Chunlin Zhang

Cross-protective immunity can account for the alternating epidemic pattern of dengue virus serotypes circulating in Bangkok

Dengue virus, the causative agent of dengue fever and its more serious manifestation dengue hemorrhagic fever, is widespread throughout tropical and subtropical regions. The virus exists as four distinct serotypes, all of which have cocirculated in Bangkok for several decades with epidemic outbreaks occurring every 8–10 years. We analyze time-series data of monthly infection incidence, revealing a distinctive pattern with epidemics of serotypes 1, 2, and 3 occurring at approximately the same time and an isolated epidemic of serotype 4 occurring in the intervening years. Phylogenetic analysis of virus samples collected over the same period shows that clade replacement events are linked to the epidemic cycle and indicates that there is an interserotypic immune reaction. Using an epidemic model with stochastic seasonal forcing showing 8- to 10-year epidemic oscillations, we demonstrate that moderate cross-protective immunity gives rise to persistent out-of-phase oscillations similar to those observed in the data, but that strong or weak cross-protection or cross-enhancement only produces in-phase patterns. This behavior suggests that the epidemic pattern observed in Bangkok is the result of cross-protective immunity and may be significantly altered by changes in the interserotypic immune reaction.

Clade Replacements in Dengue Virus Serotypes 1 and 3 Are Associated with Changing Serotype Prevalence

ABSTRACT The evolution of dengue virus (DENV) is characterized by phylogenetic trees that have a strong temporal structure punctuated by dramatic changes in clade frequency. To determine the cause of these large-scale phylogenetic patterns, we examined the evolutionary history of DENV serotype 1 (DENV-1) and DENV-3 in Thailand, where gene sequence and epidemiological data are relatively abundant over a 30-year period. We found evidence for the turnover of viral clades in both serotypes, most notably in DENV-1, where a major clade replacement event took place in genotype I during the mid-1990s. Further, when this clade replacement event was placed in the context of changes in serotype prevalence in Thailand, a striking pattern emerged; an increase in DENV-1 clade diversity was associated with an increase in the abundance of this serotype and a concomitant decrease in DENV-4 prevalence, while clade replacement was associated with a decline in DENV-1 prevalence and a rise of DENV-4. We postulate that intraserotypic genetic diversification proceeds at times of relative serotype abundance and that replacement events can result from differential susceptibility to cross-reactive immune responses.

Safety and Immunogenicity of a Tetravalent Live-attenuated Dengue Vaccine in Flavivirus Naive Children

A Phase I/II observer-blind, randomized, controlled trial evaluated the safety and immunogenicity of a dengue virus (DENV) vaccine candidate in healthy Thai infants (aged 12-15 months) without measurable pre-vaccination neutralizing antibodies to DENV and Japanese encephalitis virus. Fifty-one subjects received two doses of either DENV (N = 34; four received 1/10th dose) or control vaccine (N = 17; dose 1, live varicella; dose 2, Haemophilus influenzae type b). After each vaccine dose, adverse events (AEs) were solicited for 21 days, and non-serious AEs were solicited for 30 days; serious AEs (SAEs) were recorded throughout the study. Laboratory safety assessments were performed at 10 and 30 days; neutralizing antibodies were measured at 30 days. The DENV vaccine was well-tolerated without any related SAEs. After the second dose, 85.7% of full-dose DENV vaccinees developed at least trivalent and 53.6% developed tetravalent neutralizing antibodies ≥ 1:10 to DENV (control group = 0%). This vaccine candidate, therefore, warrants continued development in this age group (NCT00322049; clinicaltrials.gov).

Phenotypic analysis of dengue virus isolates associated with dengue fever and dengue hemorrhagic fever for cellular attachment, replication and interferon signaling ability.

Eighteen dengue viruses (DENVs) representing all four serotypes, isolated from pediatric patients at childrens hospital, Queen Sirikit National Institute of Child Health, Bangkok, Thailand exhibiting a diverse spectrum of disease ranging from uncomplicated dengue fever (DF) to severe dengue hemorrhagic fever (DHF), were tested for their ability to attach to host cells, replicate and interfere with the IFNalpha signaling pathway by interfering with signal transducer and activator of transcription 1 (STAT-1) function. Although most isolates suppressed IFNalpha-induced STAT-1 phosphorylation, our results showed no difference between DENV strains associated with DF and those associated with DHF. However, the DHF isolates tended replicate to higher titers in dendritic cells (DCs) than the DF isolates, but this ability was independent of their cell-binding capability. Our results suggest that the emergence early in infection of viruses with a high degree of replication fitness may play an important role in DENV pathogenesis.

