Greater numbers of nucleotide substitutions are introduced into the genomic RNA of bovine viral diarrhea virus during acute infections of pregnant cattle than of non-pregnant cattle

ABSTRACT: BACKGROUND: Bovine viral diarrhea virus (BVDV) strains circulating in livestock herds show significant sequence variation. Conventional wisdom states that most sequence variation arises during acute infections in response to immune or other environmental pressures. A recent study showed that more nucleotide changes were introduced into the BVDV genomic RNA during the establishment of a single fetal persistent infection than following a series of acute infections of naive cattle. However, it was not known if nucleotide changes were introduce when the virus crossed the placenta and infected the fetus or during the acute infection of the dam. METHODS: The sequence of the open reading frame (ORF) from viruses isolated from four acutely infected pregnant heifers following exposure to persistently infected (PI) calves was compared to the sequences of the virus from the progenitor PI calf and the virus from the resulting progeny PI calf to determine when genetic change was introduced. This was compared to genetic change found in viruses isolated from a pregnant PI cow and its PI calf, and in three viruses isolated from acutely infected, non-pregnant cattle exposed to PI calves. RESULTS: Most genetic changes previously identified between the progenitor and progeny PI viruses were in place in the acute phase viruses isolated from the dams six days post-exposure to the progenitor PI calf. Additionally, each progeny PI virus had two to three unique nucleotide substitutions that were introduced in crossing the placenta and infection of the fetus. The nucleotide sequence of two acute phase viruses isolated from steers exposed to PI calves revealed that six and seven nucleotide changes were introduced during the acute infection. The sequence of the BVDV-2 virus isolated from an acute infection of a PI calf (BVDV-1a) co-housed with a BVDV-2 PI calf had ten nucleotides that were different from the progenitor PI virus. Finally, twenty nucleotide changes were identified in the PI virus of a calf born to a PI dam. CONCLUSIONS: These results demonstrate that nucleotide changes are introduced into the BVDV infecting pregnant cattle at rates of 2.3 to 8 fold higher then during the acute infection of non-pregnant animals.     

Low-level expression and reversion both contribute to reactivation of herpes simplex virus drug-resistant mutants with mutations on homopolymeric sequences in thymidine kinase

Many acyclovir-resistant herpes simplex virus isolates from patients contain insertions or deletions in homopolymeric sequences in the thymidine kinase (TK) gene (tk). Viruses that have one (G8) or two (G9) base insertions in a run of seven G's (G string) synthesize low levels of active TK (TK-low phenotype), evidently via ribosomal frameshifting. These levels of TK can suffice to permit reactivation from latently infected mouse ganglia, but in a majority of ganglia, especially with the G9 virus, reactivation of virus that has reverted to the TK-positive phenotype predominates. To help address the relative contributions of translational mechanisms and reversion in reactivation, we generated viruses with a base either inserted or deleted just downstream of the G string. Both of these viruses had a TK-low phenotype similar to that of the G8 and G9 viruses but with less reversion. Both of these viruses reactivated from latently infected trigeminal ganglia, albeit inefficiently, and most viruses that reactivated had a uniformly TK-low phenotype. We also generated viruses that have one insertion in a run of six C's or one deletion in a run of five C's. These viruses lack measurable TK activity. However, they reactivated from latently infected ganglia, albeit inefficiently, with the reactivating viruses having reverted to the wild-type TK phenotype. Therefore, for G-string mutants, levels of active TK as low as 0.25% generated by translational mechanisms can suffice for reactivation, but reversion can also contribute. For viruses that lack TK activity due to mutations on other homopolymeric sequences, reactivation can occur via reversion.     

Cloning of a DNA fragment from the left-hand terminus of the adenovirus type 2 genome and its use in site-directed mutagenesis.

