Molecular Detection of Rickettsia felis and Rickettsia bellii in Mosquitoes.

To add to the limited information on Rickettsia in mosquitoes in China, we carried out a PCR survey on convenience samples of 3051 mosquitoes collected with hand nets in and around domestic dwellings in 25 provinces. Five species of mosquitoes were identified: Culex pipiens pallens (n = 1620), Aedes albopictus (806), Armigeres subalbatus (377), Anopheles sinensis (168), and Culex tritaeniorhynchus (80). A Rickettsia nested-PCR targeting the variable domain of gltA showed Rickettsia felis in four mosquito species of 16 provinces Cx. pipiens pallens (1.8%, 29/1620); Ae. albopictus (1.2%, 10/806); An. sinensis (1.2%, 2/168); and Ar. subalbatus (2.1%, 8/377). Rickettsia bellii was also widespread, occurring in 12 provinces and 2 species: Cx. pipiens pallens (4.3%, 69/1620) and An. sinensis (0.6%, 1/168). R. felis and R. bellii were found in almost similar numbers in female [1.5% (27/1809) and 1.2% (21/1809), respectively] as in male mosquitoes [1.8% (22/1242) and 4.0% (49/1242), respectively]. Our results indicated that mosquitoes in China are widely infected with R. felis, the agent of human flea-borne spotted fever, and that R. bellii can also occur outside of the Americas and its usual tick hosts.

A genetic analysis of an important hydrophobic interaction at the P22 tailspike protein N-terminal domain.

P22 bacteriophage has been studied extensively and has served as a model for many important processes such as in vivo protein folding, protein aggregation and protein-protein interactions. The trimeric tailspike protein (TSP) serves as the receptor-binding protein for the P22 bacteriophage to the bacterial host. The homotrimeric P22 tail consists of three chains of 666aa in which the first 108aa form a trimeric dome-like structure which is called the N-terminal domain (NTD) and is responsible for attachment of the tailspike protein to the rest of the phage particle structure in the phage assembly pathway. Knowledge of this interaction requires information on what amino acids are interacting in the interface and how the NTD structure is maintained. The first 23aa form the "stem peptide" which originates at the dome top and terminates at the dome bottom. It contains a hydrophobic valine patch (V8-V9-V10) located within the dome structure. It is hypothesized that the interaction between the hydrophobic valine patch located on stem peptide and the adjacent polypeptide is critical for the interchain interaction which should be important for the stability of the P22 TSP NTD itself. To test this hypothesis, each amino acid in the valine residues is substituted by an acid, a basic, and a hydrophobic amino acid. The results of such substitutions are presented as well as associated studies. The data strongly suggest that the valine patch is of critical importance in the hydrophobic interaction between stem peptide valine patch and an adjacent chain.

Antigen-Sparing and Enhanced Efficacy of Multivalent Vaccines Adjuvanted with Immunopotentiators in Chickens.

