Follow up estimation of Aedes aegypti entomological parameters and mathematical modellings

The dengue virus is a vector-borne disease transmitted by mosquito Aedes aegypti and the incidence is strongly influenced by temperature and humidity which vary seasonally. To assess the effects of temperature on dengue transmission, mathematical models are developed based on the population dynamics theory. However, depending on the hypotheses of the modelling, different outcomes regarding to the risk of epidemics are obtained. We address this question comparing two simple models supplied with model's parameters estimated from temperature-controlled experiments, especially the entomological parameters regarded to the mosquito's life cycle in different temperatures. Once obtained the mortality and transition rates of different stages comprising the life cycle of mosquito and the oviposition rate, we compare the capacity of vector reproduction (the basic offspring number) and the risk of infection (basic reproduction number) provided by two models. The extended model, which is more realistic, showed that both mosquito population and dengue risk are situated at higher values than the simplified model, even that the basic offspring number is lower.     

Human Apolipoprotein A-I Is Associated with Dengue Virus and Enhances Virus Infection through SR-BI

Diseases caused by dengue virus (DV) infection vary in severity, with symptoms ranging from mild fever to life threatening dengue hemorrhage fever (DHF) and dengue shock syndrome (DSS). Clinical studies have shown that significant decrease in the level of lipoproteins is correlated with severe illness in DHF/DSS patients. Available evidence also indicates that lipoproteins including high-density lipoprotein (HDL) and low-density lipoprotein (LDL) are able to facilitate cell entry of HCV or other flaviviruses via corresponding lipoprotein receptors. In this study, we found that pre-incubation of DV with human serum leads to an enhanced DV infectivity in various types of cells. Such enhancement could be due to interactions between serum components and DV particles. Through co-immunoprecipitation we revealed that apolipoprotein A-I (ApoA-I), the major protein component in HDL, is associated with DV particles and is able to promote DV infection. Based on that observation, we further found that siRNA knockdown of the scavenger receptor class B type I (SR-BI), the cell receptor of ApoA-I, abolished the activity of ApoA-I in enhancement of DV infection. This suggests that ApoA-I bridges DV particles and cell receptor SR-BI and facilitates entry of DV into cells. FACS analysis of cell surface dengue antigen after virus absorption further confirmed that ApoA-I enhances DV infection via promoting initial attachment of the virus to cells. These findings illustrate a novel entry route of DV into cells, which may provide insights into the functional importance of lipoproteins in dengue pathogenesis.     

The envelope glycoprotein domain III of dengue virus serotypes 1 and 2 inhibit virus entry

Dengue virus (DV) is a flavivirus and its urban transmission is maintained largely by its mosquito vectors and vertebrate host, often human. In this study, investigation was carried out on the involvement of domain III of the envelope (E) glycosylated protein of dengue virus serotypes 1 and 2 (DV-1 and DV-2 DIII) in binding to host cell surfaces, thus mediating virus entry. Domain III protein of flavivirus can also serve as an attractive target in inhibiting virus entry. The respective DV DIII proteins were expressed as soluble recombinant fusion proteins before purification through enzymatic cleavage and affinity purification. The purified recombinant DV-1 and DV-2 DIII proteins both demonstrated the ability to inhibit the entry of DV-1 and DV-2 into HepG2 cells and C6/36 mosquito cells. As such, the DV DIII protein is indeed important for the interaction with cellular receptors in both human and mosquito cells. In addition, this protein induced antibodies that completely neutralized homologous dengue serotypes although not with the same efficiency among the heterologous serotypes. This observation may be of importance when formulating a generic vaccine that is effective against all dengue virus serotypes.     

Dengue and human health: A global scenario of its occurrence,diagnosis and therapeutics

Dengue is one of the highest and rapidly spreading vector-borne viral diseases with high mortality rates. The infection causes acute febrile illness, a major public health concern in the tropics and subtropics globally. The disease is caused by an RNA virus that belongs to the Flaviviridae family. The virus is transferred to humans by the mosquito vector called Aedvrves aegypti, which is the cause of new prevalent sicknesses worldwide. These vector-borne viral diseases spread very fast and pose public health and economic challenges that deemed various prevention and control techniques. The Flavivirus genus consists of five different types of viruses starting from DENV-1 to DENV-5. Thus, the present review focuses on the origin of the virus, how the Dengue virus can be detected, infection, the morphology of the virus, its classifications as proposed by ICTV, the replication and genome of the dengue virus, translation, receptor binding, and some vaccine trial volunteers. In addition, it highlights the current challenges and limitations of effective dengue treatment.     

