Yutaka Takebe

Evolutionary Genetics of Human Enterovirus 71: Origin, Population Dynamics, Natural Selection, and Seasonal Periodicity of the VP1 Gene

ABSTRACT Human enterovirus 71 (EV-71) is one of the major etiologic causes of hand, foot, and mouth disease (HFMD) among young children worldwide, with fatal instances of neurological complications becoming increasingly common. Global VP1 capsid sequences (n = 628) sampled over 4 decades were collected and subjected to comprehensive evolutionary analysis using a suite of phylogenetic and population genetic methods. We estimated that the common ancestor of human EV-71 likely emerged around 1941 (95% confidence interval [CI], 1929 to 1952), subsequently diverging into three genogroups: B, C, and the now extinct genogroup A. Genealogical analysis revealed that diverse lineages of genogroup B and C (subgenogroups B1 to B5 and C1 to C5) have each circulated cryptically in the human population for up to 5 years before causing large HFMD outbreaks, indicating the quiescent persistence of EV-71 in human populations. Estimated phylogenies showed a complex pattern of spatial structure within well-sampled subgenogroups, suggesting endemicity with occasional lineage migration among locations, such that past HFMD epidemics are unlikely to be linked to continuous transmission of a single strain of virus. In addition, rises in genetic diversity are correlated with the onset of epidemics, driven in part by the emergence of novel EV-71 subgenogroups. Using subgenogroup C1 as a model, we observe temporal strain replacement through time, and we investigate the evidence for positive selection at VP1 immunogenic sites. We discuss the consequences of the evolutionary dynamics of EV-71 for vaccine design and compare its phylodynamic behavior with that of influenza virus.

Molecular Biology of Interleukin 4 and Interleukin 5 Genes and Biology of their Products that Stimulate B Cells, T Cells and Hemopoietic Cells

Lymphokines produced by helper T cells activated by antigen mediate numerous efTector functions of T cells. Unlike immunoglobulin or the T cell receptor, lymphokines do not bear antigen specificity and stimulate proliferation and differentiation of lymphocytes and hemopoietic cells (Arai et al. 1986). Since many lymphokines are composed of single polypeptide chains, their coding sequences can be isolated by functional expression in appropriate host cells. Using a pcD cDNA expression vector (Okayama & Berg 1983), we have developed a screening procedure employing transfection of plasmid DNAs into mammalian cells followed by assay of transfected cell supernatants for lymphokine activities of interest (Yokota et al. 1984. 1985, 1987c). Based on this expression cloning protocol, many T cell lymphokine genes have been isolated and their primary structures determined. These studies have revealed a regulatory network formed between lymphoid cells and hemopoietic cells through the action of multiple lymphokines produced by activated T cells. For example, interleukin 2 (IL-2) stimulates predominantly T cells whereas interleukin 3 (lL-3) and granulocytemacrophage colony stimulating factor (GM-CSF) stimulate hemopoietic cells. A number of molecules stimulate B cells. Among them, interleukin 4 (IL-4), interleukin 5 (IL-5) and B cell stimulatory factor 2 (BSF-2) have been chemically defined by molecular cloning (Lee et al. 1986, Noma et al. 1986, Yokota et al. !986,

On-going generation of multiple forms of HIV-1 intersubtype recombinants in the Yunnan Province of China

ObjectivesTo investigate the molecular epidemiology of HIV in Chinas Yunnan Province, where the initial HIV-1 outbreak among injecting drug users (IDU) occurred in 1989, and to analyse the genesis and interrelationship of the epidemic with that in surrounding areas. DesignA molecular epidemiological investigation was conducted among IDU in three prefectures in Yunnan Province, including Wenshan (east), Honghe (southeast) and Dehong (west). MethodsThirty-nine specimens were collected from consenting IDU in 2000–2001. The nucleotide sequences of 2.6 kb gag-RT and 340 base pair (bp) env (C2/V3) regions were determined. Phylogenetic tree and recombination breakpoint analyses were performed. ResultsThe circulating recombinant form (CRF), CRF08_BC, predominated in east Yunnan near Guangxi Province (89% in Wenshan and 81% in Honghe), whereas it was not detected in Dehong (0/14) in the west. In contrast, 71% (10/14) of the Dehong isolates were unique recombinant forms (URF), mostly between subtypes B′ (Thailand variant of subtype B) and C, with distinct profiles of recombination breakpoints. The subtype B′ accounts for the remaining 29% (4/14) of Dehong isolates. Interestingly, two Honghe isolates (2/16) shared some of the precise B′/C recombination breakpoints with CRF07_BC. ConclusionNew recombinant strains are arising continually in west Yunnan near the Myanmar border. Some appeared to be secondary recombinants derived from CRF07_BC that had further recombined with other strains. The uneven distribution of subtypes, CRF and URF, suggests the presence of independent transmission networks and clusters among IDU in Yunnan.

High prevalence of HIV-1 and hepatitis C virus coinfection among injection drug users in the southeastern region of Yunnan, China.

