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PubMed2006

Vaccination against equine influenza: quid novi?

Equine influenza virus is a leading cause of respiratory disease in the horse. Equine influenza vaccines containing inactivated virus were first developed in the 1960s. Despite their intensive use, equine influenza outbreaks still continue to occur and therefore new strategies of vaccination are necessary to improve vaccine efficacy. Numerous methods of vaccination have been evaluated and commercialised in the horse, the most recent being the cold-adapted influenza virus and poxvirus-based vaccines. As a large animal model, the horse is also a useful species in which to evaluate the potential of new generations of influenza vaccine such as live-attenuated influenza virus engineered by reverse genetics. This report details the equine immune responses conferring protection against influenza. It then undertakes a selective review of different strategies of vaccination against equine influenza that have been developed over the last two decades and discusses factors that may influence the efficacy of vaccination. Finally it outlines progress in the development of a novel vaccination strategy against equine influenza using reverse genetics.

Age Factors↗
PubMed2005

Role of N-linked glycans on bunyamwera virus glycoproteins in intracellular trafficking, protein folding, and virus infectivity.

The membrane glycoproteins (Gn and Gc) of Bunyamwera virus (BUN, family Bunyaviridae) contain three potential sites for the attachment of N-linked glycans: one site (N60) on Gn and two (N624 and N1169) on Gc. We determined that all three sites are glycosylated. Digestion of the glycoproteins with endo-beta-N-acetylglucosaminidase H (endo H) or peptide:N-glycosidase F revealed that Gn and Gc differ significantly in their glycan status and that late in infection Gc glycans remain endo H sensitive. The roles of the N-glycans in intracellular trafficking of the glycoproteins to the Golgi, protein folding, and virus replication were investigated by mutational analysis and confocal immunofluorescence. Elimination of the glycan on Gn, by changing N60 to a Q residue, resulted in the protein misfolding and failure of both Gn and Gc proteins to traffic to the Golgi complex. We were unable to rescue a viable virus by reverse genetics from a cDNA containing the N60Q mutation. In contrast, mutant Gc proteins lacking glycans on either N624 or N1169, or both sites, were able to target to the Golgi. Gc proteins containing mutations N624Q and N1169Q acquired endo H resistance. Three viable N glycosylation-site-deficient viruses, lacking glycans on one site or both sites on Gc, were created by reverse genetics. The viability of these recombinant viruses and analysis of growth kinetics indicates that the glycans on Gc are not essential for BUN replication, but they do contribute to the efficiency of virus infection.

Animals↗
PubMedDate not supplied

Generation of influenza A virus from cloned cDNAs--historical perspective and outlook for the new millenium.

Influenza virus reverse genetics has reached a level of sophistication where one can confidently generate virus entirely from cloned DNAs. The new systems makes it feasible to study the molecular mechanisms of virus replication and pathogenicity, as well as to generate attenuated live virus vaccines, gene delivery vehicles, and possibly other RNA viruses from cloned cDNAs. During the next decade, one can anticipate the translation of influenza virus reverse genetics into biomedically relevant advances.

Animals↗
PubMed1998

Transfectant influenza A viruses with long deletions in the NS1 protein grow efficiently in Vero cells.

We established a reverse genetics system for the nonstructural (NS) gene segment of influenza A virus. This system is based on the use of the temperature-sensitive (ts) reassortant virus 25A-1. The 25A-1 virus contains the NS gene from influenza A/Leningrad/134/57 virus and the remaining gene segments from A/Puerto Rico (PR)/8/34 virus. This particular gene constellation was found to be responsible for the ts phenotype. For reverse genetics of the NS gene, a plasmid-derived NS gene from influenza A/PR/8/34 virus was ribonucleoprotein transfected into cells that were previously infected with the 25A-1 virus. Two subsequent passages of the transfection supernatant at 40 degreesC selected viruses containing the transfected NS gene derived from A/PR/8/34 virus. The high efficiency of the selection process permitted the rescue of transfectant viruses with large deletions of the C-terminal part of the NS1 protein. Viable transfectant viruses containing the N-terminal 124, 80, or 38 amino acids of the NS1 protein were obtained. Whereas all deletion mutants grew to high titers in Vero cells, growth on Madin-Darby canine kidney (MDCK) cells and replication in mice decreased with increasing length of the deletions. In Vero cells expression levels of viral proteins of the deletion mutants were similar to those of the wild type. In contrast, in MDCK cells the level of the M1 protein was significantly reduced for the deletion mutants.