Effect of the Rho GTPase inhibitor-1 on the entry of dengue serotype 2 virus into EAhy926 cells

Dengue virus (DV) is the most rapidly spreading arbovirus in the world. Our previous studies indicated that Rac1, a kind of Rho GTPase, was related with the increased vascular permeability in DV infection. However, the molecular mechanisms that regulate the activity of the Rac1 pathway during DV infection is not fully understood yet. Recently, Rho-specific guanine nucleotide dissociated inhibitors (Rho GDIs), as a pivotal upstream regulator of Rho GTPase, attract our attention. To identify the role of GDI-1 in DV2 infection, the expression of GDI in Eahy926 cells was detected. Moreover, a GDI-1 down-regulated cell line was constructed to explore the correlation between GDI-1 and Rac1 and to further evaluate the function of GDI in DV life cycle. Our results indicated that DV2 infection could up-regulate GDI-1 expression, and down-regulation of GDI enhanced the activity of Rac1. In addition, down-regulated GDI-1 significantly inhibited all steps of DV2 replication cycle. GDI-1 plays an important role in DV2 infection via negatively regulating the activation of the Rac1-actin pathway. These results not only contribute to our further understanding of the pathogenesis of severe dengue but also provide further insight into the development of antiviral drugs.

     

Lethal Antibody Enhancement of Dengue Disease in Mice Is Prevented by Fc Modification

Immunity to one of the four dengue virus (DV) serotypes can increase disease severity in humans upon subsequent infection with another DV serotype. Serotype cross-reactive antibodies facilitate DV infection of myeloid cells in vitro by promoting virus entry via Fcγ receptors (FcγR), a process known as antibody-dependent enhancement (ADE). However, despite decades of investigation, no in vivo model for antibody enhancement of dengue disease severity has been described. Analogous to human infants who receive anti-DV antibodies by transplacental transfer and develop severe dengue disease during primary infection, we show here that passive administration of anti-DV antibodies is sufficient to enhance DV infection and disease in mice using both mouse-adapted and clinical DV isolates. Antibody-enhanced lethal disease featured many of the hallmarks of severe dengue disease in humans, including thrombocytopenia, vascular leakage, elevated serum cytokine levels, and increased systemic viral burden in serum and tissue phagocytes. Passive transfer of a high dose of serotype-specific antibodies eliminated viremia, but lower doses of these antibodies or cross-reactive polyclonal or monoclonal antibodies all enhanced disease in vivo even when antibody levels were neutralizing in vitro. In contrast, a genetically engineered antibody variant (E60-N297Q) that cannot bind FcγR exhibited prophylactic and therapeutic efficacy against ADE-induced lethal challenge. These observations provide insight into the pathogenesis of antibody-enhanced dengue disease and identify a novel strategy for the design of therapeutic antibodies against dengue.     

Dispersing hemipteran vectors have reduced arbovirus prevalence

A challenge in managing vector-borne zoonotic diseases in human and wildlife populations is predicting where epidemics or epizootics are likely to occur, and this requires knowing in part the likelihood of infected insect vectors dispersing pathogens from existing infection foci to novel areas. We measured prevalence of an arbovirus, Buggy Creek virus, in dispersing and resident individuals of its exclusive vector, the ectoparasitic swallow bug (Oeciacus vicarius), that occupies cliff swallow (Petrochelidon pyrrhonota) colonies in western Nebraska. Bugs colonizing new colony sites and immigrating into established colonies by clinging to the swallows’ legs and feet had significantly lower virus prevalence than bugs in established colonies and those that were clustering in established colonies before dispersing. The reduced likelihood of infected bugs dispersing to new colony sites indicates that even heavily infected sites may not always export virus to nearby foci at a high rate. Infected arthropods should not be assumed to exhibit the same dispersal or movement behaviour as uninfected individuals, and these differences in dispersal should perhaps be considered in the epidemiology of vector-borne pathogens such as arboviruses.     

