Junying Chen

Antibody-dependent enhancement of dengue virus infection inhibits RLR-mediated Type-I IFN-independent signalling through upregulation of cellular autophagy

Antibody dependent enhancement (ADE) of dengue virus (DENV) infection is identified as the main risk factor of severe Dengue diseases. Through opsonization by subneutralizing or non-neutralizing antibodies, DENV infection suppresses innate cell immunity to facilitate viral replication. However, it is largely unknown whether suppression of type-I IFN is necessary for a successful ADE infection. Here, we report that both DENV and DENV-ADE infection induce an early ISG (NOS2) expression through RLR-MAVS signalling axis independent of the IFNs signaling. Besides, DENV-ADE suppress this early antiviral response through increased autophagy formation rather than induction of IL-10 secretion. The early induced autophagic proteins ATG5-ATG12 participate in suppression of MAVS mediated ISGs induction. Our findings suggest a mechanism for DENV to evade the early antiviral response before IFN signalling activation. Altogether, these results add knowledge about the complexity of ADE infection and contribute further to research on therapeutic strategies.

Genetic analysis of the VP1 region of human enterovirus 71 strains isolated in Fuyang, China, during 2008

Enterovirus 71 (EV71) is a common cause of Hand, foot, and mouth disease (HFMD) and may also cause severe neurological diseases, such as encephalitis and poliomyelitis-like paralysis. To examine the genetic diversity of EV71, we determined and analyzed the complete VP1 sequences (891 nucleotides) from nine EV71 strains isolated in Fuyang, China. We found that nine EV71 strains isolated were over 98% homologous at the nucleotide level and 93%–100% homologous to members of the C4 subgenogroup. At the amino acid level, these Fuyang strains were 99%–100% homologous to one another, 97%–100% homologous to members of the C4 subgenogroup, and the histidine(H) at amino acid position 22 was conserved among the Fuyang strains. The results indicate that Fuyang isolates belong to genotype C4, and an H at position 22 appears to be a marker for the Fuyang strains.

Molecular Characterization and Viral Origin of the 2015 Dengue Outbreak in Xishuangbanna, Yunnan, China.

A total of 1067 serum samples were collected from febrile patients in Xishuangbanna, Yunnan, 2015. Of these, 852 cases were confirmed to be dengue NS1-positive. 76 structural protein genes were sequenced through RT-PCR based on the viral RNAs extracted from serum samples. Phylogenetic analysis revealed that all strains were classified as cosmopolitan genotype of DENV-2. After comparing with the DENV-2SS, 173 base substitutions were found in 76 sequences, resulting in 43 nonsynonymous mutations, of which 22 mutations existed among all samples. According to secondary structure prediction, 8 new possible nucelotide/protein binding sites were found and another 4 sites were lost among the 775 amino acids of DENV structural proteins as compared with DENV-2SS. Meanwhile, 6 distinct amino acid changes were found in the helix and strand regions, and the distribution of the exposed and buried regions was slightly altered. The results indicated that the epidemic dengue strains of Xishuangbanna in 2015 are most similar to the Indian strain in 2001 and the Sri Lankan strain in 2004. Moreover, it also show a very strong similarity to the epidemic strains of Fujian province in 1999 and 2010, which show that there is an internal recycling epidemic trend of DENV in China.

Dynamic constitution of the pathogens inducing encephalitis in hand, foot and mouth disease in Kunming, 2009-2011

Hand, foot and mouth disease (HFMD), caused by various viral pathogens, is an emerging infectious disease in children in Asia. Understanding the composition of these pathogens is necessary to prevent and control this disease. In the present study, the pathogens in 436 HFMD patients (from 2009 to 2011) with concurrent clinical indications of encephalitis, meningoencephalitis, or both, were defined using the semi-nested PCR. A systematic analysis of the composition of these pathogens was performed. Various enteroviruses that are capable of inducing central nervous system (CNS) damage in HFMD patients were identified, including enterovirus 71, coxsackievirus A16, and Echovirus 9. Most of these pathogens were found co-infecting the patients. The composition of the pathogens that induced CNS damage in the HFMD patients was dynamically modulated in the cases.

