Evolutionary Dynamics of Foot and Mouth Disease Virus Serotype A and Its Endemic Sub-Lineage A/ASIA/Iran-05/SIS-13 in Pakistan

Foot and mouth disease (FMD) causes severe economic losses to the livestock industry of endemic countries, including Pakistan. Pakistan is part of the endemic pool 3 for foot and mouth disease viruses (FMDV), characterized by co-circulating O, A, and Asia 1 serotypes, as designated by the world reference laboratory for FMD (WRL-FMD). FMDV serotype A lineage ASIA/Iran-05 is widespread in buffalos and cattle populations and was first reported in Pakistan in 2006. This lineage has a high turnover, with as many as 10 sub-lineages reported from Pakistan over the years. In this study, we reconstructed the evolutionary, demographic, and spatial history of serotype A and one of its sub-lineages, A/ASIA/Iran-05/SIS-13, prevalent in Pakistan. We sequenced nearly complete genomes of three isolates belonging to sub-lineage A/ASIA/Iran-05/SIS-13. We estimated recombination patterns and natural selection acting on the serotype A genomes. Source and transmission routes in Pakistan were inferred, and the clustering pattern of isolates of the SIS-13 sub-lineage were mapped on a tree. We hereby report nearly complete genome sequences of isolates belonging to sub-lineage A/ASIA/Iran-05/SIS-13, along with purported recombinant genomes, and highlight that complete coding sequences can better elucidate the endemic history and evolutionary pressures acting on long-term co-circulating FMDV strains.     

Sequence variation in the gene for the immunogenic capsid protein VP1 of foot-and-mouth disease virus type A.

The nucleotide sequences have been determined and compared from cloned cDNA genes coding for the foot-and-mouth disease virus (FMDV) immunogenic capsid protein, VP1, from eight different A subtypes: A5 Westerwald/58, A12 119ab (large plaque variant), A22 550 USSR/65, A24 Cruzeiro Brazil/55, A27 Cundinamarca Colombia/76, A32 Venezuela/70, A Venceslau Brazil/76, and A Argentina/79. We have also found sequence variations among different cDNA clones of the A5 and A24 subtypes. There are regions of nucleotide sequence within the VP1 gene that vary considerably among the subtypes as well as other regions that remain relatively constant. One highly variable region (codons 130-171) encodes amino acids previously identified as being exposed on the virus surface and constituting an important immunogenic site of the virus. There potentially exist secondary structures within the viral RNA sequences that code for this immunogenic site that could decrease the fidelity of replication at this sequence. The rapid generation of FMDV variants encouraged by such structures in the RNA could work together with various selective pressures to explain the observed accumulation of immunologically distinct viruses of the FMDV A type.     

Sequence variability of the integrase protein from a diverse collection of HIV type 1 isolates representing several subtypes

HIV-1 recombinants between viruses from different subtypes appear to be surprisingly common in several regions of the world. To detect such intersubtype recombinants that contain mosaic genomes, we have analyzed sequences from the integrase (IN)-coding region of the polymerase (pol) gene from 23 viruses of known envelope (env) subtype from South America and Africa. As defined by env sequences, these viral genomes included nine subtype A, four subtype B, three subtype C, and four subtype D viruses from group M, and three viruses from group O HIV-1. Mosaic genomes were common, with 7 mosaic genomes among the 20 group M isolates analyzed. Two of these isolates had mosaic IN-coding regions that were distinct, but that had recombination breakpoints at the same location, in the highly conserved polypurine track. Mosaic genomes were particularly common in the viruses from Kenya (five of nine), consistent with our previous prediction that there was a high frequency of intersubtype recombinants circulating in this country. The IN amino acid sequence was highly conserved among the several represented subtypes, including group O. Group M IN sequences shared 94% or greater amino acid sequence identity within a subtype and 91% or greater identity between subtypes. The most divergent M and O variant amino acid sequences differed by only 19%, and the known functional domains were conserved among all of the isolates. The high degree of genetic homogeneity among the virus isolates representing several subtypes indicates that a single drug targeted against IN might be effective for all HIV-1 infections.     