Both virus and tumor necrosis factor alpha are critical for endothelium damage in a mouse model of dengue virus-induced hemorrhage

Hemorrhage is a common clinical manifestation in dengue patients. However, the pathogenic mechanism of dengue virus (DV)-induced hemorrhage awaits clarification. We established a mouse model of DV hemorrhage using immunocompetent C57BL/6 mice by injecting DV serotype 2 strain 16681 intradermally. While inoculation of 3 x 10(9) PFU of DV induced systemic hemorrhage in all of the mice by day 3 of infection, one out of three of those injected with 4 x 10(7) to 8 x 10(7) PFU developed hemorrhage in the subcutaneous tissues. The mice that were inoculated with 4 x 10(7) to 8 x 10(7) PFU but that did not develop hemorrhage were used as a basis for comparison to explore the pathogenic mechanism of dengue hemorrhage. The results showed that mice with severe thrombocytopenia manifested signs of vascular leakage and hemorrhage. We observed that high viral titer, macrophage infiltration, and tumor necrosis factor alpha (TNF-alpha) production in the local tissues are three important events that lead to hemorrhage. Immunofluorescence staining revealed that DV targeted both endothelial cells and macrophages. In addition, the production of high levels of TNF-alpha in tissues correlated with endothelial cell apoptosis and hemorrhage. By comparing TNF-alpha(-/-) to IgH(-/-), C5(-/-), and wild-type mice, we found that TNF-alpha was important for the development of hemorrhage. In vitro studies showed that mouse primary microvascular endothelial cells were susceptible to DV but that TNF-alpha enhanced DV-induced apoptosis. Our mouse model illustrated that intradermal inoculation of high titers of DV predisposes endothelial cells to be susceptible to TNF-alpha-induced cell death, which leads to endothelium damage and hemorrhage development. This finding highlights the contribution of the innate immune response to dengue hemorrhage.     

Class II ADP-ribosylation factors are required for efficient secretion of dengue viruses

Identification and characterization of virus-host interactions are very important steps toward a better understanding of the molecular mechanisms responsible for disease progression and pathogenesis. To date, very few cellular factors involved in the life cycle of flaviviruses, which are important human pathogens, have been described. In this study, we demonstrate a crucial role for class II Arf proteins (Arf4 and Arf5) in the dengue flavivirus life cycle. We show that simultaneous depletion of Arf4 and Arf5 blocks recombinant subviral particle secretion for all four dengue serotypes. Immunostaining analysis suggests that class II Arf proteins are required at an early pre-Golgi step for dengue virus secretion. Using a horseradish peroxidase protein fused to a signal peptide, we show that class II Arfs act specifically on dengue virus secretion without altering the secretion of proteins through the constitutive secretory pathway. Co-immunoprecipitation data demonstrate that the dengue prM glycoprotein interacts with class II Arf proteins but not through its C-terminal VXPX motif. Finally, experiments performed with replication-competent dengue and yellow fever viruses demonstrate that the depletion of class II Arfs inhibits virus secretion, thus confirming their implication in the virus life cycle, although data obtained with West Nile virus pointed out the differences in virus-host interactions among flaviviruses. Our findings shed new light on a molecular mechanism used by dengue viruses during the late stages of the life cycle and demonstrate a novel function for class II Arf proteins.     

Identification of B cell epitopes of dengue virus 2 NS3 protein by monoclonal antibody

Dengue virus is a major international public health concern, and there is a lack of available effective vaccines. Virus-specific epitopes could help in developing epitope peptide vaccine. Previously, a neutralizing monoclonal antibody (mAb) 4F5 against nonstructural protein 3 (NS3) of dengue virus 2 (DV2) was developed in our lab. In this work, the B cell epitope recognized by mAb 4F5 was identified using the phage-displayed peptide library. The results of the binding assay and competitive inhibition assay indicated that the peptides, residues 460–469 (U460-469 RVGRNPKNEN) of DV2 NS3 protein, were the B cell epitopes recognized by mAb 4F5. Furthermore, the epitope peptides and a control peptide were synthesized and then immunized female BALB/c mice. ELISA analysis showed that immunization with synthesized epitope peptide elicited a high level of antibody in mice, and immunofluorescent staining showed that the antisera from fusion epitope-immunized mice also responded to DV2 NS3 protein, which further characterized the specific response of the present epitope peptide. Therefore, the present work revealed the specificity of the newly identified epitope (U460-469) of DV2 NS3 protein, which may shed light on dengue virus (DV) vaccine design, DV pathogenesis study, and even DV diagnostic reagent development.     

