Meng Ling Moi

Two cases of Zika fever imported from French Polynesia to Japan, December 2013 to January 2014

We present two cases of imported Zika fever to Japan, in travellers returning from French Polynesia, where an outbreak due to Zika virus (ZIKV) is ongoing since week 41 of 2013. This report serves to raise awareness among healthcare professionals, that the differential diagnosis of febrile and subfebrile patients with rash should include ZIKV infection, especially in patients returning from areas affected by this virus.

Autochthonous dengue fever, Tokyo, Japan, 2014.

After 70 years with no confirmed autochthonous cases of dengue fever in Japan, 19 cases were reported during August–September 2014. Dengue virus serotype 1 was detected in 18 patients. Phylogenetic analysis of the envelope protein genome sequence from 3 patients revealed 100% identity with the strain from the first patient (2014) in Japan.

Zika fever imported from Thailand to Japan, and diagnosed by PCR in the urines.

In July 2014, a Japanese traveller returning from Thailand was investigated for fever, headache, rash and conjunctivitis. Zika virus RNA was detected in his urine sample by real-time reverse transcription polymerase chain reaction. Serological tests showed cross reactivity of IgM against the dengue virus. Zika fever could be misdiagnosed or missed and should be considered in febrile patients with a rash, especially those returning from Thailand.

Common marmoset (Callithrix jacchus) as a primate model of dengue virus infection: development of high levels of viraemia and demonstration of protective immunity.

Dengue virus (DENV) causes a wide range of illnesses in humans: dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Animal models that constantly develop high levels of viraemia are required for the development of protective and preventive measures. Common marmosets (Callithrix jacchus) demonstrated high levels of viraemia after inoculation with clinical isolates of four serotypes of DENV; in particular, over 10(6) genome copies ml(-1) after inoculation with DENV-2. Non-structural protein 1 and DENV-specific IgM and IgG antibodies were consistently detected. The DENV-2 genome was detected in lymphoid organs including the lymph nodes, spleen and thymus, and also in non-lymphoid organs. DENV antigen was detected by immunohistochemistry in the liver and spleen from inoculated marmosets. Four marmosets were reinoculated with DENV-2 at 33 weeks after primary inoculation with DENV-2. The DENV-2 genome was not detected in any of these marmosets, indicating protection from a secondary infection. The results indicate that common marmosets are highly sensitive to DENV infection, and suggest that marmosets could be a reliable primate model for the evaluation of candidate vaccines.

Importation of dengue virus type 3 to Japan from Tanzania and Côte d'Ivoire.

Travelers can introduce viruses from disease-endemic to non–disease-endemic areas. Serologic and virologic tests confirmed dengue virus infections in 3 travelers returning to Japan: 2 from Tanzania and 1 from Côte d’Ivoire. Phylogenetic analysis of the envelope gene showed that 2 genetically related virus isolates belonged to dengue virus type 3 genotype III.

Involvement of the Fcγ receptor IIA cytoplasmic domain in antibody-dependent enhancement of dengue virus infection

Sub-neutralizing concentrations of antibody to dengue virus (DENV) enhance DENV infection of Fc gamma receptor-expressing cells. This phenomenon, referred to as antibody-dependent enhancement (ADE), has been hypothesized to be responsible for the severe form of DENV infection, including dengue haemorrhagic fever and dengue shock syndrome. To analyse further the mechanisms of ADE in vitro, this study introduced a series of cytoplasmic mutants into human Fc gammaRIIA. The mutated Fc gammaRIIA was then expressed on COS-7 cells to see whether these mutants could enhance DENV infection. Wild-type Fc gammaRIIA enhanced DENV infection, consistent with previous reports using Fc gammaR-positive monocytes. Disruption of the immune tyrosine activation motif (ITAM) in the cytoplasmic domain of Fc gammaRIIA or removing the sequences between the two ITAM regions eliminated ADE. These findings suggest that the specific structure of the Fc gammaRIIA cytoplasmic domain is essential for the ability of Fc gammaRIIA to mediate ADE.

Detection of Higher Levels of Dengue Viremia Using FcγR-Expressing BHK-21 Cells Than FcγR-Negative Cells in Secondary Infection but Not in Primary Infection

It has been reported that levels of viremia reflect the severity of illness in dengue virus infection. We assessed the levels of viremia in patients with primary and secondary infections, using 2 cell lines: FcγR-expressing BHK cells and FcγR-negative cells. In primary infection, virus titers were at similar levels between FcγR-expressing and FcγR-negative cells. In secondary infection, however, virus titers were ∼10 times higher in FcγR-expressing cells on days 1-6 when compared with FcγR-negative cells, indicating discrepancy in viremia titers between FcγR-expressing and FcγR-negative cells. The results suggest that dengue virus-antibody complexes with infectious capacity exist in patients with secondary infection, and these immune complexes can be detected by using FcγR-expressing cells. As it has been reported that principal target cells of dengue virus infection are FcγR-positive, monocyte/macrophage lineage cells, virus titers determined using FcγR-expressing cells may better reflect the actual viremic conditions in vivo.