&NA; The southeastern region of Yunnan province is a key site for drug trafficking and HIV‐1 infection spread from the west of Yunnan and Laos to southeastern China. To investigate the prevalence of HIV‐1 infection and hepatitis C virus (HCV) coinfection among injection drug users (IDUs) in southeastern Yunnan, three cohorts of 285 addicts, including 242 IDUs and 43 oral drug users, living in the cities of Gejiu and Kaiyuan and the county of Yanshan were studied. HIV‐1 and HCV infections were detected by enzyme‐linked immunosorbent assay and/or polymerase chain reaction. Data on the age, sex, risk behavior, drug use history, employment, ethnic background, and marriage status were obtained by interview. The overall prevalence of HIV‐1 infection was 71.9%. The rate of HCV coinfection among 138 HIV‐1‐infected IDUs was 99.3%. Most HIV‐infected IDUs were 20 to 35 years old (86.7%) and were ethnic Han (75.9%), suggesting that the epidemic in Yunnan is no longer confined to non‐Han ethnic minorities. HIV prevalence in female IDUs (81.2%) was significantly higher than in male IDUs (68.2%) (p < .05). The prevalence of HIV infection reached 68.4% after 1 year of injection drug use. Needle/syringe sharing is the major high risk factor for the spread of HIV‐1 and HCV infections. Large‐scale educational campaigns are urgently needed to reduce the spread of HIV and HCV infection in these regions.

An infectious DNA clone of HIV type 1 subtype C.

Among the 10 subtypes of the M group of human immunodeficiency virus type 1, subtype C is the most prevalent in India and may dominate worldwide in the near future; however, there has been no report on the infectious DNA clone of this subtype. We have isolated an infectious DNA clone of the 93IN101 strain of HIV-1 subtype C, which was isolated in India in 1993. MAGIC5 cells, which are derived from HeLa-CD4-LTR-beta-gal (MAGI) cells and express CCR5, were inoculated with the 93IN101 strain of HIV-1 subtype C. The genomic DNA of the infected cells was used as a template for amplification of the HIV-1 genome. The genome DNA obtained was subcloned into pBR322, and the resulting plasmid was designated as pIndie-C1. The insert of pIndie-C1 was 9680 bp in length and had an intact genomic organization with open reading frames of all structural, regulatory, and accessory proteins. Phylogenetic analysis confirmed that the nucleotide sequence of pIndie-C1 is closely related to those of HIV-1 subtype C isolated in India. Transfection of pIndie-C1 into 293T cells yielded as much virus as did pNL432, one of the most widely used HIV DNA clones. The recovered Indie-C1 virus infected MAGIC5 but not the parent MAGI cells, indicating that Indie-C1 is CCR5 tropic. Expressed Env protein was reacted efficiently with the sera of HIV-1-infected patients of India, but not of Japan. Expression of Nef and Vpr was also confirmed by immunoblotting.

The rapidly expanding CRF01_AE epidemic in China is driven by multiple lineages of HIV-1 viruses introduced in the 1990s.

Objectives:We sought to comprehensively analyze the origin, transmission patterns and sub-epidemic clusters of the HIV-1 CRF01_AE strains in China. Methods:Available HIV-1 CRF01_AE samples indentified in national molecular epidemiologic surveys were used to generate near full-length genome (NFLG) sequences. The new and globally available CRF01_AE NFLG sequences were subjected to phylogenetic and Bayesian molecular clock analyses, and combined with epidemiologic data to elucidate the history of CRF01_AE transmission in China. Results:We generated 75 new CRF01_AE NFLG sequences from various risk populations covering all major CRF01_AE epidemic regions in China. Seven distinct phylogenetic clusters of CRF01_AE were identified. Clusters 1, 2 and 3 were prevalent among heterosexuals and IDUs in southern and southwestern provinces. Clusters 4 and 5 were found primarily among MSM in major northern cities. Clusters 6 and 7 were only detected among heterosexuals in two southeast and southwest provinces. Molecular clock analysis indicated that all CRF01_AE clusters were introduced from Southeast Asia in the 1990s, coinciding with the peak of Thailands HIV epidemic and the initiation of Chinas free overseas travel policy for their citizens, which started with Thailand as the first destination country. Conclusion:Chinas HIV-1 epidemic of sexual transmissions, was initiated by multilineages of CRF01_AE strains, in contrast to the mono-lineage epidemic of B′ strain in former plasma donors and IDUs. Our study underscores the difficulty in controlling HIV-1 sexual transmission compared with parenteral transmission.