The HpaI E fragment (0-4.5 map units) of adenovirus type 2 (Ad2) DNA was cloned in the plasmid vector pBR322. Excision of the viral insert with PstI and XbaI generated a fragment which comigrated with Ad2 XbaI-E (0-3.8 map units), and this fragment was ligated to the 3.8-100 fragment generated by XbaI cleavage of the DNA of the Ad5 mutant, dl309 (N. Jones and T. Shenk, Cell 17:683-689, 1979). Transfection with the ligation products resulted in the production of progeny virus which was able to replicate on both HeLa and line 293 cells, demonstrating the biological activity of the sequences rescued from the plasmid. Small deletions were introduced around the SmaI site (map position 2.8) within the cloned viral insert, and the altered DNA sequences were reintroduced into progeny virus as described above. The mutant viruses grew well on line 293 cells but plaqued with greatly reduced efficiency on HeLa cells, exhibiting a host range phenotype similar to previously described mutants with lesions located within this region of the genome. When plasmid-derived left-end fragments containing pBR322 DNA sequences to the left of map position 0 were ligated to the 3.8-100 fragment of dl309 DNA, the infectivity of the ligation products was not reduced. However, all progeny viruses examined yielded normal-size restriction enzyme fragments from their left-hand ends, indicating that the bulk of the pBR322 DNA sequences are removed either prior to or as a consequence of the replication of the transfecting DNA molecules.     

Identification of human telomeric repeat motifs at the genome termini of human herpesvirus 7: structural analysis and heterogeneity.

Human herpesvirus 6 (HHV-6) and HHV-7 are closely related T-lymphotropic betaherpesviruses which share a common genomic organization and are composed of a single unique component (U) that is bounded by direct repeats (DRL and DRR). In HHV-6, a sequences have been identified at each end of the DR motifs, resulting in the arrangement aDRLa-U-aDRRa. In order to determine whether determine whether HHV-7 contains similar a sequences, we have sequenced the DRL-U and U-DRR junctions of HHV-7 strain JI, together with the DRR.DRL junction from the head-to-tail concatamer that is generated during productive virus infection. In addition, we have sequenced the genomic termini of an independent isolate of HHV-7. As in HHV-6, a (GGGTTA)n motif identical to the human telomeric repeat sequence (TRS) was identified adjacent to, but not at, the genome termini of HHV-7. The left genome terminus and the U-DRR junction contained a homolog of the consensus herpesvirus packaging signal, pac-1, followed by short tandem arrays of TRSs separated by single copies of a second 6-bp repeat. This organization is similar to the arrangement found at U-DRR in HHV-6 but differs from it in that the TRS arrays are considerably shorter in HHV-7. The right genome terminus and the DRL-U junction contained a homolog of the consensus herpesvirus packaging signal, pac-2, followed by longer tandem arrays of TRSs separated by single copies of either a 6-bp or a 14-bp repeat. This arrangement is considerably more complex than the simple tandem array of TRSs that is present at the corresponding genomic location in HHV-6 and corresponds to a site of both inter- and intrastrain heterogeneity in HHV-7. The presence of TRSs in lymphotropic herpesviruses from humans (HHV-6 and HHV-7), horse (equine herpesvirus 2), and birds (Marek's disease virus) is striking and suggests that these sequences may have functional or structural significance.     

Nucleic Acid Homology of Murine Type-C Viral Genes

The nucleic acid sequence homology between various murine, endogenous type-C viruses (three host range classes of BALB/c virus, the AT-124 virus, and the CCL 52 virus) and two laboratory strains of murine leukemia virus (Rauscher and Kirsten) was determined by DNA:RNA hybridization. The viral sequences exhibit varying degrees of partial homology. DNA:DNA hybridizations were performed between [³H]DNA probes prepared from N- and X-tropic BALB/c endogenous viruses and cellular DNAs from BALB/c, NIH Swiss, and AKR inbred mouse strains as well as from California feral mice and the Asian mouse subspecies Mus musculus molossinus and M. musculus castaneus. All of these strains of mice are shown to possess multiple (six to seven per haploid genome), partially related copies of type-C virogenes in their DNAs. Thermal melting profiles of the DNA:RNA and DNA:DNA hybrids suggest that the partial homology of the viral nucleic acid sequences is the result of base alterations throughout the viral genomes, rather than the loss of discrete segments of viral sequences.     

A seven-segmented influenza A virus expressing the influenza C virus glycoprotein HEF

