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PubMed2003

Reassortants of H5N1 influenza viruses recently isolated from aquatic poultry in Hong Kong SAR.

The H5N1 virus (H5N1/97) that caused the bird flu incident in Hong Kong in 1997 has not been isolated since the poultry slaughter in late 1997. But the donor of its H5 hemagglutinin gene, Goose/Guangdong/1/96-like (Gs/Gd/96-like) virus, established a distinct lineage and continued to circulate in geese in the area. In 2000, a virus from the Goose/Guangdong/1/96 lineage was isolated for the first time from domestic ducks. Subsequently, it has undergone reassortment, and these novel reassortants now appear to have replaced Gs/Gd/96-like viruses from its reservoir in geese and from ducks. The internal gene constellation is also different from H5N1/97, but these variants have the potential for further reassortment events that may allow the interspecies transmission of the virus.

Animals↗

PubMed1999

Generation and characterization of reassortant influenza A viruses propagated in serum-free cultured MDCK-SF1 cells.

The replacement of embryonated chicken eggs by tissue culture cells for the production of influenza vaccines is likely to take place in the near future. Vaccines have already been produced in Madin Darby Canine Kidney (MDCK) cells (Brands et al, in this issue) and extensively tested in phase III trials in humans (Palache et al, in this issue) and it seems a matter of time before such vaccines will become available. For this reason, the generation of high-growth reassortants of influenza A virus strains in MDCK cells has been examined. Influenza A virus reassortants of the field strains A/Taiwan/1/86, A/Johannesburg/82/96 and A/Shenzhen/227/95 (all H1N1) were generated in serum-free cultured MDCK-SF1 cells by dual infection with A/Hong Kong/2/68 (H3N2), a strain selected for its high-growth phenotype. These reassortant viruses all contained at least the matrix gene of A/Hong Kong/2/68 which apparently correlates with an improvement of the viral yield.

Animals↗

PubMed1989

Genetic relatedness of the kemerovo serogroup viruses: II. RNA-RNA blot hybridization and gene reassortment in vitro of the Great Island serocomplex.

The majority of the thirty-two Great Island serocomplex isolates examined exhibit distinct dsRNA polyacrylamide gel profiles. Yet, these viruses are closely related by blot hybridization with only two genes showing significant sequence divergence. Gene reassortment was demonstrated between selected pairs of the Great Island serocomplex viruses with two different geographic regions represented. The majority of the reassortant progeny from the cross of selected pairs resulted in progeny with multiple gene-replacements. The ability of these selected isolates to reassort confirms the close taxonomic relationship of the isolates in spite of their geographic distribution.

Electrophoresis, Polyacrylamide Gel↗

PubMed1989

Genetic relatedness of the Kemerovo serogroup viruses: III. RNA-RNA blot hybridization and gene reassortment in vitro of the Chenuda serocomplex.

The dsRNA polyacrylamide gel profiles of five Chenuda serocomplex viruses were distinct. Blot hybridization and gene reassortment in vitro studies demonstrated that the Chenuda serocomplex may be divided into three sets: Chenuda, Huacho, and Mono Lake. Genes were highly conserved among members of each set, whereas genes were not highly conserved between members of different sets. Gene reassortment was demonstrated in intra-set crosses, but inter-set crosses did not yield reassortant progeny. The taxonomic significance of these data to the Chenuda serocomplex is discussed.

Electrophoresis, Polyacrylamide Gel↗

PubMed1988

Genetic relatedness of the Kemerovo serogroup viruses: I. RNA-RNA blot hybridization and gene reassortment in vitro of the Kemerovo serocomplex.

The dsRNA profiles of the Czechoslovakian and Siberian serotypes of the Kemerovo serocomplex viruses examined were similar in agarose, while their dsRNA profiles were distinct in polyacrylamide gel. Blot hybridization studies of the Kemerovo serocomplex viruses demonstrated that the genes were highly conserved among the members within each type, but not between types. Gene reassortment in vitro was demonstrated among selected pairs of the Kemerovo serocomplex viruses by intra- and inter-typic crosses. The majority of the reassortant progeny from inter-typic crosses were single gene replacements, whereas the majority of the reassortant progeny from intra-typic crosses were multiple gene replacements suggesting that certain gene combinations were restrictive under conditions of the experiment.