We previously described that immunopotentiators, CVCVA5, increased the efficacy of H5 and H9 subtype avian influenza vaccines in chickens, ducks, and geese. In this study, we further investigated the effects of the CVCVA5 for improving the efficacy of other univalent or multivalent inactivated vaccines. The immune response administrated with half-dose of monovalent vaccine plus CVCVA5 were higher than those of one dose of monovalent vaccine without immunopotentiators as measured by levels of antibodies from serum, tears and bronchoalveolar lavage fluids, and cytokines of IFNγ and IL-4 from serum. Vaccines included the univalent vaccine of Newcastle Disease virus (ND), Egg Drop Syndrome virus (EDS), Infectious Bronchitis virus (IB), and Infectious Bursal Disease virus (IBD). The CVCVA5 also improved the immune response of both ND and IBD vaccines with less dosage. The sterile protective immunity was monitored with one- or a half-dose of adjuvanted ND vaccine or one dose of adjuvanted IBD vaccine, respectively. The improved immune efficacy was observed in a half-dose of adjuvanted bivalent vaccines compared to one dose of vaccines without CVCVA5 as measured by the antibody levels, including bivalent vaccine of ND-H9, ND-IB, and ND-IBD. The CVCVA5 also boosted the immune efficacy of the tetravalent vaccine (ND-IB-EDS-H9). A half-dose of adjuvanted commercial vaccine or 75% antigen-sparing adjuvanted vaccine elicited similar antibody levels to those of one dose non-adjuvanted commercial vaccines. The CVCVA5 improved the effect of a booster vaccination as measured by the antibody levels against H5 or H9 virus antigens, in which chickens primed with the adjuvanted ND-IB vaccines given a booster with H5-H9 bivalent vaccines without CVCVA5 using 5-day intervals. The inflammatory response may contribute to these additional effects by increasing the levels of IFNγ and IL-4 after the injection of the adjuvanted ND-IB vaccines. Results indicated that the CVCVA5 improved the serum and mucosal antibody levels, cytokine levels of the chickens given the univalent vaccine, and also improved serum antibody titers in bivalent and tetravalent vaccines. This has a potential as an improve vaccine.

Molecular Detection of Anaplasma spp. and Ehrlichia spp. in Ruminants from Twelve Provinces of China.

Anaplasma spp. and Ehrlichia spp. are tick-transmitted bacteria that are of significant economic importance as they can infect large and small ruminants and also people. There is little information on anaplasmosis and ehrlichiosis in ruminants in China. 16S rRNA FRET-qPCRs were used to screen convenience whole blood samples from 2,240 domestic ruminants in 12 provinces of China for Anaplasma spp. and Ehrlichia spp. Positive samples were further analyzed with a standard PCR for the gltA. Anaplasma spp. DNA was detected in the sheep (11.7%; 13/111), goats (81.8%; 219/270), cattle (13.2%; 241/1,830), and water buffaloes (6.9%; 2/29). Ehrlichia spp. DNA was detected in sheep (1.8%; 2/111), goats (1.1%; 3/270), and cattle (3.6%; 65/1830) but not in water buffaloes (0/29). Sequencing of gltA PCR products showed that A. marginale, A. ovis, Ehrlichia canis, and Ehrlichia sp. (JX629807) were present in ruminants from China, while the 16S rRNA FRET-qPCR sequence data indicated that there might also be A. platys, A. phagocytophilum, Anaplasma sp. BL126-13 (KJ410243), and Anaplasma sp. JC3-6 (KM227012). Our study shows that domestic ruminants from China are not uncommonly infected with a variety of Anaplasma spp. and Ehrlichia spp.

The copy number of Epstein-Barr virus latent genome correlates with the oncogenicity by the activation level of LMP1 and NF-κB.

A tumor model that Epstein-Barr virus (EBV) latent infection facilitated the tumorigenicity was previously established using the Maxi-EBV system. In the present approach, EBV-lost cell clones demonstrated significantly decreased tumorigenesis. On the other hand, the LMP1 gene in Maxi-EBV genome was replaced by that of nasopharyngeal carcinoma origin. The resultant cell line, 293-1/NL showed much lower malignancy than the original 293-EBV. The result was opposite to our expectation. The change of 293 sublineage cells for EBV harboring also got similar result. To seek the underlying reason, the copy number of EBV genome in all the cell lines was detected. The result indicated that 293-EBV contained about 4.5-fold higher EBV copies than 293-1/NL did. Parallel EBV genomes led to relatively stable copies in different 293 sublineages, suggesting the viral genome structure is a factor for the sustainability of EBV's copy number. Moreover, the LMP1 transcription in high copy-containing cells showed abnormally high level. Furthermore, the main LMP1-driven pathway, transcription factor NF-κB, was highly activated in high-copy cells. Here we first manifest by experimental model that the copy number of EBV latent genome correlates with the viral pathogenesis, which depends on the activation level of LMP1 and NF-κB. Overall, both the presence and amount of EBV genome are crucial for the viral oncogenicity.

