Molecular evolution of the major capsid protein VP1 of enterovirus 70.

Nucleotide sequences of the genome RNA encoding capsid protein VP1 (918 nucleotides) of 18 enterovirus 70 (EV70) isolates collected from various parts of the world in 1971 to 1981 were determined, and nucleotide substitutions among them were studied. The genetic distances between isolates were calculated by the pairwise comparison of nucleotide difference. Regression analysis of the genetic distances against time of isolation of the strains showed that the synonymous substitution rate was very high at 21.53 x 10(-3) substitution per nucleotide per year, while the nonsynonymous rate was extremely low at 0.32 x 10(-3) substitution per nucleotide per year. The rate estimated by the average value of synonymous and nonsynonymous substitutions (W.-H. Li, C.-C. Wu, and C.-C. Luo, Mol. Biol. Evol. 2:150-174, 1985) was 5.00 x 10(-3) substitution per nucleotide per year. Taking the average value of synonymous and nonsynonymous substitutions as genetic distances between isolates, the phylogenetic tree was inferred by the unweighted pairwise grouping method of arithmetic average and by the neighbor-joining method. The tree indicated that the virus had evolved from one focal place, and the time of emergence was estimated to be August 1967 +/- 15 months, 2 years before first recognition of the pandemic of acute hemorrhagic conjunctivitis. By superimposing every nucleotide substitution on the branches of the phylogenetic tree, we analyzed nucleotide substitution patterns of EV70 genome RNA. In synonymous substitutions, the proportion of transitions, i.e., C<==>U and G<==>A, was found to be extremely frequent in comparison with that reported on other viruses or pseudogenes. In addition, parallel substitutions (independent substitutions at the same nucleotide position on different branches, i.e., different isolates, of the tree) were frequently found in both synonymous and nonsynonymous substitutions. These frequent parallel substitutions and the low nonsynonymous substitution rate despite the very high synonymous substitution rate described above imply a strong restriction on nonsynonymous substitution sites of VP1, probably due to the requirement for maintaining the rigid icosahedral conformation of the virus.     

Fixation of mutations at the VP1 gene of foot-and-mouth disease virus. Can quasispecies define a transient molecular clock?

The number of nucleotide (nt) substitutions found in the VP1 gene (encoding viral capsid protein) between any two of 16 closely related isolates of foot-and-mouth disease virus (FMDV) has been quantified as a function of the time interval between isolations [Villaverde et al., J. Mol. Biol. 204 (1988) 771-776]. One of them (isolate C-S12) includes some replacements found in isolates that preceded it and other replacements found in later isolates. The study has revealed alternating periods of rapid evolution and of relative genetic stability of VP1. During a defined period of acute disease, the rate of fixation of replacements at the VP1 coding segment was 6 x 10(-3) substitutions per nt per year. Only small differences in the rate of evolution were observed between subsegments within the VP1 gene. The observation of a relatively constant rate of evolution during a disease episode was unexpected. We propose that such constancy may be a consequence of random sampling of mutants from the FMDV quasispecies, followed by their amplification in susceptible hosts (to generate a new quasispecies). Successive sampling and amplification events may result in a steady accumulation of mutations.     

3D gene of foot-and-mouth disease virus. Conservation by convergence of average sequences

The nucleotide sequence of the 3D (polymerase) gene of eight epidemiologically related isolates of foot-and-mouth disease virus of serotype C1 is reported. The genetic heterogeneity of 3D RNA is compared with that of the VP1-coding RNA of the same viruses. Regression lines of substitutions per nucleotide that distinguish any pair of viruses as a function of the time interval between the corresponding isolations show: (1) the slope (substitutions/nucleotide per month) is 2.1 times larger for the VP1 RNA than for the 3D RNA region; (2) the intercept with the ordinate (substitutions/nucleotide) for VP1 RNA is indistinguishable from that for 3D RNA. Thus, the average heterogeneity of the VP1-coding region is very similar to that of the 3D-coding region only among co-circulating viruses. Nine mutations and points of heterogeneity occurred within nucleotide residues 883 to 1026, which encode an amino acid segment, extremely conserved among many different RNA viruses. The results suggest that, rather than due to inherently lower mutability, the conservation of 3D genes is caused by a limitation in the fixation of substitutions in viable genomes.     