Genes, Viral↗

PubMed1988

[Genetic basis of influenza virus virulence: gene composition and virulence of reassortants between mouse-adapted and nonadapted strains from different subtypes].

Reassortment analysis of the pneumovirulence for mice marker of influenza virus has been performed. The original A/USSR/90/77 (H1H1) influenza virus strain or its mouse-adapted variant were crossed with a variant of A/Aichi/2/68 (H3N2) influenza virus highly virulent for mice. The reassortant having HA gene of the original A/USSR/90/77 virus and the other genes of the highly virulent A/Aichi/2/68 strain was avirulent for mice, whereas a similar reassortant possessing HA gene of the mouse-adapted A/USSR/90/77 strain was as virulent as A/Aichi/2/68 parent virus. The reasortant having HA and M genes of A/Aichi/2/68 and other genes of the mouse-adapted A/USSR/90/77 was moderately virulent, resembling in this respect the latter parent. The data indicates that changes in the different genes in course of viral adaptation to mice result in a differential acquisition of virulence for mice.

Adaptation, Biological↗

PubMed2005

Isolation and molecular characterization of a naturally occurring non-structural protein 5 (NSP5) gene reassortant of group A rotavirus of serotype G2P[4] with a long RNA pattern.

We report here two unusual strains of group A rotavirus, AU85 and AU102, isolated from children with diarrhea. These strains showed an unusual combination of serotype G2 and a long RNA pattern. RNA-RNA hybridization assays showed that these strains are reassortants in which a single genome segment 11 (the NSP5 gene) was derived from a Wa genogroup strain, while other 10 genome segments from a DS-1 genogroup strain. Phylogenetic analysis showed that the NSP5 gene of strain AU85 did not form cluster with Wa strain, while it belonged to the cluster of YM and other porcine strains. Phylogenetic analysis also showed that NSP5 and VP7 genes of AU85 were derived from the rotavirus circulating in the area. Both co-electrophoresis and RNA-RNA hybridization showed that AU85 and AU102 are identical strains. Moreover, the nucleotide sequence comparison between these two strains revealed that they had 100% identical NSP4, NSP5, and VP7 genes. These results suggest that AU85 was a reassortant formed relatively recently between rotaviruses belonging to the Wa and the DS-1 genogroup.

Child↗

PubMed1994

Infection of children with avian-human reassortant influenza virus from pigs in Europe.

Pigs have been proposed to act as the intermediate hosts in the generation of pandemic human influenza strains by reassortment of genes from avian and human influenza virus strains. The circulation of avian-like H1N1 influenza viruses in European pigs since 1979 and the detection of human-avian reassortants in pigs raises the question of whether these viruses actually have the potential to transmit and cause disease in humans. We now report the serologic and genetic characterization of two human influenza A viruses (A/Netherlands/5/93 [H3N2] and A/Netherlands/35/93 [H3N2]) that caused influenza in children in The Netherlands in 1993. The results show that these viruses are human-avian ressortants that were generated and currently still are circulating in European swine. This shows the pivotal role that pigs can play in the generation and transmission of avian influenza virus genes to humans and their potential to generate a new human pandemic strain.

Animals↗

PubMed1999

High-yield reassortant influenza vaccine production virus has a mutation at an HLA-A 2.1-restricted CD8+ CTL epitope on the NS1 protein.