Subtyping animal influenza virus with general multiplex RT-PCR and Liquichip high throughput (GMPLex).

This study developed a multiplex RT-PCR integrated with luminex technology to rapidly subtype simultaneously multiple influenza viruses. Primers and probes were designed to amplify NS and M genes of influenza A viruses HA gene of H1, H3, H5, H7, H9 subtypes, and NA gene of the N1 and N2 subtypes. Universal super primers were introduced to establish a multiplex RT-PCR (GM RT-PCR). It included three stages of RT-PCR amplification, and then the RT-PCR products were further tested by LiquiChip probe, combined to give an influenza virus (IV) rapid high throughput subtyping test, designated as GMPLex. The IV GMPLex rapid high throughput subtyping test presents the following features: high throughput, able to determine the subtypes of 9 target genes in H1, H3, H5, H7, H9, N1, and N2 subtypes of the influenza A virus at one time; rapid, completing the influenza subtyping within 6 hours; high specificity, ensured the specificity of the different subtypes by using two nested degenerate primers and one probe, no cross reaction occurring between the subtypes, no non-specific reactions with other pathogens and high sensitivity. When used separately to detect the product of single GM RT-PCR for single H5 or N1 gene, the GMPLex test showed a sensitivity of 10⁻5(= 280ELD50) forboth tests and the Luminex qualitative ratio results were 3.08 and 3.12, respectively. When used to detect the product of GM RT-PCR for H5N1 strain at the same time, both showed a sensitivity of 10⁻4(=2800 ELD50). The GMPLex rapid high throughput subtyping test can satisfy the needs of influenza rapid testing.

Toward the development of a plant-based vaccine against reovirus.

Transgenic lines of Arabidopsis thaliana producing recombinant TC protein were developed. The S1 gene encoding sigmaC protein of an avian reovirus (ARV) was amplified by reverse-transcription PCR (RT-PCR). The amplified product was cloned into a plant-expression vector, pE1857, with a strong promoter for expression. The resulting construct with the BAR gene cassette for bialaphos selection was designated rpE-sigmaC and was introduced into Agrobacterium tumefaciens by electroporation. Agrobacterium containing the rpE-sigmaC constructs that transform A. thaliana and transgenic plants were selected using bialaphos selection. The presence of S1 transcript in plants and their relative level of expression were determined by real time RT-PCR. Western blot analysis further confirmed the presence of sigmaC protein in the plants. Transgenic lines with high levels of sigmaC protein were selected for immunization experiments using specific-pathogen free chickens. Recombinant sigmaC protein produced in plants induced a variety of immunoglobulin G (IgG) antibody responses in chickens. Recombinant protein administered either subcutaneously or orally in birds showed significant protection against challenge. Results suggested that the recombinant sigmaC protein produced in plants has the potential for large-scale vaccination against ARV in commercial poultry.

RSV fusion (F) protein DNA vaccine provides partial protection against viral infection.

