Shyan-Song Chiou

Bovine lactoferrin inhibits Japanese encephalitis virus by binding to heparan sulfate and receptor for low density lipoprotein

Lactoferrin is a natural anti-microbial protein which affects Japanese encephalitis virus (JEV) activity. Binding of lactoferrin to cell surface expressed heparan sulfate (HS), one possible receptor for JEV, has been postulated to be the possible mechanism of anti-JEV antiviral activity. In this study, we evaluate the effects of bovine lactoferrin (bLF) against JEV infection in vitro, using both wild-type (WT) and laboratory-adapted strains. bLF inhibited the infectivity of all the JEV strains tested. In particular the infectivity of the HS-adapted JEV strains was strongly reduced, whereas the non HS-adapted JEV strains were inhibited to lesser extent. Using both HS-adapted CJN-S1 and non HS-adapted CJN-L1 viruses, the results showed that bLF inhibited the early events essential to initiate JEV infection, which includes blocking virus attachment to cellular membranes and reducing viral penetration. This anti-JEV activity was the highest using HS-adapted CJN-S1 strain on HS-expressed CHO-K1 cells. Also, binding of bLF to heparin-sepharose blocked JEV binding; and soluble HS attenuated the anti-JEV activity of bLF. The results support the premise that the interaction of bLF with cell surface expressed glycosaminoglycans, in particular the highly sulfated HS, plays an essential role in the antiviral activity of bLF. However, bLF was functional in inhibiting CJN-S1 entry into HS-deficient CHO-pgsA745 cells, and bLF-treated CHO-K1 and -pgsA745 cells also prevented non HS-adapted CJN-L1 virus entry, indicating that a non-HS pathway may be involved in bLF inhibition of JEV entry. The low-density lipoprotein receptor (LDLR), possibly involved in the entry of several RNA viruses, also binds to bLF. We found that both rLDLR and anti-LDLR antibodies reduced the effectiveness of bLF inhibition of JEV infection. This finding provided evidence to suggest that cell surface-expressed LDLR may play a role in JEV infection, especially for non HS-adapted strains.

Japanese encephalitis virus genotype replacement, Taiwan, 2009-2010.

Genotype I of Japanese encephalitis virus first appeared in Taiwan in 2008. Phylogenetic analysis of 37 viruses from pig farms in 2009–2010 classified these viruses into 2 unique subclusters of genotype I viruses and suggested multiple introductions and swift replacement of genotype III by genotype I virus in Taiwan.

Inhibition of enveloped viruses infectivity by curcumin.

Curcumin, a natural compound and ingredient in curry, has antiinflammatory, antioxidant, and anticarcinogenic properties. Previously, we reported that curcumin abrogated influenza virus infectivity by inhibiting hemagglutination (HA) activity. This study demonstrates a novel mechanism by which curcumin inhibits the infectivity of enveloped viruses. In all analyzed enveloped viruses, including the influenza virus, curcumin inhibited plaque formation. In contrast, the nonenveloped enterovirus 71 remained unaffected by curcumin treatment. We evaluated the effects of curcumin on the membrane structure using fluorescent dye (sulforhodamine B; SRB)-containing liposomes that mimic the viral envelope. Curcumin treatment induced the leakage of SRB from these liposomes and the addition of the influenza virus reduced the leakage, indicating that curcumin disrupts the integrity of the membranes of viral envelopes and of liposomes. When testing liposomes of various diameters, we detected higher levels of SRB leakage from the smaller-sized liposomes than from the larger liposomes. Interestingly, the curcumin concentration required to reduce plaque formation was lower for the influenza virus (approximately 100 nm in diameter) than for the pseudorabies virus (approximately 180 nm) and the vaccinia virus (roughly 335 × 200 × 200 nm). These data provide insights on the molecular antiviral mechanisms of curcumin and its potential use as an antiviral agent for enveloped viruses.

Formalin Inactivation of Japanese Encephalitis Virus Vaccine Alters the Antigenicity and Immunogenicity of a Neutralization Epitope in Envelope Protein Domain III.

Formalin-inactivated Japanese encephalitis virus (JEV) vaccines are widely available, but the effects of formalin inactivation on the antigenic structure of JEV and the profile of antibodies elicited after vaccination are not well understood. We used a panel of monoclonal antibodies (MAbs) to map the antigenic structure of live JEV virus, untreated control virus (UCV), formalin-inactivated commercial vaccine (FICV), and formalin-inactivated virus (FIV). The binding activity of T16 MAb against Nakayama-derived FICV and several strains of FIV was significantly lower compared to live virus and UCV. T16 MAb, a weakly neutralizing JEV serocomplex antibody, was found to inhibit JEV infection at the post-attachment step. The T16 epitope was mapped to amino acids 329, 331, and 389 within domain III (EDIII) of the envelope (E) glycoprotein. When we explored the effect of formalin inactivation on the immunogenicity of JEV, we found that Nakayama-derived FICV, FIV, and UCV all exhibited similar immunogenicity in a mouse model, inducing anti-JEV and anti-EDII 101/106/107 epitope-specific antibodies. However, the EDIII 329/331/389 epitope-specific IgG antibody and neutralizing antibody titers were significantly lower for FICV-immunized and FIV-immunized mouse serum than for UCV-immunized. Formalin inactivation seems to alter the antigenic structure of the E protein, which may reduce the potency of commercially available JEV vaccines. Virus inactivation by H2O2, but not by UV or by short-duration and higher temperature formalin treatment, is able to maintain the antigenic structure of the JEV E protein. Thus, an alternative inactivation method, such as H2O2, which is able to maintain the integrity of the E protein may be essential to improving the potency of inactivated JEV vaccines.

