Lingzhai Zhao

Severe dengue outbreak in Yunnan, China, 2013

In recent decades, the impact of dengue has increased both geographically and in intensity, and this disease is now a threat to approximately half of the worlds population. An unexpected large outbreak of dengue fever was reported in Xishuangbanna Dai Autonomous Prefecture, Yunnan Province, China, in 2013. This was the first autochthonous outbreak with a significant proportion of severe dengue cases in mainland China in a decade. According to the 2009 World Health Organization guidelines, half of the 136 laboratory confirmed cases during the epidemic were severe dengue. The clinical presentation included severe haemorrhage (such as massive vaginal and gastrointestinal bleeding), severe plasma leakage (such as pleural effusion, ascites, or hypoproteinaemia), and organ involvement (such as myocarditis and lung impairment); 21 cases eventually deteriorated to shock. During this outbreak, all severe cases occurred in adults, among whom about 43% had co-morbid conditions. Nucleic acid detection and virus isolation confirmed dengue virus serotype 3 (DENV-3) to be the pathogenic agent of this outbreak. Phylogenetic analyses of envelope gene sequences showed that these DENV-3 isolates belonged to genotype II. This finding is of great importance to understand the circulation of DENV and predict the risk of severe disease in mainland China. Here, we provide a brief report of the epidemiology, clinical manifestations, and aetiology of this dengue fever outbreak, and characterize DENV strains isolated from clinical specimens.

Isolation and characterization of dengue virus serotype 2 from the large dengue outbreak in Guangdong, China in 2014

Dengue has been well recognized as a global public health threat, but only sporadic epidemics and imported cases were reported in recent decades in China. Since July 2014, an unexpected large dengue outbreak has occurred in Guangdong province, China, resulting in more than 40000 patients including six deaths. To clarify and characterize the causative agent of this outbreak, the acute phase serum from a patient diagnosed with severe dengue was subjected to virus isolation and high-throughput sequencing (HTS). Traditional real-time RT-PCR and HTS with Ion Torrent PGM detected the presence of dengue virus serotype 2 (DENV-2). A clinical DENV-2 isolate GZ05/2014 was obtained by culturing the patient serum in mosquito C6/36 cells. The complete genome of GZ05/2014 was determined and deposited in GenBank under the access number KP012546. Phylogenetic analysis based on the complete envelope gene showed that the newly DENV-2 isolate belonged to Cosmopolitan genotype and clustered closely with other Guangdong strains isolated in the past decade. No amino acid mutations that are obviously known to increase virulence or replication were identified throughout the genome of GZ05/2014. The high homology of Guangdong DENV-2 strains indicated the possibility of establishment of local DENV-2 circulation in Guangdong, China. These results help clarify the origin of this epidemic and predict the future status of dengue in China.

Transfer of convalescent serum to pregnant mice prevents Zika virus infection and microcephaly in offspring.

Transfer of convalescent serum to pregnant mice prevents Zika virus infection and microcephaly in offspring

Delineating antibody recognition against Zika virus during natural infection

Zika virus (ZIKV) is an emerging mosquito-transmitted flavivirus that shares a considerable degree of homology with dengue virus (DENV). Here, we examined longitudinal antibody response against ZIKV during natural infection in 2 convalescent individuals. By decomposing the antibody recognition into DI/DII and DIII of the E glycoprotein, we showed their development in humans followed a spatiotemporal hierarchy. Plasma binding to DI/DII appeared to peak and wane during early infection with extensive cross-reactivity with DI/DII of DENV. Binding to DIII, however, peaked early but persisted months into the infection without detectable cross-reactivity with DIII of DENV. A clear trend of increase in DIII-specific neutralizing activity was observed over the course of infection. mAbs isolated during early infection are largely DI/DII specific, weakly neutralizing, and highly cross-reactive with DENV, while those from later infection are more diverse in recognition, potently neutralizing, and ZIKV specific. The most potent neutralizing mAb targeting the DIII provided 100% protection in mice from lethal ZIKV infection and could therefore serve as a promising candidate for antibody-based therapy and prevention. The dynamic features unveiled here will assist us to better understand the pathogenesis of ZIKV infection and inform rational design of vaccines.

