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Objective: To evaluate the potential of local mosquitoes to act as vectors for dengue transmission in Japan. Methods: Serotype 2 ThNH28/93 was used to test the dengue susceptibility profiles of Aedes flavopictus miyarai (Ae. f. miyarai), Aedes galloisi (Ae. galloisi) and Aedes albopictus (Ae. albopictus), which were collected in Japan. We used Aedes aegypti from Thailand as a positive control. The mosquitoes were infected with the virus intrathoracically or orally. At 10 or 14 days post infection, the mosquitoes were dissected and total RNA was extracted from their abdomens, thoraxes, heads and legs. Mosquito susceptibility to dengue virus was evaluated using RT-PCR with dengue virus-specific primers. Differences in the infection and mortality rates of the different mosquito species were tested using Fisher's exact probability test. Results: The infection rates for dengue virus administered intrathoracically to Ae. f. miyarai, Ae. galloisi and Aedes aegypti mosquitoes were identical by RT-PCR on Day 10 post infection. All of the body parts we tested were RT-PCR-positive for dengue virus. For the orally admin-istered virus, the infection rates in the different body parts of the Ae. f. miyarai mosquitoes were slightly higher than those of Ae. albopictus mosquitoes, but were similar to the control mosquitoes (P>0.05). The mortality rates for Ae. f. miyarai and Ae. albopictus mosquitoes were similar (P=0.19). Our data indicated that dengue virus was able to replicate and disseminate to secondary infection sites in all of the four mosquito species (Japanese and Thai). Conclusions: Ae. albopictus is a well-known candidate for dengue transmission in Japan. However, our data suggest that Ae. f. miyarai from Ishigaki Island (near Okinawa Island) and Ae. galloisi from Hokkaido (Northern Japan) should also be regarded as potential vectors for dengue transmission in these regions. Further studies on these mosquitoes should be conducted.
ABSTRACT
Objective: To evaluate the potential of local mosquitoes to act as vectors for dengue transmission in Japan. Methods: Serotype 2 ThNH28/93 was used to test the dengue susceptibility profiles of Aedes flavopictus miyarai (Ae. f. miyarai), Aedes galloisi (Ae. galloisi) and Aedes albopictus (Ae. albopictus), which were collected in Japan. We used Aedes aegypti from Thailand as a positive control. The mosquitoes were infected with the virus intrathoracically or orally. At 10 or 14 days post infection, the mosquitoes were dissected and total RNA was extracted from their abdomens, thoraxes, heads and legs. Mosquito susceptibility to dengue virus was evaluated using RT-PCR with dengue virus-specific primers. Differences in the infection and mortality rates of the different mosquito species were tested using Fisher's exact probability test. Results: The infection rates for dengue virus administered intrathoracically to Ae. f. miyarai, Ae. galloisi and Aedes aegypti mosquitoes were identical by RT-PCR on Day 10 post infection. All of the body parts we tested were RT-PCR-positive for dengue virus. For the orally administered virus, the infection rates in the different body parts of the Ae. f. miyarai mosquitoes were slightly higher than those of Ae. albopictus mosquitoes, but were similar to the control mosquitoes (P > 0.05). The mortality rates for Ae. f. miyarai and Ae. albopictus mosquitoes were similar (P = 0.19). Our data indicated that dengue virus was able to replicate and disseminate to secondary infection sites in all of the four mosquito species (Japanese and Thai). Conclusions: Ae. albopictus is a well-known candidate for dengue transmission in Japan. However, our data suggest that Ae. f. miyarai from Ishigaki Island (near Okinawa Island) and Ae. galloisi from Hokkaido (Northern Japan) should also be regarded as potential vectors for dengue transmission in these regions. Further studies on these mosquitoes should be conducted.
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Dengue virus (DENV) nonstructural protein 1 (NS1) is a highly conserved 46-kDa protein that contains 2 glycosylation sites (Asn-130 and Asn-207) and 12 conserved cysteine (Cys) residues. Here, we performed site-directed mutagenesis to generate systematic mutants of viral strain TSV01. The results of the subsequent analysis showed that an alanine substitution at the second N-linked glycan Asn-207 in NS1 delayed viral RNA synthesis, reduced virus plaque size, and weakened the cytopathic effect. Three mutants at Cys sites (Cys-4, Cys-55, Cys-291) and a C-terminal deletion (ΔC) mutant signiifcantly impaired RNA synthesis, and consequently abolished viral growth, whereas alanine mutations at Asn-130 and Glu-173 resulted in phenotypes that were similar to the wild-type (WT) virus. Further analysis showed that the Asn-207 mutation slightly delayed viral replication. These results suggest that the three conserved disulifde bonds and the second N-linked glycan in NS1 are required for DENV-2 replication.
