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PubMed1987

Analysis of mixed infection of sheep with bluetongue virus serotypes 10 and 17: evidence for genetic reassortment in the vertebrate host.

Two seronegative sheep were infected intravenously with 10(9) PFU each of bluetongue virus (BTV) serotype 10 and BTV serotype 17. One animal experienced a mild bluetongue-like disease, and both experienced a short-duration viremia and developed neutralizing immune responses to both virus serotypes. Progeny virus was isolated from venous blood from each animal by using conditions in which reassortment could not have occurred during isolation. Electropherotypes were determined for the progeny viruses from the infected sheep, yielding strikingly similar results for the two animals. In both sheep, serotype 10 dominated among the progeny, accounting for 92% of the progeny. Serotype 17 was rarely isolated and accounted for 3% of the progeny analyzed. The remaining 5% of the progeny clones were reassortant and derived genome segments from both serotypes 10 and 17. Analysis of the parental origin of genome segments in the small number of reassortant progeny analyzed suggested that selection of specific genome segments may have occurred in the infected sheep. These data indicate that reassortment of genome segments occurs, at low frequency, in sheep mixedly infected with BTV.

Animals↗

PubMed1988

Four viral genes independently contribute to attenuation of live influenza A/Ann Arbor/6/60 (H2N2) cold-adapted reassortant virus vaccines.

Clinical studies previously demonstrated that live influenza A virus vaccines derived by genetic reassortment from the mating of influenza A/Ann Arbor/6/60 (H2N2) cold-adapted (ca) donor virus with epidemic wild-type influenza A viruses are reproducibly safe, infectious, immunogenic, and efficacious in the prevention of illness caused by challenge with virulent wild-type virus. These influenza A reassortant virus vaccines also express the ca and temperature sensitivity (ts) phenotypes in vitro, but the genes of the ca virus parent which specify the ca, ts, and attenuation (att) phenotypes have not adequately been defined. To identify the genes associated with each of these phenotypes, we isolated six single-gene substitution reassortant viruses, each of which inherited only one RNA segment from the ca parent virus and the remaining seven RNA segments from the A/Korea/1/82 (H3N2) wild-type virus parent. These were evaluated in vitro for their ca and ts phenotypes and in ferrets, hamsters, and seronegative adult volunteers for the att phenotype. We found that the polymerase PA gene of the ca parent specifies the ca phenotype and that the PB2 and PB1 genes independently specify the ts phenotype. The PA, M, PB2, and PB1 genes of the ca donor virus each contribute to the att phenotype. The finding that four genes of the ca donor virus contribute to the att phenotype provides a partial explanation for the observed phenotypic stability of ca reassortant viruses following replication in humans.

Adult↗

PubMed2006

Terminal reassortment drives the quantum evolution of type III effectors in bacterial pathogens.

Many bacterial pathogens employ a type III secretion system to deliver type III secreted effectors (T3SEs) into host cells, where they interact directly with host substrates to modulate defense pathways and promote disease. This interaction creates intense selective pressures on these secreted effectors, necessitating rapid evolution to overcome host surveillance systems and defenses. Using computational and evolutionary approaches, we have identified numerous mosaic and truncated T3SEs among animal and plant pathogens. We propose that these secreted virulence genes have evolved through a shuffling process we have called "terminal reassortment." In terminal reassortment, existing T3SE termini are mobilized within the genome, creating random genetic fusions that result in chimeric genes. Up to 32% of T3SE families in species with relatively large and well-characterized T3SE repertoires show evidence of terminal reassortment, as compared to only 7% of non-T3SE families. Terminal reassortment may permit the near instantaneous evolution of new T3SEs and appears responsible for major modifications to effector activity and function. Because this process plays a more significant role in the evolution of T3SEs than non-effectors, it provides insight into the evolutionary origins of T3SEs and may also help explain the rapid emergence of new infectious agents.

Bacterial Proteins↗

PubMedDate not supplied

[The isolation and characteristics of reassortants between the Lassa and Mopeia arenaviruses].