Animals↗
PubMed2006

Making better influenza virus vaccines?

Killed and live influenza virus vaccines are effective in preventing and curbing the spread of disease, but new technologies such as reverse genetics could be used to improve them and to shorten the lengthy process of preparing vaccine seed viruses. By taking advantage of these new technologies, we could develop live vaccines that would be safe, cross-protective against variant strains, and require less virus per dose than conventional vaccines. Furthermore, pandemic vaccines against highly virulent strains such as the H5N1 virus can only be generated by reverse genetics techniques. Other technologic breakthroughs should result in effective adjuvants for use with killed and live vaccines, increasing the number of available doses. Finally, universal influenza virus vaccines seem to be within reach. These new strategies will be successful if they are supported by regulatory agencies and if a robust market for influenza virus vaccines against inter-pandemic and pandemic threats is made and sustained.

Adjuvants, Immunologic↗
PubMed2006

Arabidopsis thaliana cyclic nucleotide gated channel 3 forms a non-selective ion transporter involved in germination and cation transport.

The Arabidopsis thaliana genome contains 20 cyclic nucleotide gated channel (CNGC) genes encoding putative non-selective ion channels. Classical and reverse genetic approaches have revealed that two members of this family (CNGC2 and CNGC4) play a role in plant defence responses whereas CNGC1 and CNGC10 may participate in heavy metal and cation transport. Yet, it remains to be resolved how the ion transport attributes of CNGCs are integrated into their physiological function. In this study, CNGC3 is characterized through heterologous expression, GUS- and GFP-reporter gene fusions, and by adopting a reverse genetics approach. A CNGC3-GFP fusion protein shows that it is mainly targeted to the plasma membrane. Promoter GUS studies demonstrate CNGC3 expression predominantly in the cortical and epidermal root cells, but also a ubiquitous presence in shoot tissues. Expression of CNGC3 in yeast indicates it can function as a Na(+) uptake and a K(+) uptake mechanism. cngc3 null mutations decreased seed germination in the presence of NaCl but not KCl. Relative to the wild type, mutant seedling growth is more resistant to the presence of toxic concentrations of NaCl and KCl. The ionic composition and ion uptake characteristics of wild-type and mutant seedlings suggests that the growth advantage in these conditions may be due to restricted ion influx in mutant plants, and that CNGC3 functions in the non-selective uptake of monovalent cations in Arabidopsis root tissue.

Arabidopsis↗
PubMed2007

Separation of near full-length hepatitis C virus quasispecies variants from a complex population.

A long RT-PCR (LRP) protocol was developed recently for robust amplification of a near full-length HCV genomic sequence from clinical samples, followed by efficient cloning [Fan, X., Xu, Y., Di Biceglie, A.M., 2006. Efficient amplification and cloning of near full-length hepatitis C virus genome from clinical samples. Biochem. Biophys. Res. Commun. 346, 1163-1172]. In the present study, the LRP protocol has been estimated for its error rate and the validation by sequencing fully the near full-length HCV inserts from six recombinant clones derived from a patient sample with complex viral diversity. These sequences were compared with the near full-length HCV sequence that was generated by direct sequencing of multiple overlapped PCR products from the same sample, referred to as the population sequence. Comparative analysis confirmed the artificial nature of the PCR-assembled population sequence and identified potential domains for linked viral mutations. The data also suggested that the hypervariable region 1 (HVR1) may be a biological marker for the phenotype at the quasispecies level. These observations emphasize the significance of the use of near full-length genomic sequences for HCV genetic studies and for reverse genetic analysis using authentic quasispecies variants.

Cloning, Molecular↗
PubMed2000

Recovery of pathogenic measles virus from cloned cDNA.

