Use of type 1 /type 2 chimeric polioviruses to study determinants of poliovirus type 1 neurovirulence in a mouse model

We previously described the characteristics of a type 1/type 2 (PV-1/PV-2) chimeric poliovirus, v510, which contains the six amino acids specific for PV-2 in the B-C loop of VP1. This virus was found to be mouse-adapted, as PV-2 and in contrast with PV-1. Determinants of host range were studied in detail and are reported here. PV-1/PV-2 chimeras containing partial PV-1----PV-2 substitutions in the B-C loop of VP1 were obtained by making use of a mutagenesis cartridge on PV-1 cDNA. Analysis of mouse neurovirulence of these chimeras, when correlated with the three-dimensional structure of the v510 capsid, revealed that PV-2 residues important for mouse tropism are those which determine the particular conformation of the B-C loop of VP1 in v510. The mutation of the adenine residue at position 480 of the 5' noncoding region into a guanine residue has been shown to be an important determinant of PV-1 attenuation in monkeys. We show that introduction of this mutation in the v510 genome results in a virus which is partially attenuated for mice. This suggests that analysis of genomic determinants important for PV-1 neurovirulence could be carried out in a mouse model by making use of a mouse-adapted PV-1/PV-2 chimera.     

A point mutation in VP1 of coxsackievirus B4 alters antigenicity

While coxsackievirus infections have been linked to several autoimmune diseases, very little is known about the immunogenicity of the coxsackieviruses. Using two genetically related variants of coxsackievirus B4, CB4-P and CB4-V, the relationship between virulence and antigenicity was examined. The virulent variant, CB4-V, was shown to be more antigenic than the avirulent CB4-P variant. The increased antigenicity of CB4-V was due to a single amino acid substitution in the VP1 capsid protein (a threonine residue at amino acid position 129), a site that had been previously identified as a major determinant of viral virulence. Thr-129 of VP1 is predicted to lie within a conformational B cell epitope. In addition, a nearby linear B cell epitope spanning residues 68 to 82 of VP1 was identified as a potential serotype-specific, neutralization antigenic site. The linear and conformational B cell epitopes of coxsackievirus B4 may be analogous to antigenic sites 1 and 1B of poliovirus. To address whether the increased antigenicity of CB4-V influenced the severity of disease, mouse strains that differ in their outcome to viral infection were analyzed. Mice that developed the most severe disease and succumbed to infection were more immunoresponsive than mice that survived infection with CB4-V. The data suggest that immune-mediated mechanisms play a role in the severity of CB4-V induced disease.     

Antigenic variation of foot-and-mouth disease virus of serotype C during propagation in the field is mainly restricted to only one structural protein (VP1)

The primary structure of VP3, VP2 and VP4 capsid protein genes has been determined for six epizootiologically-related foot-and-mouth disease virus (FMDV) isolates of serotype C1, two of which presented immunogenic differences as determined by a cross-protection assay. The results obtained have been compared with those previously reported for the corresponding VP1 genes Martinez et al. (1988) Gene 62, 75-84. High rates of fixation of mutations have been estimated for the four capsid protein genes that ranged from 3.9 X 10(-4) to 4.5 X 10(-3) substitutions per nucleotide per year, with the highest values corresponding to VP1. Despite this genetic heterogeneity most of the amino acid exchanges are within the VP1 protein. Of the fourteen amino acid substitutions one was located in VP2 and two in VP3. Five out of the eleven amino acid exchanges that affected VP1 were located within residues 138-149, part of a main immunogenic site in FMDV. These results show that in the course of a foot-and-mouth disease outbreak, immunologically relevant amino acid substitutions occur mainly in viral capsid protein VP1.     

The enhanced virulence of very virulent infectious bursal disease virus is partly determined by its B-segment

Summary. There is a remarkable difference in virulence of infectious bursal disease virus (IBDV) strains ranging from sub-clinical infections for serotype 2 and cell culture adapted serotype 1 strains, to 100% mortality for very virulent serotype 1 strains in young SPF chickens. It is known that cell culture adaptation related attenuation is determined by distinct mutations in the hypervariable region of the VP2 outer capsid protein, encoded on the A-segment. Amino acid mutations in the hypervariable VP2 region however, offer no explanation for the difference in virulence of classical and very virulent serotype 1 strains. Here we show by in vitro and in vivo analysis of rescued segment reassorted IBDVs that virulence factors are not only located on the A-segment, but on the RNA Dependent RNA Polymerase (VP1) encoding B-segment as well. Insight into the virulence factors of very virulent IBDV will contribute to the improvement of live IBDV vaccines.     