Current influenza virus vaccines are prepared using high-yield reassortant virus strains obtained from a mixed infection of the new virus strain and a prototype high-yielding virus strain. The high-titered reassortant virus strain used as vaccine seed virus possesses the recent virus HA and NA and contains the internal genes from the high-growing prototype parent. We established a human CD8(+) cytotoxic T cell (CTL) line, 10-2C2, which recognizes an HLA-A2.1-restricted influenza A virus H1, H2, H3 cross-reactive T cell epitope on amino acids 122-130 of the NS1 protein, and unexpectedly we observed that there was decreased lysis of target cells infected with the A/Texas/36/91 (H1N1) vaccine virus strain compared to the lysis of target cells infected with the prototype A/PR/8/34 (H1N1) virus. RT-PCR results showed that the A/Texas vaccine virus strain contained a quasispecies. Approximately 50% of viral RNA of the NS1 gene had a nucleotide substitution that resulted in the N --> K amino acid change at the sixth position of the nonamer peptide. Current influenza vaccines are inactivated and do not contain the NS1 protein; however, future influenza vaccines may include live attenuated vaccines and with this mutation a live virus would fail to induce a CD8(+) CTL response to this epitope in individuals with HLA-A2.1, a very common allele, and potentially have reduced efficacy.

Base Sequence↗

PubMed2001

A reassortant bunyavirus isolated from acute hemorrhagic fever cases in Kenya and Somalia.

In late 1997 and early 1998, a large outbreak of hemorrhagic fever occurred in East Africa. Clinical samples were collected in Kenya and southern Somalia, and 27 of 115 (23%) hemorrhagic fever patients tested showed evidence of acute infection with Rift Valley fever (RVF) virus as determined by IgM detection, virus isolation, detection of virus RNA by reverse transcription-polymerase chain reaction (RT-PCR), or immunohistochemistry. However, two patients (one from Kenya and the other from Somalia) whose illness met the hemorrhagic fever case definition yielded virus isolates that were not RVF. Electron microscopy suggested these two virus isolates were members of the family Bunyaviridae. RT-PCR primers were designed to detect bunyavirus RNA in these samples. Regions of the S and L segments of the two isolates were successfully amplified, and their nucleotide sequences exhibited nearly complete identity with Bunyamwera virus, a mosquito-borne virus not previously associated with severe human disease. Unexpectedly, the virus M segment appeared to be reassorted, as the sequences detected exhibited 32-33% nucleotide and 28% amino acid differences relative to the corresponding M segment sequence of Bunyamwera virus. The association of this reassortant bunyavirus, proposed name Garissa virus, with severe disease is supported by the detection of the virus RNA in acute-phase sera taken from 12 additional hemorrhagic fever cases in the region.

Animals↗

PubMed1994

Recurrent circulation of single nonstructural gene substitution reassortants among human rotaviruses with a short RNA pattern.

To determine the relative frequency of intergenogroup reassortants of rotavirus in nature, we analyzed the genetic composition of 22 electrophoretically distinct stool isolates which accounted for 95.2% of stool rotaviruses with a short RNA pattern collected during 10 rotavirus seasons. These strains all showed subgroup I and G2 specificities, but two distinct hybridization patterns were observed when the probes prepared from Wa (a member of the Wa genogroup) and KUN (a member of the DS-1 genogroup) were used. Genomic RNAs from 10 strains (accounting for 64% of the field rotaviruses with short RNA pattern) hybridized exclusively to the KUN probe, and thus belonged to the DS-1 genogroup. On the other hand, genomic RNAs of the remaining 12 strains (accounting for 36% of the field rotaviruses with short RNA pattern) formed one hybrid band with the Wa probe and 10 hybrid bands with the KUN probe. Thus, they were single gene substitution intergenogroup reassortants between members of the Wa and DS-1 genogroups. They had a similar genetic constellation in that a gene segment encoding either NS35 or NS34 from a Wa-like strain was introduced into a DS-1-like genome background.

Electrophoresis, Polyacrylamide Gel↗

PubMed2004

Genetic analysis of human H2N2 and early H3N2 influenza viruses, 1957-1972: evidence for genetic divergence and multiple reassortment events.