Reassortants with a mixed phenotype were produced by combined inoculation of Vero cells with Lassa and Mopeya viruses. These reassortants produced small plaques (Mopeya virus phenotype) and were not pathogenic for newborn mice (Lassa virus phenotype). The genotype of the reassortants was studied by dot hybridization experiments on filters using cDNA-probes differentiating genome segments of these viruses. The reassortants were shown to have Mopeya virus L-RNA and Lassa virus S-RNA.

Animals↗

PubMed1988

[Properties of reassortants of human and avian influenza viruses. Reproduction in MDCK cells at suboptimum temperatures].

A series of reassortants has been constructed by crossing of UV-inactivated avian influenza virus of H3N8 subtype and live human influenza virus of H1N1 subtype, adapted to growth in continuous canine kidney cell line (MDCK). The analysis of RNA duplexes has shown that the reassortants contain HA gene of avian influenza virus whereas the other genes belong to human parent virus. The reassortants were efficiently reproduced in MDCK cells at low temperature (limiting for the avian parent virus). The data suggest that the avian virus HA gene does not hamper the reproduction of reassortant viruses in mammalian cells under the conditions unfavorable for the multiplication of avian influenza subtype H3N8 viruses.

Animals↗

PubMedDate not supplied

[Immunogenicity of attenuated reassortants of influenza A virus /Leningrad/134/47/57 (H2N2) for mice, depending on their genome].

Reassortants of cold-adapted strain A/Leningrad/134/47/57 (H2N2) and virulent strain A/PR/8/34 (H1N1), modeling genomic composition of vaccinal strains, were tried in mouse experiments. Reassortants' genomes included the major part (5, 6, or 7) from cold-adapted strain and 1 to 3 genes of virulent strain. All the tested reassortants did not differ by temperature sensitivity or cold adaptation phenotypes, did not cause the death of mice, but differed by the level of reproduction in murine lungs and by immunogenicity. Strains with genomic formulas 6/2 (HA and NA of a virulent strain) and 5/3 (M) (HA, NA, and M of a virulent strain) were characterized by the highest immunogenicity. Reassortants including, besides HA and NA, PA gene 5/3 (PA) or inheriting only HA gene from strain A/pr/8/34 (H1N1) were hyperattenuated and low immunogenic.

Animals↗

PubMed1995

Preferential selection of VP7 gene from a parent rotavirus strain (SA11) in sequential passages after mixed infection with SA11 and SA11-human rotavirus single-VP7 gene-substitution reassortants.

We studied the competitive growth among SA11-L2(G3) and its single-human VP7 gene-substitution reassortants SA11-L2/KU-R1(G1) and SA11-L2/DS1-R1(G2), which have the genetic background of SA11-L2, during sequential passages after mixed infection. When the same infectious units (m.o.i. of 5 p.f.u./cell) of SA11-L2 and a reassortant SA11-L2/KU-R1 were inoculated onto and passaged in MA104 cells, 88% of the virus clones isolated from the culture fluid at the 3rd passage belonged to G3, and all the clones from the 10th passage had G3 specificity. Even when SA11-L2/KU-R1 with titer 10 times higher than that of SA11-L2 was used in the coinfection, the predominance of clones with G3-VP7 was observed. Although G2 clones slightly surpassed G1 clones in number in the mixed culture of SA11-L2/KU-R1 and SA11-L2/DS1-R1, G3 clones predominated in the virus progeny from a mixed culture infected with the same titers of SA11-L2, SA11-L2/KU-R1, and SA11-L2/DS1-R1. However, no significant difference in viral growth was detected among SA11-L2 and the two reassortants.

Antigens, Viral↗

PubMed2005

Postreassortment changes in a model system: HA-NA adjustment in an H3N2 avian-human reassortant influenza virus.