Near full-length genomes of 15 HIV type 1 group O isolates

Human immunodeficiency virus type 1 (HIV-1) is classified into three distinct groups; M (major), N (non-M/non-O), and O (outlier). Group M strains are further subclassified into subtypes, subsubtypes, and circulating recombinant forms (CRF). While the level of genetic diversity within group O is similar to that between group M subtypes, group O has not been classified into subtypes. A previous study, based on the phylogenetic analyses of the gag p24, pol p32, and env gp160 sequences from 39 group O isolates, laid the foundation for the classification of group O subtypes. Five phylogenetic clusters, I-V, were identified that have characteristics analogous to group M subtypes. However, a complete phylogenetic analysis and classification of group O requires the availability of at least two full-length and one partial genomes for each group O phylogenetic cluster. In this study, 15 group O isolates were selected for full genome sequencing. Phylogenetic analysis of the 15 sequences with eight additional group O genomes supports the classification of three group O subtypes (I-III) and the potential existence of one CRF (IV) and at least one additional subtype (V). The group O subtypes are equidistant to each other and lack subsegments of other subtypes. The intra- and intersubtype genetic distances for group O are similar in magnitude to the corresponding distances for group M subtypes. Intersubtype recombination was identified in three of the 23 (13%) group O genomes. Formal classification of group O subtypes should be forthcoming pending the analysis of additional group O genomes and agreement of the HIV nomenclature committee.     

Evaluation of HIV type 1 group O isolates: identification of five phylogenetic clusters

HIV-1 group O strains have a level of genetic diversity similar to that of strains in group M; however, group O has not been readily classified into genetic subtypes. Phylogenetic classification of group O has been hindered by the limited sequence information available. To facilitate phylogenetic analysis, we sequenced the gag p24 (693 nt), pol p32 (864 nt), and env gp160 (approximately 2700 nt) genes from 39 group O-infected specimens. These specimens include 32 plasma samples collected in Cameroon between 1996 and 1999, 2 specimens collected in the United States, and 5 infections previously isolated in Equatorial Guinea. Phylogenetic analysis of HIV-1 group O sequences resulted in the identification of five clusters that are maintained across gag, pol, and env, generally supported by high bootstrap values, and approximately equidistant from each other. In addition to the group O clusters, several isolates branch independently and are equidistant from the other group O isolates. Cluster I comprises greater than 50% of the group O isolates and is a diverse set of isolates that is subdivided into subclusters. The average intra-, sub-, and intercluster distances for group O are similar to the corresponding distances for group M subtypes. The five group O clusters have characteristics similar to those of group M subtypes. Thus the data presented may form the basis for classification of group O into subtypes. However, full-length genomes representing each group O cluster will be required to formalize a group O subtype classification.     

Outbreaks of Foot-and-Mouth Disease in Burundi,East Africa,in 2016, Caused by Different Serotypes

Burundi is a small, densely populated country in the African Great Lakes region. In March 2016, several hundreds of cattle were reported with vesicular lesions, suggesting foot-and-mouth disease (FMD). Epithelial samples, saliva, and blood were collected in six of the affected provinces spread over the country. The overall seroprevalence of FMD virus (FMDV) in the affected herds, as determined by antibodies against FMDV non-structural proteins, was estimated at 87%. Antibodies against FMDV serotypes O (52%), A (44%), C (19%), SAT1 (36%), SAT2 (58%), and SAT3 (23%) were detected across the provinces. FMDV genome was detected in samples from five of the six provinces using rRT-PCR. FMDV was isolated from samples from three provinces: in Cibitoke province, serotypes A and SAT2 were isolated, while in Mwaro and Rutana provinces, only serotype SAT2 was isolated. In Bururi and Cankuzo provinces, the serological profile suggested a recent incursion with serotype SAT2, while in Bubanza province, the serological profile suggested past incursions with serotype O and possibly serotype SAT1. The phylogenetic assessments showed the presence of topotypes A/Africa/G-I and SAT2/IV, similarly to previously characterized virus strains from other countries in the region, suggesting a transboundary origin and necessitating a regional approach for vaccination and control of FMD.     