Phylogenic analysis of all gene segments of human H2N2 viruses isolated from 1957 to 1968 was undertaken to better understand the evolution of this virus subtype. Human H3N2 viruses isolated from 1968 to 1972 were also examined to investigate genetic events associated with their emergence in humans and to identify the putative H2N2 ancestral virus. All gene segments of human H2N2 viruses demonstrated divergent evolution into two distinct clades (I and II) among late H2N2 isolates. All gene segments of 1968 H3N2 viruses that were retained from human H2N2 viruses were most similar to clade I H2N2 genes. However, genes of both clades were found among H3N2 isolates of 1969-1971. Unique phylogenic topologies reflected multiple reassortment events among late H2N2 or H3N2 viruses that resulted in a variety of different genome constellations. These results suggest that H2N2 viruses continued to circulate after 1968 and that establishment of H3N2 viruses in humans was associated with multiple reassortment events that contributed to their genetic diversity.

Amino Acid Sequence↗

PubMed2000

Genetic characterization of H3N2 influenza viruses isolated from pigs in North America, 1977-1999: evidence for wholly human and reassortant virus genotypes.

Since 1998, H3N2 viruses have caused epizootics of respiratory disease in pigs throughout the major swine production regions of the U.S. These outbreaks are remarkable because swine influenza in North America had previously been caused almost exclusively by H1N1 viruses. We sequenced the full-length protein coding regions of all eight RNA segments from four H3N2 viruses that we isolated from pigs in the Midwestern U.S. between March 1998 and March 1999, as well as from H3N2 viruses recovered from a piglet in Canada in January 1997 and from a pig in Colorado in 1977. Phylogenetic analyses demonstrated that the 1977 Colorado and 1997 Ontario isolates are wholly human influenza viruses. However, the viruses isolated since 1998 from pigs in the Midwestern U.S. are reassortant viruses containing hemagglutinin, neuraminidase and PB1 polymerase genes from human influenza viruses, matrix, non-structural and nucleoprotein genes from classical swine viruses, and PA and PB2 polymerase genes from avian viruses. The HA proteins of the Midwestern reassortant swine viruses can be differentiated from those of the 1995 lineage of human H3 viruses by 12 amino acid mutations in HA1. In contrast, the Sw/ONT/97 virus, which did not spread from pig-to-pig, lacks 11 of these changes.

Animals↗

PubMed2002

Generation and characterization of six single VP4 gene substitution reassortant rotavirus vaccine candidates: each bears a single human rotavirus VP4 gene encoding P serotype 1A[8] or 1B[4] and the remaining 10 genes of rhesus monkey rotavirus MMU18006 or bovine rotavirus UK.

The global disease burden of rotavirus diarrhea in infants and young children has stimulated interest in the biological and clinical characteristics of these agents, leading to intensive efforts to develop a vaccine. A rhesus rotavirus (RRV)-based quadrivalent vaccine ("RotaShield") was licensed and administered to about 1 million infants and found to be highly effective. However, it was withdrawn because of a link with intussusception. This vaccine was developed according to a modified "Jennerian" approach in which one of the two major outer capsid proteins (VP7) shares neutralization specificity with one of the four epidemiologically important human rotavirus serotypes. The other outer capsid protein (VP4) is derived solely from RRV and is distinct from the VP4 of the four human rotavirus serotypes of epidemiologic importance. In an effort to further increase the immunogenicity of the existing VP7-based RRV quadrivalent vaccine, we generated three single VP4 gene substitution reassortant rotavirus candidate vaccines, each of which bears a single human rotavirus VP4 gene encoding P serotype 1A[8] or 1B[4] specificity while the remaining 10 genes are derived from the rhesus rotavirus. By incorporating one or two of these strains into the quadrivalent vaccine, a pentavalent or hexavalent RRV-based vaccine could be formulated thus providing antigenic coverage not only for VP7 serotype 1, 2, 3 and 4 but also for VP4 serotype 1A[8] or 1B[4], thus possibly augmenting its immunogenicity. Similarly, three single VP4 gene (P1A[8] or P1B[4]) substitution reassortants have also been generated in a background of 10 bovine (UK) rotavirus genes for addition to a second generation UK-based quadrivalent vaccine.