Letter to the editor: molecular genotyping of Dengue Virus Types 2 and 4 from the Guatemalan and Honduran Epidemics of 2007 using the envelope glycoprotein gene.

Eight serum specimens collected from dengue patients in Guatemala and Honduras during the Central American epidemic of 2007 were analyzed. Virus identification and serotyping performed by a nested RT-PCR assay revealed two DENV-1 isolates from Guatemala, four DENV-2 isolates, two each from Guatemala and Honduras, and two DENV-4 isolates from Honduras. Viral genotyping determined by phylogenetic analysis of the complete envelope gene sequences demonstrated that the DENV-2 isolates from Guatemala and Honduras fell into the American/Asian Genotype III, and were most closely related to DENV-2/NI/BID-V2683-1999 isolated from a dengue case in Nicaragua in 1999; and the DENV-4 F07-076 isolate from Honduras belonged to genotype II, and was most closely related to DENV-4/US/BID-V1093/1998 isolated from Puerto Rico in 1998. Our results suggest that the 2007 dengue outbreaks in Guatemala and Honduras were most likely caused by the re-emergence of earlier, indigenous DENV strains rather than by newly introduced strains and there were at least three serotypes of DENV co-circulating during the 2007 Central American epidemics.

Evidence for inter- and intra-genotypic variations in dengue serotype 4 viruses representing predominant and non-predominant genotypes co-circulating in Thailand from 1977 to 2001

In order to characterize viral genetic variation among predominant and non-predominant genotypes of Thai dengue serotype 4 viruses (DENV-4) and follow mutations that occur during virus evolution, we performed a comparative analysis of the complete genomic sequences of six DENV-4 isolates representing three genotypes (I, IIA, and III) co-circulating in Thailand over a 24-year period. The results revealed [1] remarkable genetic variation in the viral genome between predominant and non-predominant genotypes; [2] inter-genotype-specific amino acid and nucleotide mutations in most regions of the viral genome; [3] more amino acid and nucleotide substitutions in later as compared to earlier isolates for predominant genotype I strains; [4] a single nucleotide substitution at nucleotide position 77 of the 5-′NTR of two non-predominant genotype III strains that disrupted a small conserved 3′stem–loop (SL) in the cyclization sequence required for virus replication; [5] a high degree of conservation of PrM/M and NS2B proteins, and the 5′-NTR in predominant genotype I strains with no mutations observed over the 24-year period of observation; and [6] no molecular markers that appeared to correlate with disease severity. Several mutations identified in this study might have a significant impact on the persistence of virus in the population, including one in the 5′-NTR that disrupted a small, highly conserved 3′SL2 structure at the terminus of the cyclized 5′–3′ RNA sequences in two genotype III strains, and three amino acid (aa) charge change mutations in the E and NS5 proteins of genotype I strains. The conserved 3′-SL structure may be a target for antiviral drug development.

Intra-genotypic variation of predominant genotype II strains of dengue type-3 virus isolated during different epidemics in Thailand from 1973 to 2001

The prevalence of all four dengue virus (DENV) serotypes has increased dramatically in recent years in many tropical and sub-tropical countries accompanied by an increase in genetic diversity within each serotype. This expansion in genetic diversity is expected to give rise to viruses with altered antigenicity, virulence, and transmissibility. We previously demonstrated the co-circulation of multiple DENV genotypes in Thailand and identified a predominant genotype for each serotype. In this study, we performed a comparative analysis of the complete genomic sequences of 28 DENV-3 predominant genotype II strains previously collected during different DENV-3 epidemics in Thailand from 1973 to 2001 with the goal to define mutations that might correlate with virulence, transmission frequency, and epidemiological impact. The results revealed (1) 37 amino acid and six nucleotide substitutions adopted and fixed in the virus genome after their initial substitutions over nearly 30-year-sampling period, (2) the presence of more amino acid and nucleotide substitutions in recent virus isolates compared with earlier isolates, (3) six amino acid substitutions in capsid (C), pre-membrane (prM), envelope (E), and nonstructural (NS) proteins NS4B and NS5, which appeared to be associated with periods of high DENV-3 epidemic activity, (4) the highest degree of conservation in C, NS2B and the 5′-untranslated region (UTR), and (5) the highest percentage of amino acid substitutions in NS2A protein.