中国大陆地区柯萨奇病毒A组4型VP1区基因特征分析

目的 分析中国大陆地区柯萨奇病毒A组4型(Coxsackievirus A4,CV-A4)流行毒株基因特征和进化规律。方法 利用Mega6.0软件对GenBank核苷酸数据库收录的分离于中国大陆地区CV-A4毒株VP1区基因序列进行分析,构建系统进化树,并计算分离毒株核苷酸和氨基酸同源性。结果 纳入中国大陆地区CV-A4毒株合计116株,中国大陆地区16个省份CV-A4流行毒株潜在的优势基因型为F。与原型株High Point相比,中国大陆地区CV-A4毒株核苷酸和氨基酸同源性分别为79.9%~84.1%和92.5%~98.7%,收录中国大陆地区CV-A4毒株间核苷酸和氨基酸同源性分别为82.6%~100.0%和91.5%~100.0%。结论 针对中国大陆地区CV-A4毒株流行特征和进化规律,采取有效措施防控手足口病。     

一起儿童病毒性脑炎暴发肠道病毒ECHO6型分离株VP1基因特征分析

目的 对埃可病毒6型(ECHO6)福建龙岩分离株进行分子生物学分析,阐明ECHO6病毒福建龙岩分离株的分子生学特征及其与世界其它分离株的基因关系。方法 采集暴发病例脑脊液标本,用RD细胞进行分离病毒,阳性分离物采用中和试验及PCR序列分析确定其血清型别;RT-PCR扩增VP1基因全长,测定序列进行同源性及亲缘进化树分析。结果 13例细胞培养阳性分离物经中和试验及序列分析结果均为ECHO6。随机选取4株病毒株进行VP1全基因序列分析显示: 4个病毒株之间同源性＞99%;与山东病毒株E6/SD/sewage/081226/2-2同源性高于其他参考株。在遗传进化树上,4株分离病毒株同属一个分支, 为C2亚型。结论 ECHO6是引起本次福建省龙岩市长汀县儿童病毒性脑炎暴发的病原体,其基因型为C2亚型。     

Implications of a quasispecies genome structure: effect of frequent, naturally occurring amino acid substitutions on the antigenicity of foot-and-mouth disease virus.

We provide evidence that the quasispecies nature (extreme genetic heterogeneity) of foot-and-mouth disease virus is relevant to the virus evading an immune response. A monoclonal antibody neutralizing the viral infectivity (clone SD6) recognizes an epitope located around a highly conserved sequence (amino acid sequence Arg-Gly-Asp-Leu-Ala at positions 141-145) in the capsid protein VP1 of foot-and-mouth disease virus of serotype C1. The amino acid substitutions Ala-138----Thr and Leu-147----Ile (or ----Val) reduced 100-fold the binding titer of monoclonal antibody SD6 to virions or to VP1. The effect of those substitutions was quantitatively reproduced with synthetic peptides representing the relevant sequences. This provides evidence that the two chemically conservative amino acids replacements--and not other substitutions present in the virus quasispecies--are responsible for the modified interaction with neutralizing monoclonal antibody SD6. The three substitutions were fixed in the viral capsid during one occurrence of foot-and-mouth disease and, furthermore, they are of a type found frequently among independent foot-and-mouth disease virus isolates. The results implicate the extreme heterogeneity of foot-and-mouth disease virus as an important element of viral pathogenesis.     

