Circulation of endemic type 2 vaccine-derived poliovirus in Egypt from 1983 to 1993

From 1988 to 1993, 30 cases of poliomyelitis associated with poliovirus type 2 were found in seven governorates of Egypt. Because many of the cases were geographically and temporally clustered and because the case isolates differed antigenically from the vaccine strain, it was initially assumed that the cases signaled the continued circulation of wild type 2 poliovirus. However, comparison of sequences encoding the major capsid protein, VP1 (903 nucleotides), revealed that the isolates were related (93 to 97% nucleotide sequence identity) to the Sabin type 2 oral poliovirus vaccine (OPV) strain and unrelated (<82% nucleotide sequence identity) to the wild type 2 polioviruses previously indigenous to Egypt (last known isolate: 1979) or to any contemporary wild type 2 polioviruses found elsewhere. The rate and pattern of VP1 divergence among the circulating vaccine-derived poliovirus (cVDPV) isolates suggested that all lineages were derived from a single OPV infection that occurred around 1983 and that progeny from the initiating infection circulated for approximately a decade within Egypt along several independent chains of transmission. Complete genomic sequences of an early (1988) and a late (1993) cVDPV isolate revealed that their 5' untranslated region (5' UTR) and noncapsid- 3' UTR sequences were derived from other species C enteroviruses. Circulation of type 2 cVDPVs occurred at a time of low OPV coverage in the affected communities and ceased when OPV coverage rates increased. The potential for cVDPVs to circulate in populations with low immunity to poliovirus has important implications for current and future strategies to eradicate polio worldwide.     

The Jezierski papers: live polio vaccine development in colobus monkey cells but not chimpanzee cells in the Belgian Congo, 1952-1958

A reading of ten relevant papers by Alexandre Jezierski provides evidence for the only attempt in Central Africa to develop a live oral polio vaccine (OPV) from growing reference wild polio strains to 210 passages in colobus monkey tissue culture, and experimental administration to about 25 humans. Chimpanzees were used as a human model, but their tissues or kidneys were absent from the passage and production line of the proposed vaccine. Thus, the implication published by Hooper that Jezierski had produced a candidate OPV that might have contained chimpanzee viruses, possibly simian immunodeficiency virus cpz or the precursor of human immunodeficiency virus-1 group M, is incorrect.     

Comparative biochemical studies of type 3 poliovirus

A study of the biochemistry of type 3 poliovirus strains which involves the examination of the virus-coded polypeptides in infected cells and the preparation of oligonucleotide maps is reported. The polypeptide patterns were shown to be a relatively stable property of virus strains and distinguished Sabin vaccine strains from wild strains of poliovirus type 3. This approach may be of value in deciding the origin (vaccine or nonvaccine) of field isolates of poliovirus. Oligonucleotide maps were found to be sensitive indicators of differences among strains and appear to form a basis for determining genetic relationships among strains. The nucleotide maps of two viruses isolated from human cases of paralytic poliomyelitis temporally associated with the administration of attenuated vaccine suggested a vaccine origin for the strain. In one case the nucleotide map was indistinguishable from that of the vaccine strain.     

Environmental Surveillance of Poliovirus in Sewage Water around the Introduction Period for Inactivated Polio Vaccine in Japan

Environmental virus surveillance was conducted at two independent sewage plants from urban and rural areas in the northern prefecture of the Kyushu district, Japan, to trace polioviruses (PVs) within communities. Consequently, 83 PVs were isolated over a 34-month period from April 2010 to January 2013. The frequency of PV isolation at the urban plant was 1.5 times higher than that at the rural plant. Molecular sequence analysis of the viral VP1 gene identified all three serotypes among the PV isolates, with the most prevalent serotype being type 2 (46%). Nearly all poliovirus isolates exhibited more than one nucleotide mutation from the Sabin vaccine strains. During this study, inactivated poliovirus vaccine (IPV) was introduced for routine immunization on 1 September 2012, replacing the live oral poliovirus vaccine (OPV). Interestingly, the frequency of PV isolation from sewage waters declined before OPV cessation at both sites. Our study highlights the importance of environmental surveillance for the detection of the excretion of PVs from an OPV-immunized population in a highly sensitive manner, during the OPV-to-IPV transition period.     