Molecular and antigenic characterization of a highly evolved derivative of the type 2 oral poliovaccine strain isolated from sewage in Israel

An unusual, highly diverged derivative of the Sabin type 2 oral poliovaccine (OPV) strain was recovered from environmental samples during routine screening for wild polioviruses. Virus was cultivated in L20B cells and then passaged on BGM cells at 40 degrees C (RCT [reproductive capacity at supraoptimal temperature]-positive marker) to select against most OPV strains. All but 1 of 25 RCT-positive OPV-derived environmental isolates were antigenically and genetically (>99.5% VP1 sequence match) similar to the respective Sabin strains. However, isolate PV2/4568-1/ISR98 (referred to below as 4568-1) escaped neutralization with Sabin 2-specific monoclonal antibodies and cross-adsorbed sera, and had multiple nucleotide substitutions (220 of 2,646; 8.3%) in the P1 capsid region. Fourteen of the 44 associated amino acid substitutions in the capsid mapped to neutralizing antigenic sites. Neutralizing titers in the sera of 50 Israeli children 15 years old were significantly lower to 4568-1 (geometric mean titer [GMT], 47) than to Sabin 2 (GMT, 162) or to the prototype wild strain, PV2/MEF-1/EGY42 (GMT, 108). Two key attenuating sites had also reverted in 4568-1 (A(481) to G in the 5' untranslated region and the VP1 amino acid I(143) to T), and the isolate was highly neurovirulent for transgenic mice expressing the poliovirus receptor (PVR-Tg21 mice). The extensive genetic divergence of 4568-1 from the parental Sabin 2 strain suggested that the virus had replicated in one or more people for approximately 6 years. The presence in the environment of a highly evolved, neurovirulent OPV-derived poliovirus in the absence of polio cases has important implications for strategies for the cessation of immunization with OPV following global polio eradication.     

Mouse adaptation of a sub-genogroup B5 strain of human enterovirus 71 is associated with a novel lysine to glutamic acid substitution at position 244 in protein VP1

Most human enterovirus 71 (HEV71) strains infect only primates and are unable to cause clinically apparent infection in mice. Here we describe a mouse-adapted HEV71 strain that belongs to sub-genogroup B5 with increased virulence in newborn BALB/c mice. The mouse-virulent strain was initially selected by serial passage of a HEV71 clinical isolate (HEV71-B5) in Chinese hamster ovary (CHO) cells (CHO-B5), followed by serial passage in newborn mice. Virus from the fifth mouse passage was cultured twice on Vero cells and designated as MP-B5. MP-B5 induces severe disease of high mortality in newborn mice in a dose-dependent manner. Skeletal muscle is the primary site of virus replication and results in severe myositis. CHO-B5 harbours a single amino acid substitution (K(149) → I) in the VP2 capsid protein. Five additional nucleotide sequence changes were identified in MP-B5, two of which are located in the 5' UTR and the three within the open reading frame (ORF). Two of the ORF mutations resulted in deduced amino acid changes in the capsid protein VP1: S(241) → L and K(244) → E; the third ORF mutation was a synonymous C → T change at nucleotide position 6072 within the 3D polymerase gene. Infectious cDNA clone-derived mutant virus populations of HEV71 belonging to sub-genogroup B3 (CHO-26 M) that contain the VP1 mutations identified in MP-B5 were generated in order to determine the mutation(s) responsible for mouse virulence. Only viruses expressing the VP1 (K(244) → E) mutation were virulent in 5-day-old BALB/c mice, indicating that the VP1 (K(244) → E) change is the critical genetic determinant of mouse adaptation and virulence in this model.     