Influenza viruses are classified into three types: A, B, and C. The genomes of A- and B-type influenza viruses consist of eight RNA segments, whereas influenza C viruses only have seven RNAs. Both A and B influenza viruses contain two major surface glycoproteins: the hemagglutinin (HA) and the neuraminidase (NA). Influenza C viruses have only one major surface glycoprotein, HEF (hemagglutinin-esterase fusion). By using reverse genetics, we generated two seven-segmented chimeric influenza viruses. Each possesses six RNA segments from influenza virus A/Puerto Rico/8/34 (PB2, PB1, PA, NP, M, and NS); the seventh RNA segment encodes either the influenza virus C/Johannesburg/1/66 HEF full-length protein or a chimeric protein HEF-Ecto, which consists of the HEF ectodomain and the HA transmembrane and cytoplasmic regions. To facilitate packaging of the heterologous segment, both the HEF and HEF-Ecto coding regions are flanked by HA packaging sequences. When introduced as an eighth segment with the NA packaging sequences, both viruses are able to stably express a green fluorescent protein (GFP) gene, indicating a potential use for these viruses as vaccine vectors to carry foreign antigens. Finally, we show that incorporation of a GFP RNA segment enhances the growth of seven-segmented viruses, indicating that efficient influenza A viral RNA packaging requires the presence of eight RNA segments. These results support a selective mechanism of viral RNA recruitment to the budding site.     

Genetic variation of the poliovirus genome with two VPg coding units.

Amongst the picornaviruses, poliovirus encodes a single copy of the genome-linked protein, VPg wheras foot-and-mouth disease virus uniquely encodes three copies of VPg. We have previously shown that a genetically engineered poliovirus genome containing two tandemly arranged VPgs is quasi-infectious (qi) that, upon genome replication, inadvertently deleted one complete VPg sequence. Using two genetically marked viral genomes with two VPg sequences, we now provide evidence that this deletion occurs via homologous recombination. The mechanism was abrogated when the second VPg was engineered such that its nucleotide sequence differed from that of the first VPg sequence by 36%. Such genomes also expressed a qi phenotype, but progeny viruses resulted from (i) random deletions yielding single VPg coding sequences of varying length lacking the Q*G cleavage site between the VPgs and (ii) mutations in the AKVQ*G cleavage sites between the VPgs at either the P4, P1 or P1' position. These variants present a unique genetic system defining the cleavage signals recognized in 3Cpro-catalyzed proteolysis. We propose a recognition event in the cis cleavages of the polyprotein P2-P3 region, and we present a hypothesis why the poliovirus genome does not tolerate two tandemly arranged VPg sequences.     

Mutations and selection in the generation of class II histocompatibility antigen polymorphism.

A comparison of seven human DR and DC class II histocompatibility antigen beta-chain amino acid sequences indicates that the allelic variation is of comparable magnitude within the DR and DC beta-chain genes. Silent and replacement nucleotide substitutions in six DR and DC beta-chain sequences, as well as in seven murine class II sequences (three I-A beta and four I-A alpha alleles) were analyzed. The results suggest that the mutation rates are of a comparable magnitude in the nucleotide sequences encoding the first and second external domains of the class II molecules. Nevertheless, the allelic amino acid replacements are predominantly located in the first domains. We conclude that a conservative selective pressure acts on the second domains, whereas in many positions in the first domains replacement substitutions are selectively neutral or maybe even favoured. Thus, the difference between the first and second domains as regards the number of amino acid replacements is mainly due to selection.     

Synethesis and integration of viral DNA in chicken cells at different time after infection with various multiplicities of avian oncornavirus.

To see if integration of the provirus resulting from RNA tumor virus infection is limited to specific sites in the cell DNA, the variation in the number of copies of virus-specific DNA produced and integrated in chicken embryo fibroblasts after RAV-2 infection with different multiplicities has been determined at short times, long times, and several transfers after infection. The number of copies of viral DNA in cells was determined by initial hybridization kinetics of single-stranded viral complementary DNA with a moderate excess of cell DNA. The approach took into account the different sizes of cell DNA and complementary DNA in the hybridization mixture. It was found that uninfected chicken embryo fibroblasts have approximately seven copies, part haploid genome of DNA sequences homologous to part of the Rous-association virus 2 (RAV-2) genome. Infection with RAV-2 adds additional copies, and different sequences, of RAV -2- specific DNA. By 13 h postinfection, there are 3 to 10 additional copies per haploid genome. This number can not be increased by increasing the multiplicity of infection, and stays relatively constant up to 20 h postinfection, when some of the additional viral DNA is integrated. Between 20 and 40 h postinfection, the cells accumulated up to 100 copies per haploid genome of viral DNA. Most of these are unintegrated. This number decreases with cell transfer, until cells are left with one to three copies of additional viral DNA sequences per haploid genome, of which most are integrated. The finding that viral infection causes the permanent addition of one to three copies of integrated viral DNA, despite the cells being confronted with up to 100 copies per haploid genome after infection, is consistent with a hypothesis that chicken cells contain a limited number of specific integration sites for the oncornavirus genome.     

