Molecular surveillance of dengue in Semarang, Indonesia revealed the circulation of an old genotype of dengue virus serotype-1

Abstract

Dengue disease is currently a major health problem in Indonesia and affects all provinces in the country, including Semarang Municipality, Central Java province. While dengue is endemic in this region, only limited data on the disease epidemiology is available. To understand the dynamics of dengue in Semarang, we conducted clinical, virological, and demographical surveillance of dengue in Semarang and its surrounding regions in 2012. Dengue cases were detected in both urban and rural areas located in various geographical features, including the coastal and highland areas. During an eight months' study, a total of 120 febrile patients were recruited, of which 66 were serologically confirmed for dengue infection using IgG/IgM ELISA and/or NS1 tests. The cases occurred both in dry and wet seasons. Majority of patients were under 10 years old. Most patients were diagnosed as dengue hemorrhagic fever, followed by dengue shock syndrome and dengue fever. Serotyping was performed in 31 patients, and we observed the co-circulation of all four dengue virus (DENV) serotypes. When the serotypes were correlated with the severity of the disease, no direct correlation was observed. Phylogenetic analysis of DENV based on Envelope gene sequence revealed the circulation of DENV-2 Cosmopolitan genotype and DENV-3 Genotype I. A striking finding was observed for DENV-1, in which we found the co-circulation of Genotype I with an old Genotype II. The Genotype II was represented by a virus strain that has a very slow mutation rate and is very closely related to the DENV strain from Thailand, isolated in 1964 and never reported in other countries in the last three decades. Moreover, this virus was discovered in a cool highland area with an elevation of 1,001 meters above the sea level. The discovery of this old DENV strain may suggest the silent circulation of old virus strains in Indonesia.

Figure 1. Study area around the Semarang region.

Colored areas represent the three districts involved in this study. Black dots represent the locations were the cases occurred.

Figure 2. Dengue cases monthly distribution (A) and patients' age (B).

All serologically positive dengue patients were grouped according to their hospital admission date and ages.

Figure 3. Dengue virus serotype distribution (A) and the altitude of dengue cases (B).

Serotypes were determined using both conventional RT-PCR and real-time RT-PCR as described in the Method section. The altitudes of the locations where the cases occurred were recorded using altimeter.

Figure 4. MCC (Maximum Clade Credibility) tree of DENV-1 genotype I and II generated by bayesian inference method as implemented in BEAST using GTR evolution model and gamma parameter rates from the E-protein sequences.

The color of branches indicated the rate of evolution of each isolate with blue line for slow rate and red line for faster rate, with values range of 2.72±2.1×10⁻⁴. The red labels indicated the isolates from Semarang; the green labels indicated other isolates from Indonesia. The dots in the node indicated the posterior probability of that particular cluster, with large yellow dots indicated posterior probability >0.75, medium orange dots for posterior probability between 0.75–0.5, and small red dots for posterior probability <0.5.

Figure 5. MCC (Maximum Clade Credibility) tree of DENV-2 genotype Cosmopolitan generated by bayesian inference method as implemented in BEAST using GTR evolution model and gamma parameter rates from the E-protein sequences.

The color of branches indicated the rate of evolution of each isolate with blue line for slow rate and red line for faster rate, with values range of 11.66±2.1×10⁻⁴. The red labels indicated the isolates from Semarang; the green labels indicated other isolates from Indonesia. The dots in the node indicated the posterior probability of that particular cluster, with large yellow dots indicated posterior probability >0.75, medium orange dots for posterior probability between 0.75–0.5, and small red dots for posterior probability <0.5.

Figure 6. MCC (Maximum Clade Credibility) tree of DENV-3 genotype I generated by bayesian inference method as implemented in BEAST using GTR evolution model and gamma parameter rates from the E-protein sequences.

The color of branches indicated the rate of evolution of each isolate with blue line for slow rate and red line for faster rate. The red labels indicated the isolates from Semarang; the green labels indicated other isolates from Indonesia. The dots in the node indicated the posterior probability of that particular cluster, with large yellow dots indicated posterior probability >0.75, medium orange dots for posterior probability between 0.75–0.5, and small red dots for posterior probability <0.5.