Mapping of attenuating sequences of an avirulent poliovirus type 2 strain.

A mouse model for poliomyelitis was used to identify genomic sequences that attenuate neurovirulence of poliovirus strain P2/P712. This type 2 strain is avirulent in primates and mice yet grows as well as virulent strains in cell culture. The approach used was to exchange portions of the genome of the mouse-virulent P2/Lansing strain with the corresponding region from P2/P712 to identify sequences that could attenuate Lansing neurovirulence in mice. A full-length infectious cDNA of P2/P712 was assembled and used to construct recombinants between P2/P712 and P2/Lansing. The results of neurovirulence testing of 11 recombinants indicated that strong attenuating determinants are located in the 5' noncoding region of P2/P712 and a region encoding capsid protein VP1 and 2Apro, 2B, and part of 2C. An attenuating determinant was further localized to between nucleotides 456 and 628 of P2/P712. A third sequence from P2/P712, nucleotides 752 to 2268, encoding VP4, VP2, and part of VP3, was weakly attenuating. The sequence from nucleotide 4454, approximately halfway through the 2C-coding region, to the end of the P2/P712 genome did not contain attenuating determinants. Nucleotide sequence analysis revealed that P2/P712 differs from the type 2 Sabin vaccine strain by only 22 nucleotides. Six differences lead to amino acid changes in the coding region, and four differences are in the 5' noncoding region. These studies show that, like the type 1 and type 3 Sabin vaccine strains, the attenuated type 2 strain P712 contains multiple attenuating sequences, including strongly attenuating sequences in the 5' noncoding region of the genome.     

Identification of a consistent pattern of mutations in neurovirulent variants derived from the sabin vaccine strain of poliovirus type 2.

Complete nucleotide sequencing of the RNAs of two unrelated neurovirulent isolates of Sabin-related poliovirus type 2 revealed that two nucleotides and one amino acid (amino acid 143 in the major capsid protein VP1) consistently departed from the sequences of the nonneurovirulent poliovirus type 2 712 and Sabin vaccine strains. This pattern of mutation appeared to be a feature common to all neurovirulent variants of poliovirus type 2.     

Construction of viable deletion and insertion mutants of the Sabin strain of type 1 poliovirus: function of the 5'' noncoding sequence in viral replication.

A number of deletion and insertion sequences were introduced into the 5' noncoding sequence (742 nucleotides long) of the genome of the Sabin strain of type 1 poliovirus by using an infectious cDNA clone of the virus strain. The genomes of all three poliovirus serotypes contained highly homologous sequences (nucleotide positions 509 to 639) as well as highly variable sequences (positions 640 to 742) in the 5' noncoding region. The viability of mutant viruses was tested by transfecting mutant cDNA clones into African green monkey kidney cells and then estimating the plaque sizes displayed on the cells. The results suggested that the highly variable sequence next to the VP4 coding region did not play an important role, at least in the in vitro culture system used, that the loci of highly conserved nucleotide sequences were not always expected to be the genome regions essential for viral replication, that the sequence between positions 564 and 599 carried genetic information to maintain the efficiency of certain steps in viral replication, and that the sequence between positions 551 to 563 might play an essential role in viral replication. Four-base deletion or insertion mutations were introduced into relatively variable sequences in the genome region upstream of position 509. The results suggest that variable sequences do not always indicate that the corresponding genome regions are less important. Apparent revertants (large-plaque variants) were easily generated from one of the viable mutants with the small-plaque phenotype. The determination of nucleotide sequences of the revertant genomes revealed the second mutation site. The results suggested that the different loci at around positions 200 and 500 might specifically interact with each other. This interaction may result in the formation of a functional structure that influences the efficiency of certain steps in the viral replication.     

Complete nucleotide sequence of wild-type hepatitis A virus: comparison with different strains of hepatitis A virus and other picornaviruses.

