Evolutionary study and phylodynamic pattern of human influenza A/H3N2 virus in Indonesia from 2008 to 2010 - Fig 4

<p><b>The Bayesian Skyline plot of HA (A) and (B) NA genes</b>. The <i>x</i>-axis represents the time (mm/yy) and the <i>y</i>-axis represents a measure of relative genetic diversity (N_et, where the N_e is the effective population size and t the generation time from infected host to infected host). Black line represents the mean value while the 95% confidence limits shown in blue line.</p

MCC tree from Bayesian analysis of HA gene of H3N2 viruses originated from Indonesia and other countries.

<p>Indonesian samples are illustrated in different colored branches according its geographical origin. The numbers I, II and III represent major lineages of the virus. Insert figure is the color-coded geographical origin.</p

Evolutionary study and phylodynamic pattern of human influenza A/H3N2 virus in Indonesia from 2008 to 2010

<div><p>Influenza viruses are by nature unstable with high levels of mutations. The sequential accumulation of mutations in the surface glycoproteins allows the virus to evade the neutralizing antibodies. The consideration of the tropics as the influenza reservoir where viral genetic and antigenic diversity are continually generated and reintroduced into temperate countries makes the study of influenza virus evolution in Indonesia essential. A total of 100 complete coding sequences (CDS) of Hemagglutinin (HA) and Neuraminidase (NA) genes of H3N2 virus were obtained from archived samples of Influenza-Like Illness (ILI) surveillance collected from 2008 to 2010. Our evolutionary and phylogenetic analyses provide insight into the dynamic changes of Indonesian H3N2 virus from 2008 to 2010. Obvious antigenic drift with typical ‘ladder-like’ phylogeny was observed with multiple lineages found in each year, suggesting co-circulation of H3N2 strains at different time periods. The mutational pattern of the Indonesian H3N2 virus was not geographically related as relatively low levels of mutations with similar pattern of relative genetic diversity were observed in various geographical origins. This study reaffirms that the existence of a particular lineage is most likely the result of adaptation or competitive exclusion among different host populations and combination of stochastic ecological factors, rather than its geographical origin alone.</p></div

Indonesian map with color-coded geographical origin.

<p>Indonesian map with color-coded geographical origin.</p

Rates of nucleotide substitution and TMRCA of the HA and NA genes.

<p>Rates of nucleotide substitution and TMRCA of the HA and NA genes.</p

MCC tree from Bayesian analysis of NA gene of H3N2 viruses originated from Indonesia and other countries.

<p>Indonesian samples are illustrated in different colored branches according its geographical origin. The numbers I, II and III represent major lineages of the virus. Insert figure is the color-coded geographical origin.</p

Mfold RNA structure prediction for the complete 3’UTR of DENV-3 prototype strain H87 (KU050695) and the neurotropic DENV-3 201610225 (KY863456).

<p>Stop codon (UAA) is indicated with red font in both RNA structure just upstream nucleotide 10268. Nucleotide insertion and changes in isolate 201610225 are indicated with yellow and red circles, respectively. The positions of the nucleotide changes are indicated according to 201610225 nucleotide numbering with H87 nucleotide numbering in parentheses. The two stem-loops (SLI and SLII), two dumbbells (DBI and DBII), and small hairpin 3’ stem-loop (SHP-3’SL) structures are shown.</p

Amino acid substitutions between 201610225, DENV-3 prototype strain (H87), and DENV-3 Indonesian historical strain (Sleman/78 and den3_88) genome sequence.

<p>Amino acid substitutions between 201610225, DENV-3 prototype strain (H87), and DENV-3 Indonesian historical strain (Sleman/78 and den3_88) genome sequence.</p

Isolation and complete genome analysis of neurotropic dengue virus serotype 3 from the cerebrospinal fluid of an encephalitis patient

<div><p>Although neurological manifestations associated with dengue viruses (DENV) infection have been reported, there is very limited information on the genetic characteristics of neurotropic DENV. Here we describe the isolation and complete genome analysis of DENV serotype 3 (DENV-3) from cerebrospinal fluid of an encephalitis paediatric patient in Jakarta, Indonesia. Next-generation sequencing was employed to deduce the complete genome of the neurotropic DENV-3 isolate. Based on complete genome analysis, two unique and nine uncommon amino acid changes in the protein coding region were observed in the virus. A phylogenetic tree and molecular clock analysis revealed that the neurotropic virus was a member of Sumatran-Javan clade of DENV-3 genotype I and shared a common ancestor with other isolates from Jakarta around 1998. This is the first report of neurotropic DENV-3 complete genome analysis, providing detailed information on the genetic characteristics of this virus.</p></div

Isolation of DENV-3 from CSF and serum samples of an encephalitis patient in Vero and C6/36 mosquito cell cultures.

<p>Phase contrast light microscopy of uninfected monolayer Vero cells (A); infected Vero cells at day 5 after inoculation with CSF sample (B); and infected Vero cells at day 10 after inoculation with serum sample (C). Indirect immunofluorescent assay (IFA) staining of uninfected C6/36 mosquito cells (D); infected C6/36 mosquito cells at third passage of inoculation with CSF sample (E). For IFA staining, cells were stained with mouse anti-DENV-3 monoclonal antibody (clone 5D4), followed by FITC-conjugated goat anti-mouse IgG. Evans blue was used as a counterstain (red). The arrows in B and C point to cytopathic effect (CPE), while the arrows in E point to DENV-infected cells.</p