Genetic variability and functional implication of the long control region in HPV-16 variants in Southwest China

HPV-16 long control region (LCR) has been shown to be the most variable region of the HPV-16 genome and may play important roles in viral persistence and the development of cervical cancer. This study aimed to assess the risk of HPV-16 LCR variants for cervical cancer in women of Southwest China. 2146 cervical scrapings of volunteer outpatients and 74 cervical cancer tissues were screened.14 entire HPV-16 LCRs from asymptomatic carriers and 34 entire HPV-16 LCRs from cervical cancer patients were successfully amplified and sequenced to align to others described. 58 different point mutations were detected in 54 nucleotide sites of HPV-16 LCR. G7193T and G7521A variants, accounting for 100% of the infections, were predicted to locate at the binding site for FOXA1 and SOX9, respectively. A7730C variant which showed a high mutation frequency in cervical cancer was predicted to be a binding site for the cellular transcription factor PHOX2A. In addition, phylogenetic analysis displayed a high prevalence of A lineage in HPV-16 LCR in this Southwest China population. This study may help understanding of the intrinsic geographical relatedness and the correlations between LCR mutations and the development of carcinogenic lesions in Southwest China population. And it provides useful data for the further study of the biological function of HPV-16 LCR variants.

Complete genome analysis of dengue virus type 3 isolated from the 2013 dengue outbreak in Yunnan, China

In the past few decades, dengue has spread rapidly and is an emerging disease in China. An unexpected dengue outbreak occurred in Xishuangbanna, Yunnan, China, resulting in 1331 patients in 2013. In order to obtain the complete genome information and perform mutation and evolutionary analysis of causative agent related to this largest outbreak of dengue fever. The viruses were isolated by cell culture and evaluated by genome sequence analysis. Phylogenetic trees were then constructed by Neighbor-Joining methods (MEGA6.0), followed by analysis of nucleotide mutation and amino acid substitution. The analysis of the diversity of secondary structure for E and NS1 protein were also performed. Then selection pressures acting on the coding sequences were estimated by PAML software. The complete genome sequences of two isolated strains (YNSW1, YNSW2) were 10,710 and 10,702 nucleotides in length, respectively. Phylogenetic analysis revealed both strain were classified as genotype II of DENV-3. The results indicated that both isolated strains of Xishuangbanna in 2013 and Laos 2013 stains (KF816161.1, KF816158.1, LC147061.1, LC147059.1, KF816162.1) were most similar to Bangladesh (AY496873.2) in 2002. After comparing with the DENV-3SS (H87) 62 amino acid substitutions were identified in translated regions, and 38 amino acid substitutions were identified in translated regions compared with DENV-3 genotype II stains Bangladesh (AY496873.2). 27(YNSW1) or 28(YNSW2) single nucleotide changes were observed in structural protein sequences with 7(YNSW1) or 8(YNSW2) non-synonymous mutations compared with AY496873.2. Of them, 4 non-synonymous mutations were identified in E protein sequences with (2 in the β-sheet, 2 in the coil). Meanwhile, 117(YNSW1) or 115 (YNSW2) single nucleotide changes were observed in non-structural protein sequences with 31(YNSW1) or 30 (YNSW2) non-synonymous mutations. Particularly, 14 single nucleotide changes were observed in NS1 sequences with 4/14 non-synonymous substitutions (4 in the coil). Selection pressure analysis revealed no positive selection in the amino acid sites of the genes encoding for structural and non-structural proteins. This study may help understand the intrinsic geographical relatedness of dengue virus 3 and contributes further to research on their infectivity, pathogenicity and vaccine development.