First detection of the Africa/Caribbean/Latin American subtype of Culex flavivirus in Asian country, Taiwan

Culex flavivirus (CxFV), a member of the genus flavivirus, is a novel insect-specific flaviviruses that can be divided into two subtypes, the cytopathic Asia/U.S. and the noncytopathic Africa/Caribbean/Latin American subtypes. The CxFV circulates in several Asian countries, and here we conducted the first study investigating CxFV in Taiwan. A total of 14,016 mosquitoes were collected between 2010 and 2012 and 3.4% (6/179) of the pools were CxFV-positive. The phylogenetic analyses indicate that the Taiwan isolates are closely related to the Africa/Caribbean/Latin American subtype, but form an independent cluster. In the cytology experiments, the CxFV Taiwan isolate infected only mosquito cells and caused cell-cell fusion that might be associated with a unique glycine residue at position 117 within the envelope protein, which is shared with the cytopathic effect-causing Asia/US subtype. This study marks the first time the Africa/Caribbean/Latin American subtype of CxFV has been identified in an Asian country and grouped into a novel cluster.

Mouse Mammary Tumor Virus-Like Nucleotide Sequences in Canine and Feline Mammary Tumors

ABSTRACT Mouse mammary tumor virus (MMTV) has been speculated to be involved in human breast cancer. Companion animals, dogs, and cats with intimate human contacts may contribute to the transmission of MMTV between mouse and human. The aim of this study was to detect MMTV-like nucleotide sequences in canine and feline mammary tumors by nested PCR. Results showed that the presence of MMTV-like env and LTR sequences in canine malignant mammary tumors was 3.49% (3/86) and 18.60% (16/86), respectively. For feline malignant mammary tumors, the presence of both env and LTR sequences was found to be 22.22% (2/9). Nevertheless, the MMTV-like LTR and env sequences also were detected in normal mammary glands of dogs and cats. In comparisons of the MMTV-like DNA sequences of our findings to those of NIH 3T3 (MMTV-positive murine cell line) and human breast cancer cells, the sequence similarities ranged from 94 to 98%. Phylogenetic analysis revealed that intermixing among sequences identified from tissues of different hosts, i.e., mouse, dog, cat, and human, indicated the MMTV-like DNA existing in these hosts. Moreover, the env transcript was detected in 1 of the 19 MMTV-positive samples by reverse transcription-PCR. Taken together, our study provides evidence for the existence and expression of MMTV-like sequences in neoplastic and normal mammary glands of dogs and cats.

Reduced neutralizing antibody titer against genotype I virus in swine immunized with a live-attenuated genotype III Japanese encephalitis virus vaccine

A shift in prevalence from Japanese encephalitis virus (JEV) genotype III (GIII) to GI virus has been observed in several Asian countries. Genotype I virus was first detected in Taiwan in 2008, and became the dominant JEV island-wide within a year. We conducted a serosurvey using swine serum specimens from multiple counties in Taiwan following the transmission season in 2009 and results showed 67-100% of JEV seropositive swine showed evidence of GI virus infection. The envelope (E) protein is a structural protein that elicits protective neutralizing antibodies (Nt Ab). The GIII at222 (a live-attenuated swine vaccine) virus E protein differs at eight amino acid residues (E-123, E-129, E-138, E-176, E-209, E-222, E-327 and E-366) from that of the GI TC2009-1 strain (isolated in Taiwan in 2009). Twenty piglets were vaccinated with two doses of at222 vaccine, and serum specimens were collected to evaluate the strain-specific Nt Ab titer against GIII at222, GIII CJN, and GI TC2009-1 viruses. Seropositivity rates (Nt Ab titer≥1:10) and geometric mean titers (GMT) were similar against at222 and CJN viruses. However, sera from swine vaccinated with at222 were least potently neutralizing against GI TC2009-1 virus. The estimated protective threshold against GI virus was observed only when the PRNT50 against at222 virus was ≥1:320. Thus, our current study indicates that the live-attenuated at222 swine vaccine can be partially protective against GI virus, and suggests that the efficacy of GIII swine vaccines currently used may require a comprehensive reevaluation in the field.

Bovine Lactoferrin Inhibits Dengue Virus Infectivity by Interacting with Heparan Sulfate, Low-Density Lipoprotein Receptor, and DC-SIGN

Bovine lactoferrin (bLF) presents in milk and has been shown to inhibit several viral infections. Effective drugs are unavailable for the treatment of dengue virus (DENV) infection. In this study, we evaluated the antiviral effect of bLF against DENV infection in vivo and in vitro. Bovine LF significantly inhibited the infection of the four serotypes of DENV in Vero cells. In the time-of-drug addition test, DENV-2 infection was remarkably inhibited when bLF was added during or prior to the occurrence of virus attachment. We also revealed that bovine LF blocks binding between DENV-2 and the cellular membrane by interacting with heparan sulfate (HS), dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN), and low-density lipoprotein receptors (LDLR). In addition, bLF inhibits DENV-2 infection and decreases morbidity in a suckling mouse challenge model. This study supports the finding that bLF may inhibit DENV infection by binding to the potential DENV receptors.

Virulence of Japanese Encephalitis Virus Genotypes I and III, Taiwan

The virulence of genotype I (GI) Japanese encephalitis virus (JEV) is under debate. We investigated differences in the virulence of GI and GIII JEV by calculating asymptomatic ratios based on serologic studies during GI- and GIII-JEV endemic periods. The results suggested equal virulence of GI and GIII JEV among humans.