Natural variants of the Sabin type 1 vaccine strain of poliovirus and correlation with a poliovirus neutralization site

Independent substitution mutations have been detected in capsid polypeptide VP1 of the type 1 oral poliovirus vaccine isolated from normal infant vaccine recipients. These mutations map at amino acid residues 142 and 147 of VP1, a region only minimally hydrophilic. A synthetic peptide, corresponding to residues 141 to 147 of VP1 was synthesized, conjugated to a carrier polypeptide of bovine serum albumin. The conjugate was found to elicit a weak poliovirus neutralizing antibody response. It was also capable of priming the immune system for the production of IgG-type antibodies able to neutralize greater than 99.999% of infectious type 1 virus. It is suggested that region 141 to 147 of VP1 may be involved in neutralization of the virus and that the mutants may have accumulated by antibody selection.     

Antigenic regions of poliovirus type 3/Sabin capsid proteins recognized by human sera in the peptide scanning technique.

We used the peptide scanning technique to identify regions of poliovirus type 3/Sabin capsid proteins that bind antibodies from human immune sera. Several reactive regions were seen in VP1, VP2, and VP3 while peptides resembling VP4 did not bind antibodies. Peptides derived from sequences of the previously known antigenic sites 1 and 3 were recognized to a moderate degree. Peptides imitating the four loops in the closed ends of the beta barrels or the alpha helical CD insertions of VP1, VP2 or VP3, whether exposed in the crystal structure or not, all represented major reactivity in the scans. In VP1 several additional reactive regions were found in the amino terminal quarter of the protein, which is buried in the crystal structure, and in a partially exposed region close to but separated from the carboxy terminus. In VP2 the nonexposed peak activities clustered in a bridge-like structure spanning from the outer to the inner surface of the capsid shell. Likewise, most of the novel antigenic regions of VP3 clustered in an internal location and partially composed of beta sheets with a conserved amino acid sequence. Whether any of the novel antigenic sites is capable of inducing neutralizing antibodies is not known.     

Induction by synthetic peptides of broadly reactive, type-specific neutralizing antibody to poliovirus type 3

A region of virus capsid protein VP1 located 89-100 amino acids from the N-terminus has been proposed to comprise a major antigenic site involved in the neutralization of poliovirus type 3. Synthetic peptides 10-18 amino acids in length, containing all or part of this sequence, were tested for their ability to induce antiviral antibodies. Rabbits, but not guinea pigs or mice, immunized with the most active peptide, developed hightitered, type-specific, neutralizing antibodies for a wide range of poliovirus type 3 strains. Consistent with the broad type specificity of the antibody response was the observation that amino acids 89-100 of VP1 are highly conserved among different poliovirus type 3 strains. This sequence thus appears to provide, at least in part, a molecular basis for serotype antigenic specificity. Individual amino acids from 93 to 98 within this sequence were shown to be important for the neutralization of virus by antipeptide sera by examination of the ability of the sera to neutralize laboratory-derived poliovirus type 3 mutants with known single amino acid substitutions in the proposed antigenic site.     

Characterization of Mengo virus neutralization epitopes.

A set of four monoclonal antibodies which neutralized the infectivity of Mengo virus was used to select 20 non-neutralizable (escape) mutants. Altered amino acids were identified by sequence analyses of the capsid-coding regions of the mutant virus genomes. Mutations were found predominantly in proteins VP2 and VP3, while mutations in VP1 were detected only as second mutations. The Mengo virus VP2 mutations at amino acid residues 2144, 2145, 2147, and 2148 align with site Nlm II in human rhinovirus-14 and site 2 in polioviruses 1 and 3. The mutation at 2075 as well as those at 3057, 3061, and 3068 in VP3 correspond to site 3 in poliovirus. These alignments notwithstanding, the results of cross-neutralization experiments indicate the existence of a single composite neutralization site on the Mengo virion. Considering the three-dimensional structure of the Mengo capsid, the amino acids which are altered in the escape mutants are all exposed on the outer surface and none are found in the "pit," the probable site for binding of a cellular receptor. The VP3 mutations are located in the VP3 "knob" and the VP2 mutations on a nearby ridge. Together these mutations define a set of epitopes within a single composite antigenic determinant which forms a crescent-shaped area around the three-fold icosahedral axes of the Mengo virion.     