Reverse genetics technology so far established for measles virus (MeV) is based on the Edmonston strain, which was isolated several decades ago, has been passaged in nonlymphoid cell lines, and is no longer pathogenic in monkey models. On the other hand, MeVs isolated and passaged in the Epstein-Barr virus-transformed marmoset B-lymphoblastoid cell line B95a would retain their original pathogenicity (F. Kobune et al., J. Virol. 64:700-705, 1990). Here we have developed MeV reverse genetics systems based on the highly pathogenic IC-B strain isolated in B95a cells. Infectious viruses were successfully recovered from the cloned cDNA of IC-B strain by two different approaches. One was simple cotransfection of B95a cells, with three plasmids each encoding the nucleocapsid (N), phospho (P), or large (L) protein, respectively, and their expression was driven by the bacteriophage T7 RNA polymerase supplied by coinfecting recombinant vaccinia virus vTF7-3. The second approach was transfection with the L-encoding plasmid of a helper cell line constitutively expressing the MeV N and P proteins and the T7 polymerase (F. Radecke et al., EMBO J. 14:5773-5784, 1995) on which B95a cells were overlaid. Virus clones recovered by both methods possessed RNA genomes identical to that of the parental IC-B strain and were indistinguishable from the IC-B strain with respect to growth phenotypes in vitro and the clinical course and histopathology of experimentally infected cynomolgus monkeys. Thus, the systems developed here could be useful for studying viral gene functions in the context of the natural course of MeV pathogenesis.

Animals↗
PubMed1997

Creation of amantadine resistant clones of influenza type A virus using a new transfection procedure.

M2, the spliced segment of the matrix (M) gene of influenza A virus, is an integral membrane protein which functions as an ion channel both when the virus is in the host endosome and during protein processing in the trans-Golgi network. Amantadine inhibits replication of influenza A virus by blocking the activity of this ion channel. Reverse genetics were used to generate amantadine resistant virus mutants by introducing mutations into the M gene of cold adapted (ca) A/AA/6/60, an amantadine sensitive virus. The site directed mutagenesis involved substitutions at amino acids 27, 30 and 31, sites hypothesized to be responsible for resistance to this drug in several other influenza A viruses. This M gene was then transfected into wt A/AA/6/60, an amantadine sensitive virus, via electroporation. The desired transfectants were selected for replication in the presence of amantadine. Using this newly devised reverse genetics system to rescue a mutated gene in its homologous wild type background not only establishes the identity of amino acid mutations necessary for the establishment of amantadine resistance but will also allow us to study other mutations in the M gene without gene constellation effects. Resistance to amantadine in wt A/AA/6/60 can also occur naturally if the viruses are grown in the presence of amantadine. These spontaneously generated resistant clones contained point mutations at amino acid 30 or 31 of M2.

Amantadine↗
PubMed2006

Envelope exchange for the generation of live-attenuated arenavirus vaccines.

Arenaviruses such as Lassa fever virus cause significant mortality in endemic areas and represent potential bioterrorist weapons. The occurrence of arenaviral hemorrhagic fevers is largely confined to Third World countries with a limited medical infrastructure, and therefore live-attenuated vaccines have long been sought as a method of choice for prevention. Yet their rational design and engineering have been thwarted by technical limitations. In addition, viral genes had not been identified that are needed to cause disease but can be deleted or substituted to generate live-attenuated vaccine strains. Lymphocytic choriomeningitis virus, the prototype arenavirus, induces cell-mediated immunity against Lassa fever virus, but its safety for humans is unclear and untested. Using this virus model, we have developed the necessary methodology to efficiently modify arenavirus genomes and have exploited these techniques to identify an arenaviral Achilles' heel suitable for targeting in vaccine design. Reverse genetic exchange of the viral glycoprotein for foreign glycoproteins created attenuated vaccine strains that remained viable although unable to cause disease in infected mice. This phenotype remained stable even after extensive propagation in immunodeficient hosts. Nevertheless, the engineered viruses induced T cell-mediated immunity protecting against overwhelming systemic infection and severe liver disease upon wild-type virus challenge. Protection was established within 3 to 7 d after immunization and lasted for approximately 300 d. The identification of an arenaviral Achilles' heel demonstrates that the reverse genetic engineering of live-attenuated arenavirus vaccines is feasible. Moreover, our findings offer lymphocytic choriomeningitis virus or other arenaviruses expressing foreign glycoproteins as promising live-attenuated arenavirus vaccine candidates.

