FPV-vectored AIV vaccines in ducks.

Revaccination with Marek's disease vaccines induces productive infection and superior immunity.

The most common lymphoproliferative disease in chickens is Marek's disease (MD), which is caused by the oncogenic herpesvirus Marek's disease virus (MDV). The emergence of hypervirulent pathotypes of MDV has led to vaccine failures, which have become common and which have resulted in serious economic losses in some countries, and a revaccination strategy has been introduced in practice. The mechanism by which revaccination invokes superior immunity against MD is unknown. After field trials which showed that revaccination provided protection superior to that provided by a single vaccination were performed, experiments were conducted to explore the interaction between revaccinated chickens and MDV. The results showed that the chickens in the revaccination groups experienced two consecutive productive infections but that the chickens in the single-vaccination groups experienced one productive infection, demonstrating that revaccination of viruses caused the chickens to have productive and then latent infections. Revaccination of the virus induced in the chickens a higher and a longer temporary expansion of the CD8(+), CD4(+), and CD3(+) T-lymphocyte subpopulations, stronger peripheral blood lymphocyte proliferative activity; and higher levels of neutralizing antibody than single vaccination. These findings disagree with the postulate that MDV antigens persist, stimulate the immune system, and maintain a high level immunity after vaccination. The suppression of productive infection by maternal antibodies in chickens receiving the primary vaccination and a lower level of productive infection in the revaccination groups challenged with MDV were observed. The information obtained in this study suggests that the productive infection with revaccinated MDV in chickens plays a crucial role in the induction of superior immunity. This finding may be exploited for the development of a novel MD vaccine that results in the persistence of the antigen supply and that maintains a high level of immunity and may also have implications for other viral oncogenic diseases in humans and animals.

[Matrine and anti-tumor drugs in inhibiting the growth of human lung cancer cell line].

OBJECTIVE: To study the activities of matrine and anti-tumor drugs on SPCA/I human lung adenocarcinoma cell line. METHODS: Suppression effects of different concentrations of matrine and matrine combined with anti-tumor drugs on lung cancer cells were measured by methyl thiazolyl tetrazolium (MTT) colorimetric assay. RESULTS: Different concentrations of matrine could inhibit the growth of SPCA/I human lung adenocarcinoma cells and there was a positive correlation between the inhibition rate and the drug concentration. Different concentrations of matrine combined with anti-tumor drugs had higher growth inhibition rate than anti-tumor drugs alone. CONCLUSION: Matrine has direct growth suppression effect on SPCA/I human lung adenocarcinoma cells and matrine combined with anti-tumor drugs shows a significant synergistic effect on tumor cells.