Complete nucleotide sequence of a strain of coxsackie B4 virus of human origin that induces diabetes in mice and its comparison with nondiabetogenic coxsackie B4 JBV strain

The E2 strain of coxsackie B4 virus (CB4), which is of human origin, can induce a diabetes-like syndrome in mice. The cDNA of the genome of the E2 strain was cloned and sequenced. The E2 viral genome was found to comprise 7,396 bases, which appear to encode a polyprotein of 2,183 amino acids with an overall similarity of 94.91% to nondiabetogenic CB4 prototype JBV strain. The E2 genome is organized like other enteroviruses. It has a 5′ noncoding region of 744 nucleotides, a single long open translational reading frame starting at nucleotide 745 and extending to nucleotide 7293, a 3′ noncoding region of 100 nucleotides, and a poly (A) tract. Ge-nomic sequence comparison of the E2 and JBV strains showed 1,369 nucleotide substitutions in the genome of the E2 strain, most of which are single and silent. There were 111 resultant amino acid changes arising from some of these substitutions, including 82 amino acid changes in the noncapsid proteins, and 29 amino acid changes in the capsid proteins VP1, VP2, VP3, and VP4, which showed 11, 13, 4, and 1 substitution(s), respectively. Noncapsid protein P2-C showed eight amino acid substitutions. On the basis of the sequence comparison of E2 and JBV strains of CB4, we suggest that some of the amino acid changes in the capsid and noncapsid proteins of the E2 strain may be involved in the determination of its diabetogenicity. © 1994 Wiley-Liss, Inc.     

A new antigenic site of poliovirus recognized by an intertypic cross-neutralizing monoclonal antibody

A monoclonal antibody (mAb 7J6) neutralizing poliovirus type 2 (PV2) and poliovirus type 1 (PV1) was obtained after immunization of BALB/c mice with infectious PV2, strain MEF-1. Preincubation of mAb 7J6 with PV1 inhibited its binding to PV2 and vice versa. Neutralization-resistant variants of PV2 and PV1 were selected. Nucleotide sequencing of the RNAs of some variants revealed mutations in the loop of amino acid residues 239 to 245 in VP2 and in the loop of amino acid residues 195 to 207 in VP3. This is the first evidence that these two loops contribute to a neutralization antigenic site (N-Ag) for poliovirus. Moreover, this new site on PV2 induced intertypic cross-neutralizing antibodies.     

Selection and characterization of an acid-resistant mutant of serotype O foot-and-mouth disease virus

Foot-and-mouth disease virus (FMDV) loses infectivity and immunogenicity due to its disassembly in culture environments below pH 6.8. To study the molecular basis of viral resistance to acid-induced disassembly and improve the acid stability of inactivated FMD vaccines during the manufacturing process, type O FMDV mutants with increased resistance to acid inactivation were selected, and the genes encoding their capsid proteins were sequenced. Three amino acid substitutions (VP1 N17D, VP2 D86A, and VP4 S73N) were found in all of the mutants. When these substitutions were introduced into seven infectious FMDV clones alone or combined, a single amino acid substitution in the VP1 protein, N17D, which also appears in type C FMDV acid-resistant mutants, was found to be responsible for the increased resistance to acid inactivation for type O FMDV. In addition, although viral fitness was reduced under standard culture conditions, viral growth kinetics and virulence were not significantly altered in the rescued mutant virus rN17D with the VP1 N17D substitution. Importantly, the N17D substitution could confer improved immunogenicity to the mutant virus rN17D under acidic conditions compared with its parental virus O/YS/CHA/05. These results demonstrate that the N17D substitution in VP1 is the molecular determinant of the acid-resistant phenotype in type O FMDV, indicating the potential for use of this substitution to improve the acid stability of inactivated FMD vaccines during the vaccine production process.     

Characterization of mutations in the VP(1) region of Sabin strain type 1 polioviruses isolated from vaccine-associated paralytic poliomyelitis cases in Iran.

