Recombinants between attenuated and virulent strains of poliovirus type 1: derivation and characterization of recombinants with centrally located crossover points

Recombinants with a centrally located crossover point were selected from crosses between poliovirus type 1 strains and intertypic (type 3/type 1) recombinants. Two such recombinants were characterized in some detail. In one of them (v1/a1-6), the 5' half of the genome was derived from a virulent type 1 strain, while the 3' half came from an attenuated type 1 strain. The genome of the other recombinant (a1/v1-7) had the reverse organization, with the 5' and 3' halves being derived from the type 1 attenuated and virulent strains, respectively. As deduced from the RNase T1 oligonucleotide maps, the a1/v1-7 genome also had a relatively short centrally located insert of the poliovirus type 3 origin. Both recombinants exhibited ts phenotypes. The RNA phenotypes of the recombinants corresponded to that of the parent donating the 3' half of the genome, v1/a1-6 and a1/v1-7 expressing RNA- and RNA +/- characters, respectively. Despite being a ts RNA- virus, v1/a1-6 proved to be neurovirulent when injected intracerebrally into Cercopithecus aethiops monkeys, although it exhibited a somewhat diminished level of pathogenicity as compared to its virulent type 1 parent. Recombinant a1/v1-7 behaved as an attenuated strain. These data supported our previous conclusion drawn from the experiments with intertypic poliovirus recombinants that the attenuated phenotype of poliovirus depends largely on the structure of the 5' half of its genome, although mutations of the 3' half may alleviate the virulence of the virus to a degree.     

Recombinants of Mahoney and Sabin strain poliovirus type 1: analysis of in vitro phenotypic markers and evidence that resistance to guanidine maps in the nonstructural proteins

A neutralizing monoclonal antibody-resistant variant of the Sabin vaccine strain of poliovirus type 1 and a guanidine-resistant variant of the virulent parent Mahoney strain were derived. The two variants were used, in a coinfection, to generate recombinant virus containing both resistance markers. Recombinants appeared on the order of 1.0 PFU for every 10(4) total PFU. Two independently derived recombinant viruses were isolated. Each isolate contained the P1 (structural protein) gene region of the Sabin strain virus and the P3 (nonstructural replicase protein) gene region of the Mahoney strain virus. The recombinant virus phenotypes were compared with certain characteristic in vitro phenotypes of both parents. It was found that the slow growth in cell culture, temperature sensitivity, and virion surface charge characteristics of the Sabin virus mapped entirely to the structural protein gene region whereas the phenotype of the actinomycin D sensitivity of the Sabin virus mapped to the gene region specifying the nonstructural replication proteins. Sequence analysis of the recombinant RNA revealed that the crossover occurred 3' of nucleotide 3919. This result showed that the resistance of poliovirus mutants to growth in 2 mM guanidine hydrochloride maps in the 3'-terminal region of the viral genome specifying the nonstructural proteins.     

Attenuated influenza B virus recombinants obtained by crossing of B/England/2608/76 virus with a cold-adapted B/Leningrad/14/17/55 strain

Crossing of an attenuated influenza B virus strain (B/Leningrad/14/17/55) passaged at a low temperature with a virulent influenza B virus strain (B/England/2608/76) yielded recombinants similar in the antigenic specificity of their haemagglutinin (HA) and neuraminidase (NA) to B/England/2608/76 strain, but possessing an RCT37.5 marker alike to the attenuated donor. Analysis of the genome composition of 2 recombinants has shown that they inherited genes coding for P (1, 2, 3) and M (7) proteins from the attenuated parent, but genes coding for HA (4), NA (6), NP (5) and NS (8) proteins from the virulent parent. All recombinants proved to be areactogenic for adult volunteers with no pre-existing antibody to the corresponding HA (less than or equal to 8); however, they had a reduced immunogenicity as compared to parent viruses.     

Neurovirulence of influenza virus in mice I. Neurovirulence of recombinants between virulent and avirulent virus strains