The complete nucleotide sequence of wild-type hepatitis A virus (HAV) HM-175 was determined. The sequence was compared with that of a cell culture-adapted HAV strain (R. Najarian, D. Caput, W. Gee, S.J. Potter, A. Renard, J. Merryweather, G.V. Nest, and D. Dina, Proc. Natl. Acad. Sci. USA 82:2627-2631, 1985). Both strains have a genome length of 7,478 nucleotides followed by a poly(A) tail, and both encode a polyprotein of 2,227 amino acids. Sequence comparison showed 624 nucleotide differences (91.7% identity) but only 34 amino acid differences (98.5% identity). All of the dipeptide cleavage sites mapped in this study were conserved between the two strains. The sequences of these two HAV strains were compared with the partial sequences of three other HAV strains. Most amino acid differences were located in the capsid region, especially in VP1. Whereas changes in amino acids were localized to certain portions of the genome, nucleotide differences occurred randomly throughout the genome. The most extensive nucleotide homology between the strains was in the 5' noncoding region (96% identity for cell culture-adapted strains versus wild type; greater than 99% identity among cell culture-adapted strains). HAV proteins are less homologous with those of any other picornavirus than the latter proteins are when compared with each other. When the sequences of wild-type and cell culture-adapted HAV strains are compared, the nucleotide differences in the 5' noncoding region and the amino acid differences in the capsid region suggest areas that may contain markers for cell culture adaptation and for attenuation.     

Mapping of sequences required for mouse neurovirulence of poliovirus type 2 Lansing.

Intracerebral inoculation of mice with poliovirus type 2 Lansing induces a fatal paralysis, while most other poliovirus strains are unable to cause disease in the mouse. To determine the molecular basis for Lansing virus neurovirulence, we determined the complete nucleotide sequence of the Lansing viral genome from cloned cDNA. The deduced amino acid sequence was compared with that of two mouse-avirulent strains. There are 83 amino acid differences between the Lansing and Sabin type 2 strain and 179 differences between the Lansing and Mahoney type 1 strain scattered throughout the genome. To further localize Lansing sequences important for mouse neurovirulence, four intertypic recombinants were isolated by exchanging DNA restriction fragments between the Lansing 2 and Mahoney 1 infectious poliovirus cDNA clones. Plasmids were transfected into HeLa cells, and infectious recombinant viruses were recovered. All four recombinant viruses, which contained the Lansing capsid region and different amounts of the Mahoney genome, were neurovirulent for 18- to 21-day-old Swiss-Webster mice by the intracerebral route. The genome of neurovirulent recombinant PRV5.1 contained only nucleotides 631 to 3413 from Lansing, encoding primarily the viral capsid proteins. Therefore, the ability of Lansing virus to cause paralysis in mice is due to the viral capsid. The Lansing capsid sequence differs from that of the mouse avirulent Sabin 2 strain at 32 of 879 amino acid positions: 1 in VP4, 5 in VP2, 4 in VP3, and 22 in VP1.     

我国登革3型病毒广西80-2株基因组全序列分析

对我国登革 3型病毒 80 2株基因组进行全序列测定 ,为了解其基因组结构与功能的关系提供依据 .根据登革 3型病毒H87株的序列设计并合成引物 ,应用RT PCR和RACE法 ,对 80 2株基因组RNA进行扩增、克隆测序后获得我国登革 3型病毒广西株基因组序列 .该株病毒基因组全长10 696nt ,不含poly(A)尾 ,4种碱基数分别为A :3 4 3 7,C :2 2 15,G :2 773 ,U :2 2 71.包含一个读码框架 ,自 95至 10 2 67位 ,共 10 170个碱基 ,编码 3 3 90个氨基酸 ,5′和 3′非编码区长度分别为 94nt和4 3 2nt.与H 87株比较 ,核苷酸和氨基酸序列同源性均在 99%以上 ,有 2 8个碱基发生改变 ,其中 2 6个碱基突变发生在读码框架内 ,碱基转换 18个 ,颠换 10个 ;碱基突变引起 14个氨基酸的改变 .80 2株与H87株病毒的基因组全序列同源性高 ,变异度小 .     

Human rhinovirus 2: complete nucleotide sequence and proteolytic processing signals in the capsid protein region.

cDNA clones representing the entire genome of human rhinovirus 2 have been obtained and used to determine the complete nucleotide sequence. The genome consists of 7102 nucleotides and possesses a long open reading frame of 6450 nucleotides; this reading frame is initiated 611 nucleotides from the 5' end and stops 42 nucleotides from the polyA tract. The N-terminal sequences of three of the viral capsid proteins have been elucidated, thus defining the positions of three cleavage sites on the polyprotein. The extensive amino acid sequence homology with poliovirus and human rhinovirus 14 enabled the other cleavage sites to be predicted. Cleavages in the 3' half of the molecule appear to take place predominantly at Gln-Gly pairs, whereas those in the 5' half (including the capsid proteins) are more heterogeneous.     

Phylogenetic relationships of aquatic birnaviruses based on deduced amino acid sequences of genome segment A cDNA.