2010年山东省环境污水中脊髓灰质炎病毒的监测及其基因特征分析

本研究在山东省开展了脊髓灰质炎病毒(Poliovirus,PV)的外环境监测,从济南、临沂两地采集污水标本,浓缩处理后进行病毒分离,对分离到的PV采用中和试验进行血清定型,并对其VP1及3D区进行序列测定,分析其基因突变和重组情况。2010年,共采集污水标本32份,PV阳性10份,阳性率31.3%;分离到18株PV(PV1型3株,PV2型9株,PV3型6株),均为疫苗相关株,VP1完整编码区核苷酸变异数在0~4个之间,在3株PV2型病毒和4株PV3型病毒的基因组中发现重组;对VP1区影响神经毒力的减毒位点分析发现,PV1型病毒中有1株在nt 2 749发生突变(A→G),PV2型病毒中有1株在nt2 908发生A→G突变,3株在nt2 909发生U→C突变,6株PV3型病毒全部在nt2 493发生C→U突变。环境污水中可以分离到PV,其基因重组率和主要减毒位点的回复突变率较高,未发现脊灰野毒株和疫苗衍生株脊灰病毒(Vaccine-derived poliovirus,VDPV)。     

Biological Characteristics of Cloned Populations of Herpes Simplex Virus Types 1 and 2

By using cloned types 1 and 2 herpes simplex virus, obtained by selecting large and small plaques produced by material from human lesions, studies were performed to compare properties between preparations of each type. Regarding the rate of inactivation by ultraviolet light, no differences were found between the two antigenic types and none between the preparations obtained from either type. In contrast, type 1 preparations were found to be more readily inactivated at 45 C than type 2. Plaque size of cloned preparations changed by passage in cell culture. A broader range of plaque sizes was obtained, and average plaque size was larger. After 20 passages, preparations obtained from different types gave rise to one of three kinds of cytopathic effect. The cytopathic effect produced by type 1 preparations remained as before 20 passages and consisted of round cells in a compact central mass. For type 2, two kinds of cytopathic effect were seen in cloned preparations. This consisted of aggregates of round cells (seen in preparations before 20 passages) or of large, loose aggregates of round cells of various sizes. Results from neutralization studies using virus before and after 20 passages in cell culture versus antisera prepared against live or ultraviolet-inactivated virus showed no differences between cloned preparations obtained from a given type.     

Two amino acid mutations in the capsid protein of type 2 porcine circovirus (PCV2) enhanced PCV2 replication in vitro and attenuated the virus in vivo

Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS) in pigs. To identify potential genetic determinants for virulence and replication, we serially passaged a PCV2 isolate 120 times in PK-15 cells. The viruses harvested at virus passages 1 (VP1) and 120 (VP120) were biologically, genetically, and experimentally characterized. The PCV2 VP120 virus replicated in PK-15 cells to a titer similar to that of the PK-15 cell line-derived nonpathogenic PCV1 but replicated more efficiently than PCV2 VP1 with a difference of about 1 log unit in the titers. The complete genomic sequences of viruses at passages 0, 30, 60, 90, and 120 were determined. After 120 passages, only two nucleotide mutations were identified in the entire genome, and both were located in the capsid gene: the mutations were located at nucleotide positions 328 (C328G) and 573 (A573C). The C328G mutation, in which a proline at position 110 of the capsid protein changed to an alanine (P110A), occurred at passage 30 and remained in the subsequent passages. The second mutation, A573C, resulting in a change from an arginine to a serine at position 191 (R191S), appeared at passage 120. To experimentally characterize the VP120 virus, 31 specific-pathogen-free pigs were randomly divided into three groups. Ten pigs in group 1 received phosphate-buffered saline as negative controls. Each pig in group 2 (11 pigs) was inoculated intramuscularly and intranasally with 10(4.9) 50% tissue culture infective doses (TCID(50)) of PCV2 VP120. Each pig in group 3 (10 pigs) was similarly inoculated with 10(4.9) TCID(50) of PCV2 VP1. Viremia was detected in 9 of 10 pigs in the PCV2 VP1 group with a mean duration of 3 weeks, but in only 4 of 11 pigs in the PCV2 VP120 group with a mean duration of 1.6 weeks. The PCV2 genomic copy numbers in serum in the PCV2 VP1 group were significantly higher than those in the PCV2 VP120 group (P < 0.0001). Gross and histopathologic lesions in pigs inoculated with PCV2 VP1 were more severe than those inoculated with PCV2 VP120 at both day 21 and 42 necropsies (P = 0.0032 and P = 0.0274, respectively). Taken together, the results from this study indicated that the P110A and R191S mutations in the capsid of PCV2 enhanced the growth ability of PCV2 in vitro and attenuated the virus in vivo. This finding has important implications for PCV2 vaccine development.     