Dengue Virus Infection-Enhancing Activity in Serum Samples with Neutralizing Activity as Determined by Using FcγR-Expressing Cells

Background Progress in dengue vaccine development has been hampered by limited understanding of protective immunity against dengue virus infection. Conventional neutralizing antibody titration assays that use FcγR-negative cells do not consider possible infection-enhancement activity. We reasoned that as FcγR-expressing cells are the major target cells of dengue virus, neutralizing antibody titration assays using FcγR-expressing cells that determine the sum of neutralizing and infection-enhancing activity, may better reflect the biological properties of antibodies in vivo. Methods and Findings We evaluated serum samples from 80 residents of a dengue endemic country, Malaysia, for neutralizing activity, and infection-enhancing activity at 1∶10 serum dilution by using FcγR-negative BHK cells and FcγR-expressing BHK cells. The serum samples consisted of a panel of patients with acute DENV infection (31%, 25/80) and a panel of donors without acute DENV infection (69%, 55/80). A high proportion of the tested serum samples (75%, 60/80) demonstrated DENV neutralizing activity (PRNT50≥10) and infection-enhancing activity. Eleven of 18 serum samples from patients with acute secondary DENV infection demonstrated neutralizing activity to the infecting serotype determined by using FcγR-negative BHK cells (PRNT50≥10), but not when determined by using FcγR-expressing cells. Conclusion Human serum samples with low neutralizing activity determined by using FcγR-negative cells showed DENV infection-enhancing activity using FcγR-expressing cells, whereas those with high neutralizing activity determined by using FcγR-negative cells demonstrate low or no infection-enhancing activity using FcγR-expressing cells. The results suggest an inverse relationship between neutralizing antibody titer and infection-enhancing activity, and that neutralizing activity determined by using FcγR-expressing cells, and not the activity determined by using FcγR-negative cells, may better reflect protection to DENV infection in vivo.

Development of an antibody-dependent enhancement assay for dengue virus using stable BHK-21 cell lines expressing FcγRIIA

Dengue virus (DENV) causes a wide range of symptoms, from mild febrile illness, dengue fever (DF), to severe life threatening illness, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Subneutralizing concentrations of antibody to DENV enhance DENV infection in Fc gammaR positive cells. This phenomenon is known as antibody-dependent enhancement (ADE). ADE is considered to be a risk factor for DHF and DSS. To develop an ADE assay for DENV, two stable BHK-21 cell lines were established that express Fc gammaRIIA (BHK-Fc gammaRIIA). The BHK-Fc gammaRIIA cell lines were used in an ADE assay with monoclonal antibody (4G2) to DENV, and DENV antibody-positive human sera. Virus growth was quantified directly in BHK-Fc gammaRIIA cells with a standard plaque assay procedure. ADE was detected with monoclonal antibody (4G2) to DENV. ADE was also detected with DENV antibody-positive human sera, but not with DENV antibody-negative human sera. The new ADE assay using BHK-Fc gammaRIIA cells is simple and practical, and is useful for defining the role of ADE in the pathogenesis of DENV infection.

Discrepancy in Dengue Virus Neutralizing Antibody Titers between Plaque Reduction Neutralizing Tests with Fcγ Receptor (FcγR)-Negative and FcγR-Expressing BHK-21 Cells

ABSTRACT Protective immunity against dengue virus (DENV) is best reflected by the presence of neutralizing antibodies. The conventional plaque reduction neutralizing test (PRNT) is performed using Fcγ receptor (FcγR)-negative cells. Because FcγR plays a key role in antibody-dependent enhancement, we examined neutralizing antibody titers of mouse monoclonal antibodies and human serum samples in PRNTs using FcγRIIA-negative and FcγRIIA-expressing BHK cells. There was a discrepancy in dengue virus neutralizing antibody titers between PRNTs using FcγRIIA-negative versus FcγRIIA-expressing BHK cells. Neutralizing antibody titers to DENV-1 and DENV-2 tested with monoclonal antibodies, and with most of the human serum samples, were higher in assays using BHK cells than those using FcγRIIA-expressing BHK cells. The results suggest that neutralizing antibody titers determined using FcγRIIA-expressing cells may better reflect the protective capacity of anti-DENV antibodies, as the major target cells of DENV infection are FcγR-positive cells.