Slow resolution of inflammation in severe adult dengue patients

BackgroundThe pathogenesis of severe dengue has not been fully elucidated. The inflammatory response plays a critical role in the outcome of dengue disease.MethodsIn this study, we investigated the levels of 17 important inflammation mediators in plasma collected from mild or severe adult dengue patients at different time points to understand the contribution of inflammation to disease severity and to seek experimental evidence to optimize the existing clinical treatment strategies. Patients were simply classified as mild and severe dengue according to the 2009 WHO classification. Plasma was collected on day 3-5, 6-7, 8-10 and 14-17 of illness. Levels of 17 inflammation mediators including TNF-α, IL-1α, IFN-γ, IL-6, IFN-α, MIF, IL-10, IL-1RA, IL-8, IP-10, MCP-1, RANTES, GRO, eotaxin-1, sICAM-1 and sVCAM-1 were determined by a multiplex Luminex® system. Different trends of inflammation mediators throughout the disease were compared between mild and severe patients.ResultsInflammation mediators including IL-1α, IFN-γ, IL-10, IL-8, IP-10, MCP-1 and sVCAM-1 displayed significant differences on day 8-10 of illness between mild and severe dengue patients. Their concentrations were higher in severe patients than mild ones at the same time points. Moreover, those cytokines decreased gradually in mild patients but not in severe patients.ConclusionOur results revealed the coexistence of excessive inflammatory response and slow resolution of inflammation in severe adult dengue patients. Hence suppression and/or pro-resolution of inflammation could be a potential therapeutic approach for treatment of severe dengue.

Epidemiological and Virological Characterizations of the 2014 Dengue Outbreak in Guangzhou, China.

Dengue used to be recognized as an imported and sporadic disease in China. Since June 2014, an unexpected large dengue outbreak has attacked Guangzhou, China, resulting in more than 40,000 cases. Among the 1,942 laboratory-confirmed hospitalized dengue cases, 121 were diagnosed as severe dengue according to the 2009 WHO guideline, and 2 patients finally died. Laboratory diagnosis and virus isolation demonstrated that the majority (96%) cases were caused by dengue virus serotype 1 (DENV-1), and the others by serotype 2 (DENV-2). 14 DENV strains were isolated from the sera of acute-phase dengue patients during this outbreak, and the complete envelope (E) gene of 12 DENV-1 strains and two DENV-2 strains were determined using RT-PCR assay. Phylogenetic analysis based on the E gene revealed the DENV-1 strains isolated during the outbreak belonged to genotype I and V, respectively. These isolates formed three clades. DENV-2 isolates were assigned to the same clade belonging to genotype cosmopolitan. These strains isolated in 2014 were closely related to the isolates obtained from the same province, Guangdong, in 2013. No amino acid mutations known to increase virulence were identified throughout the E protein of isolates in 2014. These results indicate that dengue is turning into endemic in Guangdong, China, and extensive seroepidemiological investigation and mosquito control measures are critically needed in the future.

Characterization of the contemporary Zika virus in immunocompetent mice

Na-Na Zhanga,b,y, Min Tianc,y, Yong-Qiang Denga,d,y, Jia-Nan Haoe,y, Hong-Jiang Wang, Xing-Yao Huang, Xiao-Feng Li, Yu-Guang Wang, Ling-Zhai Zhao, Fu-Chun Zhang, and Cheng-Feng Qin Department of Virology, Beijing Institute of Microbiology and Epidemiology, Beijing, China; Guangxi Medical University, Nanning, China; Beijing Traditional Medicine Chinese Hospital, Capital Medical University, Beijing, China; State Key Laboratory of Pathogen and Biosecurity, Beijing, China; Anhui Medical University, Hefei, China; Guangzhou Eighth People’s Hospital, Guangzhou Medical University, Guangzhou, Guangdong, China