Effect of passage in culture on a clone of BALB/c 3T3 cells transformed by simian virus 40.

Most simian virus 40 (SV40)-transformed BALB/c 3T3 clones employed for biochemical studies have been used without regard to passage level. To determine whether virus-induced properties are stable as a function of passage, we have extensively characterized one transformed clone, FNE, which was isolated after SV40 infection BALB/c 3T3 cells in factor-free medium. From the initial testing at passage 5 and for at least 50 subsequent subcultures, the cells stably maintained many transformed growth properties, including high saturation density, morphology, colony formation on contact-inhibited monolayers, tumorigenicity, and synthesis of viral-specific RNA. However, other properties varied as a function of passage. There was a slight decrease in viral genome equivalents per cell from 1.1 copy/cell at passage 5 to 0.7 copies at passage 40. Initially, the cells were negative for all type C virus; however, cells carried at low density for 13 to 20 passages (65 to 100 generations) began to release an endogenous type C virus that then persisted in the culture. Spontaneous release of type C virus did not occur in control BALB/c 3T3 cells carried under identical culture conditions for 90 passages. When the cultures were releasing type C viruses they stained uniformly and brightly positive for SV40 tumor (T) antigen by immunofluorescence, whereas T antigen staining was variable at early passage. These experiments suggest that subtle but perhaps important differences in viral gene expression can occur as a function of passage; they also demonstrate the importance of evaluating the interactions between SV40 and endogenous type C viruses.     

Development and characterization of a live varicella vaccine (Oka strain)

A live varicella vaccine (Oka strain) was developed by serial passage of the Oka strain isolated in our laboratory, in human embryonic lung cells (11 times at 34 C) and guinea pig embryo cells (12 times at 37 C). It is slightly temperature sensitive at 39 C and shows a higher ratio of infectivity in guinea pig embryo cells to infectivity in human embryo cells than wild-type strains. The DNA digest with Hpa I enzyme of the Oka strain contained one unique fragment (K), although its mobility differed only slightly from that of the corresponding fragment of wild-type strains. Studies with clinical varicella zoster virus (VZV) isolates from vaccinees indicated that tests on the ratio of infectivity in guinea pig embryo fibroblasts (GPEF) to that in human embryo fibroblasts (HuEF) and the profile of the DNA digest with Hpa I are useful for differentiation of the vaccine strain from wild-type strains. The vaccine virus showed stable immunogenicity during at least 15 further repeated passages in human diploid cells, a character which seems helpful for production of a large quantity of vaccine virus for practical use.     

Reversion to neurovirulence of the live-attenuated Sabin type 3 oral poliovirus vaccine

The complete nucleotide sequence has been determined of a strain of poliovirus type 3, P3/119, isolated from the central nervous system of a victim of fatal vaccine-associated poliomyelitis. Comparison of this sequence with those obtained previously for the Sabin type 3 vaccine, P3/Leon 12a1b and its neurovirulent progenitor, P3/Leon/37, reveals that these three strains are on a direct geneaological lineage and therefore that P3/119 is a bona fide revertant of the vaccine. P3/119 differs in sequence from its attenuated vaccine parent at just seven positions. Only one of these differences, a mutation from U to C at position 472 in the presumed noncoding region of the genome, is a back mutation to the wild type sequence. Of the six other differences, three give rise to coding changes in virus structural proteins, two are silent changes in the major open reading frame of the genome and one affects the 3'-terminus just prior to the poly A tract. These differences indicate that there are three possible types of molecular change which could, singly or collectively, result in attenuation and reversion to neurovirulence of the Sabin type 3 vaccine.     