In our previous studies we described the postreassortment changes in the hemagglutinin (HA) of H2N1, H3N1, H4N1 and H13N1 influenza A virus reassortants with HAs derived from avian viruses and low-functional neuraminidase (NA) of a human parent virus A/USSR/90/77 (H1N1). The changes involved amino acid substitutions that increased the negative local charge in the vicinity of the receptor-binding pocket and decreased the affinity of HA to sialic acid receptors. In the present report we describe the studies performed with H3N2 reassortant viruses having HA of A/Duck/Ukraine/1/63 (H3N8) virus and NA of A/Aichi/2/68 (H3N2) virus. Amino acid changes in the HA gene registered in virus variants selected in the course of serial passages resulted in a decrease in the affinity to sialic acid-containing substrates and cell receptors. However, the decrease was less expressed than in the reassortants containing the low-functional NA of N1 subtype described in our earlier studies, and the amino acid changes were not necessarily associated with an increase of negative charge. In one passage variant an amino acid substitution in NA was detected. The relevance of these results for the evolution of the H3N2 virus of the 1968 pandemic is discussed.

Amino Acid Substitution↗

PubMed2005

Synthesis of infectious in vitro transcripts from Cassia yellow blotch bromovirus cDNA clones and a reassortment analysis with other bromoviruses in protoplasts.

Cassia yellow blotch virus (CYBV), genus Bromovirus, was isolated from the Australian native legume, Cassia pleurocarpa, in western Queensland, and its host range was found to be distinct from other bromoviruses. In this study, CYBV was shown to infect systemically and efficiently a model plant species, Arabidopsis thaliana, as we recently reported for another bromovirus, Spring beauty latent virus (SBLV). We constructed full-length cDNA clones of CYBV genomic RNAs from which infectious in vitro transcripts can be transcribed, and determined their complete nucleotide sequences. CYBV RNA3 contains the box B motif in the intercistronic region, but lacks the subgenomic promoter-like sequence in the 5' noncoding region, as does Brome mosaic virus (BMV). To understand relationships among bromoviruses, we generated reassortants between CYBV and three other bromoviruses, BMV, SBLV and Cowpea chlorotic mottle virus. We found that all reassortants between BMV and CYBV accumulated viral RNAs to detectable levels in protoplasts of Nicotiana benthamiana, even when RNAs 1 and 2, which encode the replication proteins 1a and 2a, respectively, were heterologous. Sequence comparison and reassortment experiments of CYBV and other bromoviruses demonstrated that CYBV is closely related to BMV.

Bromovirus↗

PubMed2006

Genetic characterization of novel reassortant H1N2 influenza A viruses isolated from pigs in southeastern China.

In December 2004, three influenza H1N2 viruses were isolated from lung samples of pigs that had died from respiratory disease on a farm in southeastern China. To determine the genetic characterization and probable origin, one of the three isolates, A/Swine/Zhejiang/1/2004 (Sw/ZJ/1/2004), was genetically analyzed. Sw/ZJ/1/2004 was a reassortant with an NA gene most closely related to the corresponding gene from a human-like H3N2 virus circulating in 1995. The remaining seven genes were most closely related to those from the classical swine H1N1 virus. Sw/ZJ/1/2004 appeared to be a novel reassortant H1N2 virus that was genetically distinguishable from other H1N2 viruses found in pigs worldwide. The isolation of Sw/ZJ/1/2004 provided further evidence for pigs serving as a "mixing vessel" for the generation of new reassortant genotypes of influenza viruses and emphasizes the importance of reinforcing influenza virus surveillance in pigs in China.

Amino Acid Sequence↗

PubMed2002

Prevalence of, and antigenic variation in, serotype G10 rotaviruses and detection of serotype G3 strains in diarrheic calves: implications for the origin of G10P11 or P11 type reassortant asymptomatic strains in newborn children in India.