The neurovirulence of influenza A/WSN (HONI) virus in mice was studied using recombinants between neurovirulent WSN and nonneurovirulent A/Hong Kong (HK, H3N2) viruses. Parental derivation of genes in recombinants were analyzed by the electrophoresis of viral RNA in urea-polyacrylamide gel. The neurovirulence was tested by intracerebral inoculation of recombinants into mice. It was found that five large genes, P₃, P₁, P₂, HA, and NP, were not essential for the virulence, because recombinants having these five genes derived from HK parent were virulent. Recombinants in which any of three remaining WSN genes, NA, M, and NS, was replaced with the one from HK parent, failed to kill mice. Therefore, these three genes were responsible for the difference of neurovirulence between the two virus strains. However, when tested in mice immunosuppressed by the administration of cyclophosphamide, recombinants containing either M or NS protein from HK parent were virulent, but viruses containing HK neuraminidase were still avirulent. Viruses containing HK neuraminidase appeared incapable of multiplying in the mouse brain, while those containing either M or NS protein derived from HK virus multiplied to a limited extent. It was suggested that WSN neuraminidase was the principal determining factor of the neurovirulence of WSN virus, without which no virus multiplication occurred, while M and probably NS proteins of WSN virus played a role of helper or accessory virulence factor(s), enabling the efficient virus replication.     

Translation deficiency of the Sabin type 3 poliovirus genome: association with an attenuating mutation C472----U

Previous studies have shown that the genome of Sabin type 3 poliovaccine strain (P3/Leon 12a1b) possesses a diminished translation efficiency as compared to genomes of closely related neurovirulent strains, the neurovirulent progenitor (P3/Leon/37), or a revertant (P3/119/70) of the vaccine (Y.V. Svitkin, S.V. Maslova, and V.I. Agol, 1985, Virology 147, 243-252). Here we attempted to evaluate the contribution of each mutation in the genome of the vaccine to this translation deficiency. Recombinants between P3/Leon 12a1b and P3/Leon/37 or P3/119/70 were constructed in vitro and their RNAs were translated in a cell-free system derived from Krebs-2 cells. The results show that of 10 nucleotide differences between the genomes of P3/Leon 12a1b and P3/Leon/37 9 have minor or no effect on translation and that the only mutation of significance is C472----U which is known to reduce the neurovirulence of the virus. Reversion from uridine to cytosine at position 472 in type 3 poliovaccine upon replication in the human gut resulted in an increase of both translation efficiency of polio RNAs and neurovirulence of corresponding strains. The data provide evidence for a common nucleotide sequence regulatory element for protein synthesis of the virus and its neurovirulence. In vitro translation assays may therefore prove to be useful for detection of attenuating mutations in the 5' noncoding region of poliovirus genome. The apparent involvement of the translation mechanism in the expression of neurovirulent or attenuated phenotype of poliovirus is briefly discussed.     

Infectious cDNA clone of attenuated Langat tick-borne flavivirus (strain E5) and a 3' deletion mutant constructed from it exhibit decreased neuroinvasiveness in immunodeficient mice

Forty-five years ago a naturally attenuated tick-borne flavivirus, Langat (LGT) strain TP21, was recovered from ticks in Malaysia. Subsequently, it was tested as a live attenuated vaccine for virulent tick-borne encephalitis viruses. In a large clinical trial its attenuation was confirmed but there was evidence of a low level of residual virulence. Thirty-five years ago further attenuation of LGT TP21 was achieved by multiple passages in eggs to yield mutant E5. To study the genetic determinants of the further attenuation exhibited by E5 and to allow us to manipulate the genome of this virus for the purpose of developing a satisfactory live attenuated tick-borne flavivirus vaccine, we recovered infectious E5 virus from a full-length cDNA clone. The recombinant E5 virus (clone 651) recovered from a full-length infectious cDNA clone was more attenuated in immunodeficient mice than that of its biologically derived E5 parent. Increase in attenuation was associated with three amino acid substitutions, two located in the structural protein E and one in nonstructural protein NS4B. Subsequently an even greater degree of attenuation was achieved by creating a viable 320 nucleotide deletion in the 3'-noncoding region of infectious full-length E5 cDNA. This deletion mutant was not cytopathic in simian Vero cells and it replicated to lower titer than its E5-651 parent. In addition, the E5 3' deletion mutant was less neuroinvasive in SCID mice than its E5-651 parent. Significantly, the deletion mutant proved to be 119,750 times less neuroinvasive in SCID mice than its progenitor, LGT strain TP21. Despite its high level of attenuation, the E5 3' deletion mutant remained highly immunogenic and intraperitoneal (ip) inoculation of 10 PFU induced complete protection in Swiss mice against subsequent challenge with 2000 ip LD50 of the wild-type LGT TP21.     