The present study was conducted to investigate the feasibility and efficacy of a RSV F DNA vaccine incorporated with a mucosal adjuvant. Two DNA vaccine vectors (DRF-412 and DRF-412-P) were developed containing residues 412-524 of the RSV F gene. These antigenic regions were cloned into the phCMV1 DNA vaccine vector. One of the DNA vaccine vectors, DRF-412, contained the ctxA(2)B region of the cholera toxin gene as a mucosal adjuvant. The in vitro expressions of these DNA vectors were confirmed in Cos-7 cells by indirect immunofluorescence and Western blot analyses. In vivo expression of the cloned gene was further confirmed in mouse muscle tissue by immunohistological analysis. The active transcription of the RSV F gene in mouse muscle cells was confirmed by RT-PCR. The purified DRF-412 and DRF-412-P DNA vectors were used to immunize mice by intramuscular injections. Our results indicated that DRF-412 and DRF-412-P vaccine vectors were as effective as live RSV in inducing neutralization antibody, systemic Ab (IgG, IgG1, IgG2a, and IgG2b) responses, and mucosal antibody responses (Ig A). The Th1 (TNF-alpha, IL-12p70, IFN-gamma, IL-2) and Th2 (IL-10, IL-6) cytokine profiles were analyzed after stimulation of spleen cells from mice immunized with purified RF-412 protein. We observed that mice inoculated with vector DRF-412 induced a higher mixed Th1/Th2 cytokine immune response than DRF-412-P. Reverse transcriptase and quantitative real-time PCR (qRT-PCR) revealed that mice immunized with the DRF-412 vector contained less viral RNA in lung tissue and the lung immunohistology study confirmed that mice immunized with DRF-412 had better protection than those immunized with the DRF-412-P vector. These results indicate that the RSV DRF-412 vaccine vector, which contains the cholera toxin subunit ctxA2B as a mucosal adjuvant may provide a better DNA vaccination strategy against RSV.

Integrity of a recombinant hemagglutinin protein of an avian influenza virus.

An open reading frame representing cDNA from a hemagglutinin (HA) encoding gene of a low pathogenic avian influenza virus (AIV) subtype H10N7 was cloned in the pNMT1-TOPO vector under the control of thiamine response promoter. This construct was designated as pNMT1-HA. The pNMT1-HA construct was transformed into Schizosaccharomyces pombe for expression of HA antigen. The correct expression of recombinant HA protein was confirmed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The level of expression of recombinant HA protein was approximately 0.2% of total soluble protein. Purified yeast-derived recombinant HA protein showed hemagglutination activity. The 2-D and 3-D scanning images of recombinant HA protein were observed with an atomic force microscope (AFM). The structural integrity of the HA protein under AFM and hemagglutination activity provided support that the recombinant HA protein may be suitable for development of AIV subunit vaccine for mass administration to poultry.

Identification of a pyridoxine (pyridoxamine) 5'-phosphate oxidase from Arabidopsis thaliana.

Pyridoxine (pyridoxamine) 5'-phosphate oxidase (PPOX) catalyzes the oxidative conversion of pyridoxamine 5'-phosphate (PMP) or pyridoxine 5'-phosphate (PNP) to pyridoxal 5'-phosphate (PLP). The At5g49970 gene of Arabidopsis thaliana shows homology to PPOX's from a number of organisms including the Saccharomyces cerevisiae PDX3 gene. A cDNA corresponding to putative A. thaliana PPOX (AtPPOX) was obtained using reverse transcriptase-polymerase chain reaction and primers landing at the start and stop codons of At5g49970. The putative AtPPOX is 530 amino acid long and predicted to contain three distinct parts: a 64 amino acid long N-terminal putative chloroplast transit peptide, followed by a long Yjef_N domain of unknown function and a C-terminal Pyridox_oxidase domain. Recombinant proteins representing the C-terminal domain of AtPPOX and AtPPOX without transit peptide were expressed in E. coli and showed PPOX enzyme activity. The PDX3 knockout yeast deficient in PPOX activity exhibited sensitivity to oxidative stress. Constructs of AtPPOX cDNA of different lengths complemented the PDX3 knockout yeast for oxidative stress. The role of the Yjef_N domain of AtPPOX was not determined, but it shows homology with a number of conserved hypothetical proteins of unknown function.

Effect of in ovo administered reovirus vaccines on immune responses of specific-pathogen-free chickens.

