The antiviral activities of Houttuynia cordata (H. cordata) aqueous extract against dengue virus serotype 2 (DEN‐2), strain 16681, were evaluated in this study. The results showed that pre‐ and post‐incubation of H. cordata extract (10–100 µg/mL) with HepG2 cells significantly reduced intracellular DEN‐2 RNA production correlating with the decrease in dengue protein expression. In the direct blocking mode, the extract bound with DEN‐2 and strongly inhibited the intracellular viral RNA replication with an effective dose (EC50) of 0.8 µg/mL. Concentrations as low as 10–40 µg/mL of H. cordata extract also exhibited protective effect on virion release from infected LLC‐MK2 cells. High‐performance liquid chromatography analysis of H. cordata extract indicated that hyperoside was the predominant bioactive compound, and was likely to play a role in this inhibition. The extract was also shown to have no genotoxic effect on human blood cells. PRACTICAL APPLICATIONS Houttuynia cordata is recognized as medicinal vegetable consumed by people in East and Southeast Asia. It has multilateral activities in health promotion and regulation of inflammatory reactions induced by several pathogens. Dengue viral infection is a global health problem since licensed vaccines or specific antiviral drug treatments are not yet available. The current study provides scientific data to support the phytomedicinal properties of H. cordata aqueous extract on anti‐dengue virus in three modes of action (prevention, treatment and virucidal). A major constituent in the extract, hyperoside, seems to be the bioactive compound effectively acting against dengue infection. H. cordata aqueous extract was shown to be nontoxic and hyperoside may be considered a lead compound possessing drug potential for further development.

Variants of wild-type dengue serotype 2 (DEN-2) virus containing nucleotide substitutions at positions 14, 15, or 57 in the 5′ NCR were constructed by PCR-mediated site-directed mutagenesis. All three viruses containing a single point substitution demonstrated attenuation phenotype as evidenced by decreases replication and plaque size in cell culture assay. All three variants were less neurovirulent in newborn mice compared to the wild type. The mutants were immunogenic in adult mice immunogenicity and maintained stable replication characteristics following passage in mice. The variant viruses were competent for replication in Aedes aegypi mosquito vector, albeit at lower levels of infection and dissemination in the mosquito than the wild-type Den-2 16681 virus. Although all of the viruses, including the wild type, were found transmissible in mosquito life cycles, they were found subsequentially decreased in efficiency of infection, transmission, and dissemination rates along the mosquito generations and all of them remained genetically stable.

A C57U nucleotide mutation in a predicted RNA stem structure (nt 11-16/56-61) of the 59 non-coding region (59NCR) of dengue 2 (DEN-2) 16681 virus is partially attenuating, but unstable during serial passage of certain candidate DEN-2 PDK-53-based vaccine viruses containing this mutation. Here, 11 different mutations (one or more point substitution and/or deletion) between nt 54 and 70 in the 59NCR of the pD2/IC-30P-A (16681) infectious clone are described. Four mutants were infectious. Three mutants with single point substitutions replicated well in cell culture and exhibited variable neurovirulence in mice. Constructs containing multiple substitutions or any deletions failed to produce infectious viruses. Unexpectedly, a double C57U+G58C mutant replicated as efficiently as D2/IC-30P-A virus, and was more neurovirulent for newborn ICR mice. Thus, despite its predicted additional disruption of the RNA stem structure, the engineered contiguous secondary G58C mutation caused reversion of the partially attenuated phenotype caused by the 59NCR-C57U mutation.

Insertion of green fluorescent protein (GFP) encoding-gene into virus genes has provided a valuable tool for flavivirus research. This study aimed to develop dengue virus (DENV) replicons expressing GFP reporter that would provide a fast in vitro system to analyze functional roles of specific DENV sequences in viral replication. Two classes of recombinant replicon constructs were generated; one was a RNA-launched replicon with a GFP gene directly inserted into a fulllength DENV genome (FL-DENV/GFP), and the other consisted of 4 types of DNA-launched DENV subgenomic replicons with GFP replacement at various structural genes (Δ-DENV/GFP). The FL-DENV/GFP resulted in GFP expression in transfected cells with no viable DENV being recovered from the transfection. The Δ-DENV/GFP constructs with partial structural gene deletion (ΔC-, ΔCprM/M-, ΔprM/M-, or ΔE-) expressed bright and long lasting GFP. The GFP expression intensity in living cells correlated well with the level of RNA replication. Various mutations in the 5′noncoding region of DENV-2 previously shown to be important genetic determinants for virus replication and mouse virulence were incorporated into the 5 different replicon constructs. Characterizations of 29 mutants demonstrated that these replicons can provide a useful platform for a quick and powerful in vitro system to analyze genetic determinants of DENV replication. These constructs can also be useful for development of vectors expressing foreign genes for various researches.

This study aimed to generate a stable cell line harboring subgenomic dengue virus replicon and a green fluorescent gene (DENV/GFP) for a cell-based model to screen anti-DENV compounds. The gene-encoding envelope protein of DENV-2 was deleted and then replaced with fragments of the GFP gene and a foot-and-mouth-disease virus 2A-derived cleavage site. The human cytomegalovirus immediate early and antisense hepatitis delta virus ribozyme sequences were added at the 5′-and 3′-ends. An internal ribosome entry site and neomycin resistance genes were placed upstream and next to the NS1 gene. The recombinant plasmids were propagated in a mammalian cell line. A stable cell line with the brightest green fluorescent protein and the highest viral protein and RNA expression was selected from six clones. The clone was then examined for effectiveness in an antiviral drug screening assay with compounds isolated from the local plants using two known antiviral agents as controls. Two novel flavones, PMF and TMF, were discovered having DENV-inhibitory properties. The data were validated by a conventional plaque titration assay. The results indicate that this newly developed cell line is efficient for use as a cell-based model for primary screening of anti-DENV compounds.