Genetic variability of isolates of pandemic influenza A virus H1N1 isolated in Russia in 2009

Complete nucleotide sequence of the genome segments encoding the surface glycoproteins, hemagglutinin, and neuraminidase of influenza A virus H1N1 derived from the patients with influenza in the context of pandemic (H1N1) 2009 was determined out of 14 isolates of pandemic influenza. The philogenetic analysis of these sequences demonstrated their genetic similarity to the corresponding genes of the pandemic influenza virus A (H1N1) 2009 isolates obtained in other countries; each gene homology was greater than 99%. Neuraminidase mutations causing virus resistance to oseltamivir and other neuraminidase inhibitors, known from the literature, were not detected. The hemagglutinin gene mutation D222G was found in 4 isolates from autopsy material. In the hemagglutinin of pandemic A/Salekhard/01/2009(H1N1) isolate a mutation G155E leading to the increase in viral replication in developing chick embryos was detected. The nature and frequency of nucleotides substitutions within HA and NA genes were determined in the current research.     

Fixation of mutations in the viral genome during an outbreak of foot-and-mouth disease: heterogeneity and rate variations

Rates of fixation of mutations during the evolution of the foot-and-mouth disease virus (FMDV) C₁ in nature have been estimated by hybridization of viral RNA to cloned cDNAs representing defined FMDV genome segments, and comparison of the selected RNAs by T₁ RNase oligonucleotide fingerprinting. Values ranged from <0.04 × 10⁻² to 4.5 × 10⁻² substitutions per nucleotide per year (s/nt/yr), depending on the time period and the genomic segment considered. Rates for viral structural protein genes were up to sixfold higher than for nonstructural protein genes. Values in excess of 10⁻² s/nt/yr have been measured for the RNA region that encodes VP1–VP3. The nucleotide sequences of the major immunogenic region of capsid protein VP1 have been determined for six new FMDV C₁ isolates, and they are compared with the two previously known sequences of FMDV C₁ (C-S8 and C₁-O). Both oligonucleotide fingerprinting of selected RNA fragments and direct nucleotide sequencing demonstrate that genetic heterogeneity exists among three viruses isolated on the same day, introducing a significant indetermination in the evaluation of fixation rates of mutations. During the FMDV C₁ outbreak, amino acid substitutions did occur that are known to affect the immunological properties of the virus. The proportion of mutations between two viral RNAs does not increase significantly with the time elapsed between the two isolations, suggesting a cocirculation of multiple, related, nonidentical FMDVs (‘evolving quasispecies’) as the mode of evolution of this agent.     

Attenuated influenza virus construct with enhanced hemagglutinin protein expression

Influenza A viruses encoding an altered viral NS1 protein have emerged as promising live attenuated vaccine platforms. A carboxy-terminal truncation in the NS1 protein compromises its interferon antagonism activity, making these viruses attenuated in the host yet still able to induce protection from challenge with wild-type viruses. However, specific viral protein expression by NS1-truncated viruses is known to be decreased in infected cells. In this report, we show that recombinant H5N1 and H1N1 influenza viruses encoding a truncated NS1 protein expressed lower levels of hemagglutinin (HA) protein in infected cells than did wild-type viruses. This reduction in HA protein expression correlated with a reduction in HA mRNA levels in infected cells. NS1 truncation affected the expression of HA protein but not that of the nucleoprotein (NP). This segment specificity was mapped to the terminal sequences of their specific viral RNAs. Since the HA protein is the major immunogenic component in influenza virus vaccines, we sought to restore its expression levels in NS1-truncated viruses in order to improve their vaccine efficacy. For this purpose, we generated an NS1-truncated recombinant influenza A/Puerto Rico/8/34 (rPR8) virus carrying the G3A C8U "superpromoter" mutations in the HA genomic RNA segment. This strategy retained the attenuation properties of the recombinant virus but enhanced the expression level of HA protein in infected cells. Finally, mice immunized with rPR8 viruses encoding a truncated NS1 protein and carrying the G3A C8U mutations in the HA segment demonstrated enhanced protection from wild-type virus challenge over that for mice vaccinated with an rPR8 virus encoding the truncated NS1 protein alone.     

Evolutionary pattern of the hemagglutinin gene of influenza B viruses isolated in Japan: cocirculating lineages in the same epidemic season.