Previous studies have shown predominant association of G10P11 type bovine rotavirus-derived reassortant strains with asymptomatic infections in newborn children in India. To understand the epidemiological and genetic basis for the origin of these strains in humans, the relative frequencies of different serotypes among bovine rotaviruses (BRVs) isolated from southern, western and central regions of the country were determined by subgroup and serotype analysis as well as nucleotide (nt) sequence analysis of the genes encoding the outer capsid proteins VP4 and VP7. Since the human G10P11 asymptomatic neonatal strain I321 possessed NSP1 from a human rotavirus, to determine its genetic origin in the bovine strains, comparative analysis of partial gene sequences from representative G10P11 strains was also carried out. The following observations were of great epidemiological significance, (i) G10P11 strains predominated in all the three regions with frequencies ranging between 55.6% and 85.2%. In contrast to the high prevalence of G6 strains in other countries, only one G6 strain was detected in this study and G8 strains represented 5.8% of the isolates, (ii) among the G10 strains, in serotyping ELISA, four patterns of reactivity were observed that appeared to correlate with the differences in electropherotypic patterns and amino acid (aa) sequence of the VP7, (iii) surprisingly, strains belonging to serotype G3 were detected more frequently (10.7%) than those of serotypes G6 and G8 combined, while strains representing the new serotype (G15) were observed in a single farm in Bangalore, and (iv) about 3.9% of the isolates were nontypeable as they exhibited high cross-reactivity to the serotyping MAbs used in the study. Comparative analysis of the VP7 gene sequence from the prototype G3 MAb-reactive bovine strain J63 revealed greatest sequence relatedness (87.6% nt and 96.0% aa) with that of serotype G3 rhesus-monkey strain RRV. It also exhibited high sequence homology with the VP7 from several animal and animal rotavirus-related human G3 strains (Simian SA11; equine ERV316 and FI-14; canine CU-1 and K9; porcine 4F; Feline Cat2 and human HCR3, YO and AU1). Partial nucleotide sequence analysis of the NSP1 gene of J63 showed greatest nt sequence homology (95.9%) to the NSP1 gene allele of the Indian G8 strain, isolated from a diarrheic child, which is likely to have been transmitted directly from cattle and 92.6% homology to that of the bovine G8 strain A5-10 suggesting the likely origin of J63 by gene reassortment between a bovine G8 strain and a G3 animal strain. Prevalence of G10P11 strains in cattle and G10P11 or P11 type reassortant strains in asymptomatic neonates as well as detection of G8P[1] strains in diarrheic children support our hypothesis for bidirectional transmission of rotaviruses between humans and cattle and origin of novel strains catalyzed by the age-old traditions and socio-economic conditions in India.

Amino Acid Sequence↗

PubMed1995

Differentiation of cognate dsRNA genome segments of bluetongue virus reassortants by temperature gradient gel electrophoresis.

The analysis of reassortant viruses has been a valuable tool in the investigation of protein interaction and function in double-stranded (ds) RNA virus research. The differentiation of cognate dsRNA genome segments of reassortants is conventionally achieved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). However, due to a high degree of sequence homology among different bluetongue virus (BTV) serotypes, it is not uncommon to find that certain cognate dsRNA segments cannot be differentiated by SDS-PAGE. Temperature gradient gel electrophoresis (TGGE) has been shown to be a much more sensitive method of differentiating RNA or DNA fragments of high sequence homology. Here we report the preliminary application of TGGE in analysis of genomic reassortants of two BTV serotypes, 1 and 23. While six out of ten genome segments between BTV-1 and BTV-23 could not be resolved by SDS-PAGE, all of them were differentiated by TGGE. The ability of TGGE to distinguish between dsRNA segments of high sequence homology may also make it useful in the search for BTV genes responsible for defined characteristics, such as virulence, by differentiating wild-type and mutated gene segments of viruses displaying altered phenotypes.

Bluetongue virus↗

PubMed2006

Effects of the potency and composition of the multivalent human-bovine (WC3) reassortant rotavirus vaccine on efficacy, safety and immunogenicity in healthy infants.