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Objective To compare the humoral immune response of BALB/c mice immunized by recombinant plasmids PeDNA3.1-M-NS1 and pcDNA3.1-N-NS1.Methods Dengue type 2 virus(DENV2)NS1 gene were constructed two partial sequences(1-413 bp)of the pcDNA3.1 eukaryotic plasmids and pET28a(+)plasmid for prokaryotic expression,identification,purification and quantification.The BALB/c mice were immunized by pcDNA3.1-M-NS1,pcDNA3.1-N-NS1 recombinant plasmids with adjuvant.Each animal received a primary inoculation and two boosts at 1-week intervals.Then the blood samples of BALB/c mice were collected from different experiment groups at day 7,14,28 and 56,respectively after first immunization.The specific IgM/IgG antibodies for NS1 protein in serum were confirmed by indirect ELISA.And then the activities of the specific protective antibody were determined by cytopathic effect inhibition(CPEI).Results Construction of the pET28a(+)-NS1 m/pET28a(+)-NS1n prokaryotic expression plasmid,SDS-PAGE analysis showed that,NS1 gene partial sequence was expressed,both the relative molecular weight of about 22.3×103:Western blot showed that the protein can bind anti-His tag monoclonal antibody;byNi affinity chromatographywith apurity of 92% protein,on the C6/36 cell toxicity,and can be used ELASA detection.The results showed that the levels of specific IgM/IgG antibody and neutralizing antibody activities were increased in pcDNA3.1-M-NS1 booster immunization group than other groups.The result had been observed longer duration of antibody level in peDNA3.1-M-NS1 booster immunization group.Conclusion Humoral immune response were significantly different between pcDNA3.1-M-NS1 and pcDNA3.1-N-NS1 recombinant plasmid immunized mice groups.
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Objective To compare humoral immune response of BALB/c mice immunized by recombinant plasmids poDNA3.1-BE and pcDNA3.1-NE. Methods The BALB/c mice were immunized by recombinant plasmids pcDNA3.1-BE, pcDNA3.1-NE with adjuvant. Each animal received a primary inoculation and two boosts at 2-week intervals. Then the blood samples of BALB/c mice were collected from different experiment groups at day 14, 28, 42, 70 and 98, respectively after first immunization. The specific IgM/IgG antibodies for E protein in serum were confirmed by indirect ELISA. And then the activities of the specific neutralizing antibody were determined by cytopathic effect inhibition (CPEI). Results The levels of specific IgM/IgG antibodies and neutralizing antibody activities were increased in PODNA3.1-BE enhance immunization group than that of other groups. The result had been observed longer duration of antibody level in pcDNA3.,-BE enhance immunization group. Conclusion Humoral immune response were significant differences between recombinant plasmid pcDNA3.1-BE and PcDNA3.1-NE immunized mice groups.
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Objective:BALB/C mice model infected with DV2 NGC strain was evaluated and dynamic patterns of cytokines were investigated.Methods:The dynamic changes of cytokines were observed in the infected BALB/C mice model by indirect ELISA was observed.Results:The dynamic levels of IL-4、IFN-? in the plasma of BALB/C mice after primary and secondary infected DV2 NGC strain were different.The results showed that the levels of IL-4 were significantly increasing in the early primary infected BALB/C mice while that of IFN-? were lower;In secondary infection ,IL-4 reached a peak on the first day( 4294 668 ? 349 038 pg/ml) and then the levels of IFN-? were higher on the forth and eleventh day.The concentrations of the cytokines produced infectious animals had closely relationship with the doses of DV2.Conclusion:Infection eliciting a dominant humonral immune response induced a high expression of Th2-related cytokines,whereas those show some appearance of Th1 cytokines.Th1 response,as described late for the disease infected with DV2 NGC strain,played a role while it inhibited Th2 response to some extend.Thus,it was responsible to recovery.Th cell and the cytokines were critic to the DV infective diseases.
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Objective:BALB/C mice model infected with different Dengue Type 2 virus (DV 2) strains was evaluated and dynamic patterns of humoral immune responses caused by the various viral strains were investigated.Methods:The BALB/C mice were multisubcutaneouly injected by different DV 2 strains. The dynamic changes of the anti-DV 2 IgM/IgG antibody were observed in the infected BALB/C mice by the indrect ELISA and the viremia was observed.Results:The productions of specific antibodies in the infected BALB/C mice after primary and secondary injection with different DV 2 strains showed some difference. Howerer, BALB/C mice infected by the DV 2 B strain from a DHF patient showed the prolonged viremia.Conclusion:The dynamic curve of specific antibody and the viremia induced by various DV 2 strain (DV 2 NGC strain.V.DV 2 B strain) were different in some existence.