The unexpectedly low efficacy of influenza vaccine during school outbreaks of influenza B virus in the spring of 1987 in Japan was probably attributable to a poor antibody response of vaccinees to the epidemic viruses. An antigenic analysis of the causative B viruses isolated in 1987 and 1988 showed much variation in hemagglutination inhibition patterns. The nucleotide sequences that code for the HA1 domain of B/Fukuoka/c-27/81, B/Ibaraki/2/85, B/Nagasaki/1/87, and B/Yamagata/16/88 viruses were determined and compared with those of the previously reported hemagglutinin genes. The nucleotide sequences of the hemagglutinin gene of a new variant, B/Yamagata/16/88, had only 93.4% homology with those of two other viruses from the same epidemic. An analysis of nucleotide and amino acid substitutions of the hemagglutinin genes of influenza B viruses revealed that new and some old variants could cocirculate in the same epidemic. A phylogenetic tree constructed by the neighbor-joining method allowed estimation of an evolutionary rate of 2.3 x 10(-3) synonymous (silent) substitutions per nucleotide site per year in the hemagglutinin gene.     

The low evolutionary rate of human T-cell lymphotropic virus type-1 confirmed by analysis of vertical transmission chains

The evolutionary rate of the human T-cell lymphotropic virus type-1 (HTLV-1) is considered to be very low, in strong contrast to the related human retrovirus HIV. However, current estimates of the HTLV-1 rate rely on the anthropological calibration of phylogenies using assumed dates of human migration events. To obtain an independent rate estimate, we analyzed two variable regions of the HTLV-1 genome (LTR and env) from eight infected families. Remarkable genetic stability was observed, as only two mutations in LTR (756 bp) and three mutations in env (522 bp) occurred within the 16 vertical transmission chains, including one ambiguous position in each region. The evolutionary rate in HTLV-1 was then calculated using a maximum-likelihood approach that used the highest and lowest possible times of HTLV-1 shared ancestry, given the known transmission histories. The rates for the LTR and env regions were 9.58 x 10(-8)-1.25 x 10(-5) and 7.84 x 10(-7) -2.33 x 10(-5)nucleotide substitutions per site per year, respectively. A more precise estimate was obtained for the combined LTR-env data set, which was 7.06 x 10(-7)-1.38 x 10(-5)substitutions per site per year. We also note an interesting correlation between the occurrence of mutations in HTLV-1 and the age of the individual infected.     

Amino Acids Transitioning of 2009 H1N1pdm in Taiwan from 2009 to 2011

A swine-origin influenza A was detected in April 2009 and soon became the 2009 H1N1 pandemic strain (H1N1pdm). The current study revealed the genetic diversity of H1N1pdm, based on 77 and 70 isolates which we collected, respectively, during the 2009/2010 and 2010/2011 influenza seasons in Taiwan. We focused on tracking the amino acid transitioning of hemagglutinin (HA) and neuraminidase (NA) genes in the early diversification of the virus and compared them with H1N1pdm strains reported worldwide. We identified newly emerged mutation markers based on A/California/04/2009, described how these markers shifted from the first H1N1pdm season to the one that immediately followed, and discussed how these observations may relate to antigenicity, receptor-binding, and drug susceptibility. It was found that the amino acid mutation rates of H1N1pdm were elevated, from 9.29×10⁻³ substitutions per site in the first season to 1.46×10⁻² in the second season in HA, and from 5.23×10⁻³ to 1.10×10⁻² in NA. Many mutation markers were newly detected in the second season, including 11 in HA and 8 in NA, and some were found having statistical correlation to disease severity. There were five noticeable HA mutations made to antigenic sites. No significant titer changes, however, were detected based on hemagglutination inhibition tests. Only one isolate with H275Y mutation known to reduce susceptibility to NA inhibitors was detected. As limited Taiwanese H1N1pdm viruses were isolated after our sampling period, we gathered 8,876 HA and 6,017 NA H1N1pdm sequences up to April 2012 from NCBI to follow up the dynamics of mentioned HA mutations. While some mutations described in this study seemed to either settle in or die out in the 2011–2012 season, a number of them still showed signs of transitioning, prompting the importance of continuous monitoring of this virus for more seasons to come.     

Intrahost human immunodeficiency virus type 1 evolution is related to length of the immunocompetent period.