Delayed and highly specific antibody response to nonstructural protein 1 (NS1) revealed during natural human ZIKV infection by NS1-based capture ELISA

BackgroundZika virus (ZIKV) had spread rapidly in the past few years in southern hemisphere where dengue virus (DENV) had caused epidemic problems for over half a century. The high degree of cross-reactivity of Envelope (E) protein specific antibody responses between ZIKV and DENV made it challenging to perform differential diagnosis between the two infections using standard ELISA method for E protein.MethodsUsing an IgG capture ELISA, we investigated the kinetics of nonstructural protein 1 (NS1) antibody response during natural ZIKV infection and the cross-reactivity to NS1 proteins using convalescent sera obtained from patients infected by either DENV or ZIKV.ResultsThe analyses of the sequential serum samples from ZIKV infected individuals showed NS1 specific Abs appeared 2 weeks later than E specific Abs. Notably, human sera from ZIKV infected individuals did not contain cross-reactivity to NS1 proteins of any of the four DENV serotypes. Furthermore, four out of five NS1-specific monoclonal antibodies (mAbs) isolated from ZIKV infected individuals did not bind to DENV NS1 proteins. Only limited amount of cross-reactivity to ZIKV NS1 was displayed in 108 DENV1 immune sera at 1:100 dilution.ConclusionsThe high degree of NS1-specific Abs in both ZIKV and DENV infection revealed here suggest that NS1-based diagnostics would significantly improve the differential diagnosis between DENV and ZIKV infections.

Dengue immune sera enhance Zika virus infection in human peripheral blood monocytes through Fc gamma receptors.

Antibody dependent enhancement (ADE) has most often been associated with dengue virus (DENV). Studies using leukemia cell lines suggest that DENV specific antibodies can enhance Zika virus (ZIKV) infectivity, and vice versa. To examine the mechanisms of ADE of ZIKV infection in primary human cells, we assessed 40 serum samples obtained from convalescent DENV-1 or DENV-3 infected subjects. All sera tested exhibited high binding potency, while modest or none neutralization activities against ZIKV. Primary CD14+ monocytes, rather than B and T cells in peripheral blood mononuclear cells (PBMCs), were found to be the mediators of the enhancement of ZIKV infectivity by DENV immune sera. Monocyte-derived immature dendritic cells (DCs), but not mature DCs were highly permissive to ZIKV infection, whereas neither immature nor mature DCs could mediate enhanced ZIKV infection in the presence of DENV immune sera. In addition, antibody blocking of either FcγRI (CD64), or FcγRII (CD32), or FcγRIII (CD16) resulted in diminished ADE of ZIKV infection. Our findings provide an improved understanding of the pathogenesis of ZIKV infection, and inform rational vaccine design.

Zika virus shedding in the stool and infection through the anorectal mucosa in mice

Zika virus (ZIKV) has elicited global concern due to its unique biological features, unusual transmission routes, and unexpected clinical outcomes. Although ZIKV transmission through anal intercourse has been reported in humans, it remains unclear if ZIKV is detectable in the stool, if it can infect the host through the anal canal mucosa, and what the pathogenesis of such a route of infection might be in the mouse model. Herein, we demonstrate that ZIKV RNA can be recovered from stools in multiple mouse models, as well as from the stool of a ZIKV patient. Remarkably, intra-anal (i.a.) inoculation with ZIKV leads to efficient infection in both Ifnar1 −/− and immunocompetent mice, characterized by extensive viral replication in the blood and multiple organs, including the brain, small intestine, testes, and rectum, as well as robust humoral and innate immune responses. Moreover, i.a. inoculation of ZIKV in pregnant mice resulted in transplacental infection and delayed fetal development. Overall, our results identify the anorectal mucosa as a potential site of ZIKV infection in mice, reveal the associated pathogenesis of i.a. infection, and highlight the complexity of ZIKV transmission through anal intercourse.