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PubMed1982

Reassortant virus derived from avian and human influenza A viruses is attenuated and immunogenic in monkeys.

An influenza A reassortant virus that contained the hemagglutinin and neuraminidase genes of a virulent human virus, A/Udorn/72 (H3N2), and the six other influenza A virus genome segments from an avirulent avian virus, A/Mallard/New York/6750/78 (H2N2), was evaluated for its level of replication is squirrel monkeys and hamsters. In monkeys, the reassortant virus was as attenuated and as restricted in its level of replication in the upper and lower respiratory tract as its avian influenza virus parent. Nonetheless, infection with the reassortant induced significant resistant to challenge with virulent human influenza virus. In hamsters, the reassortant virus replicated to a level intermediate between that of its parents. These findings suggest that the nonsurface antigen genes of the avian parental virus are the primary determinants of restriction of replication of the reassortant virus in monkeys. Attenuation of the reassortant virus for primates is achieved by inefficient functioning of the avian influenza genes in primate cells, while antigenic specificity of the human influenza virus is provided by the neuraminidase and hemagglutinin genes derived from the human virus. This approach could lead to the development of a live influenza A virus vaccine that is attenuated for man if the avian influenza genes are similarly restricted in human cells.

Animals↗

PubMed1985

Nucleoprotein and membrane protein genes are associated with restriction of replication of influenza A/Mallard/NY/78 virus and its reassortants in squirrel monkey respiratory tract.

An avian influenza A virus, A/Mallard/NY/6750/78(H2N2), was restricted in in replication in the respiratory tract of squirrel monkeys. Avian-human influenza A reassortant viruses possessing the six RNA segments coding for nonsurface proteins (i.e., internal genes) of this avian virus were as restricted in replication in squirrel monkeys as their avian influenza parent. These findings indicated that restriction of replication of the avian influenza virus is a function of one or more of its internal genes. For an investigation of which of the avian influenza genes was responsible for restricted replication in the respiratory tract of primates, reassortant viruses were produced that contained human influenza virus surface antigens from the A/Udorn/72(H3N2) virus and one or more of the internal genes derived from the avian influenza virus parent. Avian-human reassortant influenza A viruses containing only the nucleoprotein or matrix protein RNA segment from the avian influenza virus parent were as restricted in their growth as an avian-human influenza reassortant virus containing each of the six avian influenza internal genes. In addition, an avian-human influenza reassortant virus possessing only the avian RNA 1 and nonstructural genes (which by themselves do not specify restricted replication) manifested a significant reduction of virus replication in squirrel monkey tracheas. Thus, the avian nucleoprotein and matrix genes appear to play a major role in the host range restriction exhibited by the A/Mallard/78 virus and its reassortants, but the combination of RNA 1 and nonstructural genes also contributes to restriction of replication.

Animals↗

PubMed1985

Reassortant rotaviruses as potential live rotavirus vaccine candidates.

A series of reassortants was isolated from coinfection of cell cultures with a wild-type animal rotavirus and a "noncultivatable" human rotavirus. Wild-type bovine rotavirus (UK strain) was reassorted with human rotavirus strains D, DS-1, and P; wild-type rhesus rotavirus was reassorted with human rotavirus strains D and DS-1. The D, DS-1, and P strains represent human rotavirus serotypes 1, 2, and 3, respectively. Monospecific antiserum (to bovine rotavirus, NCDV strain) or a set of monoclonal antibodies to the major outer capsid neutralization glycoprotein, VP7 (of the rhesus rotavirus), was used to select for reassortants with human rotavirus neutralization specificity. This selection technique yielded many reassortants which received only the gene segment coding for the major neutralization protein from the human rotavirus parent, whereas the remaining genes were derived from the animal rotavirus parent. Single human rotavirus gene substitution reassortants of this sort represent potential live vaccine strains.

Animals↗

PubMed1997

Genetic exchange by recombination or reassortment is infrequent in natural populations of a tripartite RNA plant virus.