The antigenic diversity threshold theory predicts that antigenic sites of human immunodeficiency virus type 1, such as the V3 region of the external glycoprotein gp120, evolve more rapidly during the symptom-free period in individuals progressing to AIDS than in those who remain asymptomatic for a long time. To test this hypothesis, genomic RNA sequences were obtained from the sera of 44 individuals at seroconversion and 5 years later. The mean number of nonsynonymous nucleotide substitutions in the V3 region of the viruses circulating in 31 nonprogressors (1.1 x 10(-2) +/- 0.1 x 10(-2) per site per year) was higher than the corresponding value for 13 progressors (0.66 x 10(-2) +/- 0.1 x 10(-2) per site per year) (P < 0.01), while no difference between the mean numbers of synonymous substitutions in the two groups was seen (0.37 x 10(-2) +/- 0.1 x 10(-2) and 0.51 x 10(-2) +/- 0.2 x 10(-2) per site per year for nonprogressors and progressors, respectively; P > 0.1). The mean ratios of synonymous nucleotide p distance to nonsynonymous p distance were 0.35 for nonprogressors and 0.62 for progressors. The number of nonsynonymous substitutions was not associated with virus load or virus phenotype, which are established predictors of disease progression, but correlated strongly with the duration of the immunocompetent period (r2 = 0.41; P = 0.001). This indicates that there is no causative relationship between intrahost evolution and CD4+ cell decline. Our data suggest that intrahost evolution in human immunodeficiency virus type 1 infection is driven by selective forces, the strength of which is related to the duration of the immunocompetent period.     

Antigenic structure of the influenza B virus hemagglutinin: nucleotide sequence analysis of antigenic variants selected with monoclonal antibodies.

We report here the complete nucleotide sequence of the hemagglutinin (HA) gene of influenza B virus B/Oregon/5/80 and, through comparative sequence analysis, identify amino acid substitutions in the HA1 polypeptide responsible for the antigenic alterations in laboratory-selected antigenic variants of this virus. The complete nucleotide sequence of the B/Oregon/5/80 HA gene was established by a combination of chemical sequencing of a full-length cDNA clone and dideoxy sequencing of the virion RNA. The nucleotide sequence is very similar to previously reported influenza B virus HA gene sequences and differs at only nine nucleotide positions from the B/Singapore/222/79 HA gene (Verhoeyen et al., Nucleic Acids Res. 11:4703-4712, 1983). The nucleotide sequences of the HA1 portions of the HA genes of 18 laboratory-selected antigenic variants were determined by the dideoxy method. Comparison of the deduced amino acid sequences of the parental and variant HA1 polypeptides revealed 16 different amino acid substitutions at nine positions. All amino acid substitutions resulted from single-point mutations, and no double mutants were detected, demonstrating that as in the influenza A viruses, single amino acid substitutions are sufficient to alter the antigenicity of the HA molecule. Many of the amino acid substitutions in the variants occurred at positions also observed to change in natural drift strains. The substitutions appear to identify at least two immunodominant regions which correspond to proposed antigenic sites A and B on the influenza A virus H3 HA.     

Characterization of the Influenza A H5N1 Viruses of the 2008-09 Outbreaks in India Reveals a Third Introduction and Possible Endemicity

Widespread infection of highly pathogenic avian influenza A H5N1 was reported from backyard and commercial poultry in West Bengal (WB), an eastern state of India in early 2008. Infection gradually spread to Tripura, Assam and Sikkim, the northeastern states, with 70 outbreaks reported between January 2008 and May 2009. Whole genome sequence analysis of three isolates from WB, one isolate from Tripura along with the analysis of hemagglutinin (HA) and neuraminidase (NA) genes of 17 other isolates was performed during this study. In the HA gene phylogenetic tree, all the 2008-09 Indian isolates belonged to EMA3 sublineage of clade 2.2. The closest phylogenetic relationship was found to be with the 2007-09 isolates from Bangladesh and not with the earlier 2006 and 2007 Indian isolates implying a third introduction into the country. The receptor-binding pocket of HA1 of two isolates from WB showed S221P mutation, one of the markers predicted to be associated with human receptor specificity. Two substitutions E119A (2 isolates of WB) and N294S (2 other isolates of WB) known to confer resistance to NA inhibitors were observed in the active site of neuraminidase. Several additional mutations were observed within the 2008-09 Indian isolates indicating genetic diversification. Overall, the study is indicative of a possible endemicity in the eastern and northeastern parts of the country, demanding active surveillance specifically in view of the critical mutations that have been observed in the influenza A H5N1 viruses.     