甲型肝炎减毒活疫苗(H2株)快速繁殖株不同代次全基因序列比较

为探索甲型肝炎减毒活疫苗 (H2株 )细胞适应的分子机制 ,将甲型肝炎减毒活疫苗毒株(HAVH2K7)在人胚肺二倍体细胞KMB17上快速连续系列传代增强适应 ,繁殖周期由原来的 2 8d缩短为 14d ,连续传代后抗原滴度和感染性滴度不断增加 ,传至 2 2代抗原滴度达 1∶10 2 4 ,感染性滴度lgCCID50 (每ml)为 7 83 .分别将第 6代和第 2 2代病毒用AC PCR法和PCR法扩增 .扩增片段分别与pGEM T载体连接得到重组质粒 ,测定cDNA插入片段的序列 .对 2个不同代次全基因序列及氨基酸序列比较分析表明 ,HAVH2K7适应至第 6代时 ,整个基因组有 6个核苷酸变异 ,全部位于编码区内 ,变异率为 0 0 7% ,导致 3个氨基酸变化 ,分别位于VP2 (A S) ;2C(N H) ;3A(R C) .适应至第 2 2代时 ,整个基因组出现 18个核苷酸突变 ,变异率为 0 2 4 % ,13个是该代次产生的 ,变异最大区域 5′端非编码区 (5′UTR)有 5个核苷酸变异 .编码区突变导致 7个氨基酸变化 ,其中 4个氨基酸变化是该代次在 6代基础上特有的变异 (2C ,Q P ;3A ,A S ;3C ,T A ;3D ,V G) .2C区是编码区变异最多区域 ,共有 4个核苷酸突变 ,在 6代变异基础上出现 3个新突变 ,导致 1个氨基酸变异 .说明 5′UTR的2C区变异对病毒的翻译效率、感染性滴度提高具有重要作用 .     

Extreme heterogeneity in populations of vesicular stomatitis virus.

Vesicular stomatitis virus (VSV) sequence evolution and population heterogeneity were examined by T1 oligonucleotide mapping. Individual clones isolated from clonal pools of wild-type Indiana serotype VSV displayed identical T1 maps. This was observed even after one passage at high concentrations of the potent viral mutagen 5-fluorouracil. Under low-multiplicity passage conditions, the consensus T1 fingerprint of this virus remained unchanged after 523 passages. Interestingly, however, individual clones from this population (passage 523) differed significantly from each other and from consensus sequence. When virus population equilibria were disrupted by high-multiplicity passage (in which defective interfering particle interference is maximized) or passage in the presence of mutagenic levels of 5-fluorouracil, rapid consensus sequence evolution occurred and extreme population heterogeneity was observed (with some members of these population differing from others at hundreds of genome positions). A limited sampling of clones at one stage during high-multiplicity passages suggested the presence of at least several distinct master sequences, the related subpopulations of which exhibit at least transient competitive fitness within the total virus population (M. Eigen and C.K. Biebricher, p. 211-245, in E. Domingo, J.J. Holland, P. Ahlquist, ed., RNA Genetics, vol. 3, 1988). These studies further demonstrate the important role of selective pressure in determining the genetic composition of RNA virus populations. This is true under equilibrium conditions in which little consensus sequence evolution is observed owing to stabilizing selection as well as under conditions in which selective pressure is driving rapid RNA virus genome evolution.     

Properties of Venezuelan Equine Encephalomyelitis Virus Accompanying Attenuation In Vitro

Virus obtained during serial plaque passage of the virulent parent egg seed (PES) of the Trinidad strain of Venezuelan equine encephalomyelitis (VEE) virus produced only large plaques during either 3 serial plaque passages in chick fibroblasts or 10 plaque passages in L cells, and was lethal for mice by the intraperitoneal route. Virus showing these characteristics was designated the stable large-plaque (Ls) type. In contrast, virus obtained during serial plaque passage of the attenuated 9t strain in chick fibroblasts formed only very small plaques and was not lethal for mice by the intraperitoneal route. Virus showing these properties was designated the stable small-plaque (Ss) type. Under other passage conditions, however, large-plaque virus that yielded about 90% large and 10% small plaques was obtained; this virus was designated the unstable large or Lu type because it differed from the Ls type, which yielded only large plaques. The Lu type continued to yield the same ratio of large to small plaques for several plaque-to-plaque passages. In addition, small-plaque virus that yielded both large and small plaques and that showed a reduced capability to infect mice was also recovered. This virus was designated the unstable small or Su type because it differed from the Ss type in its higher level of virulence and in its plaque-forming properties. Thus, based upon the properties of virulence for mice and plaque size, four viral types could be discerned. The evidence suggests that serial passage in cell culture imposed environmental pressures that sequentially selected the following viral types: Ls, Lu, Su, and Ss.     

