The Fifth Residue of the Coat Protein of Turnip Mosaic Virus Is Responsible for Long-Distance Movement in a Local-Lesion Host and Aphid Transmissibility in a Systemic Host.

HC-Pro and coat protein (CP) genes of a potyvirus facilitate cell-to-cell movement and are involved in the systemic movement of the viruses. The interaction between HC-Pro and CP is mandatory for aphid transmission. Two turnip mosaic virus (TuMV) isolates, RC4 and YC5, were collected from calla lily plants in Taiwan. The virus derived from the infectious clone pYC5 cannot move systemically in Chenopodium quinoa plants and loses aphid transmissibility in Nicotiana benthamiana plants, like the initially isolated virus. Sequence analysis revealed that two amino acids, P5 and A206, of YC5 CP uniquely differ from RC4 and other TuMV strains. Recombination assay and site-directed mutagenesis revealed that the fifth residue of leucine (L) at the N-terminal region of the CP (TuMV-RC4), rather than proline (P) (TuMV-YC5), is critical to permit the systemic spread in C. quinoa plants. Moreover, the single substitution mutant YC5-CPP5L became aphid transmissible, similar to RC4. Fluorescence microscopy revealed that YC5-GFP was restricted in the petioles of inoculated leaves, whereas YC5-CPP5L-GFP translocated through the petioles of inoculated leaves, the main stem, and the petioles of the upper uninoculated leaves of C. quinoa plants. In addition, YC5-GUS was blocked at the basal part of the petiole connecting to the main stem of the inoculated C. quinoa plants, whereas YC5-CPP5L-GFP translocated to the upper leaves. Thus, a single amino acid, the residue L5 at the N-terminal region right before the 6DAG8 motif, is critical for the systemic translocation ability of TuMV in a local lesion host and for aphid transmissibility in a systemic host.

Analysis of 10 Environmental Allergen Components of the American Cockroach in Taiwan.

BACKGROUND: Cockroaches are important sources of indoor airborne allergens. The American cockroach (Periplaneta americana) is the second leading inhalant allergen causing allergic airway diseases in Taiwan. We previously reported a difference in pathogenicity of different allergen components from American cockroaches. OBJECTIVE: To analyze the environmental profile of American cockroach allergen components. METHODS: Polyclonal antibodies were generated to recombinant American cockroach allergens, Per a 1 through Per a 10. The levels of each allergen in (1) whole-body extracts and feces from American cockroaches and in (2) fresh-frozen 6-month-old and 12-month-old dead American cockroaches were evaluated by immunoblotting and quantified. Levels of allergen components from patients' household dust samples were determined by competition enzyme-linked immunosorbent assay. RESULTS: Per a 1, 2, and 10 proteins were present predominantly in roach feces, whereas other allergen components were found predominantly in roach bodies. There was a time-dependent decrease in total levels of some allergen proteins. Although levels of Per a 4, 5, 6, and 9 significantly decreased to less 20% of the basal level, there was no significant change in levels of Per a 2, 7, and 10 after 1-year decomposition. The most abundant allergen components in 20 dust samples from patients' houses were Per a 9, Per a 10, and Per a 2. CONCLUSION: The concentration of 10 American cockroach allergen components differed in the environment. Per a 2 and Per a 10 can be used as markers of long-term environmental cockroach control and Per a 9 as current status of control in patients' houses.

Generation of transgenic watermelon resistant to Zucchini yellow mosaic virus and Papaya ringspot virus type W.

Zucchini yellow mosaic virus (ZYMV) and Papaya ringspot virus type W (PRSV W) are major limiting factors for production of watermelon worldwide. For the effective control of these two viruses by transgenic resistance, an untranslatable chimeric construct containing truncated ZYMV coat protein (CP) and PRSV W CP genes was transferred to commercial watermelon cultivars by Agrobacterium-mediated transformation. Using our protocol, a total of 27 putative transgenic lines were obtained from three cultivars of 'Feeling' (23 lines), 'China baby' (3 lines), and 'Quality' (1 line). PCR and Southern blot analyses confirmed that the chimeric construct was incorporated into the genomic DNA of the transformants. Greenhouse evaluation of the selected ten transgenic lines of 'Feeling' cultivar revealed that two immune lines conferred complete resistance to ZYMV and PRSV W, from which virus accumulation were not detected by Western blotting 4 weeks after inoculation. The transgenic transcript was not detected, but small interfering RNA (siRNA) was readily detected from the two immune lines and T(1) progeny of line ZW 10 before inoculation, indicating that RNA-mediated post-transcriptional gene silencing (PTGS) is the underlying mechanism for the double-virus resistance. The segregation ratio of T(1) progeny of the immune line ZW10 indicated that the single inserted transgene is nuclearly inherited and associated with the phenotype of double-virus resistance as a dominant trait. The transgenic lines derived from the commercial watermelon cultivars have great potential for control of the two important viruses and can be implemented directly without further breeding.