Aquatic birnaviruses, such as infectious pancreatic necrosis virus (IPNV), cause serious diseases in a variety of fish species used worldwide in aquaculture and have also been isolated from a variety of healthy fish and shellfish species. These viruses exhibit a high degree of antigenic heterogeneity and variation in biological properties such as pathogenicity, host range, and temperature of replication. To better understand genetic and biological diversity among these viruses, the nucleotide and deduced amino acid sequences were determined from cDNA of the large open reading frame (ORF) of genome segment A of the 9 type strains of Serogroup A and 4 other representative strains of Serotype A1, the predominant serotype in the United States. In addition, nucleotide and deduced amino acid sequences were determined for the VP2 coding region of a variety of isolates representing 5 of the 9 serotypes. VP2 is the major outer capsid protein of aquatic birnaviruses. RT-PCR was used to amplify a 2904 bp cDNA fragment including all but a few bp of the large ORF of genome segment A or a 1611 bp fragment representing the entire VP2 coding region. Nucleotide and deduced amino acid sequences were determined from the PCR products. Pairwise comparisons were made among our data and 2 other aquatic birnavirus sequences previously published. Several hypervariable regions were identified within the large ORF. The most divergent pair of viruses exhibited a similarity of 80.1% in the deduced amino acid sequence encoded by the large ORF. Genomic relationships revealed in a phylogenetic tree constructed from comparison of the deduced amino acid sequences of the large ORF demonstrated that these viruses were clustered into several genogroups. Phylogenetic comparison of the deduced amino acid sequences of the VP2 coding region of 28 aquatic birnavirus isolates, including the type strains of all 9 serotypes, demonstrated 6 genogroups, some of which were comprised of several genotypes. The most divergent pair of viruses exhibited a similarity of 81.2% in the deduced amino acid sequence from the VP2 coding region. In contrast to previous studies of much shorter genomic sequences within the C-terminus-pVP2/NS junction coding region, these genogroups based on the entire large ORF or the VP2 coding region generally correlated with geographical origin and serological classification. Isolates from the major Canadian serotypes were more closely related to the European isolates than to isolates from the United States.     

Sequence diversity within the reovirus S2 gene: reovirus genes reassort in nature, and their termini are predicted to form a panhandle motif.

To better understand genetic diversity within mammalian reoviruses, we determined S2 nucleotide and deduced sigma 2 amino acid sequences of nine reovirus strains and compared these sequences with those of prototype strains of the three reovirus serotypes. The S2 gene and sigma 2 protein are highly conserved among the four type 1, one type 2, and seven type 3 strains studied. Phylogenetic analyses based on S2 nucleotide sequences of the 12 reovirus strains indicate that diversity within the S2 gene is independent of viral serotype. Additionally, we found marked topological differences between phylogenetic trees generated from S1 and S2 gene nucleotide sequences of the seven type 3 strains. These results demonstrate that reovirus S1 and S2 genes have distinct evolutionary histories, thus providing phylogenetic evidence for lateral transfer of reovirus genes in nature. When variability among the 12 sigma 2-encoding S2 nucleotide sequences was analyzed at synonymous positions, we found that approximately 60 nucleotides at the 5' terminus and 30 nucleotides at the 3' terminus were markedly conserved in comparison with other sigma 2-encoding regions of S2. Predictions of RNA secondary structures indicate that the more conserved S2 sequences participate in the formation of an extended region of duplex RNA interrupted by a pair of stem-loops. Among the 12 deduced sigma 2 amino acid sequences examined, substitutions were observed at only 11% of amino acid positions. This finding suggests that constraints on the structure or function of sigma 2, perhaps in part because of its location in the virion core, have limited sequence diversity within this protein.     

Nucleotide sequence and genome organization of canine parvovirus.

The genome of a canine parvovirus isolate strain (CPV-N) was cloned, and the DNA sequence was determined. The entire genome, including ends, was 5,323 nucleotides in length. The terminal repeat at the 3' end of the genome shared similar structural characteristics but limited homology with the rodent parvoviruses. The 5' terminal repeat was not detected in any of the clones. Instead, a region of DNA starting near the capsid gene stop codon and extending 248 base pairs into the coding region had been duplicated and inserted 75 base pairs downstream from the poly(A) addition site. Consensus sequences for the 5' donor and 3' acceptor sites as well as promotors and poly(A) addition sites were identified and compared with the available information on related parvoviruses. The genomic organization of CPV-N is similar to that of feline parvovirus (FPV) in that there are two major open reading frames (668 and 722 amino acids) in the plus strand (mRNA polarity). Both coding domains are in the same frame, and no significant open reading frames were apparent in any of the other frames of both minus and plus DNA strands. The nucleotide and amino acid homologies of the capsid genes between CPV-N and FPV were 98 and 99%, respectively. In contrast, the nucleotide and amino acid homologies of the capsid genes for CPV-N and CPV-b (S. Rhode III, J. Virol. 54:630-633, 1985) were 95 and 98%, respectively. These results indicate that very few nucleotide or amino acid changes differentiate the antigenic and host range specificity of FPV and CPV.     