Viral Population Diversity during Co-Infection of Foot-And-Mouth Disease Virus Serotypes SAT1 and SAT2 in African Buffalo in Kenya

African buffalo are the natural reservoirs of the SAT serotypes of foot-and-mouth disease virus (FMDV) in sub-Saharan Africa. Most buffalo are exposed to multiple FMDV serotypes early in life, and a proportion of them become persistently infected carriers. Understanding the genetic diversity and evolution of FMDV in carrier animals is critical to elucidate how FMDV persists in buffalo populations. In this study, we obtained oropharyngeal (OPF) fluid from naturally infected African buffalo, and characterized the genetic diversity of FMDV. Out of 54 FMDV-positive OPF, 5 were co-infected with SAT1 and SAT2 serotypes. From the five co-infected buffalo, we obtained eighty-nine plaque-purified isolates. Isolates obtained directly from OPF and plaque purification were sequenced using next-generation sequencing (NGS). Phylogenetic analyses of the sequences obtained from recombination-free protein-coding regions revealed a discrepancy in the topology of capsid proteins and non-structural proteins. Despite the high divergence in the capsid phylogeny between SAT1 and SAT2 serotypes, viruses from different serotypes that were collected from the same host had a high genetic similarity in non-structural protein-coding regions P2 and P3, suggesting interserotypic recombination. In two of the SAT1 and SAT2 co-infected buffalo identified at the first passage of viral isolation, the plaque-derived SAT2 genomes were distinctly grouped in two different genotypes. These genotypes were not initially detected with the NGS from the first passage (non-purified) virus isolation sample. In one animal with two SAT2 haplotypes, one plaque-derived chimeric sequence was found. These findings demonstrate within-host evolution through recombination and point mutation contributing to broad viral diversity in the wildlife reservoir. These mechanisms may be critical to FMDV persistence at the individual animal and population levels, and may contribute to the emergence of new viruses that have the ability to spill-over to livestock and other wildlife species.     

O型口蹄疫病毒蛋白酶编码序列及氨基酸序列变化的分析

目的与方法从GenBank中下载了19株O型口蹄疫病毒的Lab、3C、3D基因,结合本实验室收录的FMDVOA/58Lab、3C、3D的序列信息,分析比较了三种FMDV蛋白酶(Lpro,3Cpro,3Dpol)的编码序列和氨基酸序列。结果这FMDV具有的蛋白酶的编码序列在突变上没有显著差异(P>0.05),而在氨基酸序列的突变上存在着显著差异(P<0.01)。通过多重比较发现Lpro与3Cpro和3Dpol在氨基酸突变上存在差异(P<0.05),而3Cpro和3Dpol之间突变差异不显著。结论利用Swiss-pdbViewer蛋白质分析软件模拟三种蛋白酶中各个突变热点氨基酸残基的替换,发现突变热点不影响三种蛋白酶的空间结构。这表明病毒基因组在复制过程中的突变只是FMDV变异的原始动力,而FMDV变异的方向性进化动力是感染FMDV的宿主细胞对病毒蛋白酶功能造成的选择压力。     

Cross-Serotype Reactivity of ELISAs Used to Detect Antibodies to the Structural Proteins of Foot-and-Mouth Disease Virus

Antibodies to the foot-and-mouth disease virus (FMDV) capsid induced by infection or vaccination can provide serotype-specific protection and be measured using virus neutralization tests and viral structural-protein (SP-)ELISAs. Separate tests are needed for each serotype, but cross-serotype reactions complicate serotyping. In this study, inter-serotypic responses were quantified for five SP-ELISA formats by testing 294 monovalent mainly bovine sera collected following infection, vaccination, or vaccination and infection with one of five serotypes of FMDV. Over half of the samples, representing all three immunization categories, scored positive for at least one heterologous serotype and some scored positive for all serotypes tested. A comparative approach to identifying the strongest reaction amongst serotypes O, A and Asia 1 improved the accuracy of serotyping to 73–100% depending on the serotype and test system, but this method will be undermined where animals have been infected and/or vaccinated with multiple FMDV serotypes. Preliminary studies with stabilized recombinant capsid antigens of serotypes O and A that do not expose internal epitopes showed reduced cross-reactivity, supporting the hypothesis that capsid integrity can affect the serotype-specificity of the SP-ELISAs. The residual cross-reactivity associated with capsid surface epitopes was consistent with the evidence of cross-serotype virus neutralization.     

