Chuan-Liang Kao

Infection of Human Dendritic Cells by Dengue Virus Causes Cell Maturation and Cytokine Production

Dengue virus (DV) infection is a major problem in public health. It can cause fatal diseases such as Dengue hemorrhagic fever and Dengue shock syndrome. Dendritic cells (DC) are professional APCs required for establishing a primary immune response. Here, we investigated the role of human PBMC-derived DC in DV infection. Using different techniques, including plaque assay, flow cytometry analysis, nested RT-PCR, and confocal microscope and electron microscope examinations, we show that DV can enter cultured human DC and produce virus particles. After entrance, DV could be visualized in cystic vesicles, vacuoles, and the endoplasmic reticulum. The DV-infected DC also showed proliferation and hypertrophy of the endoplasmic reticulum as well as the swollen mitochondria. In addition, the DV-stimulated DC could express maturation markers such as B7-1, B7-2, HLA-DR, CD11b, and CD83. Furthermore, the infection of DC by DV induced production of TNF-α and IFN-α, but not IL-6 and IL-12. Although DC underwent spontaneous apoptosis in the absence of feeding cytokines, this process appeared to be delayed after DV infection. Our observations provide important information in understanding the pathogenesis of DV infection.

Antibodies to Envelope Glycoprotein of Dengue Virus during the Natural Course of Infection Are Predominantly Cross-Reactive and Recognize Epitopes Containing Highly Conserved Residues at the Fusion Loop of Domain II

ABSTRACT The antibody response to the envelope (E) glycoprotein of dengue virus (DENV) is known to play a critical role in both protection from and enhancement of disease, especially after primary infection. However, the relative amounts of homologous and heterologous anti-E antibodies and their epitopes remain unclear. In this study, we examined the antibody responses to E protein as well as to precursor membrane (PrM), capsid, and nonstructural protein 1 (NS1) of four serotypes of DENV by Western blot analysis of DENV serotype 2-infected patients with different disease severity and immune status during an outbreak in southern Taiwan in 2002. Based on the early-convalescent-phase sera tested, the rates of antibody responses to PrM and NS1 proteins were significantly higher in patients with secondary infection than in those with primary infection. A blocking experiment and neutralization assay showed that more than 90% of anti-E antibodies after primary infection were cross-reactive and nonneutralizing against heterologous serotypes and that only a minor proportion were type specific, which may account for the type-specific neutralization activity. Moreover, the E-binding activity in sera of 10 patients with primary infection was greatly reduced by amino acid replacements of three fusion loop residues, tryptophan at position 101, leucine at position 107, and phenylalanine at position 108, but not by replacements of those outside the fusion loop of domain II, suggesting that the predominantly cross-reactive anti-E antibodies recognized epitopes involving the highly conserved residues at the fusion loop of domain II. These findings have implications for our understanding of the pathogenesis of dengue and for the future design of subunit vaccine against DENV as well.

An External Loop Region of Domain III of Dengue Virus Type 2 Envelope Protein Is Involved in Serotype-Specific Binding to Mosquito but Not Mammalian Cells

ABSTRACT Dengue virus (DV) is a flavivirus and infects mammalian cells through mosquito vectors. This study investigates the roles of domain III of DV type 2 envelope protein (EIII) in DV binding to the host cell. Recombinant EIII interferes with DV infection to BHK21 and C6/36 cells by blocking dengue virion adsorption to these cells. Inhibition of EIII on BHK21 cells was broad with no serotype specificity; however, inhibition of EIII on C6/36 cells was relatively serotype specific. Soluble heparin completely blocks binding of EIII to BHK21 cells, suggesting that domain III binds mainly to cell surface heparan sulfates. This suggestion is supported by the observation that EIII binds very weakly to gro2C and sog9 mutant mammalian cell lines that lack heparan sulfate. In contrast, heparin does not block binding of EIII to mosquito cells. Furthermore, a synthetic peptide that includes amino acids (aa) 380 to 389 of EIII, IGVEPGQLKL, inhibits binding of EIII to C6/36 but not BHK21 cells. This peptide corresponds to a lateral loop region on domain III of E protein, indicating a possible role of this loop in binding to mosquito cells. In summary, these results suggest that EIII plays an important role in binding of DV type 2 to host cells. In addition, EIII interacts with heparan sulfates when binding to BHK21 cells, and a loop region containing aa 380 to 389 of EIII may participate in DV type 2 binding to C6/36 cells.