Complete genomic characterization of an intertypic Sabin 3/Sabin 2 capsid recombinant

The genetic properties of strain K/2002, isolated from fecal samples of a 7-month-old child who had received his first oral poliovirus vaccine (OPV) dose at the age of 3 months, are described. Preliminary sequencing characterization of isolate K/2002 revealed an S3/S2 recombination event at the 3' end of the VP1 coding region. A recombination event resulted in the introduction of six Sabin 2 amino acid residues in a Sabin 3 genomic background. Furthermore, mutations associated with loss of the attenuated phenotype of Sabin 3 strains have been identified in the genome of isolate K/2002. The data presented here emphasize the need for careful planning of vaccination strategies, which involve stopping OPV administration in regions that are certified to be polio-free.     

The nucleotide sequence of poliovirus type 3 leon 12 a1b: comparison with poliovirus type 1.

The complete nucleotide sequence of the genome of the Sabin vaccine strain of poliovirus type 3 (P3/Leon 12 a1 b) has been determined from cDNA cloned in E. coli. The genome comprises a 5' non-coding region of 742 nucleotides, a large open reading frame of 6618 nucleotides (89% of the sequence) and a 3' non-coding region of 72 nucleotides. There is 77.4% base-sequence homology and 89.6% predicted amino-acid homology between types 1 and 3. Conservation of all glutamine-glycine and tyrosine-glycine cleavage sites suggests a mechanism of polyprotein processing similar to that established for poliovirus type 1.     

A comparison of complete genome sequences of the attenuated RC-HL strain of rabies virus used for production of animal vaccine in Japan, and the parental Nishigahara strain

In order to establish the molecular basis of the pathogenicity of the attenuated RC-HL strain of rabies virus used for the production of animal vaccine in Japan, the complete genome sequence of this strain was determined and compared with that of the parental Nishigahara strain which is virulent for adult mice. The viral genome of both strains was composed of 11,926 nucleotides. The nucleotide sequences of the two genomes showed a high homology of 98.9%. The homology of the G gene was lower than those of N, P, M and L genes at both nucleotide and deduced amino acid levels, and the percentage of radical amino acid substitutions on the G protein was the highest among the five proteins. These findings raise the possibility that the structure of the G protein is the most variable among the five proteins of the two strains. Furthermore, we found two clusters of amino acid substitutions on the G and L proteins. The relevance of these clusters to the difference in the pathogenicity between the two strains is discussed.     

Natural genetic exchanges between vaccine and wild poliovirus strains in humans

In a previous study of poliovirus vaccine-derived strains isolated from patients with vaccine-associated paralytic poliomyelitis (VAPP) (9, 11), we reported that a high proportion (over 50%) of viruses had a recombinant genome. Most were intertypic vaccine/vaccine recombinants. However, some had restriction fragment length polymorphism (RFLP) profiles different from those of poliovirus vaccine strains. We demonstrate here that five such recombinants, of 88 VAPP strains examined, carried sequences of wild (nonvaccine) origin. To identify the parental wild donor of these sequences, we used RFLP profiles and nucleotide sequencing to look for similarity in the 3D polymerase-coding region of 61 wild, cocirculating poliovirus isolates (43 type 1, 16 type 2, and 2 type 3 isolates). In only one case was the donor identified, and it was a wild type 1 poliovirus. For the other four vaccine/wild recombinants, the wild parent could not be identified. The possibility that the wild sequences were of a non-poliovirus-enterovirus origin could not be excluded. Another vaccine/wild recombinant, isolated in Belarus from a VAPP case, indicated that the poliovirus vaccine/wild recombination is not an isolated phenomenon. We also found wild polioviruses (2 of 15) carrying vaccine-derived sequences in the 3' moiety of their genome. All these results suggest that genetic exchanges with wild poliovirus and perhaps with nonpoliovirus enteroviruses, are also a natural means of evolution for poliovirus vaccine strains.     