Phylogenetic subtypes of human T-lymphotropic virus type I and their relations to the anthropological background.

Isolates of human T-lymphotropic virus type I (HTLV-I) were phylogenetically analyzed from native inhabitants in India and South America (Colombia and Chile) and from Ainu (regarded as pure Japanese descendants from the preagricultural "Jomon" period). Their genomes were partially sequenced together with isolates from Gabon in central Africa and from Ghana in West Africa. The phylogenetic tree was constructed from the sequence data obtained and those of previously reported HTLV-I isolates and simian T-lymphotropic virus type I (STLV-I) isolates. The heterogeneity of HTLV-I was recently recognized, and one major type, generally called the "cosmopolitan" type, contained Japanese, Caribbean, and West African isolates. The phylogenetic tree constructed in the present study has shown that this cosmopolitan type can be further grouped into three lineages (subtypes A, B, and C). Subtype A consists of some Caribbean, two South American, and some Japanese isolates, including that from the Ainu, in addition to an Indian isolate, and subtype B consists of other Japanese isolates in addition to another Indian isolate, suggesting that there might be at least two ancestral lineages of the Japanese HTLV-I. Subtype A implies a close connection of the Caribbean and South American natives with the Japanese and thereby a possible migration of the lineage to the American continent via Beringia in the Paleolithic era. Subtype C consists of the West African and other Caribbean isolates, indicating that not all but part of the Caribbean strains directly originated from West Africa probably during the period of slave trade. The tree also has shown that the HTLV-I isolate from Gabon in central Africa forms a cluster with STLV-I from a chimpanzee, suggesting a possible interspecies transmission between man and the chimpanzee in the past. No specific clustering was observed in the tree in relation to manifestations of the disease such as adult T-cell leukemia and HTLV-I-related neurological disorders. Thus, the topology of the phylogenetic tree reflects the movement of people carrying the virus in the past.     

HIV type 1 subtype A epidemic in injecting drug user (IDU) communities in Iran

HIV-1 C2-V3 subgenomic regions of the env gene from Iranian seropositive injecting drug users (IDUs) living in Mashhad have been analyzed to evaluate molecular and phylogenetic relationships with IDUs living in Tehran and identify possible common founder virus isolates. The results show that the viral sequences of the Iranian IDUs are strongly related and form a single cluster within the A subtype related to African Ugandan/Kenyan sub-Saharan isolates. Pairwise nucleotide alignment shows higher average divergence values within the Mashhad group than the Tehran group. Furthermore, the Mashhad sequences show much less conserved amino acid residues in the V3 loop than the Tehran sequences. These data represent the first macro-analysis of the HIV-1 molecular evolution in the Iran and Middle East epidemics and may be extremely relevant to guide the development and implementation of diagnostic as well as preventive/therapeutic approaches in this region.     

Trans-Encapsidation of Foot-and-Mouth Disease Virus Genomes Facilitates Escape from Neutralizing Antibodies

Foot-and-mouth disease is an economically devastating disease of livestock caused by foot-and-mouth disease virus (FMDV). Vaccination is the most effective control measure in place to limit the spread of the disease; however, the success of vaccination campaigns is hampered by the antigenic diversity of FMDV and the rapid rate at which new strains emerge that escape pre-existing immunity. FMDV has seven distinct serotypes, and within each serotype are multiple strains that often induce little cross-protective immunity. The diversity of FMDV is a consequence of the high error rate of the RNA-dependent RNA polymerase, accompanied by extensive recombination between genomes during co-infection. Since multiple serotypes and strains co-circulate in regions where FMDV is endemic, co-infection is common, providing the conditions for recombination, and also for other events such as trans-encapsidation in which the genome of one virus is packaged into the capsid of the co-infecting virus. Here, we demonstrate that the co-infection of cells with two FMDVs of different serotypes results in trans-encapsidation of both viral genomes. Crucially, this facilitates the infection of new cells in the presence of neutralizing antibodies that recognize the capsid that is encoded by the packaged genome.     