BACKGROUND: The live-attenuated oral polio vaccine used to interrupt poliovirus transmission is genetically unstable. Reversion of some attenuating mutations, which normally occurs during vaccine strain replication in some recipients, and can rarely cause vaccine-associated paralytic poliomyelitis (VAPP). The poliovirus eradication program designed by the World Health Organization (WHO) includes immunization with OPV in addition to careful surveillance of all acute-flaccid paralysis (AFP) cases. OBJECTIVES: In Iran we last isolated imported wild poliovirus in 2000 and the immunization coverage was 100% in 2002. During 2001, there were three AFP cases with residual paralysis from which Sabin-like type 1 polioviruses were isolated in our national polio laboratory. STUDY DESIGN: The complete VP(1) region of the three isolates was sequenced and amino acid substitutions associated with these neurovirulent isolates were recorded. RESULTS: These isolates had either 4, 2 or 1 nucleotide substitution(s) in the VP(1) region, corresponding to amino acid change in the VP(1) of isolate 1 of either (H-[149]->Y), (T-[106]->A) or (I-[90]->L), respectively. CONCLUSIONS: Surveillance of the VAPP cases in countries where endemic transmission has recently ceased increases our understanding of the important neurovirulent mutations in vaccine-strain isolates and assists in planning the next step in the eradication program in these countries.     

Isolation of recombinant type 2 vaccine-derived poliovirus (VDPV) from a Nigerian child

A type 2 vaccine-derived poliovirus (VDPV), differing from Sabin 2 at 2.5% (22/903) of VP1 nucleotide (nt) positions, was isolated from an incompletely immunized 21-month-old Nigerian child who developed acute flaccid paralysis in 2002. Sequences upstream of nt position 620 (within the 5'-untranslated region [5'-UTR]) and downstream of nt position 5840 (in the 3C(pro) region) were derived from species C enteroviruses unrelated to the oral poliovirus vaccine (OPV) strains. The two substitutions associated with the attenuated phenotype had either recombined out (A(481)-->G in the 5'-UTR) or reverted (Ile(143)-->Thr in VP1). The VDPV isolate had lost the temperature sensitive phenotype of Sabin 2 and it was antigenically distinct from the parental OPV strain, having amino acid substitutions in or near neutralizing antigenic sites 1 and 3. The date of the initiating OPV dose, calculated from the number of synonymous substitutions in the capsid region, was estimated to be approximately 16 to 18 months before onset of paralysis, a finding inconsistent with the most recent mass OPV campaign (conducted 12 days before onset of paralysis) as being the source of infection. Although no related type 2 VDPVs were detected in Nigeria or elsewhere, the VDPV was found in an area where conditions favor VDPV emergence and spread.     

Virulence of swine vesicular disease virus is determined at two amino acids in capsid protein VP1 and 2A protease

To identify the genetic determinants of virulence for swine vesicular disease virus, a panel of recombinant and site-directed mutant viruses were constructed from cDNA clones of a virulent J1′73 strain and an avirulent H/3′76 strain. Initial studies mapped the genetic determinants of virulence to either or both of the two sites at nucleotide (nt) 2842, encoding VP1-132, and nt 3355, encoding 2A-20. To determine their relative importance with regard to virulence, viruses mutated at either of these two sites from the avirulent to the virulent genotype and vice versa were tested in pigs. Viruses, mutated at nt 2842 to the virulent genotype (vSVLS104MJ1) or mutated at nt 3355 to the virulent genotype (vSVLS201MJ1), slightly recovered virulence but were very weak compared with viruses with site-directed mutations at both sites (vSVLS104/201MJ1). On the other hand, viruses, mutated at nt 2842 to the avirulent genotype (vSVLS104M00) or mutated at nt 3355 to the avirulent genotype (vSVLS201M00), did not have attenuated virulence. Sequence analysis of viruses recovered from inoculated pigs revealed that reversion at nt 3355 to the virulent genotype occurred in pigs which had been inoculated with vSVLS201M00. These results suggested that both amino acids determined the virulent phenotype, but that the 2A-20 site might be the major determinant for virulence.     