Animals↗
PubMed1997

Inhibitory effects of chronic ethanol consumption on cellular immune responses to hepatitis C virus core protein are reversed by genetic immunizations augmented with cytokine-expressing plasmids.

Chronic hepatitis C viral (HCV) infection is a major clinical problem in alcoholics with liver disease and may result from ethanol effects on the host immune response. To experimentally assess such effects, the DNA-based immunization approach was used to produce humoral and cellular immune responses against the HCV core protein in mice. Mice were fed an ethanol or isocaloric pair-fed control liquid diet followed by immunizations with HCV core DNA-expressing constructs. Chronic ethanol feeding was found to inhibit Th cell and CTL activities and substantially reduced cytokine secretion as well. In addition, a switch from Th1 to Th0 subtype was observed in proliferating CD4+ T cells derived from chronic ethanol-fed mice. These immunosuppressive effects were directly due to ethanol since crossover experiments to an isocaloric control diet restored the defects in cellular immunity. Furthermore, we determined if coadministration of an IL-2 or GM-CSF DNA expression plasmid with a plasmid expressing the HCV core protein (pHCV2-2) would reverse the inhibitory effects of chronic ethanol feeding on cellular immune responses. Coimmunization of chronic ethanol-fed mice with either IL-2 or GM-CSF expression plasmids restored cellular immunity and induced CD4+ inflammatory T cell and CD8+ CTL responses comparable with control mice immunized with pHCV2-2 alone. These studies provide evidence of how chronic ethanol feeding may effect cellular immune responses to a viral structural protein in the context of genetic immunization.

Adjuvants, Immunologic↗
PubMed2006

Establishment of a Nipah virus rescue system.

Nipah virus (NiV), a paramyxovirus, was first discovered in Malaysia in 1998 in an outbreak of infection in pigs and humans and incurred a high fatality rate in humans. Fruit bats, living in vast areas extending from India to the western Pacific, were identified as the natural reservoir of the virus. However, the mechanisms that resulted in severe pathogenicity in humans (up to 70% mortality) and that enabled crossing the species barrier were not known. In this study, we established a system that enabled the rescue of replicating NiVs from a cloned DNA by cotransfection of a constructed full-length cDNA clone and supporting plasmids coding virus nucleoprotein, phosphoprotein, and polymerase with the infection of the recombinant vaccinia virus, MVAGKT7, expressing T7 RNA polymerase. The rescued NiV (rNiV), by using the newly developed reverse genetics system, showed properties in vitro that were similar to the parent virus and retained the severe pathogenicity in a previously established animal model by experimental infection. A recombinant NiV was also developed, expressing enhanced green fluorescent protein (rNiV-EGFP). Using the virus, permissibility of NiV was compared with the presence of a known cellular receptor, ephrin B2, in a number of cell lines of different origins. Interestingly, two cell lines expressing ephrin B2 were not susceptible for rNiV-EGFP, indicating that additional factors are clearly required for full NiV replication. The reverse genetics for NiV will provide a powerful tool for the analysis of the molecular mechanisms of pathogenicity and cross-species infection.

Animals↗
PubMed2002

Sex reversal of genetic females (XX) induced by the transplantation of XY somatic cells in the medaka, Oryzias latipes.

In order to investigate the function of gonadal somatic cells in the sex differentiation of germ cells, we produced chimera fish containing both male (XY) and female (XX) cells by means of cell transplantation between blastula embryos in the medaka, Oryzias latipes. Sexually mature chimera fish were obtained from all combinations of recipient and donor genotypes. Most chimeras developed according to the genetic sex of the recipients, whose cells are thought to be dominant in the gonads of chimeras. However, among XX/XY (recipient/donor) chimeras, we obtained three males that differentiated into the donor's sex. Genotyping of their progeny and of strain-specific DNA fragments in their testes showed that, although two of them produced progeny from only XX spermatogenic cells, their testes all contained XY cells. That is, in the two XX/XY chimeras, germ cells consisted of XX cells but testicular somatic cells contained both XX and XY cells, suggesting that the XY somatic cells induced sex reversal of the XX germ cells and the XX somatic cells. The histological examination of developing gonads of XX/XY chimera fry showed that XY donor cells affect the early sex differentiation of germ cells. These results suggest that XY somatic cells start to differentiate into male cells depending on their sex chromosome composition, and that, in the environment produced by XY somatic cells in the medaka, germ cells differentiate into male cells regardless of their sex chromosome composition.