Molecular characterization of 39 HIV isolates representing group M (subtypes A-G) and group O: sequence analysis of gag p24, pol integrase,and env gp41

A panel of genetically diverse, well-characterized human immunodeficiency type 1 (HIV-1) virus isolates is a valuable tool for standardizing nucleic acid-based tests. Since HIV quantitative assays target different regions of the genome, thorough molecular characterization is useful for evaluating the effect of genetic variation on viral load assay performance. In this study, 39 virus isolates were examined. The representative HIV-1 group M subtypes A-G and group O strains included 30 members of the Walter Reed Army Institute of Research (WRAIR) clade panel. Though this panel is currently being used in laboratories around the world, only limited sequence information is available on most of these isolates. In this communication, gag p24 (gag), pol integrase (pol IN), and env gp41 immunodominant (IDR) region sequences were characterized to assess the level of genetic diversity and to verify group/subtype. The panel was composed of 37 group M and two group O strains.     

2011-2014年福建省手足口病相关病原柯萨奇病毒A组4型的分子流行病学分析

目的 分析福建省手足口病患者中柯萨奇病毒A组4型(Coxsackievirus A4,CVA4)的流行病学、基因变异及遗传进化等特征.方法 应用细胞培养方法获得病毒分离株,RT-PCR方法进行分子分型及扩增CVA4血清型完整VP1区全长序列,并进行序列变异及遗传进化分析.结果 2011-2014年,从福建省手足口病其它肠道病毒病例标本中分子分型33例CVA4病例,在407例的其它肠道病毒病例中的构成比为8.1％,其中2012年31例,2014年2例.CVA4病例在性别、年龄组分布特征,与一般手足口病流行特征相比,没有特异性.VP1区序列差异比较表明,2011-2014年福建省CVA4分离株之间VP1核苷酸及氨基酸序列相似性分别在92.6％～100％、95.7％～100％;与原型株及国外地方株的核苷酸序列相似性较低,分别为83.7％～85.4％、82.1％～89.1％,氨基酸序列相似性分别为96.1％～99.0％、90.4％～99.6％;与国内地方株核苷酸及氨基酸序列相似性较高,分别为87.9％～99.2％、96.1％～100％.进化树结果表明福建CVA4分离株与原型株及国外代表株的遗传距离远,与国内代表株的遗传距离近.福建CVA4分离株在进化树的分布呈多个分支.结论 CVA4已成为近年来福建省除EV71、CVA16、CVA6、CVA10外导致手足口病的又一重要病原体.福建CVA4分离株与国内地方株同进化共循环,在福建省各个地区同时流行多条亲缘关系接近的传播链.     

Phylogenetic analysis of Japanese encephalitis virus: envelope gene based analysis reveals a fifth genotype, geographic clustering, and multiple introductions of the virus into the Indian subcontinent

We report the analysis of the complete nucleotide sequence for the Indian isolate (P20778; Genbank Accession number AF080251) of Japanese encephalitis virus (JEV). The phylogenetic tree topology obtained using thirteen complete genome sequences of JEV was reproduced with the envelope, NS1, NS3, and NS5 genes and revealed extensive divergence between the two Indian strains included. A more exhaustive analysis of JEV evolution using 107 envelope sequences available for isolates from different geographic locations worldwide revealed five distinct genotypes of JEV, displaying a minimum nucleotide divergence of 7% with high bootstrap support values. The tree also revealed overall clustering of strains based on geographic location, as well as multiple introductions of JEV into the Indian subcontinent. Nonsynonymous nucleotide divergence rates of the envelope gene estimated that the ancestor common to all JEV genotypes arose within the last three hundred years.