Human TLR3 recognizes dengue virus and modulates viral replication in vitro

The elicitation of large amount inflammatory cytokine in serum has been developed as the cause of the plasma leakage in dengue fever (DF)/dengue haemorrhagic fever (DHF) infection. Virus recognition in innate immunity is the key. The Toll‐like receptors (TLRs) play an important role in pathogen recognition towards cytokine induction among several viruses; however, the role of TLRs on innate immune recognition against DENV remains unclear. This study aims at the interaction between dengue virus (DENV) and human TLRs at the incipient stage of infection in vitro. Our experiment reveals that stably expression of TLR3, 7, 8 on HEK293 enables IL‐8 secretion after DENV recognition. By the model of human monocytic cells U937, we demonstrated the trigger of IL‐8 after viral recognition of human monocytic cell is primary through TLR3 following endosomal acidification. Silencing of TLR3 in U937 cells significantly blocks the DENV‐induced IL‐8 production. Besides, the interaction is further corroborated by colocalization of TLR3 and DENV RNA upon DENV internalization. Furthermore, in this study we found the expression of TLR3 can mediate strong IFN‐α/β release and inhibit DENV viral replication significantly, thus limit the cytopathic effect.

Enterovirus-Induced miR-141 Contributes to Shutoff of Host Protein Translation by Targeting the Translation Initiation Factor eIF4E

Viruses rely on the host translation machinery to complete their life cycles. Picornaviruses use an internal ribosome entry site to initiate cap-independent protein translation and in parallel host cap-dependent translation is shut off. This process is thought to occur primarily via cleavage of host translation initiation factors eIF4GI and eIF4GII by viral proteases. Here we describe another mechanism whereby miR-141 induced upon enterovirus infection targets the cap-dependent translation initiation factor, eIF4E, for shutoff of host protein synthesis. Knockdown of miR-141 reduces viral propagation, and silencing of eIF4E can completely reverse the inhibitory effect of the miR-141 antagomiR on viral propagation. Ectopic expression of miR-141 promotes the switch from cap-dependent to cap-independent translation. Moreover, we identified a transcription factor, EGR1, which is partly responsible for miR-141 induction in response to enterovirus infection. Our results suggest that upregulation of miR-141 upon enterovirus infection can facilitate viral propagation by expediting the translational switch.

Detection of Dengue Virus Replication in Peripheral Blood Mononuclear Cells from Dengue Virus Type 2-Infected Patients by a Reverse Transcription-Real-Time PCR Assay

ABSTRACT While dengue virus is thought to replicate in mononuclear phagocytic cells in vivo, attempts to detect it in peripheral blood mononuclear cells (PBMC) by virus isolation or antigen detection have had variable and generally low rates. In this study, we developed a reverse transcription (RT)-real-time PCR assay to quantify positive- and negative-sense RNA of dengue virus type 2 within the cells. The assay includes an RT step using either sense or antisense primer followed by a real-time PCR step using the designed primers and probe, which target a capsid region highly conserved in dengue virus type 2 strains. It can be used to monitor the dynamic change of intracellular dengue virus RNA species during the course of infection. When this assay is employed in quantification of dengue virus RNA species in PBMC from 10 patients infected with dengue virus type 2, both positive- and negative-sense dengue RNA can be detected, indicating that dengue virus is actively replicating in PBMC in vivo. Moreover, the amounts of negative-sense dengue virus RNA in PBMC correlate very well with the viral load of dengue virus in plasma, suggesting that quantification of negative-sense dengue virus RNA in PBMC may provide another indicator of dengue virus replication in vivo. Use of this convenient, sensitive, and accurate method of quantification in clinical samples from patients with different disease severity would further our understanding of the pathogenesis of dengue.

Laboratory-based surveillance and molecular epidemiology of influenza virus in Taiwan.