Animals↗

PubMed2000

Emergence of H3N2 reassortant influenza A viruses in North American pigs.

In late summer through early winter of 1998, there were several outbreaks of respiratory disease in the swine herds of North Carolina, Texas, Minnesota and Iowa. Four viral isolates from outbreaks in different states were analyzed, both antigenically and genetically. All of the isolates were identified as H3N2 influenza viruses with antigenic profiles similar to those of recent human H3 strains. Genotyping and phylogenetic analysis demonstrated that the four swine viruses had emerged through two different pathways. The North Carolina isolate is the product of genetic reassortment between human and swine influenza viruses, while the others arose from reassortment of human, swine and avian viral genes. The hemagglutinin genes of the four isolates were all derived from the human H3N2 virus circulating in 1995. It remains to be determined if either of these recently emerged viruses will become established in the pigs in North America and whether they will become an economic burden.

Animals↗

PubMed1995

Is rotavirus a population of reassortants?

Rotaviruses appear to exist as heterogeneous populations of reassortants and related variants. This phenomenon is consistent with the quasispecies concept of RNA viruses, and suggests that gene reassortment is a crucial evolutionary mechanism for this segmented virus. It also explains puzzling epidemiological findings and controversial results of vaccine-efficacy studies.

Animals↗

PubMed2005

Serotype diversity and reassortment between human and animal rotavirus strains: implications for rotavirus vaccine programs.

The development of rotavirus vaccines that are based on heterotypic or serotype-specific immunity has prompted many countries to establish programs to assess the disease burden associated with rotavirus infection and the distribution of rotavirus strains. Strain surveillance helps to determine whether the most prevalent local strains are likely to be covered by the serotype antigens found in current vaccines. After introduction of a vaccine, this surveillance could detect which strains might not be covered by the vaccine. Almost 2 decades ago, studies demonstrated that 4 globally common rotavirus serotypes (G1-G4) represent >90% of the rotavirus strains in circulation. Subsequently, these 4 serotypes were used in the development of reassortant vaccines predicated on serotype-specific immunity. More recently, the application of reverse-transcription polymerase chain reaction genotyping, nucleotide sequencing, and antigenic characterization methods has confirmed the importance of the 4 globally common types, but a much greater strain diversity has also been identified (we now recognize strains with at least 42 P-G combinations). These studies also identified globally (G9) or regionally (G5, G8, and P2A[6]) common serotype antigens not covered by the reassortant vaccines that have undergone efficacy trials. The enormous diversity and capacity of human rotaviruses for change suggest that rotavirus vaccines must provide good heterotypic protection to be optimally effective.

Animals↗

PubMed2005

A hexavalent human rotavirus-bovine rotavirus (UK) reassortant vaccine designed for use in developing countries and delivered in a schedule with the potential to eliminate the risk of intussusception.

There is an urgent need for a rotavirus vaccine, because up to 592,000 infants and young children <5 years old die each year from rotavirus diarrhea, predominantly in the developing countries. We have developed a tetravalent human-bovine rotavirus (UK) reassortant vaccine with VP7 (G) specificity for serotypes 1, 2, 3, and 4, which has been shown to be safe, immunogenic, and effective in preventing severe rotavirus diarrhea. However, because of the emergence of VP7 (G) serotype 9 as an epidemiologically important serotype and the importance of VP7 (G) serotype 8 in focal areas, we are planning to add human-bovine (UK) reassortants with G8 and G9 specificity to the tetravalent vaccine, thereby formulating a "designed" hexavalent vaccine for universal use. In addition, we propose that the vaccine be administered orally in a 2-dose schedule, with the first dose given at 0-4 weeks of age and the second dose given at 4-8 weeks of age, when infants are relatively refractory to developing intussusception, thereby avoiding the age period when naturally occurring intussusception is most prevalent (i.e., ages 3-4 months through age 9 months). In this way, there may be the potential to eliminate or at least significantly decrease the risk of intussusception associated with rotavirus vaccination.

Administration, Oral↗