The type 2 dengue virus envelope protein interacts with small ubiquitin-like modifier-1 (SUMO-1) conjugating enzyme 9 (Ubc9)

Dengue viruses are mosquito-borne flaviviruses and may cause the life-threatening dengue hemorrhagic fever and dengue shock syndrome. Its envelope protein is responsible mainly for the virus attachment and entry to host cells. To identify the human cellular proteins interacting with the envelope protein of dengue virus serotype 2 inside host cells, we have performed a screening with the yeast-two-hybrid-based “Functional Yeast Array”. Interestingly, the small ubiquitin-like modifier-1 conjugating enzyme 9 protein, modulating cellular processes such as those regulating signal transduction and cell growth, was one of the candidates interacting with the dengue virus envelope protein. With co-precipitation assay, we have demonstrated that it indeed could interact directly with the Ubc9 protein. Site-directed mutagenesis has demonstrated that Ubc9 might interact with the E protein via amino acid residues K51 and K241. Furthermore, immunofluorescence microscopy has shown that the DV2E-EGFP proteins tended to progress toward the nuclear membrane and co-localized with Flag-Ubc9 proteins around the nuclear membrane in the cytoplasmic side, and DV2E-EGFP also shifted the distribution of Flag-Ubc9 from evenly in the nucleus toward concentrating around the nuclear membrane in the nucleic side. In addition, over-expression of Ubc9 could reduce the plaque formation of the dengue virus in mammalian cells. This is the first report that DV envelope proteins can interact with the protein of sumoylation system and Ubc9 may involve in the host defense system to prevent virus propagation.     

Mutagenesis of the DI/DIII linker in dengue virus envelope protein impairs viral particle assembly

The dengue virus (DV) envelope (E) protein is important in mediating viral entry and assembly of progeny virus during cellular infection. Domains I and III (DI and DIII, respectively) of the DV E protein are connected by a highly conserved but poorly ordered region, the DI/DIII linker. Although the flexibility of the DI/DIII linker is thought to be important for accommodating the structural rearrangements undergone by the E protein during viral entry, the function of the linker in the DV infectious cycle is not well understood. In this study, we performed site-directed mutagenesis on conserved residues in the DI/DIII linker of the DV2 E protein and showed that the resulting mutations had little or no effect on the entry process but greatly affected virus assembly. Biochemical fractionation and immunofluorescence microscopy experiments performed on infectious virus as well as in a virus-like particle (VLP) system indicate that the DI/DIII linker mutants express the DV structural proteins at the sites of particle assembly near the ER but fail to form infectious particles. This defect is not due to disruption of E's interaction with prM and pr in immature and mature virions, respectively. Serial passaging of the DV2 mutant E-Y299F led to the identification of a mutation in the membrane-proximal stem region of E that fully compensates for the assembly defect of this DI/DIII linker mutant. Together, our results suggest a critical and previously unidentified role for the E protein DI/DIII linker region during the DV2 assembly process.     

Structural and functional analysis of methylation and 5'-RNA sequence requirements of short capped RNAs by the methyltransferase domain of dengue virus NS5

The N-terminal 33 kDa domain of non-structural protein 5 (NS5) of dengue virus (DV), named NS5MTase(DV), is involved in two of four steps required for the formation of the viral mRNA cap (7Me)GpppA(2'OMe), the guanine-N7 and the adenosine-2'O methylation. Its S-adenosyl-l-methionine (AdoMet) dependent 2'O-methyltransferase (MTase) activity has been shown on capped (7Me+/-)GpppAC(n) RNAs. Here we report structural and binding studies using cap analogues and capped RNAs. We have solved five crystal structures at 1.8 A to 2.8 A resolution of NS5MTase(DV) in complex with cap analogues and the co-product of methylation S-adenosyl-l-homocysteine (AdoHcy). The cap analogues can adopt several conformations. The guanosine moiety of all cap analogues occupies a GTP-binding site identified earlier, indicating that GTP and cap share the same binding site. Accordingly, we show that binding of (7Me)GpppAC(4) and (7Me)GpppAC(5) RNAs is inhibited in the presence of GTP, (7Me)GTP and (7Me)GpppA but not by ATP. This particular position of the cap is in accordance with the 2'O-methylation step. A model was generated of a ternary 2'O-methylation complex of NS5MTase(DV), (7Me)GpppA and AdoMet. RNA-binding increased when (7Me+/-)GpppAGC(n-1) starting with the consensus sequence GpppAG, was used instead of (7Me+/-)GpppAC(n). In the NS5MTase(DV)-GpppA complex the cap analogue adopts a folded, stacked conformation uniquely possible when adenine is the first transcribed nucleotide at the 5' end of nascent RNA, as it is the case in all flaviviruses. This conformation cannot be a functional intermediate of methylation, since both the guanine-N7 and adenosine-2'O positions are too far away from AdoMet. We hypothesize that this conformation mimics the reaction product of a yet-to-be-demonstrated guanylyltransferase activity. A putative Flavivirus RNA capping pathway is proposed combining the different steps where the NS5MTase domain is involved.     