Two hundred seventeen field isolates of cucumber mosaic cucumovirus (CMV), sampled from 11 natural populations, were typed by RNase protection assay (RPA) using probes from the genomic RNAs of strains in subgroup I and in subgroup II of CMV strains. Most (85%) of the analyzed isolates belonged to subgroup I. For these subgroup I isolates, only two clearly different RPA patterns, A and B, were found for each of four probes representing RNA1, RNA2, and each of the two open reading frames in RNA3. On the basis of these RPA patterns for each probe, different haplotypes were defined. The frequency composition for these haplotypes differed for the various analyzed populations, with no correlation with place or year of sampling. This genetic structure corresponds to a metapopulation with local extinctions and recolonizations. Most subgroup I isolates (73%) belonged to haplotypes with RPA pattern A (type 1) or B (type 2) for all four probes. A significant fraction of subgroup I isolates (16%) gave evidence of mixed infections with these two main types, from which genetic exchange could occur. Genetic exchange by segment reassortment was seen to occur: the fraction of reassortant isolates was 4%, reassortment did not occur at random, and reassortants did not become established in the population. Thus, there is evidence of selection against reassortment between types 1 and 2 of subgroup I isolates. Aphid transmission experiments with plants doubly infected with type 1 and type 2 isolates gave further evidence that reassortment is selected against in CMV. Genetic exchange by recombination was detected for RNA3, for which two RPA probes were used. Recombinant isolates amounted to 7% and also did not become established in CMV populations. Sequence analyses of regions of RNA1, RNA2, and RNA3 showed that there are strong constraints to maintain the encoded sequence and also gave evidence that these constraints may have been different during divergence of types 1 and 2 and, later on, during diversification of these two types. Constraints to the evolution of encoded proteins may be related to selection against genetic exchange. Our data, thus, do not favor current hypotheses that explain the evolution of multipartite viral genomes to promote genetic exchange.

Genome, Viral↗

PubMed1995

High resolution genome typing and genomic reassortment events of rice dwarf Phytoreovirus.

Genomic reassortment of rice dwarf Phytoreovirus (RDV) was experimentally demonstrated for the first time in plant reoviruses. Combinations of two genomic variants, most of the genomic segments of which could be distinguished by a high resolution polyacrylamide gel electrophoresis, were used to produce genomic reassortants. After artificial mixed injection of two of three isolates (RDV-S, RDV-AI, and RDV-AN) into the insect vector Nephotettix cincticeps, rice seedlings were sequentially inoculated and the genomic origin of the viruses present in the infected plants was examined by electrophoresis. The progeny virus population contained either one or both of the respective genomic segments from the parents. Genomic segments reassorted randomly except for genome segment 1 (S1) and S9. S9 of RDV-S was mostly excluded in the reassortants in both the insects and the infected plants when it was mixed with RDV-AI or RDV-AN. On the other hand, S9 reassorted randomly in most of the virus populations in infected plants when RDV-AI and RDV-AN were co-injected into insects. When RDV-S and RDV-AI were mixed, S1 from RDV-S was present more frequently in the infected plants although both parental S1's were present in equimolar amounts in insects.

Animals↗

PubMed2003

Attenuation of a human H9N2 influenza virus in mammalian host by reassortment with an avian influenza virus.

In order to develop a surrogate virus strain for production of an inactivated influenza vaccine against a human H9N2 virus, A/Hong Kong/1073/99 (HK1073: H9N2) was co-infected in embryonated chicken eggs with an apathogenic avian influenza virus, A/Duck/Czechoslovakia/56 (Dk/Cz: H4N6), for gene segment reassortment. Multiple-gene reassortants obtained were examined for replication in mammalian hosts in vitro and in vivo by infecting MDCK cells and by intranasal administration to hamsters, respectively. A 2-6 gene reassortant with both surface glycoproteins of HK1073 origin and the rest of Dk/Cz origin, HK/CZ-13, was shown to replicate poorly in the mammalian hosts both in vivo and in vitro comparing with HK1073, although this reassortant replicated as efficiently as each parental strain in embryonated eggs. No sequence difference was observed in the HA1 region between HK1073 and HK/CZ-13, indicating that the reassortant would be equivalent in its immunogenicity to the parental HK1073 strain when it is used as an inactivated vaccine. A virus strain with attenuation in mammalian hosts is preferable for production of an H9 vaccine, since it should reduce the risk of manufacturing-related infections of employees during the vaccine production. HK/CZ-13 can therefore be a surrogate strain for production of an inactivated vaccine as well as diagnostic antigens in case of a possible future pandemic caused by an HK1073-like H9 influenza virus.