Effectiveness and safety of oral lactococci-based vaccine encoding triple common allergens to prevent airway allergy in mice.

Allergic airway disease is the most common chronic airway inflammatory disorder in developed countries. House dust mite, cockroach, and mold are the leading allergens in most tropical and subtropical countries, including Taiwan. As allergen avoidance is difficult for patients allergic to these perennial indoor allergens, allergen-specific immunotherapy (ASIT) is the only available allergen-specific and disease-modifying treatment. However, for patients sensitized to multiple allergens, ASIT using each corresponding allergen is cumbersome. In the present study, we developed a recombinant L. lactis vaccine against the three most common indoor aeroallergens and investigated its effectiveness for preventing respiratory allergy and safety in mice. Three recombinant clones of Der p 2 (mite), Per a 2 (roach), and Cla c 14 (mold) were constructed individually in pNZ8149 vector and then electroporated into host strain L.lactis NZ3900. BALB/c mice were fed with the triple vaccine 5 times per week for 4 weeks prior to sensitization. The effectiveness and safety profile were then determined. Oral administration of the triple vaccine significantly alleviated allergen-induced airway hyper-responsiveness in the vaccinated mice. The allergen-specific IgG2a was upregulated. IL-4 and IL-13 mRNA expressions as well as inflammatory cell infiltration in the lungs decreased significantly in the vaccinated groups. No body weight loss or abnormal findings in the liver and kidneys were found in any of the groups of mice. This is the first report to describe a triple-aeroallergen vaccine using a food-grade lactococcal expression system. We developed a convenient oral delivery system and intend to extend this research to develop a vaccination that can be self-administered at home by patients.

Recombinant Lactococcus lactis Expressing Ling Zhi 8 Protein Ameliorates Nonalcoholic Fatty Liver and Early Atherogenesis in Cholesterol-Fed Rabbits.

Atherosclerosis is an inflammatory disease characterized by lipid deposits in the subendothelial space leading to severe inflammation. Nonalcoholic fatty liver disease (NAFLD) shares several risk factors with atherosclerosis, including dyslipidemia, type 2 diabetes mellitus, and metabolic syndrome, all of which lead to lipid deposition in the liver causing inflammation and fibrosis. Several clinical trials have shown that certain Chinese herbal medicines with anti-inflammatory effects can be used as adjuvant therapy to prevent the development of cardiovascular events and liver disease. Ling Zhi 8 (LZ8) is an immunomodulatory protein isolated from a medicinal mushroom and has been well documented to possess a broad range of pharmacological properties. This study aimed to evaluate the protective effects of recombinant Lactococcus lactis expressing LZ8 protein on NAFLD and atherogenesis in a cholesterol-fed rabbit model. Twelve rabbits were divided into three groups and fed with syrup only, L. lactis vehicle, or recombinant L. lactis-LZ8 once a day on weekdays for five weeks, respectively. The gene expression of IL-1ß in the aorta was significantly suppressed after oral administration of L. lactis-LZ8. Moreover, in hematoxylin and eosin staining of the aorta, the intima-medial thickness was decreased, and foam cells were significantly reduced in the subendothelial space. LZ8 also inhibited the expression of IL-1ß in the liver, decreased fat droplet deposits and infiltration of inflammatory cells, and improved liver function by decreasing liver enzymes in an animal model. Our results suggest that the Lactococcus-expressing LZ8 appears to be a promising medicine for improving both NAFLD and early atherogenesis owing to its anti-inflammatory effect. Furthermore, it is available as a low-cost food-grade product.

A single amino acid of niapro of papaya ringspot virus determines host specificity for infection of papaya.

Most strains of Papaya ringspot virus (PRSV) belong to type W, causing severe loss on cucurbits worldwide, or type P, devastating papaya in tropical areas. While the host range of PRSV W is limited to plants of the families Chenopodiaceae and Cucuribitaceae, PRSV P, in addition, infects plants of the family Caricaceae (papaya family). To investigate one or more viral genetic determinants for papaya infection, recombinant viruses were constructed between PRSV P-YK and PRSV W-CI. Host reactions to recombinant viruses indicated that the viral genomic region covering the C-terminal region (142 residues) of NIaVPg, full NIaPro, and N-terminal region (18 residues) of NIb, is critical for papaya infection. Sequence analysis of this region revealed residue variations at position 176 of NIaVPg and positions 27 and 205 of NIaPro between type P and W viruses. Host reactions to the constructed mutants indicated that the amino acid Lys27 of NIaPro determines the host-specificity of PRSV for papaya infection. Predicted three-dimensional structures of NIaPros of parental viruses suggested that Lys27 does not affect the protease activity of NIaPro. Recovery of the infected plants from certain papaya-infecting mutants implied involvement of other viral factors for enhancing virulence and adaptation of PRSV on papaya.