BACKGROUND: Rotavirus gastroenteritis, which causes substantial infant mortality and morbidity worldwide, is a vaccine-preventable disease. The purpose of this study was to evaluate different compositions and potencies (vaccine virus titers) of a live multivalent human-bovine (WC3) reassortant rotavirus vaccine in order to select the potency and composition of the vaccine for further development. METHODS: The efficacy, safety, and immunogenicity of a G1, G2, G3, G4, and P1A pentavalent composition at three different potencies, a G1, G2, G3, G4 quadrivalent composition, and a P1A monovalent composition of an oral human-bovine (WC3) reassortant rotavirus vaccine were compared in a blinded, placebo-controlled trial conducted between 1998 and 2001 enrolling 1,946 healthy Finnish infants 2-8 months of age. RESULTS: All potencies of the pentavalent and quadrivalent vaccines were efficacious (58-74%) against wild-type rotavirus gastroenteritis of any severity and 100% protective against severe rotavirus disease caused by vaccine G-serotypes through the first rotavirus season post-vaccination. The monovalent P1A vaccine was 53% efficacious against moderate-and-severe rotavirus gastroenteritis. Protection against rotavirus gastroenteritis of any severity was demonstrated through two and three rotavirus seasons for all vaccine compositions. After the third dose, the percentage of infants with >or=3-fold rise in baseline serum neutralizing antibody titers against G1 ranged from 62% to 86% for recipients of the pentavalent vaccine, depending on the potency. The incidence of fever, irritability, vomiting, and diarrhea did not significantly differ between vaccine and placebo groups. A 7-month-old male developed intussusception 9 days after the first dose of the low-potency pentavalent vaccine. CONCLUSIONS: Based on the results of this trial, a pentavalent composition (G1, G2, G3, G4, and P1A) of human-bovine (WC3) reassortant rotavirus vaccine with a potency similar to that of the middle-potency pentavalent vaccine ( approximately 8 x 10(6) plaque-forming units/dose) was selected for further development.

Animals↗

PubMed2004

Reassortment and evolution of current human influenza A and B viruses.

During the 2001-2002 influenza season, human influenza A (H1N2) reassortant viruses were detected globally. The hemagglutinin (HA) of these H1N2 viruses was similar to that of the A/New Caledonia/20/99 (H1N1) vaccine strain both antigenically and genetically, while their neuraminidase (NA) was antigenically and genetically related to that of recent human influenza H3N2 reference viruses such as A/Moscow/10/99. All six internal genes of the H1N2 reassortants originated from an H3N2 virus. After being detected only in eastern Asia during the past 10 years, Influenza B/Victoria/2/87 lineage viruses reappeared in many countries outside of Asia in 2001. Additionally, reassortant influenza B viruses possessing an HA similar to that of B/Shandong/7/97, a recent B/Victoria/2/87 lineage reference strain, and an NA closely related to that of B/Sichuan/379/99, a recent B/Yamagata/16/88 lineage reference strain, were isolated globally and became the predominant influenza B epidemic strain. The current influenza vaccine is expected to provide good protection against H1N2 viruses because it contains A/New Caledonia/20/99 (H1N1) and A/Panama/2007/99 (H3N2) like viruses whose H1 HA or N2 NA are antigenically similar to those of recent circulating H1N2 viruses. On the other hand, widespread circulation of influenza B Victoria lineage viruses required inclusion of a strain from this lineage in influenza vaccines for the 2002-2003 season.

Evolution, Molecular↗

PubMedDate not supplied

Protective responses in mice to vaccination with multiply administered cold-adapted influenza vaccine reassortants and wild-type viruses.

Protective responses to influenza vaccine reassortants derived from the cold-adapted (ca) donor strains A/Leningrad/134/17/57 and B/USSR/60/69 and wild-type epidemic viruses were studied in two strains of mice. Preliminary experiments revealed that, when mixtures of three viruses were inoculated intranasally to mice with 50 microliters containing 10(6) EID50 per 200 microliters (10(5.4) EID50 per mouse), interference between strains did not occur. However, interference with the growth of the influenza reassortant B/60/32/R took place if its concentration in the mixture was reduced to 10(5) (10(4.4) per mouse) or if it was inoculated at 10(6) EID50 (10(5.4) per mouse) in the presence of the influenza reassortant R/34 and two other influenza A epidemic strains; interference was unrelated to serological responses to infection with B/60/32/R. Despite evidence of interference, mice inoculated with the same mixtures in two identical doses, three weeks apart, were able to clear a challenge from each of seven homotypic and heterotypic influenza A and B strains. Heterotypic clearance of influenza A challenge viruses was greater following mixed infection, indicating that common determinants within the surface antigen glycoproteins contributed to immune responses which were broader than could be expected to be induced by parenteral vaccination.