Analysis of the evolution and variation of the human influenza A virus nucleoprotein gene from 1933 to 1990.

This study examined the evolution and variation of the human influenza virus nucleoprotein gene from the earliest isolates to the present. Phylogenetic reconstruction of the most parsimonious evolutionary path connecting 49 nucleoprotein sequences yielded a single lineage. The average calculated rate of mutation was 3.6 nucleotide substitutions per year (2.3 x 10(-3) substitutions per site per year). Thirty-two percent of these mutations resulted in amino acid substitutions, and the remainder were silent mutations. Analysis of virus isolates from China and elsewhere showed no significant differences in their rate of evolution, genetic diversity, or mean survival time. The nearly constant rate of change was maintained through the two antigenic shifts, and there were no obvious changes in the number or types of mutations associated with the changes in the surface proteins. A detailed comparison of the changes that have occurred on the main evolutionary path with those that have occurred on the side branches of the phylogenetic tree was made. This showed that while 35% of the mutations on the side branches resulted in amino acid changes, only 21% of those on the main path affected the protein sequence. These results suggest that although the rate of change of the human influenza virus nucleoprotein is much higher than that previously described for avian influenza viruses, there are measurable constraints on the evolution of the surviving virus lineage. Comparison of the nucleoproteins of virus isolates adapted to chicken embryos with the nucleoproteins of those grown only in MDCK cells revealed no consistent differences between the virus pairs. Thus, although the nucleoprotein is known to be critical for host specificity, its adaptation to growth in eggs apparently involves no immediate selective pressures, such as are found with hemagglutinin.     

Molecular and antigenic characterization of reassortant H3N2 viruses from turkeys with a unique constellation of pandemic H1N1 internal genes

Triple reassortant (TR) H3N2 influenza viruses cause varying degrees of loss in egg production in breeder turkeys. In this study we characterized TR H3N2 viruses isolated from three breeder turkey farms diagnosed with a drop in egg production. The eight gene segments of the virus isolated from the first case submission (FAV-003) were all of TR H3N2 lineage. However, viruses from the two subsequent case submissions (FAV-009 and FAV-010) were unique reassortants with PB2, PA, nucleoprotein (NP) and matrix (M) gene segments from 2009 pandemic H1N1 and the remaining gene segments from TR H3N2. Phylogenetic analysis of the HA and NA genes placed the 3 virus isolates in 2 separate clades within cluster IV of TR H3N2 viruses. Birds from the latter two affected farms had been vaccinated with a H3N4 oil emulsion vaccine prior to the outbreak. The HAl subunit of the H3N4 vaccine strain had only a predicted amino acid identity of 79% with the isolate from FAV-003 and 80% for the isolates from FAV-009 and FAV-0010. By comparison, the predicted amino acid sequence identity between a prototype TR H3N2 cluster IV virus A/Sw/ON/33853/2005 and the three turkey isolates from this study was 95% while the identity between FAV-003 and FAV-009/10 isolates was 91%. When the previously identified antigenic sites A, B, C, D and E of HA1 were examined, isolates from FAV-003 and FAV-009/10 had a total of 19 and 16 amino acid substitutions respectively when compared with the H3N4 vaccine strain. These changes corresponded with the failure of the sera collected from turkeys that received this vaccine to neutralize any of the above three isolates in vitro.     

Identification of Low- and High-Impact Hemagglutinin Amino Acid Substitutions That Drive Antigenic Drift of Influenza A(H1N1) Viruses