Oral edible plant vaccine containing hypoallergen of American cockroach major allergen Per a 2 prevents roach-allergic asthma in a murine model.

BACKGROUND: American cockroaches (Periplaneta americana) are an important indoor allergen source and a major risk factor for exacerbations and poor control of asthma. We previously reported that allergen components from American cockroaches exhibit varying levels of pathogenicity. Sensitization to major American cockroach allergen, Per a 2, correlated with more severe clinical phenotypes among patients with allergic airway diseases. MATERIALS AND METHODS: In this study, we examined whether oral plant vaccine-encoding full-length Per a 2 clone-996 or its hypoallergenic clone-372 could exert a prophylactic role in Per a 2-sensitized mice. The cDNAs coding Per a 2-996 and Per a 2-372 were inserted into TuMV vector and expressed in Chinese cabbage. Adult female BALB/c mice were fed with the cabbage extracts for 21 days and subsequently underwent two-step sensitization with recombinant Per a 2. RESULTS: Per a 2-specific IgE measured by in-house ELISA in the sera of Per a 2-372-treated groups were significantly lower than in the control groups after allergen challenge but not the Per a 2-996-treated group. Moreover, Per a 2-372 vaccine markedly decreased airway hyper-responsiveness and infiltration of inflammatory cells into the lungs, as well as reduced mRNA expression of IL-4 and IL-13 in comparison with the control mice. CONCLUSION: Our data suggest that oral administration of edible plant vaccine encoding Per a 2 hypo-allergen may be used as a prophylactic strategy against the development of cockroach allergy.

Engineered Mild Strains of Papaya ringspot virus for Broader Cross Protection in Cucurbits.

ABSTRACT Papaya ringspot virus (PRSV) HA5-1, a mild mutant of type P Hawaii severe strain (PRSV P-HA), has been widely used for the control of PRSV type P strains in papaya, but did not provide practical protection against PRSV type W strains in cucurbits. In order to widen the protection effectiveness against W strains, chimeric mild strains were constructed from HA5-1 to carry the heterologous 3' genomic region of a type W strain W-CI. Virus accumulation of recombinants and their crossprotection effectiveness against W-CI and P-HA were investigated. In horn melon and squash plants, the recombinant carrying both the heterologous coat protein (CP) coding region and the 3' untranslated region (3'UTR), but not the heterologous CP coding region alone, significantly enhanced the protection against W-CI. The heterologous 3'UTR alone is critical for the enhancement of the protection against W-CI in horn melon, but not in zucchini squash. In papaya, the heterologous CP coding region or 3'UTR alone, but not both together, significantly reduced the effectiveness of cross protection against P-HA. Our recombinants provide broader protection against both type W and P strains in cucurbits; however, the protective effectiveness is also affected by virus accumulation, the organization of the 3' genomic region, and host factors.

Comparative reactions of recombinant papaya ringspot viruses with chimeric coat protein (CP) genes and wild-type viruses on CP-transgenic papaya.

Transgenic papaya cultivars SunUp and Rainbow express the coat protein (CP) gene of the mild mutant of papaya ringspot virus (PRSV) HA. Both cultivars are resistant to PRSV HA and other Hawaii isolates through homology-dependent resistance via post-transcriptional gene silencing. However, Rainbow, which is hemizygous for the CP gene, is susceptible to PRSV isolates from outside Hawaii, while the CP-homozygous SunUp is resistant to most isolates but susceptible to the YK isolate from Taiwan. To investigate the role of CP sequence similarity in overcoming the resistance of Rainbow, PRSV HA recombinants with various CP segments of the YK isolate were constructed and evaluated on Rainbow, SunUp and non-transgenic papaya. Non-transgenic papaya were severely infected by all recombinants, but Rainbow plants developed a variety of symptoms. On Rainbow, a recombinant with the entire CP gene of YK caused severe symptoms, while recombinants with only partial YK CP sequences produced a range of milder symptoms. Interestingly, a recombinant with a YK segment from the 5' region of the CP gene caused very mild, transient symptoms, whereas recombinants with YK segments from the middle and 3' parts of the CP gene caused prominent and lasting symptoms. SunUp was resistant to all but two recombinants, which contained the entire CP gene or the central and 3'-end regions of the CP gene and the 3' non-coding region of YK, and the resulting symptoms were mild. It is concluded that the position of the heterologous sequences in the recombinants influences their pathogenicity on Rainbow.