Polymorphism in the Interleukin-4 Promoter Affects Acquisition of Human Immunodeficiency Virus Type 1 Syncytium-Inducing Phenotype

ABSTRACT The emergence of syncytium-inducing (SI) variants of human immunodeficiency virus type 1 (HIV-1) in infected individuals is an indicator of poor prognosis and is often correlated with faster CD4+ cell depletion and rapid disease progression. Interleukin-4 (IL-4) is a pleiotropic cytokine with various immune-modulating functions including induction of immunoglobulin E (IgE) production in B cells, down-regulation of CCR5 (a coreceptor for HIV-1 non-SI [NSI] strains), and up-regulation of CXCR4 (a coreceptor for HIV-1 SI variants). Here we show that homozygosity of a polymorphism in the IL-4 promoter region, IL-4 −589T, is correlated with increased rates of SI variant acquisition in HIV-1-infected individuals in Japan. This mutation was also shown to be associated with elevated serum IgE levels in HIV-1-infected individuals, especially in those at advanced stages of disease. In contrast, neither a triallele polymorphism in IL-10, another Th2 cytokine, nor a biallele polymorphism in the RANTES promoter affected acquisition of the SI phenotype. This finding suggested that IL-4-589T increases IL-4 production in the human body and thus accelerates the phenotypic switch of HIV-1 from NSI to SI and possibly disease progression of AIDS.

The unique HCV genotype distribution and the discovery of a novel subtype 6u among IDUs co-infected with HIV-1 in Yunnan, China†‡

The Yunnan province is the epicenter of HIV‐1 epidemics in China and a center for drug trafficking to the other parts of the world. In six prefectures of this province, a total of 132 IDUs were recruited to determine the sero‐prevalence of HCV and HIV‐1 and the positive rates were 93.94% and 68.18%, respectively (P < 0.001). Co‐infection with HCV and HIV‐1 was found among 89 IDUs, of whom several HCV fragments were amplified and sequenced. Sequences of the HCV 5′NCR‐C and NS5B region were determined from 82 IDUs. Phylogenetic analyses showed consistent genotyping among 80 IDUs. Among them HCV genotypes 1a, 1b, 3a, 3b, 6a, 6n, and a tentatively assigned novel 6u subtype were found in 1 (1.25%), 16 (20%), 19 (23.75%), 24 (30%), 4 (5%), 9 (11.25%) and 7 (8.75%) individuals, respectively. In two IDUs, genotyping results were discordant, suggesting mixed HCV infections or recombination. The proportion of patients with HCV 1b tended to decrease from the north to south and from the east to west in this province. Genotype 3 and 6 strains were more frequent in the southern prefectures. The novel subtype 6u strains were only detected in Dehong which borders Myanmar. Our findings showed a unique pattern of HCV genotype distribution, which is similar to that in the southeastern Asian countries but distinct from that among the general population in China. Routes of drug trafficking and the resulting high prevalence of HIV‐1 infection may have contributed to this pattern of HCV genotype distribution. J. Med. Virol. 80: 1142–1152, 2008.

Identification and Characterization of a New Class of Human Immunodeficiency Virus Type 1 Recombinants Comprised of Two Circulating Recombinant Forms, CRF07_BC and CRF08_BC, in China

ABSTRACT We identified a new class of human immunodeficiency virus type 1 (HIV-1) recombinants (00CN-HH069 and 00CN-HH086) in which further recombination occurred between two established circulating recombinant forms (CRFs). These two isolates were found among 57 HIV-1 samples from a cohort of injecting drug users in eastern Yunnan Province of China. Informative-site analysis in conjunction with bootscanning plots and exploratory tree analysis revealed that these two strains were closely related mosaics comprised of CRF07_BC and CRF08_BC, which are found in China. The genotype screening based on gag-reverse transcriptase sequences of 57 samples from eastern Yunnan identified 47 CRF08_BC specimens (82.5%), 5 CRF07_BC specimens (8.8%), and 3 additional specimens with the novel recombinant structure. These new “second-generation” recombinants thus constitute a substantial proportion (5 of 57; 8.8%) of HIV-1 strains in this population and may belong to a new but yet-undefined class of CRF. This might be the first example of CRFs recombining with each other, leading to the evolution of second-generation inter-CRF recombinants.

DNA vaccine-encapsulated virus-like particles derived from an orally transmissible virus stimulate mucosal and systemic immune responses by oral administration

Delivery of foreign genes to the digestive tract mucosa by oral administration of nonreplicating gene transfer vectors would be a very useful method for vaccination and gene therapy. However, there have been few reports on suitable vectors. In the present study, we found that plasmid DNA can be packaged in vitro into a virus-like particle (VLP) composed of open reading frame 2 of hepatitis E virus, which is an orally transmissible virus, and that these VLPs can deliver this foreign DNA to the intestinal mucosa in vivo. The delivery of plasmid DNA to the mucosa of the small intestine was confirmed by the results of immunohistochemical analyses using an expression plasmid encoding human immunodeficiency virus env (HIV env) gp120. After oral administration of VLPs loaded with HIV env cDNA, significant levels of specific IgG and IgA to HIV env in fecal extracts and sera were found. Moreover, mice used in this study exhibited cytotoxic T-lymphocyte responses specific to HIV env in the spleen, Payers patches and mesenteric lymph nodes. These findings suggest that VLPs derived from orally transmissible viruses can be used as vectors for delivery of genes to mucosal tissue by oral administration for the purpose of DNA vaccination and gene therapy.