Defined recombinants of poliovirus and coxsackievirus: sequence-specific deletions and functional substitutions in the 5'-noncoding regions of viral RNAs

We describe the isolation of a variant of a polio--coxsackie recombinant virus (PCV110) containing a genomic RNA with a chimeric 5'-noncoding region. The variant virus [designated PCV110(1)] has growth and biosynthetic properties that are quite different from the original, temperature-sensitive isolate of the recombinant virus [designated PCV110(4)]. Nucleotide sequencing of the 5'-noncoding region of RNA from PCV110(1) revealed a 4-base deletion within the substituted coxsackievirus region of the chimeric genome that may contribute to the loss of temperature sensitivity of this variant recombinant virus. In addition, we have generated new recombinant viruses that contain (1) coxsackievirus sequences within the N66-N627 region of the poliovirus genome and (2) coxsackievirus sequences substituted from N1-N627 in the poliovirus genome. These recombinant viruses are not temperature sensitive for growth at 37 degrees and have biosynthetic properties similar to those of wild-type poliovirus. Our results provide evidence that replicase recognition signals encoded in the 5' noncoding regions of enterovirus genomic RNAs are not strictly sequence specific.     

Biochemical characterization of an aphthovirus type 0(1) strain campos attenuated for cattle by serial passages in chicken embryos

The biochemical properties of a virulent and an attenuated strain of foot-and-mouth disease virus (FMDV) Type 0(1) Campos (0(1)C) were compared in order to establish differences that could account for their altered biological functions. The avirulent strain (0(1)C-O/E) was derived from the virulent strain 0(1)C by serial passages in chicken embryos. Analysis of the RNase T1-generated oligonucleotides of the viral RNA through one- and two-dimensional (2D) gel electrophoresis (fingerprints) revealed a few changes in the genome structure of the 0(1)C-O/E strain compared to the wild type strain. In addition there was a significant decrease in the length of the poly(C) rich tract of the 0(1)C-O/E RNA. All virion structural proteins, except VP4, their precursors, and the viral RNA polymerase (p56a) show charge differences. In addition a significant decrease in the apparent molecular weight of polypeptide p100 (primary translational product from the 3' end region of the genome) of the attenuated strain was observed.     

Studies on the attenuation of the Sabin type 3 oral polio vaccine

The genetic basis of attenuation of the poliovirus type 3 vaccine strain P3/Leon 12a1b has been investigated by comparing the nucleotide sequence of this strain with that of its neurovirulent progenitor P3/Leon/37 and by constructing recombinants between these two viruses using infectious cDNAs. Preliminary results suggest that attenuation is caused by just two point mutations, one occurring in the 5' non-coding region and the other causing an amino acid change in coat protein VP3.     

Correlation of pathogenicity and gene constellation of influenza A viruses. II. Highly neurovirulent recombinants derived from non-neurovirulent or weakly neurovirulent parent virus strains.

Mixed infections with various nonneurovirulent or weakly neurovirulent influenza A strains yielded recombinants that were highly neurovirulent for mice. A correlation was detected between gene constellation and neurovirulence of these recombinants. For the recombination pair FPV/A2-England the Pol 1 and Ptra genes had to be derived from the human strain; while the HA and/or M gene has to be from FPV in order to obtain highly neurovirulent recombinants. For the FPV/PR8 pair only the Pol 1 gene needs to be derived from PR8 while the HA gene had to be from FPV in order to get highly neurovirulent recombinants. The derivation of the other genes does not appear to be important in this respect. Recombinants between FPV and the A2-Singapore influenza strain do not exhibit significant neurovirulence suggesting that at least one parent strain should be adapted to grow in mice in order to obtain neurovirulent recombinants. In addition, there is a correlation between pneumovirulence and growth in mouse kidney cells, but neurovirulence for mice and pathogenicity for chickens was not correlated. The data presented here demonstrate in principle the possiblity of an increase in pathogenicity in recombinants derived from less or nonpathogenic parent viruses.     

Genomic sequence of an infectious bursal disease virus isolate from Zambia: classical attenuated segment B reassortment in nature with existing very virulent segment A

We determined the complete nucleotide sequence of an infectious bursal disease (IBD) virus (IBDV) isolate (designated KZC-104) from a confirmed IBD outbreak in Lusaka in 2004. The genome consisted of 3,074 and 2,651 nucleotides in the coding regions of segments A and B, respectively. Alignment of both nucleotide and deduced amino acid sequences and phylogenetic analysis revealed that the genome segment A of KZC-104 was derived from a very virulent (VV) strain, whereas its segment B was derived from a classical attenuated strain. On BLAST search, the full-length segment A and B sequences showed 98 % nucleotide sequence identity to the VV strain D6948 and 99.8 % nucleotide sequence identity to the classical attenuated strain D78. This is a unique IBDV reassortant strain that has emerged in nature, involving segment B of a cell-culture-adapted attenuated vaccine.     