Administration, Intranasal↗

PubMed2001

Direct evidence for genome segment reassortment between concurrently-circulating human rotavirus strains.

Extensive heterogeneity in electropherotypes observed among group A human rotaviruses has been considered as a result of two major mechanisms; i.e., the accumulation of point mutations and genetic reassortment between concurrently-circulating strains. However, no evidence was reported thus far indicating that any one of field isolates of rotavirus was formed by direct reassortment of concurrently circulating two parental strains. Comparison of the genome of human rotavirus specimens collected over a six year period by electropherotyping and by the sequencing of selected gene segments identified two reassortants that were generated in nature between strains circulating co-dominantly in the same epidemic season. This is the first report directly showing that at least some part of electrophoretic diversity observed among rotavirus strains was explained by genetic reassortment between strains concurrently circulating in the human population. This supports the hypothesis that genetic reassortment among co-circulating strains operates as a key mechanism for the genetic variability of rotaviruses in nature.

Base Sequence↗

PubMed2006

Molecular characterization of a reassortant virus derived from Lassa and Mopeia viruses.

In this article we describe two new complete genomic sequences of Old World Arenaviruses: the Mopeia (MOP) virus and the reassortant MOP/LAS virus, clone 29, or ML29. This reassortant has the large (L) RNA from MOP virus and the small (S) RNA from Lassa (LAS) virus, Josiah strain. Recent studies showed that the ML29 virus is not pathogenic for mice, guinea pigs, or macaques, can completely protect guinea pigs from Lassa virus, and elicit vigorous cell-mediated immunity in immunized monkeys (Lukashevich, I. S., Patterson, J., Carrion, R., Moshkoff, D., Ticer, A., Zapata, J., Brasky, K., Geiger, R., Hubbard, G. B., Bryant, J., and Salvato, M. S., J Virol 79, 13934-13942, 2005). This is a molecular characterization of a reassortant virus, which has been put forward as a live attenuated vaccine candidate against Lassa Fever. Sequence analysis of this reassortant virus revealed 5 non-conservative amino acid substitutions that distinguished it from the parental LAS and MOP viruses. Three substitutions were found outside the conserved RNA-dependent RNA polymerase (RdRp) motifs. A fourth substitution was located between the glycoprotein (GPC)-cleavage site and the putative fusion peptide of GP2. The nucleocapsid protein (NP) contained a fifth substitution in the carboxyl-terminal region of the protein. Two mutations were found within each non-coding terminus of the L segment and one mutation was located in the 3' non-coding region of the S segment of the MOP/LAS virus. ML29 mutations in its genomic termini may have implications for the genetic stability and replication efficiency of ML29 reassortant.

Amino Acid Sequence↗

PubMed2005

Simple and rapid strategy for genetic characterization of influenza B virus reassortants.

Genetic reassortment of influenza viruses is widely used for creating viruses with specific phenotypes. Reassortment of two influenza viruses, each with eight RNA segments potentially yields as many as 256 gene segment combinations. Therefore, confirmation that progeny viruses possess genomes corresponding to the specified phenotypes can be laborious and time-consuming. To establish a convenient method for genotyping influenza virus reassortants, we adapted single-strand conformation polymorphism analysis (SSCP) using standard laboratory equipment. By varying the concentration of polyacrylamide between 4-6% and the concentration of glycerol between 5-8% in the gel, together with adding PCR primers to the DNA sample during the denaturing step, optimal conditions can be found for SSCP with little effort. The described method has high accuracy and reliability, and provides a tool for rapid, cost-effective genetic screening and assessment of the purity and genetic stability of the reassortant viruses. This method should be useful in basic research applications and in preparing reassortant viruses for vaccine use.