We investigated the effect of in ovo administered reovirus vaccines on the immune responses of specific-pathogen-free chickens. T-cell mitogenic responses to concanavalin A were numerically lower at 9 and 12 days of age and significantly lower at 6 days of age in birds vaccinated with a commercial reovirus vaccine compared with unvaccinated birds or birds vaccinated with an experimental reovirus-antibody complex vaccine. There were no significant differences in proportions of subpopulations of helper (CD4+CD8-) or cytotoxic (CD4-CD8+) T cells except at 12 days of age, when the percentages of CD4-CD8+ cells in the two vaccinated groups were statistically higher than in the nonvaccinated group. B-cell populations were not different among vaccine groups except at 9 days of age, when the vaccinated groups had the highest level of B cells. This commercial reovirus vaccine should not be given in ovo to embryos having little or no maternal antibody, otherwise immunosuppression may occur in the chicks. The addition of the antibody complex to the vaccine prevented this T-cell immunosuppression.

Influence of a reovirus-antibody complex vaccine on efficacy of Marek's disease vaccine administered in ovo.

Two experiments determined the influence of an experimental reovirus-antibody complex vaccine on Mareks disease virus (MDV) vaccine when used in ovo. Designs were the same except that specific-pathogen-free (SPF) broiler eggs were used in Experiment 1 and commercial broiler eggs with maternal antibodies against reovirus were used in Experiment 2. At 18 days of incubation, embryos were separated into four groups and inoculated with either diluent, MDV vaccine, reovirus-antibody complex vaccine, or a combination of reovirus-antibody complex and MDV vaccine. At 5 days of age, half the chickens in each group were challenged with MDV. At 7 wk old, all were euthanatized, weighed, and examined. At 7 days of age, remaining chickens in each group were challenged with reovirus. At 21 days old, chickens were euthanatized and weighed. No vaccine adversely affected hatchability or posthatch mortality in SPF or commercial chickens. There were no significant differences in protection against reovirus challenge when vaccines were used separately or in combination, and lesion scores were nearly identical in all vaccinated groups in both experiments. However, percentage of protection against reovirus was lower in Experiment 2, indicating an adverse effect of maternal immunity on efficacy of the reovirus vaccine. There were no significant differences in protection against MDV when the vaccines were used separately or combined. Severity of MDV lesions was nearly identical in all vaccinated groups in both experiments. However, the combination of vaccines gave numerically lower protection against MDV than MDV vaccine alone. Use of a larger number of birds, as in field conditions, may result in statistically lower protection for the vaccine combination. Large field trials are needed to determine the potential of the reovirus-antibody complex vaccine.

Safety and efficacy of an experimental reovirus vaccine for in ovo administration.

A commercial reovirus vaccine alone or experimental reovirus vaccine plus antibody complex were inoculated into 18-day-old specific pathogen free (SPF) broiler embryos at 0.1 of the recommended chick dose. The following groups were used: group 1A was not vaccinated or challenged; group 1B was not vaccinated, but was challenged with virulent reovirus; group 2 received the vaccine complexed with 1/4 dilution of antiserum; group 3 received the vaccine with 1/8 dilution of antiserum; group 4 received the vaccine with 1/16 dilution of antiserum, and group 5 received vaccine alone. At 1, 3, 6, 9, and 12 days of age, serum was collected and antibody against avian reovirus was analyzed by enzyme-linked immunosorbent assay (ELISA). At the same times, spleens were collected and vaccine virus detected by inoculating chicken embryo fibroblasts (CEFs) and examining for cytopathic effect. At 15 days of age, chickens in groups 2-5 were challenged with reovirus. At 22 days of age, birds were euthanatized and weighed. Efficacy of the vaccines was based on safety, percent protection, and antibody response. In ovo vaccination with the commercial or experimental vaccines did not adversely affect hatchability of SPF chickens. The vaccine complexed with antibody resulted in significantly less posthatch mortality (3.7%) when compared to mortality of chickens that received vaccine alone (17%). Both vaccine virus recovery and antibody response were delayed at least 3 days in birds receiving the experimental vaccines. In evo administration of reovirus antibody complex vaccines provided at least 70% protection. The experimental reovirus-antibody complex vaccines were safe and efficacious when given in ovo to SPF broiler embryos.