Animals↗
PubMed1999

Addition of a missense mutation present in the L gene of respiratory syncytial virus (RSV) cpts530/1030 to RSV vaccine candidate cpts248/404 increases its attenuation and temperature sensitivity.

Respiratory syncytial virus (RSV) cpts530/1030 is an attenuated, temperature-sensitive subgroup A vaccine candidate derived previously from cold-passaged RSV (cpRSV) by two sequential rounds of chemical mutagenesis and biological selection. Here, cpts530/1030 was shown to be highly attenuated in the upper and lower respiratory tracts of seronegative chimpanzees. However, evaluation in seropositive children showed that it retains sufficient replicative capacity and virulence to preclude its direct use as a live attenuated vaccine. Nucleotide sequence analysis of the genome of cpts530/1030 showed that it had acquired two nucleotide substitutions (compared to its cpts530 parent), both of which were in the L gene: a silent mutation at nucleotide position 8821 (amino acid 108) and a missense mutation at nucleotide position 12458 resulting in a tyrosine-to-asparagine change at amino acid 1321, herein referred to as the 1030 mutation. It also contained the previously identified 530 missense mutation at nucleotide 10060 in the L gene. The genetic basis of attenuation of cpts530/1030 was defined by the introduction of the 530 and 1030 mutations into a cDNA clone of cpRSV, from which recombinant RSV was derived and analyzed to determine the contribution of each mutation to the temperature sensitivity (ts) and attenuation (att) phenotypes of cpts530/1030. The 530 mutation, derived from cpts530, was previously shown to be responsible for the ts and att phenotypes of that virus. In the present study, the 1030 mutation was shown to be responsible for the increased temperature sensitivity of cpts530/1030. In addition, the 1030 mutation was shown to be responsible for the increased level of attenuation of cpts530/1030 in the upper and lower respiratory tracts of mice. The 530 and 1030 mutations were additive in their effects on the ts and att phenotypes. It was possible to introduce the 1030 mutation, but not the 530 mutation, into an attenuated vaccine candidate with residual reactogenicity in very young infants, namely, cpts248/404, by use of reverse genetics. The inability to introduce the 530 mutation into the cpts248/404 virus was shown to be due to its incompatibility with the 248 missense mutation at the level of L protein function. The resulting rA2cp248/404/1030 mutant virus was more temperature sensitive and more attenuated than the cpts248/404 parent virus, making it a promising new RSV vaccine candidate created by use of reverse genetics to improve upon an existing vaccine virus.

Animals↗
PubMed2001

Rescue of rabies virus from cloned cDNA and identification of the pathogenicity-related gene: glycoprotein gene is associated with virulence for adult mice.

In order to identify the viral gene related to the pathogenicity of rabies virus, we tried to establish a reverse genetics system of the attenuated RC-HL strain, which causes nonlethal infection in adult mice after intracerebral inoculation. A full-length genome plasmid encoding the complete antigenomic cDNA of the RC-HL strain and helper plasmids containing cDNAs of the complete open reading frame of the N, P, and L genes, respectively, were constructed. After transfection of these plasmids into BHK-21 cells infected with the T7 RNA polymerase-expressing vaccinia virus, infectious rabies virus with almost the same biological properties as those of the wild-type RC-HL strain was rescued. Using this reverse genetics system of the RC-HL strain, we generated a chimeric virus with the open reading frame of the glycoprotein gene from the parent Nishigahara strain, which kills adult mice after intracerebral inoculation, in the background of the RC-HL genome. Since the chimeric virus killed adult mice following intracerebral inoculation, it became evident that the open reading frame of the glycoprotein gene is related to the pathogenicity of the Nishigahara strain for adult mice.

Animals↗
PubMed2006

[Molecular mechanism affecting route of transmission for H9N2 subtype AIV].