Nucleotide Sequence of the Structural Protein-Encoding Region of Foot-and-Mouth Disease Virus A22-India

Nucleotide sequence of the structural protein-encoding region of foot-and-mouth disease virus (FMDV) A22-India 17/77 was determined using non-radioisotopic technique. Comparison of nucleotide and deduced amino acid sequence with A22-Iraq 24/64 revealed 175 synonymous (silent) and 42 non-synonymous nucleotide changes resulting in 34 amino acid substitutions along the capsid proteins (VP1–VP4). Out of the 4 structural proteins VP4 is highly conserved. The highly variable and immunodominant protein VP1 showed 47% of the total amino acid substitutions. VP2 and VP3 contain 38.2% and 14.7% of the amino acid substitutions, respectively. The VP1-based phylogenetic analysis of 18 different type A viruses including A22-India 17/77 divided them in to two broad genetic groups (Asian and European/South American), and each group is further subdivided in to two separate genotypes. A22-India 17/77, A22-Iraq 24/64 and A22-Azerbaijan/65 formed one genotype and the 4 Chinese strains formed a separate genotype in the Asian group of viruses. In the European/ South American group, A-Argentina/87 represents one genotype and the remaining 10 strains formed the second genotype in this group.     

Full-length sequence analysis of four IBDV strains with different pathogenicities

Characterization of field isolate 9109, Lukert, Edgar cell culture-adapted (CCA), and Edgar chicken embryo-adapted (CEA) serotype 1 IBDV strains using full-length genomic sequences is reported. IBDV genomic segments A and B were sequenced and the nucleotide and deduced amino acid (aa) sequences were compared with previously reported full-length sequenced IBDV strains. We found that the viral protein VPX and amino acid sequences between aa 202-451 and 210-473 of VP2 but not the entire VP2 protein are the best representatives of the entire IBDV genome. The greatest variability was found in the VP2 and 5' non-coding region of segment B among IBDV strains. The deduced amino acid sequences of the VP1 protein varies in length among the strains analyzed. The RNA-dependent, RNA-polymerase motifs within VP1 and the VP5 protein were highly conserved among isolates. Although within the VP2 processing site, amino acid sequence of Lukert was similar to the classical while the Edgar CCA, and CEA were more similar to the very virulent strains, it was determined that these strains have sequence characteristics of the classical strains. In addition, close relatedness between Lukert, Edgar CCA and CEA was observed. Although phylogenetic analysis of the VP1, VP3, and VP4 proteins indicated that 9109 is a classical type virus, this isolate shares unique amino acid changes with very virulent strains within the same proteins. Phylogenetic analysis of the 3' and 5' non-coding regions of segment A revealed that 9109 is more similar to the very virulent strains compared to the classical strains. In the VP2 protein, several amino acids were conserved between variant E and 9109 strains. Thus, it appears that 9109 isolate has characteristics of classical, very virulent, and variant strains. Our analysis indicates that although VPX amino acid comparison may be initially useful for molecular typing, full-length genomic sequence analysis is essential for thorough molecular characterization as partial sequences may not designate a particular strain as very virulent, classical, or variant.     

Molecular and structural basis of hemagglutination in mengovirus.

The molecular and structural basis of mengovirus hemagglutination (HA) was investigated by the comparison of nucleotide sequences of the entire capsid coding regions of an HA+ variant, two HA- mutants, 205 and 280, and two HA+ revertants of 205. The mutants were selected after acridine mutagenesis of mengovirus-37A, a heat-stable and HA+ variant that is neurotropic in mice. HA+ revertants of mutant 205 were isolated from brain tissue of mice inoculated with mutant 205. The nucleotide sequences were determined by consensus RNA sequencing using genomic RNA templates from purified virions. Two nucleotide differences were observed in the VP1 coding region of the RNA genomes of mutants 205 and 280 in comparison to the RNA sequences of 37A and the revertants. Interpretation of these data predict substitutions of two consecutive amino acids at residues 1231 (K to R) and 1232 (P to S) of VP1 which form part of the H-I loop of VP1 found at the icosahedral fivefold axis. Analysis of the amino acid substitutions in the context of the three-dimensional structure of the mengovirus-M capsid indicated that hemagglutination most likely involves residues found at the icosahedral fivefold axis and probably does not involve the residues that form the putative cellular receptor binding site (the "pit"). Eleven amino acid differences were observed between the structural proteins of mengovirus-M and 37A, five in VP1, three in VP2, and three in VP3.     

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.