Determining phenotype from genetic data is a fundamental challenge. Identification of emerging antigenic variants among circulating influenza viruses is critical to the vaccine virus selection process, with vaccine effectiveness maximized when constituents are antigenically similar to circulating viruses. Hemagglutination inhibition (HI) assay data are commonly used to assess influenza antigenicity. Here, sequence and 3-D structural information of hemagglutinin (HA) glycoproteins were analyzed together with corresponding HI assay data for former seasonal influenza A(H1N1) virus isolates (1997–2009) and reference viruses. The models developed identify and quantify the impact of eighteen amino acid substitutions on the antigenicity of HA, two of which were responsible for major transitions in antigenic phenotype. We used reverse genetics to demonstrate the causal effect on antigenicity for a subset of these substitutions. Information on the impact of substitutions allowed us to predict antigenic phenotypes of emerging viruses directly from HA gene sequence data and accuracy was doubled by including all substitutions causing antigenic changes over a model incorporating only the substitutions with the largest impact. The ability to quantify the phenotypic impact of specific amino acid substitutions should help refine emerging techniques that predict the evolution of virus populations from one year to the next, leading to stronger theoretical foundations for selection of candidate vaccine viruses. These techniques have great potential to be extended to other antigenically variable pathogens.     

海南省2016-2018年A(H1N1)pdm09亚型流感病毒基因特性分析

2009年A(H1N1)pdm09亚型流感病毒在墨西哥暴发,之后在全世界流行。为了解海南省2016-2018年A(H1N1)pdm09亚型流感病毒流行态势,分析血凝素(HA)与神经氨酸酶(NA)基因遗传进化特征与变异情况,本研究从中国流感监测信息系统获取海南省2016-2018年流感病毒病原学监测数据,选取5家流感监测网络实验室分离鉴定的37株A(H1N1)pdm09亚型流感毒株进行HA与NA基因测序,利用MEGA 10.1.8构建HA与NA基因种系进化树,并分析其氨基酸变异情况。结果显示,2016-2018年共出现3次A(H1N1)pdm09亚型流感病毒活动高峰。2017年10月份以后的分离株(4/8)与2018年大部分分离株(21/22)独立于疫苗株A/Michigan/45/2015聚为一个小支,发生20余处HA与NA氨基酸位点变异。与疫苗株A/California/7/2009(2010-2016)相比,2016-2018年流感病毒分离株在HA基因抗原决定簇上发生7处氨基酸变异并有一个潜在糖基化位点,未发现HA基因受体结合位点变异与NA基因耐药性变异。本研究提示,2016-2018年,A(H1N1)pdm09亚型流感病毒逐步发生规律性进化,氨基酸变异频率有增加趋势,今后应持续加强流感病毒病原学监测,密切追踪A(H1N1)pdm09亚型流感病毒基因变异情况,为科学防控提供理论依据。     

Genetic variability and molecular evolution of the human respiratory syncytial virus subgroup B attachment G protein

Human respiratory syncytial virus (HRSV) is the most important cause of acute respiratory disease in infants. Two major subgroups (A and B) have been identified based on antigenic differences in the attachment G protein. Antigenic variation between and within the subgroups may contribute to reinfections with these viruses by evading the host immune responses. To investigate the circulation patterns and mechanisms by which HRSV-B viruses evolve, we analyzed the G protein genetic variability of subgroup B sequences isolated over a 45-year period, including 196 Belgian strains obtained over 22 epidemic seasons (1982 to 2004). Our study revealed that the HRSV-B evolutionary rate (1.95 x 10(-3) nucleotide substitutions/site/year) is similar to that previously estimated for HRSV-A (1.83 x 10(-3) nucleotide substitutions/site/year). However, natural HRSV-B isolates appear to accommodate more drastic changes in their attachment G proteins. The most recent common ancestor of the currently circulating subgroup B strains was estimated to date back to around the year 1949. The divergence between the two major subgroups was calculated to have occurred approximately 350 years ago. Furthermore, we have identified 12 positively selected sites in the G protein ectodomain, suggesting that immune-driven selective pressure operates in certain codon positions. HRSV-A and -B strains have similar phylodynamic patterns: both subgroups are characterized by global spatiotemporal strain dynamics, where the high infectiousness of HRSV permits the rapid geographic spread of novel strain variants.     