Analysis of genome determinants of virulence and setting-up of a library of full-size genome copies of the attenuated virus strain of the porcine reproductive and respiratory syndrome virus North American type

Primary genome structures of 3 variants of the NADC-8 North American virulent strain of porcine reproductive and respiratory syndrome virus (PRRSV) were compared for the purpose of detecting any potential genetic virulence determinants of genus Arterivirus. Apart from the virulent variant, we also investigated the attenuated variant, obtained after 251 passages in cell culture, and the intermediate variant isolated from a pig after a partial reversion of the attenuated virus. The attenuated variant genome acquired a 3-nucleotide deletion and 50 mutations versus its virulent precursor. A comparison of the attenuated and intermediary virus variants denoted 8 nucleotide mutations entailing substitutions of 6 amino acids in 3 open reading frames (ORF1a, ORF1b and ORF6). A 32-clone library was constructed in the pACYC177 plasmid vector, which comprised full-size copies of the genome of the NADC-8 attenuated variant strain (251), virus PPCC, for the purpose of experimentally verifying the functional role of the obtained mutations. Full-size analogues ((+)-chain of RNA) of the viral genome, comprising the CAP-structures and polyadenylated ones were obtained in vitro on the basis of the cloned DNA. Seven of the 8 analyzed clones of the viral genome were infected and their insertion into the MARC-145 cell resulted in obtaining of infectious PRRSVs. Four of the constructed recombinant viruses had delayed growth parameters, and 3 of them were similar to the parental strain. The described technology (inverse genetics) would make it possible to introduce changes into the viral genome in applied and fundamental research of Arteriviruses.     

Amino acid substitutions in the structural or nonstructural proteins of a vaccine strain of equine arteritis virus are associated with its attenuation

Comparative sequence analysis of a series of strains of equine arteritis virus (EAV) of defined virulence for horses, ranging from the horse-adapted virulent Bucyrus (VB) strain to a fully attenuated vaccine strain derived from it, identified 13 amino acid substitutions associated with attenuation. These include 4 substitutions in the replicase proteins and 9 in the structural proteins. Using reverse genetic techniques, these amino acid substitutions were introduced into a virulent infectious cDNA clone pEAVrVBS derived from the VB strain of EAV. Inoculation of horses with the recombinant viruses clearly demonstrated that changes in either the replicase (nsp1, nsp2 and nsp7) or structural proteins (GP2, GP4, GP5 and M) resulted in attenuation of the virulent VB strain. The recombinant virus with substitutions in the structural proteins was more attenuated than the recombinant virus with substitutions only in the replicase proteins.     

Correlation of mutations and recombination with growth kinetics of poliovirus vaccine strains

Attenuated strains of Sabin poliovirus vaccine replicate in the human gut and, in rare cases, may cause vaccine-associated paralytic poliomyelitis (VAPP). The genetic instability of Sabin strains constitutes one of the main causes of VAPP, a disease that is most frequently associated with type 3 and type 2 Sabin strains, and more rarely with type 1 Sabin strains. In the present study, the growth phenotype of eight oral poliovirus vaccine (OPV) isolates (two non-recombinants and six recombinants), as well as of Sabin vaccine strains, was evaluated using two different assays, the reproductive capacity at different temperatures (Rct) test and the one-step growth curve test in Hep-2 cells at two different temperatures (37°C and 40°C). The growth phenotype of isolates was correlated with genomic modifications in order to identify the determinants and mechanisms of reversion towards neurovirulence. All of the recombinant OPV isolates showed a thermoresistant phenotype in the Rct test. Moreover, both recombinant Sabin-3 isolates showed significantly higher viral yield than Sabin 3 vaccine strain at 37°C and 40°C in the one-step growth curve test. All of the OPV isolates displayed mutations at specific sites of the viral genome, which are associated with the attenuated and temperature-sensitive phenotype of Sabin strains. The results showed that both mutations and recombination events could affect the phenotype traits of Sabin derivatives and may lead to the reversion of vaccinal strains to neurovirulent ones. The use of phenotypic markers along with the genomic analysis may shed additional light on the molecular determinants of the reversed neurovirulent phenotype of Sabin derivatives.     