Efficient Assembly and Secretion of Recombinant Subviral Particles of the Four Dengue Serotypes Using Native prM and E Proteins

Background

Flavivirus infected cells produce infectious virions and subviral particles, both of which are formed by the assembly of prM and E envelope proteins and are believed to undergo the same maturation process. Dengue recombinant subviral particles have been produced in cell cultures with either modified or chimeric proteins but not using the native forms of prM and E.

Methodology/Principal Findings

We have used a codon optimization strategy to obtain an efficient expression of native viral proteins and production of recombinant subviral particles (RSPs) for all four dengue virus (DV) serotypes. A stable HeLa cell line expressing DV1 prME was established (HeLa-prME) and RSPs were analyzed by immunofluorescence and transmission electron microscopy. We found that E protein is mainly present in the endoplasmic reticulum (ER) where assembly of RSPs could be observed. Biochemical characterization of DV1 RSPs secretion revealed both prM protein cleavage and homodimerization of E proteins before their release into the supernatant, indicating that RSPs undergo a similar maturation process as dengue virus. Pulse chase experiment showed that 8 hours are required for the secretion of DV1 RSPs. We have used HeLa-prME to develop a semi-quantitative assay and screened a human siRNA library targeting genes involved in membrane trafficking. Knockdown of 23 genes resulted in a significant reduction in DV RSP secretion, whereas for 22 others we observed an increase of RSP levels in cell supernatant.

Conclusions/Significance

Our data describe the efficient production of RSPs containing native prM and E envelope proteins for all dengue serotypes. Dengue RSPs and corresponding producing cell lines are safe and novel tools that can be used in the study of viral egress as well as in the development of vaccine and drugs against dengue virus.     

Passive Protection Assay of Monoclonal Antibodies Against Dengue Virus in Suckling Mice

Dengue fever and dengue hemorrhagic fever/dengue shock syndrome are highly infectious diseases caused by dengue virus (DV). Specific monoclonal antibodies (mAbs) against DV are vital for diagnosis, pathological studies, and passive immune therapy. In this study, purified DV serotype 2 (DV2) was used as antigen and BALB/c mice were immunized to induce specific antibodies. We established five hybridoma cell lines, called 78#, 1E7, 7F7, 8F12, and 8H1, respectively, and evaluated them by enzyme-linked immunosorbent assay, indirect immunofluorescence assay, Western blot, plaque reduction neutralization test, and suckling mice protection assay. Lines 78#, 1E7, 7F7, and 8F12 showed a neutralizing effect, and lines 78#, 1E7, 8F12, and 8H1 recognized envelope glycoprotein of DV2. Among them, lines 78# and 8F12 had stronger neutralizing ability in vitro and could protect some suckling mice from virus challenge. Our results demonstrate that immunization with purified virion is efficient for the production of specific neutralizing mAbs against DV2, and these mAbs could be useful tools for studying or treating DV infection.     

Natural vertical transmission of dengue viruses by Aedes aegypti in Bolivia

The natural transmission of dengue virus from an infected female mosquito to its progeny, namely the vertical transmission, was researched in wild caught Aedes aegypti during an important outbreak in the town of Santa Cruz de la Sierra, Bolivia. Mosquitoes were collected at the preimaginal stages (eggs, larvae and pupae) then reared up to adult stage for viral detection using molecular methods. Dengue virus serotypes 1 and 3 were found to be co-circulating with significant higher prevalence in male than in female mosquitoes. Of the 97 pools of Ae. aegypti (n = 635 male and 748 female specimens) screened, 14 pools, collected in February-May in 2007, were found positive for dengue virus infection: five DEN-1 and nine DEN-3. The average true infection rate (TIR) and minimum infection rate (MIR) were respectively 1.08% and 1.01%. These observations suggest that vertical transmission of dengue virus may be detected in vectors at the peak of an outbreak as well as several months before an epidemic occurs in human population.     