ABSTRACT A laboratory-based surveillance network of 11 clinical virological laboratories for influenza viruses was established in Taiwan under the coordination of the Center for Disease Control and Prevention (CDC), Taiwan. From October 2000 to March 2004, 3,244 influenza viruses were isolated, including 1,969 influenza A and 1,275 influenza B viruses. The influenza infections usually occurred frequently in winter in the northern hemisphere. However, the influenza seasonality in Taiwan was not clear during the four seasons under investigation. For example, the influenza A viruses peaked during the winters of 2001, 2002, and 2003. However, some isolated peaks were also found in the summer and fall (June to November) of 2001 and 2002. An unusual peak of influenza B also occurred in the summer of 2002 (June to August). Phylogenetic analysis shows that influenza A isolates from the same year were often grouped together. However, influenza B isolates from the year 2002 clustered into different groups, and the data indicate that both B/Victoria/2/87-like and B/Yamagata/16/88-like lineages of influenza B viruses were cocirculating. Sequence comparison of epidemic strains versus vaccine strains shows that many vaccine-like Taiwanese strains were circulating at least 2 years before the vaccine strains were introduced. No clear seasonality of influenza reports in Taiwan occurred in contrast to other more continental regions.

Polymerase chain reaction to detect human cytomegalovirus in livers of infants with neonatal hepatitis

Neonatal hepatitis is closely related to human cytomegalovirus infection in Taiwan, a conclusion based on serological and urine culture studies. To obtain more direct evidence further relating cytomegalovirus to the pathogenesis of neonatal hepatitis, the cytomegalovirus genome was studied in the liver tissues of 50 infants with neonatal hepatitis using the polymerase chain reaction (PCR). Liver tissues from 26 infants with biliary atresia and another 30 infants and children with diagnoses other than neonatal hepatitis, cholestasis, or hepatitis were also studied for comparison. Sequences from the immediate early gene 1 and 2 regions were used as primers. The liver tissues from 23 (46%) of the 50 infants with neonatal hepatitis were positive for cytomegalovirus genome, whereas those of 2 of the 26 infants with biliary atresia and none of the liver tissues from 30 infants and children without neonatal hepatitis were positive for cytomegalovirus genome, by PCR. The results of PCR correlated well with that of serology and urine culture. This study provides further evidence of cytomegalovirus in the pathogenesis of neonatal hepatitis.

Viral etiology of intussusception in Taiwanese childhood.

BACKGROUND Adenovirus infection and lymphoid hyperplasia have been associated with childhood intussusception. However, the extent of other viruses involved in this condition remains unclear. This prospective study investigates the relationship between some lymphotropic viruses and current childhood intussusception. METHODS Patients with intussusception encountered in a pediatric emergency department in a recent 3-year period were studied. Healthy infants and toddlers of comparable age served as controls. Throat and rectal viral cultures were performed in patients and controls. Viral antibodies against adenovirus, cytomegalovirus, human herpesvirus (HHV)-6, HHV-7 and Epstein-Barr virus (EBV) were tested in paired sera from the patients. Acute stage serum from each patient and mesenteric lymph nodes from patients requiring surgery were studied for the presence of adenovirus genome by PCR. RESULTS Twenty-seven of 61 (44.3%) intussusception patients, but only 2 of 52 (3.8%) healthy controls shed nonenteric adenovirus in throat and rectal specimens (P < 0.001). Of the 27 (74.1%) patients who shed adenovirus, 20 were older than 1 year old, whereas only 1 of 15 (6.7%) similarly aged patients in a previous study from the same area three decades ago did so (P = 0.001). Among 43 patients with available paired sera, acute primary viral infection was found in 17 (39.5%) by adenovirus, 4 (9.3%) by HHV-6, 5 (11.6%) by HHV-7, 2 (4.7%) by EBV and none by cytomegalovirus. Multiple viral infections occurred in 6 patients. Adenovirus genome was detected in 4 of 9 mesenteric lymph nodes and in only 3 of 60 (5%) acute phase sera. CONCLUSIONS Primary nonenteric adenovirus infection contributes to current childhood intussusception. Acute primary HHV-6, HHV-7 and EBV infections also play etiologic roles.

Microbiologic Characteristics, Serologic Responses, and Clinical Manifestations in Severe Acute Respiratory Syndrome, Taiwan

The genome of one Taiwanese severe acute respiratory syndrome-associated coronavirus (SARS-CoV) strain (TW1) was 29,729 nt in length. Viral RNA may persist for some time in patients who seroconvert, and some patients may lack an antibody response (immunoglobulin G) to SARS-CoV >21 days after illness onset. An upsurge of antibody response was associated with the aggravation of respiratory failure.