Studies on the recombination between RNA genomes of poliovirus: the primary structure and nonrandom distribution of crossover regions in the genomes of intertypic poliovirus recombinants

A series of intertypic (type 3/type 1) poliovirus recombinants was obtained whose crossover sites were expected to be located in the middle of the viral genome, between the loci encoding type-specific antigenic properties, on the 5' side, and an altered sensitivity to guanidine, on the 3' side. The primary structures of the crossover regions in the genomes of these recombinants were determined by the primer extension method. The length of the crossover sites (the uninterrupted sequences shared by the recombinant and both parental genomes that are flanked, in the recombinant RNAs, by two heterotypic segments) varied between 2 and 32 nucleotides, but the majority of the sites were 5 nucleotides long or shorter. The crossover sites were nonrandomly distributed over the presumably available genome region: only a single such site was found within the gene for polypeptide 2A, whereas an apparent clustering of the crossover sites was encountered in other genomic segments. When the crossover sites were superimposed on a model of the secondary structure of the relevant region of the viral RNA molecule, a pattern consistent with the previously proposed mechanism of poliovirus recombination (L.I. Romanova, V.M. Blinov, E.A. Tolskaya, E.G. Viktorova, M.S. Kolesnikova, E.I. Guseva, and V.I. Agol (1986) Virology 155, 202-213) was observed. It is suggested that the nonrandom distribution of the crossover sites in the genomes of intertypic poliovirus recombinants was due to two factors: the existence of preferred sites for recombination, and selection against recombinants with a lowered level of viability.     

A family of closely related bovine viral diarrhea virus recombinants identified in an animal suffering from mucosal disease: new insights into the development of a lethal disease in cattle

Induction of lethal mucosal disease (MD) in cattle is linked to the generation of cytopathogenic (cp) bovine viral diarrhea virus (BVDV) in animals persistently infected with a noncytopathogenic BVDV. In most cases the cp variants are generated by recombination with cellular or viral sequences. BVDV was obtained from the serum of an MD animal and propagated in tissue culture without plaque purification. Analysis of cDNA clones established from RNA of these cells showed that apparently a variety of different viral RNAs were present. Seven of the cDNA clones contained a cellular sequence coding for light chain 3 (LC3) of microtubule-associated proteins 1A and 1B. This insertion had already been found in the cp virus JaCP obtained from the same animal and isolated by plaque purification. Analysis of further plaque-purified cp viruses showed that the diseased animal contained a family of closely related cp BVDV recombinants. A set of viruses with different duplications of viral sequences in their genomes and a variety of defective viral RNAs with deletions were found that all contained the LC3* insertion. For all the recombinants the 3' recombination sites and, in all but one case, also the 5' recombination sites between cellular and viral sequence were identical. Variation between the individual deduced genome structures resulted from different duplications or deletions of viral sequences located upstream of the cellular insertion. These results suggest that within the animal a primary recombinant with a genome containing the LC3* insertion was generated. In a trimming process a set of secondary virus recombinants was generated from this hypothetical primary recombined RNA. These secondary recombinants display genome structures that represent variations of the basic scheme already present in the primary recombinant. Apparently this trimming process that finally led to an outbreak of MD lasted a long time since recombined RNA with the basic genome structure of the cp viruses could be demonstrated in samples already taken a long time before outbreak of the disease.     

JE Nakayama/JE SA14-14-2 virus structural region intertypic viruses: biological properties in the mouse model of neuroinvasive disease

A molecular clone of Japanese encephalitis (JE) virus Nakayama strain was used to create intertypic viruses containing either the 5'-C-prM-E or the prM-E region of the attenuated JE SA14-14-2 virus in the JE Nakayama background. These two intertypic JE viruses, JE-X/5'CprME(S) and JE-X/prME(S), respectively, generally resembled the parental JE virus in cell culture properties. Similar to virus derived from the JE Nakayama molecular clone (JE-XJN), JE-X/prME(S) was highly neuroinvasive and neurovirulent for young adult mice, whereas JE-X/5'CprME(S) was attenuated for neuroinvasiveness and only partially attenuated for neurovirulence. Immunization of young mice with JE-X/5'CprME(S) virus elicited neutralizing antibodies against JE Nakayama virus and conferred protection against encephalitis following challenge with JE Nakayama virus. The sequence of the JE-X/5'CprME(S) virus differed from that of JE-X/prME(S) virus at two nucleotides in the 5' UTR, 3 amino acid positions in the capsid protein, 4 positions in the prM protein and 1 in the envelope protein. For JE-X/prME(S) virus, the 4 differences in prM and the single substitution in the envelope represented reversions to the sequence of JE Nakayama virus. Overall, this study reveals that molecular determinants associated with the prM-E region of the attenuated JE SA14-14-2 virus are insufficient by themselves to confer an attenuation phenotype upon JE Nakayama virus. This suggests a role for determinants in the 5' UTR and/or the capsid protein of the JE SA 14-14-2 virus genome in influencing the virulence properties of the JE Nakayama virus in the mouse model.