Administration, Intranasal↗

PubMed2006

Lack of transmission of H5N1 avian-human reassortant influenza viruses in a ferret model.

Avian influenza A H5N1 viruses continue to spread globally among birds, resulting in occasional transmission of virus from infected poultry to humans. Probable human-to-human transmission has been documented rarely, but H5N1 viruses have not yet acquired the ability to transmit efficiently among humans, an essential property of a pandemic virus. The pandemics of 1957 and 1968 were caused by avian-human reassortant influenza viruses that had acquired human virus-like receptor binding properties. However, the relative contribution of human internal protein genes or other molecular changes to the efficient transmission of influenza viruses among humans remains poorly understood. Here, we report on a comparative ferret model that parallels the efficient transmission of H3N2 human viruses and the poor transmission of H5N1 avian viruses in humans. In this model, an H3N2 reassortant virus with avian virus internal protein genes exhibited efficient replication but inefficient transmission, whereas H5N1 reassortant viruses with four or six human virus internal protein genes exhibited reduced replication and no transmission. These findings indicate that the human virus H3N2 surface protein genes alone did not confer efficient transmissibility and that acquisition of human virus internal protein genes alone was insufficient for this 1997 H5N1 virus to develop pandemic capabilities, even after serial passages in a mammalian host. These results highlight the complexity of the genetic basis of influenza virus transmissibility and suggest that H5N1 viruses may require further adaptation to acquire this essential pandemic trait.

Animals↗

PubMed2002

Intercontinental circulation of human influenza A(H1N2) reassortant viruses during the 2001-2002 influenza season.

Reassortant influenza A viruses bearing the H1 subtype of hemagglutinin (HA) and the N2 subtype of neuraminidase (NA) were isolated from humans in the United States, Canada, Singapore, Malaysia, India, Oman, Egypt, and several countries in Europe during the 2001-2002 influenza season. The HAs of these H1N2 viruses were similar to that of the A/New Caledonia/20/99(H1N1) vaccine strain both antigenically and genetically, and the NAs were antigenically and genetically related to those of recent human H3N2 reference strains, such as A/Moscow/10/99(H3N2). All 6 internal genes of the H1N2 reassortants examined originated from an H3N2 virus. This article documents the first widespread circulation of H1N2 reassortants on 4 continents. The current influenza vaccine is expected to provide good protection against H1N2 viruses, because it contains the A/New Caledonia/20/99(H1N1) and A/Moscow/10/99(H3N2)-like viruses, which have H1 and N2 antigens that are similar to those of recent H1N2 viruses.

Antigens, Viral↗

PubMed1999

Bunyavirus superinfection and segment reassortment in transovarially infected mosquitoes.

Rapid evolution of bunyaviruses may occur by RNA segment reassortment between closely related viruses. Reassortment between viruses occurs in dually infected mosquitoes when two different viruses are simultaneously ingested or when the second virus is ingested within 2 days of the first virus. By 3 days after oral infection, interference to superinfection occurs, thus limiting the potential for evolution. Aedes triseriatus mosquitoes can also be transovarially infected (TI+) with LaCrosse (LAC) virus. In these studies, the potential for oral superinfection of TI+ mosquitoes was assessed. Approximately 20% of mosquitoes TI+ with either a temperature-sensitive LAC virus or wild-type (wt) LAC virus became superinfected by ingesting blood meals containing wt LAC or snowshoe hare (SSH) viruses. LAC virus TI+ mosquitoes superinfected with SSH virus were detected by blot hybridization or RT-PCR. Viruses from these mosquitoes were plaque purified and genotyped using RT-PCR. Reassortant genomes were detected in 2.3% of the viruses genotyped, and 4.0% of the genomes tested were diploid for one genome segment.

Aedes↗