Estimation of the neurovirulence of poliovirus by non-radioisotope molecular analysis to quantify genomic changes

Mutant analysis by polymerase chain reaction and restriction enzyme cleavage (MAPREC) has been developed for poliovirus to determine quantitatively for the presence of genomic changes in particular nucleotide sequences correlate with the characteristic of neurovirulence for monkeys. Currently the MAPREC is scheduled to be used as a routine safety test for oral poliomyelitis vaccine (OPV). Radioisotopes (RI) are used in MAPREC for quantitative determinations, a circumstance likely to limit its use. We investigated the possibility of developing a modified MAPREC, which did not require the use of radioisotopes, and developed a procedure designated NON-RI MAPREC. Conventional MAPREC and NON-RI MAPREC were then used in a series of studies in which analyses were performed on Sabin type 1 and Sabin type 3 attenuated vaccine polioviruses prepared under various conditions. Under the experimental conditions used, the stability of the genome of type 1 virus was shown to be markedly greater than that of the type 3 virus, and the frequency of mutants was observed to vary in relation to both the virus strain and the virus inoculum used. The results of the studies relating to the two analytical procedures used indicated that the reproducibility of both methods was of a similarly high order, but that MAPREC had a somewhat broader range of sensitivity than NON-RI MAPREC. As the quantity of genomic changes in OPV relating to neurovirulent properties are within the range of detection by NON-RI MAPREC, this procedure can be used as a quality control test for OPV.     

火鸡疱疹病毒在鸡胚成纤维细胞上有效代次的测定及其免疫原性恢复的研究

本试验证明,火鸡疱疹病毒(HVT)FC126株在鸡胚成纤维细胞(CEF)传55代的病毒,仍有预防马立克氏病(MD)的效力,至第70代效力明显下降。若将HVT CEF第35代病毒复归火鸡连续传4代,能明显恢复其免疫原性,用C细胞传代则有效代次可达到75代。因而,此方法能有效地解决免疫原性下降的种毒的更新问题。本试验还为该种毒和疫苗的有效代次的使用范围提供了可靠依据。     

Spread of vaccine-derived poliovirus from a paralytic case in an immunodeficient child: an insight into the natural evolution of oral polio vaccine

Sabin strains used in the manufacture of oral polio vaccine (OPV) replicate in the human organism and can give rise to vaccine-derived polioviruses. The increased neurovirulence of vaccine derivatives has been known since the beginning of OPV use, but their ability to establish circulation in communities has been recognized only recently during the latest stages of the polio eradication campaign. This important observation called for studies of their emergence and evolution as well as extensive surveillance to determine the scope of this phenomenon. Here, we present the results of a study of vaccine-derived isolates from an immunocompromised poliomyelitis patient, the contacts, and the local sewage. All isolates were identified as closely related and slightly evolved vaccine derivatives with a recombinant type 2/type 1 genome. The strains also shared several amino acid substitutions including a mutation in the VP1 protein that was previously shown to be associated with the loss of attenuation. Another mutation in the VP3 protein resulted in altered immunological properties of the isolates, possibly facilitating virus spread in immunized populations. The patterns and rates of the accumulation of synonymous mutations in isolates collected from the patient over the extended period of excretion suggest either a substantially nonuniform rate of mutagenesis throughout the genome, or, more likely, the strains may have been intratypic recombinants between coevolving derivatives with different degrees of divergence from the vaccine parent. This study provides insight into the early stages of the establishment of circulation by runaway vaccine strains.     