Molecular evolution of a type 1 wild-vaccine poliovirus recombinant during widespread circulation in China

Type 1 wild-vaccine recombinant polioviruses were isolated from poliomyelitis patients in China from 1991 to 1993. We compared the sequences of 34 recombinant isolates over the 1,353-nucleotide (nt) genomic interval (nt 2480 to 3832) encoding the major capsid protein, VP1, and the protease, 2A. All recombinants had a 367-nt block of sequence (nt 3271 to 3637) derived from the Sabin 1 oral poliovirus vaccine strain spanning the 3'-terminal sequences of VP1 (115 nt) and the 5' half of 2A (252 nt). The remaining VP1 sequences were closely (up to 99.5%) related to those of a major genotype of wild type 1 poliovirus endemic to China up to 1994. In contrast, the non-vaccine-derived sequences at the 3' half of 2A were more distantly related (<90% nucleotide sequence match) to those of other contemporary wild polioviruses from China. The vaccine-derived sequences of the earliest (April 1991) isolates completely matched those of Sabin 1. Later isolates diverged from the early isolates primarily by accumulation of synonymous base substitutions (at a rate of approximately 3.7 x 10(-2) substitutions per synonymous site per year) over the entire VP1-2A interval. Distinct evolutionary lineages were found in different Chinese provinces. From the combined epidemiologic and evolutionary analyses, we propose that the recombinant virus arose during mixed infection of a single individual in northern China in early 1991 and that its progeny spread by multiple independent chains of transmission into some of the most populous areas of China within a year of the initiating infection.     

Deciphering human immunodeficiency virus type 1 transmission and early envelope diversification by single-genome amplification and sequencing

Accurate identification of the transmitted virus and sequences evolving from it could be instrumental in elucidating the transmission of human immunodeficiency virus type 1 (HIV-1) and in developing vaccines, drugs, or microbicides to prevent infection. Here we describe an experimental approach to analyze HIV-1 env genes as intact genetic units amplified from plasma virion RNA by single-genome amplification (SGA), followed by direct sequencing of uncloned DNA amplicons. We show that this strategy precludes in vitro artifacts caused by Taq-induced nucleotide substitutions and template switching, provides an accurate representation of the env quasispecies in vivo, and has an overall error rate (including nucleotide misincorporation, insertion, and deletion) of less than 8 x 10(-5). Applying this method to the analysis of virus in plasma from 12 Zambian subjects from whom samples were obtained within 3 months of seroconversion, we show that transmitted or early founder viruses can be identified and that molecular pathways and rates of early env diversification can be defined. Specifically, we show that 8 of the 12 subjects were each infected by a single virus, while 4 others acquired more than one virus; that the rate of virus evolution in one subject during an 80-day period spanning seroconversion was 1.7 x 10(-5) substitutions per site per day; and that evidence of strong immunologic selection can be seen in Env and overlapping Rev sequences based on nonrandom accumulation of nonsynonymous mutations. We also compared the results of the SGA approach with those of more-conventional bulk PCR amplification methods performed on the same patient samples and found that the latter is associated with excessive rates of Taq-induced recombination, nucleotide misincorporation, template resampling, and cloning bias. These findings indicate that HIV-1 env genes, other viral genes, and even full-length viral genomes responsible for productive clinical infection can be identified by SGA analysis of plasma virus sampled at intervals typical in large-scale vaccine trials and that pathways of viral diversification and immune escape can be determined accurately.     

Human glyceraldehyde-3-phosphate dehydrogenase pseudogenes: Molecular evolution and a possible mechanism for amplification

We screened two human genomic libraries and isolated 14 different clones, designated λG1 and EG1-EG13, homologous to human glyceraldehyde-3-phosphate dehydrogenase (GAPD) cDNA. Subcloning and sequencing these recombinant phages led us to classify them as five different pseudogenes (ψG1–ψG5). All these sequences show such features typical of processed pseudogenes as numerous mutations, insertions, and deletions. The identity of numerous mutated sites among these pseudogenes and the presence of two Alu sequences flanking both ends of ψG1 suggest that GAPD pseudogenes originated from a unique reverse transcribed mRNA followed by gene duplication. The rate of nucleotide substitutions per site per year for known GAPD functional genes is low both for the synonymous substitutions (1.87×10⁻⁹) and for the nonsynonymous substitutions (0.12￠10⁻⁹) and indicates that the GAPD cDNA sequence is well conserved not only at the amino acid level, but also at the nucleotide level. The rate of nucleotide substitutions per site per year for GAPD pseudogenes shows a higher value (5.9×10⁻⁹) and suggests that these pseudogenes do not have any functional role. This work was supported by grants from the Consiglio Nazionale delle Ricerche and the Ministero Pubblica Istruzione (Rome, Italy). Special acknowledgment is given to the “Progetto Finalizzato Ingegneria Genetica e Basi Molecolari delle Malattie Ereditarie.”