A conceptual model for optimizing vaccine coverage to reduce vector-borne infections in the presence of antibody-dependent enhancement

Background

Many vector-borne diseases co-circulate, as the viruses from the same family are also transmitted by the same vector species. For example, Zika and dengue viruses belong to the same Flavivirus family and are primarily transmitted by a common mosquito species Aedes aegypti. Zika outbreaks have also commonly occurred in dengue-endemic areas, and co-circulation and co-infection of both viruses have been reported. As recent immunological cross-reactivity studies have confirmed that convalescent plasma following dengue infection can enhance Zika infection, and as global efforts of developing dengue and Zika vaccines are intensified, it is important to examine whether and how vaccination against one disease in a large population may affect infection dynamics of another disease due to antibody-dependent enhancement.

Methods

Through a conceptual co-infection dynamics model parametrized by reported dengue and Zika epidemic and immunological cross-reactivity characteristics, we evaluate impact of a hypothetical dengue vaccination program on Zika infection dynamics in a single season when only one particular dengue serotype is involved.

Results

We show that an appropriately designed and optimized dengue vaccination program can not only help control the dengue spread but also, counter-intuitively, reduce Zika infections. We identify optimal dengue vaccination coverages for controlling dengue and simultaneously reducing Zika infections, as well as the critical coverages exceeding which dengue vaccination will increase Zika infections.

Conclusion

This study based on a conceptual model shows the promise of an integrative vector-borne disease control strategy involving optimal vaccination programs, in regions where different viruses or different serotypes of the same virus co-circulate, and convalescent plasma following infection from one virus (serotype) can enhance infection against another virus (serotype). The conceptual model provides a first step towards well-designed regional and global vector-borne disease immunization programs.

     

The mannose receptor mediates dengue virus infection of macrophages

Macrophages (MØ) and mononuclear phagocytes are major targets of infection by dengue virus (DV), a mosquito-borne flavivirus that can cause haemorrhagic fever in humans. To our knowledge, we show for the first time that the MØ mannose receptor (MR) binds to all four serotypes of DV and specifically to the envelope glycoprotein. Glycan analysis, ELISA, and blot overlay assays demonstrate that MR binds via its carbohydrate recognition domains to mosquito and human cell–produced DV antigen. This binding is abrogated by deglycosylation of the DV envelope glycoprotein. Surface expression of recombinant MR on NIH3T3 cells confers DV binding. Furthermore, DV infection of primary human MØ can be blocked by anti-MR antibodies. MR is a prototypic marker of alternatively activated MØ, and pre-treatment of human monocytes or MØ with type 2 cytokines (IL-4 or IL-13) enhances their susceptibility to productive DV infection. Our findings indicate a new functional role for the MR in DV infection.     

Assessing the suitability for Aedes albopictus and dengue transmission risk in China with a delay differential equation model

Dengue is considered non-endemic to mainland China. However, travellers frequently import the virus from overseas and local mosquito species can then spread the disease in the population. As a consequence, mainland China still experiences large dengue outbreaks. Temperature plays a key role in these outbreaks: it affects the development and survival of the vector and the replication rate of the virus. To better understand its implication in the transmission risk of dengue, we developed a delay differential equation model that explicitly simulates temperature-dependent development periods and tested it with collected field data for the Asian tiger mosquito, Aedes albopictus. The model predicts mosquito occurrence locations with a high accuracy (Cohen’s κ of 0.78) and realistically replicates mosquito population dynamics. Analysing the infection dynamics during the 2014 dengue outbreak that occurred in Guangzhou showed that the outbreak could have lasted for another four weeks if mosquito control interventions had not been undertaken. Finally, we analyse the dengue transmission risk in mainland China. We find that southern China, including Guangzhou, can have more than seven months of dengue transmission per year while even Beijing, in the temperate north, can have dengue transmission during hot summer months. The results demonstrate the importance of using detailed vector and infection ecology, especially when vector-borne disease transmission risk is modelled over a broad range of climatic zones.