Mutations in the 5’ NTR and the Non-Structural Protein 3A of the Coxsackievirus B3 Selectively Attenuate Myocarditogenicity

The 5’ non-translated region (NTR) is an important molecular determinant that controls replication and virulence of coxsackievirus B (CVB)3. Previous studies have reported many nucleotide (nt) sequence differences in the Nancy strain of the virus, including changes in the 5’ NTR with varying degrees of disease severity. In our studies of CVB3-induced myocarditis, we sought to generate an infectious clone of the virus for routine in vivo experimentation. By determining the viral nt sequence, we identified three new nt substitutions in the clone that differed from the parental virus strain: C97U in the 5’ NTR; a silent mutation, A4327G, in non-structural protein 2C; and C5088U (resulting in P1449L amino acid change) in non-structural protein 3A of the virus leading us to evaluate the role of these changes in the virulence properties of the virus. We noted that the disease-inducing ability of the infectious clone-derived virus in three mouse strains was restricted to pancreatitis alone, and the incidence and severity of myocarditis were significantly reduced. We then reversed the mutations by creating three new clones, representing 1) U97C; 2) G4327A and U5088C; and 3) their combination together in the third clone. The viral titers obtained from all the clones were comparable, but the virions derived from the third clone induced myocarditis comparable to that induced by wild type virus; however, the pancreatitis-inducing ability remained unaltered, suggesting that the mutations described above selectively influence myocarditogenicity. Because the accumulation of mutations during passages is a continuous process in RNA viruses, it is possible that CVB3 viruses containing such altered nts may evolve naturally, thus favoring their survival in the environment.     

Evolution of the Sabin strain of type 3 poliovirus in an immunodeficient patient during the entire 637-day period of virus excretion

A 20-year-old female hypogammaglobulinemic patient received monotypic Sabin 3 vaccine in 1962. The patient excreted type 3 poliovirus for a period of 637 days without developing any symptoms of poliomyelitis, after which excretion appeared to have ceased spontaneously. The evolution of Sabin 3 throughout the entire period of virus excretion was studied by characterization of seven sequential isolates from the patient. The isolates were analyzed in terms of their antigenic properties, virulence, sensitivity for growth at high temperatures, and differences in nucleotide sequence from the Sabin type 3 vaccine. The isolates followed a main lineage of evolution with a rate of nucleotide substitution that was very similar to that estimated for wild-type poliovirus during person-to-person transmission. There was a delay in the appearance of antigenic variants compared to sequential type 3 isolates from healthy vaccines, which could be one of the possible explanations for the long-term excretion of virus from the patient. The distribution of mutations in the isolates identified regions of the virus possibly involved in adaptation for growth in the human gut and virus persistence. None of the isolates showed a full reversion of the attenuated and temperature-sensitive phenotypes of Sabin 3. Information of this sort will help in the assessment of the risk of spread of virulent polioviruses from long-term excretors and in the design of therapies to stop long-term excretion. This will make an important contribution to the decision-making process on when to stop vaccination once wild poliovirus has been eradicated.     

Genetic and fitness changes accompanying adaptation of an arbovirus to vertebrate and invertebrate cells

The alternating host cycle and persistent vector infection may constrain the evolution of arboviruses. To test this hypothesis, eastern equine encephalitis virus was passaged in BHK or mosquito cells, as well as in alternating (both) host cell passages. High and low multiplicities were used to examine the effect of defective interfering particles. Clonal BHK and persistent mosquito cell infections were also evaluated. Fitness was measured with one-step growth curves and competition assays, and mutations were evaluated by nucleotide sequencing and RNA fingerprinting. All passages and assays were done at 32 degrees C to eliminate temperature as a selection factor. Viruses passaged in either cell type alone exhibited fitness declines in the bypassed cells, while high-multiplicity and clonal passages caused fitness declines in both types of cells. Bypassed cell fitness losses were mosquito and vertebrate specific and were not restricted to individual cell lines. Fitness increases occurred in the cell line used for single-host-adaptation passages and in both cells for alternately passaged viruses. Surprisingly, single-host-cell passage increased fitness in that cell type no more than alternating passages. However, single-host-cell adaptation resulted in more mutations than alternating cell passages. Mosquito cell adaptation invariably resulted in replacement of the stop codon in nsP3 with arginine or cysteine. In one case, BHK cell adaptation resulted in a 238-nucleotide deletion in the 3' untranslated region. Many nonsynonymous substitutions were shared among more than one BHK or mosquito cell passage series, suggesting positive Darwinian selection. Our results suggest that alternating host transmission cycles constrain the evolutionary rates of arboviruses but not their fitness for either host alone.