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.     

A recombinant virus between the Sabin 1 and Sabin 3 vaccine strains of poliovirus as a possible candidate for a new type 3 poliovirus live vaccine strain.

Biological tests including the monkey neurovirulence test performed on recombinants between the virulent Mahoney and attenuated Sabin 1 strains of type 1 poliovirus indicated that the genome region encoding mainly the viral capsid proteins had little correlation with the neurovirulence or attenuation phenotype of the virus. The results suggested that new vaccine strains of type 2 and type 3 polioviruses may be constructed in vitro by replacing the sequence encoding the antigenic determinants in viral capsid proteins of the Sabin 1 genome by the corresponding sequences of the type 2 and type 3 genome, respectively. Accordingly, we constructed recombinants between the Sabin 1 and Sabin 3 strains of poliovirus in which genome sequences of the Sabin 1 strain encoding most or all capsid proteins were replaced by the corresponding genome sequences of the Sabin 3 strain. One of the recombinant viruses thus constructed was fully viable and showed antigenicity and immunogenicity identical to those of type 3 poliovirus. The monkey neurovirulence tests and in vitro phenotypic marker tests (temperature sensitivity of growth, sodium bicarbonate concentration dependency of growth under agar overlay, and size of plaque) were performed on the recombinant virus. The stability of the virus in regard to the temperature sensitivity phenotype was also tested. The results suggested that the recombinant virus is a possible candidate for a new type 3 poliovirus vaccine strain.     

Long-term excretion of vaccine-derived poliovirus by a healthy child

A child was found to be excreting type 1 vaccine-derived poliovirus (VDPV) with a 1.1% sequence drift from Sabin type 1 vaccine strain in the VP1 coding region 6 months after he was immunized with oral live polio vaccine. Seventeen type 1 poliovirus isolates were recovered from stools taken from this child during the following 4 months. Contrary to expectation, the child was not deficient in humoral immunity and showed high levels of serum neutralization against poliovirus. Selected virus isolates were characterized in terms of their antigenic properties, virulence in transgenic mice, sensitivity for growth at high temperatures, and differences in nucleotide sequence from the Sabin type 1 strain. The VDPV isolates showed mutations at key nucleotide positions that correlated with the observed reversion to biological properties typical of wild polioviruses. A number of capsid mutations mapped at known antigenic sites leading to changes in the viral antigenic structure. Estimates of sequence evolution based on the accumulation of nucleotide changes in the VP1 coding region detected a "defective" molecular clock running at an apparent faster speed of 2.05% nucleotide changes per year versus 1% shown in previous studies. Remarkably, when compared to several type 1 VDPV strains of different origins, isolates from this child showed a much higher proportion of nonsynonymous versus synonymous nucleotide changes in the capsid coding region. This anomaly could explain the high VP1 sequence drift found and the ability of these virus strains to replicate in the gut for a longer period than expected.     

Strong inclination toward transition mutation in nucleotide substitutions by poliovirus replicase

A viable insertion mutant of the Sabin strain of type 1 poliovirus was constructed. The mutant carried an insertion sequence of 72 nucleotides at nucleotide position 702 in the 5' non-coding region (742 nucleotides long) of the genome of the Sabin strain. This mutant showed a small-plaque phenotype, as compared with the parental virus. Indeed, the final yield of the mutant in a single cycle of infection was tenfold fewer than that of the parental virus. Many large-plaque variants that are easily generated from the insertion mutant appeared to regain efficient viral replication and have single nucleotide changes. All nucleotide changes observed were limited to within three nucleotides of an AUG sequence in the insertion sequence. The result indicates strongly that the AUG sequence itself in this genome region functions in reducing the plaque size of the parental Sabin type 1 virus. The insertion mutant with a small-plaque phenotype may be the first in vitro mutant of poliovirus whose viability is lowered only by a primary sequence inserted into the 5' non-coding region of the genome. Base substitutions to alter the AUG sequence should largely be the result of errors of the virus-specific replicase, since variants with base substitutions must be subject to only minimum selection pressure. Accordingly, nucleotide sequence analysis of the genome region containing the AUG sequence was performed on a number of genomes of large-plaque variants to investigate types of nucleotide substitutions caused by characteristic errors in RNA replication. Only one transversion mutation was detected in the genomes of 44 independently isolated large-plaque variants with single base changes in the AUG sequence. This result suggests strongly that transition mutations occur predominantly as nucleotide substitutions caused by characteristic errors of poliovirus replicase.