The available evidence suggests that H9 subtype avian influenza virus (AIV) did not circulate in Chicken flocks in China until the early 1990s. However, the pandemic of H9 subtype AI, which started in summer of 1998, spread very rapidly to more than 20 provinces within several months. Obviously, the virus responsible for the 1998 pandemic was quite different from the virus isolated in early 1990s. In order to investigate the molecular mechanism affecting the route of transmission for H9N2 AIVs, strains of A/Chicken/Guangdong/SS/94 (H9N2) (SS) and A/Chicken/ Shanghai/F/98 (H9N2) (F) were compared in their route of transmission. SS strain representing the earlier strain was isolated in chickens in Guangdong province in 1994, whereas F strain was isolated in Shanghai during 1998 pandemic. The findings suggested that F strain could transmitted in chickens by direct contact and by aerosol route. Whereas SS strain only by direct contact, and neither of two viruses by fecal contact. The cDNAs derived from the HA and NA genes of SS strain were cloned into vector pHW2000 to construct two transcription/expression plasmids respectively, and the cDNAs derived from 8 genes of F strain was done in the same way. Three recombinants were generated by reverse genetics: RF7/SSHA with the HA gene from SS strain and the remaining seven genes from F strain, RF7/SSNA with the NA gene from SS strain and the remaining seven genes from F strain, and RF7/SSHA/SSNA with the HA and NA genes of SS strain and the remaining six genes from F strain. In order to identify three recombinants, a total of seven genes from them were amplified by using PCR with universal primer pairs of H9N2 influenza virus and sequenced. In addition, three recombinants were characterized by HA and HI tests and sequence analysis. The results indicated that three recombinants were successfully rescued by reverse genetics. To determine the genes associated with the ability to transmit by aerosol route in chickens, a set of transmission experiments were designed. Groups of three chickens were inoculated with equal dose of virus by oral, intratracheal and intranasal routes. Each group was placed in direct, aerosol or fecal contact with three uninoculated chickens. Virus isolation and identification showed that only the RF7/SSHA recombinant was transmitted from inoculated to uninoculated chickens by aerosol route, whereas three recombinants were transmitted by direct contact, but not by fecal contact. The results were further confirmed by HI test of serum samples from uninoculated chickens. The data suggest that the NA gene might be the major determinant of the ability of aerosol transmission for H9N2 subtype AIVs in chickens.

Animals↗
PubMed1999

Generation of influenza A viruses entirely from cloned cDNAs.

We describe a new reverse-genetics system that allows one to efficiently generate influenza A viruses entirely from cloned cDNAs. Human embryonic kidney cells (293T) were transfected with eight plasmids, each encoding a viral RNA of the A/WSN/33 (H1N1) or A/PR/8/34 (H1N1) virus, flanked by the human RNA polymerase I promoter and the mouse RNA polymerase I terminator-together with plasmids encoding viral nucleoprotein and the PB2, PB1, and PA viral polymerases. This strategy yielded >1 x 10(3) plaque-forming units (pfu) of virus per ml of supernatant at 48 hr posttransfection. The addition of plasmids expressing all of the remaining viral structural proteins led to a substantial increase in virus production, 3 x 10(4)-5 x 10(7) pfu/ml. We also used reverse genetics to generate a reassortant virus containing the PB1 gene of the A/PR/8/34 virus, with all other genes representing A/WSN/33. Additional viruses produced by this method had mutations in the PA gene or possessed a foreign epitope in the head of the neuraminidase protein. This efficient system, which does not require helper virus infection, should be useful in viral mutagenesis studies and in the production of vaccines and gene therapy vectors.

Animals↗
PubMed2003

Development of new generation rinderpest vaccines.

Veterinary science has benefited much from the advances in biotechnology during the past 20 years. New and improved diagnostic techniques for infectious diseases have been developed and new and highly effective vaccines to prevent such diseases have been introduced and more have been, or are about to be, field-tested. The latest development in negative strand virology, reverse genetics, the ability to rescue live virus from a DNA copy of the RNA genome, is being used to address questions concerning virus pathogenicity at the molecular level and to produce "marker" vaccines, i.e. vaccines that allow serological identification of all vaccinated animals. Such a vaccine would greatly benefit the continuing campaign for the global eradication of rinderpest since it would then be possible, by serological means, to detect wild type virus circulating in local areas or regions where it is still necessary to vaccinate and where the vaccination levels are below those required to eliminate the virus. Here we describe different approaches we have taken to produce such a vaccine using reverse genetics to add a marker to the existing and widely used Plowright rinderpest vaccine.

Animals↗