Development of a peptide ELISA to discriminate vaccine-induced immunity from natural infection of hepatitis A virus in a phase IV study

Hepatitis A virus (HAV) is a highly infectious agent that causes acute liver disease. The infection can trigger the production of antibodies against the structural and non-structural proteins of HAV. Nonetheless, vaccination with an HAV vaccine leads to the production of a primary antibody against the structural proteins. Because the non-structural proteins are only produced during active virus replication, there is no or very little antibody production against the non-structural proteins. However, the current commercial immunoassay cannot distinguish between antibodies produced during natural infection and those from vaccination against HAV. In our study, six immune-dominant epitopes from the non-structural proteins were designed, synthesized, linked together and cloned into pGEX-5X-1 plasmid. The recombinant protein was expressed in E. coli and purified by Ni2+-coated magnetic agarose beads. Then the purified recombinant protein was used as an ELISA antigen to detect antibodies for HAV non-structural proteins in serum samples. Seventy-seven attenuated and 89 inactivated vaccinated samples collected from our previous phase IV study of HAV vaccines were detected by peptide ELISA developed in this study. The mean OD450 value for the vaccination samples and acute infection samples were 0.529 (0.486 for the attenuated group and 0.567 for the inactivated group) and 1.187, respectively. According to the receiver operating characteristic (ROC) curve, the sensitivity and specificity of the peptide ELISA were 93.80% and 91.00%, respectively. This peptide ELISA was confirmed to discriminate vaccine-induced immunity from natural infection of HAV in a phase IV study with high sensitivity and specificity.

Molecular characterization of the viral structural gene of the first dengue virus type 1 outbreak in Xishuangbanna, a border area of China, Burma and Laos

In the context of recent arbovirus epidemics, dengue fever is becoming a greater concern around the world. In August 2017, Xishuangbanna, which is a border city of China, Burma and Laos, had its first major dengue outbreak. A total of 156 serum samples from febrile patients were collected; 97 DENV positive serum samples were screened out, and viral RNAs were successfully and directly extracted, including 77 cases from China and 20 cases from Myanmar. Phylogenetic analysis revealed that all of the strains were classified as DENV-1. There are eight epidemic dengue strains from Myanmar and 74 from Jinghong, Xishuangbanna, that were classified as cluster 1, which are the most similar to the strain of China Guangzhou 2011. There are three epidemic strains from Xishuangbanna Mengla that were classified as cluster 2, which have the closest relationship to the strain of China Hubei 2014. However, there are 12 epidemic strains from Myanmar that were classified as cluster 3, which have the closest relationship to the strain of Laos from 2008, which shows that there is a recycling epidemic trend of DENV in China. There were 236 mutations in the base, which caused 31 nonsynonymous mutations in the DENV structural protein C/prM/E genes when the strain of Xishuangbanna and Myanmar were compared with the DENV-1SS. There is no clear homologous recombination signal among these stains. Homology modeling possibly predicted a three-dimensional structure of the structural protein of these strains and revealed that they had the same three-dimensional structure and all had five predicted protein binding sites, but there are differences in binding site 434 (DENV-1SS: Thr434, DV-Jinghong: Ser434, DV-Myanmar: Ser434, DV-Mengla: Ser434). The results of the molecular clock phylogenetic and demographic reconstruction analysis show that DENV-1 became highly diversified in 1972 followed by a slightly decreased period until 2017. In conclusion, our study lays the foundation for studying the global evolution and prevalence of DENV. Author Summary Dengue fever (DF) is a mosquito-borne illness caused by a flavivirus. Human infections with Dengue virus (DENV) could cause fever, cutaneous rash and malaise. Xishuangbanna, which is located in the southwestern Yunnan Province and is a border city with China, Burma and Laos, was reported to have outbreak of DENV in 2013 and 2015 with different types. However, there was a large outburst of dengue in May 2017. To understand the genetic characterization, potential source and evolution of the virus, 156 serum samples were analyzed. We focused on: (i) Phylogenetic analysis of the structural protein genes sequences; (ii) Mutation, recombination analysis and predicted protein binding sites of the structural protein genes; (iii) Molecular clock and demographic reconstruction of global dengue virus serotype 1(DENV-1). Our results indicated that this is the first outbreak of DENV-1 in Xishuangbanna, dengue epidemic strains on the Burma border of China show diversification, we found a virulence site changed from I to T(amino acid position: 440), which may lead to weakened virulence of the epidemic strains. We found that the evolution of DENV-1 is dominated by regional evolution. What’s more, DENV-1 became highly diversified in 1972 followed by a slightly decreased period until 2017.