Base Composition↗

PubMed2006

A multiplex RT-PCR method for screening of reassortant live influenza vaccine virus strains.

A system based on reverse transcription polymerase chain reaction (RT-PCR) of the RNA genome was established to identify genetic composition of influenza viruses generated by reassortment between an attenuated donor virus and virulent wild type virus. The primers were designed, by multiple sequence alignment of variable regions, specific for cold-adapted donor virus HTCA-A 101, as compared to other influenza A viruses. The specificity of each primer set was confirmed and the primers were combined to perform RT-PCR in multiplex manner. The multiplex PCR was adopted to distinguish the 6:2 reassortant viruses containing six internal genome segments of attenuated donor virus and two surface antigens of virulent strain from the wild type viruses. The method allowed us to optimize the reassorting process on a routine basis and to confirm the selection of reassortant clones efficiently. The method is suitable for analyzing the contribution of specific gene segments for growth and attenuating characteristics and for generation of live attenuated vaccine by annual reassortment.

Adaptation, Physiological↗

PubMed2002

Preparation and characterisation of attenuated cold-adapted influenza A reassortants derived from the A/Leningrad/134/17/57 donor strain.

The development of a rapid cell culture method for the preparation of cold-adapted (ca) influenza A reassortant viruses is described and compared with a currently used egg method. Mixtures of the ca donor A/Leningrad/134/17/57-ca (A/Len/17) and A/Beijing/32/92 (A/Beij/32), a recent H3N2 epidemic strain, were used to co-infect chicken embryo kidney (CEK) cell cultures; reassortant progeny were selected using an infectious centre assay. The assay was capable of detecting interference where the infectivity ratio for A/Len/17 and A/Beij/32 was 1:7. Progeny viruses were characterised genetically by amplification of defined regions within each of the six internal genes by PCR and identification of the products by restriction enzyme analysis. Reassortants were also tested for ca and temperature-sensitive (ts) phenotype and the identity of the surface antigens. The infectious centre assay was shown to be an effective method for isolating reassortant progeny that possessed the haemagglutinin and neuraminidase surface antigens of A/Beij/32. Reassortants with the six internal genes derived from the donor strain and possessing the ca and ts phenotype were readily obtained when (a) an infectivity ratio of 1:49 was used and (b) two plaque-to-plaque isolations of progeny virus were made after growth at 25 degrees C and one at 34 degrees C, both in the presence of antiserum to the donor strain.

Animals↗

PubMed2003

Identification of parental origin of cognate dsRNA genome segment(s) of rotavirus reassortants by constant denaturant gel electrophoresis.

Rotaviruses are the single most important etiologic agents of severe diarrhea in infants and young children worldwide. They possess a triple capsid morphology and a genome of 11 segments of double-stranded (ds) RNA. During the course of the development of various live, attenuated reassortant rotavirus vaccines, we often experienced difficulty in identifying the parental origin of certain genome segment(s) of a reassortant vaccine candidate. Various assays have been utilized for determination of the parental origin of reassortant virus genes, including polyacrylamide gel electrophoresis (PAGE), DNA and/or RNA hybridization assays and gene sequence analysis. The traditional PAGE is simple and easy to perform, however, it is common to find that certain cognate dsRNA segment(s) cannot be differentiated by this assay due to a high degree of sequence homology among different rotavirus strains. Constant denaturant gel electrophoresis (CDGE) is one of several methods that have been used to screen DNA fragments for small sequence changes or point mutations. By using the CDGE, we were successful in partially denaturing rotavirus dsRNA thereby changing the physical properties of the genome segment(s) in the gel and thus differentiating the cognate genome segment(s) of rotavirus reassortants. The CDGE provides a simple and reliable assay system for identification of parental gene origins of a rotavirus reassortant.