Comparison analysis of microRNAs in response to dengue virus type 2 infection between the Vero cell-adapted strain and its source, the clinical C6/36 isolated strain

To elucidate the differences in microRNAs during dengue virus infection between Vero cell-adapted strain (DENV-2-Vero) and its source, the clinical C6/36 isolated strain (DENV-2-C6/36), a comparison analysis was performed in Vero cells by high throughput sequencing. The results showed that the expression of 16 known and 3 novel miRNAs exhibited marked differences. 5 known miRNAs were up-regulated in DENV-2-C6/36 group, while 11 known microRNAs were down-regulated in DENV-2-Vero group. The GO enrichment and KEGG pathway analysis showed that there was a distinct difference in regulating viral replication between two strains. In DENV-2-Vero infection group, significantly enriched GO terms included virion attachment to host cells, viral structural protein/genome processing and packaging. Meanwhile, the regulation of cell death and apoptosis between two groups were different in the early stage of infection. KEGG enrichment analysis showed that DENV-2-C6/36 infection induced more intense regulation of immune-related pathways, including Fc gamma R-mediated phagocytosis, etc. DENV-2-Vero infection could partially alleviate the immune defense of Vero cells compared with DENV-2-C6/36. The results indicated that the distinct microRNA changes induced by two DENV-2 strains may be partly related to their infective abilities. Our data provide useful insights that help elucidate the host-pathogen interactions following DENV infection.

Molecular Characterization of Dengue Virus Serotype 2 Cosmospolitan Genotype From 2015 Dengue Outbreak in Yunnan, China

In 2015, a dengue outbreak with 1,067 reported cases occurred in Xishuangbanna, a city in China that borders Burma and Laos. To characterize the virus, the complete genome sequence was obtained and phylogenetic, mutation, substitution and recombinant analyses were performed. DENV-NS1 positive serum samples were collected from dengue fever patients, and complete genome sequences were obtained through RT-qPCR from these serum samples. Phylogenetic trees were then constructed by maximum likelihood phylogeny test (MEGA7.0), followed by analysis of nucleotide mutation and amino acid substitution. The recombination events among DENVs were also analyzed by RDP4 package. The diversity analysis of secondary structure for translated viral proteins was also performed. The complete genome sequences of four amplified viruses (YNXJ10, YNXJ12, YNXJ13, and YNXJ16) were 10,742, 10,742, 10,741, and 10,734 nucleotides in length, and phylogenetic analysis classified the viruses as cosmopolitan genotype of DENV-2. All viruses were close to DENV Singapore 2013 (KX380828.1) and the DENV China 2013 (KF479233.1). In comparison to DENV-2SS (M29095), the total numbers of base substitutions were 712 nt (YNXJ10), 809 nt (YNXJ12), 772 nt (YNXJ13), and 841 nt (YNXJ16), resulting in 109, 171, 130, and 180 amino acid substitutions in translated regions, respectively. In addition, compared with KX380828.1, there were 44, 105, 64, and 116 amino acid substitutions in translated regions, respectively. The highest mutation rate occurred in the prM region, and the lowest mutation rate occurred in the NS4B region. Most of the recombination events occurred in the prM, E and NS2B/3 regions, which corresponded with the mutation frequency of the related portion. Secondary structure prediction within the 3,391 amino acids of DENV structural proteins showed there were 7 new possible nucleotide-binding sites and 6 lost sites compared to DENV-2SS. In addition, 41 distinct amino acid changes were found in the helix regions, although the distribution of the exposed and buried regions changed only slightly. Our findings may help to understand the intrinsic geographical relatedness of DENV-2 and contributes to the understanding of viral evolution and its impact on the epidemic potential and pathogenicity of DENV.