Electrophoresis, Polyacrylamide Gel↗

PubMed1992

Interspecies transmission and reassortment of influenza A viruses in pigs and turkeys in the United States.

Genetic reassortment between influenza A viruses in humans and in animals and birds has been implicated in the appearance of new pandemics of human influenza. To determine whether such reassortment has occurred in the United States, the authors compared the genetic origins of gene segments of 73 swine influenza virus isolates (1976-1990), representing 11 states, and 11 turkey virus isolates (1980-1989), representing eight states. The host origin of gene segments encoding the internal proteins of H1N1 swine and turkey influenza viruses was identified by developing a dot-blot assay. All gene segments of swine influenza viruses were characteristic of influenza virus genes from that species, indicating that pigs may not be frequent participants in interspecies genetic exchange and reassortment of influenza viruses in the United States. In contrast, 73% of the turkey influenza virus isolates contained genes of swine origin. One turkey isolate was a reassortant having three genes characteristic of avian influenza virus and three of swine origin. These findings document a high degree of genetic exchange and reassortment of influenza A viruses in domestic turkeys in the United States. The molecular biologic techniques used by the authors should aid future epidemiologic studies of influenza pandemics.

Animals↗

PubMed2004

Infant immune response to human rotavirus serotype G1 vaccine candidate reassortant WI79-9: different dose response patterns to virus surface proteins VP7 and VP4.

BACKGROUND: Rotavirus is the leading cause of morbidity from gastroenteritis in the developed world and the leading cause of mortality from viral gastroenteritis (estimated 600000 deaths) worldwide. G1 is the most prevalent human serotype. Reassortant rotavirus between simian rotavirus RRV or bovine rotavirus WC3 and human strain rotaviruses have been extensively tested as candidate vaccines. Rotavirus (RV) reassortant strain WI79-9 consists of a human (strain WI79) G1 serotype VP7 surface protein on a bovine (strain WC3) background. It is a key component of a pentavalent (G1, G2, G3, G4 and P1) WC3 reassortant vaccine candidate, RotaTeq, now being tested in Phase III clinical trials. METHODS: We studied 84 infants between the ages of 2 and 8 months who received 3 oral doses of WI79-9. Serum neutralizing antibody was measured to the human (WI79 serotype P1 G1) and bovine (WC3 serotype P7 G6) parent RV after each dose. A significant response was defined as a > or =3-fold rise in antibody titer between the predose and postdose sera. RESULTS: In two separate cohorts of vaccinees given three doses of WI79-9 reassortant rotavirus, 68 to 75% of infants demonstrated a significant response to WC3 (VP4, P7) after Dose 1, fewer (24 to 39%) responses were detected after Dose 2 and rare (0 to 4%) additional responses occurred after Dose 3. The cumulative response rate to WC3 after three doses was 95% in both trials. In contrast 23 to 37% had a significant response to WI79 (VP7, G1) after Dose 1, and 57 to 61% had a significant response after Dose 2. Additional significant responses after Dose 3 led to a cumulative response of 70 to 84%. CONCLUSION: Two doses of G1 reassortant WI79 were necessary to induce significant antibody responses to human G1 (VP7) antigen in >50% of infants. Three doses were required to achieve significant antibody responses to VP7 in >70% of infants.

Animals↗

PubMed2003

Structures of rotavirus reassortants demonstrate correlation of altered conformation of the VP4 spike and expression of unexpected VP4-associated phenotypes.

Numerous prior studies have indicated that viable rotavirus reassortants containing structural proteins of heterologous parental origin may express unexpected phenotypes, such as changes in infectivity and immunogenicity. To provide a structural basis for alterations in phenotypic expression, a three-dimensional structural analysis of these reassortants was conducted. The structures of the reassortants show that while VP4 generally maintains the parental structure when moved to a heterologous protein background, in certain reassortants, there are subtle alterations in the conformation of VP4. The alterations in VP4 conformation correlated with expression of unexpected VP4-associated phenotypes. Interactions between heterologous VP4 and VP7 in reassortants expressing unexpected phenotypes appeared to induce the conformational alterations seen in VP4.

Animals↗

PubMed2003

Generation and characterization of an H9N2 cold-adapted reassortant as a vaccine candidate.

H9N2 subtype avian influenza viruses have been identified in avian species worldwide, and infections in pigs were confirmed in Hong Kong in 1998. Subsequently, H9N2 viruses were isolated from two children in Hong Kong in 1999, and five human infections were reported from China, raising the possibility that H9N2 viruses pose a potential pandemic threat for humans. These events prompted us to develop a vaccine candidate to protect humans against this subtype of influenza A viruses. Reassortant H1N1 and H3N2 human influenza A viruses with the six internal gene segments of A/Ann Arbor/6/60 (H2N2)(AA) cold-adapted (ca) virus have been tested extensively in humans and have proved to be attenuated and safe as live virus vaccines. Using classical genetic reassortment, we generated a reassortant that contains the hemagglutinin and neuraminidase genes from A/chicken/Hong Kong/G9/97 (H9N2) and six internal gene segments from the AAca virus. The G9/AAca reassortant virus exhibits the ca phenotype and the temperature-sensitive phenotypes of the AAca virus and was attenuated in mice. The reassortant virus was immunogenic and protected mice from wild-type H9N2 virus challenge. The G9/AAca virus bears the in vitro and in vivo phenotypes specified by the AAca virus and will be evaluated as a potential vaccine candidate in humans.

Acclimatization↗

PubMed1993

Production and characterization of VP4/VP7 reassortant swine rotaviruses derived from Gottfried and OSU parental strains.

The ability of viral glycoproteins (VP) VP4/VP7 reassortant swine rotaviruses (RV) to induce cross-neutralizing antibody against parental serotypes was investigated in guinea pigs. Using selective culture conditions, we produced 10 reassortant viruses that contained gene segment 4 of the OSU RV strain and gene segment 9 of the Gottfried RV strain. These reassortant RV grew to high titer in cell culture and were neutralized by monospecific antisera against both parental RV strains. The reassortant RV were chemically inactivated with binary ethylenimine, adjuvanted with aluminum hydroxide, and used to produce antisera in guinea pigs. The hyperimmune antisera had high neutralization titer against both parent RV strains. These results indicate that several of the reassortant RV may be capable of inducing neutralizing antibodies to VP4 and VP7 and may have future use as bivalent vaccine strains.

Animals↗

PubMed1998

Genetic reassortment among viruses causing hantavirus pulmonary syndrome.

In order to determine the frequency and characteristics of reassortment among viruses causing hantavirus pulmonary syndrome (HPS), mixed infections were initiated in tissue culture by using two closely related strains of Sin Nombre virus, CC107 (from eastern California) and NMR11 (from New Mexico), which share the same species of rodent host in nature, the deer mouse (Peromyscus maniculatus). Potential reassortant virus plaques were screened by multiplex RT-PCR, using primers specific for individual genome segments of each strain. Reassortant viruses involving the M and S segments and, to a lesser extent, the L segment were detected in 8.5% of 294 progeny plaques tested. In addition, approximately 30% of the progeny virus plaques appeared to contain S or M segments originating from both parental virus strains, i.e., they were diploid. Most of these diploid virus genotypes were not stable, becoming either reassortant or parental virus strains upon plaque-to-plaque virus passage. In contrast to the results above, only one virus reassortant and four diploids were observed among 163 progeny virus plaques from mixed infections between Sin Nombre virus NMR11 and the genetically more distant Black Creek Canal virus, an HPS-causing virus from Florida, which has the cotton rat (Sigmodon hispidus) as its natural host.

Animals↗