Effect of Helper Virus on the Number of Murine Sarcoma Virus DNA Copies in Infected Mammalian Cells

Cell lines of four mammalian species were each examined for the number of Moloney murine sarcoma virus (M-MSV) DNA copies in total cellular DNA after M-MSV transformation. Sarcoma-positive, leukemia-negative (S+L-) M-MSV-transformed cells were compared to M-MSV-transformed cells infected with a replicating leukemia virus. Both unfractionated M-MSV complementary DNA and complementary DNA representing the MSV-specific and the MSV-murine leukemia virus-common regions of the M-MSV genome were hybridized to total cellular DNA of various species. DNAs of mouse, cat, dog, and human S+L-cells contained from less than one to a few proviral M-MSV DNA copies per haploid genome. In contrast, helper virus-coinfected, M-MSV-producing cells of each species showed a 3- to 10-fold increase in M-MSV proviral DNA over that found in corresponding S+L- cells. MSV-specific and MSV-murine leukemia virus-common nucleotide sequences were each increased to a similar degree. A corresponding examination of cellular DNA of leukemia virus-infected normal or S+L- mammalian cells was performed to establish the resulting number of leukemia proviral DNA copies. The infection of normal or S+L- mammalian cells with several leukemia-type viruses that did not have nucleotide sequences closely related to the cell before infection resulted in the appearance of one to three corresponding leukemia proviral DNA copies.     

Variability of human immunodeficiency virus type 1 group O strains isolated from Cameroonian patients living in France.

Human immunodeficiency virus type 1 (HIV-1) nucleotide sequences encoding p24Gag and the Env C2V3 region were obtained from seven patients who were selected on the basis of having paradoxical seronegativity on a subset of HIV enzyme-linked immunosorbent assay detection kits and having atypical Western blot (immunoblot) reactivity. Sequence analyses showed that all of these strains were more closely related to the recently described Cameroonian HIV isolates of group O (HIV-1 outlier) than to group M (HIV-1 major). All seven patients had Cameroonian origins but were living in France at the time the blood samples were taken. Characterization of a large number of group M strains has to date revealed eight distinct genetic subtypes (A to H). Genetic distances between sequences from available group O isolates were generally comparable to those observed in M intersubtype sequence comparisons, showing that the group O viruses are genetically very diverse. Analysis of sequences from these seven new viral strains, combined with the three previously characterized group O strains, revealed few discernable phylogenetic clustering patterns among the 10 patients' viral sequences. The level of diversity among group O sequences suggests that they may have a comparable (or greater) age than the M group sequences, although for unknown reasons, the latter group dispersed first and is the dominant lineage in the pandemic.     

The influenza A virus PB2, PA, NP, and M segments play a pivotal role during genome packaging

The genomes of influenza A viruses consist of eight negative-strand RNA segments. Recent studies suggest that influenza viruses are able to specifically package their segmented genomes into the progeny virions. Segment-specific packaging signals of influenza virus RNAs (vRNAs) are located in the 5' and 3' noncoding regions, as well as in the terminal regions, of the open reading frames. How these packaging signals function during genome packaging remains unclear. Previously, we generated a 7-segmented virus in which the hemagglutinin (HA) and neuraminidase (NA) segments of the influenza A/Puerto Rico/8/34 virus were replaced by a chimeric influenza C virus hemagglutinin/esterase/fusion (HEF) segment carrying the HA packaging sequences. The robust growth of the HEF virus suggested that the NA segment is not required for the packaging of other segments. In this study, in order to determine the roles of the other seven segments during influenza A virus genome assembly, we continued to use this HEF virus as a tool and analyzed the effects of replacing the packaging sequences of other segments with those of the NA segment. Our results showed that deleting the packaging signals of the PB1, HA, or NS segment had no effect on the growth of the HEF virus, while growth was greatly impaired when the packaging sequence of the PB2, PA, nucleoprotein (NP), or matrix (M) segment was removed. These results indicate that the PB2, PA, NP, and M segments play a more important role than the remaining four vRNAs during the genome-packaging process.     

Influence of enhancer sequences on thymotropism and leukemogenicity of mink cell focus-forming viruses.

Oncogenic mink cell focus-forming (MCF) viruses, such as MCF 247, show a positive correlation between the ability to replicate efficiently in the thymus and a leukemogenic phenotype. Other MCF viruses, such as MCF 30-2, replicate to high titers in thymocytes and do not accelerate the onset of leukemia. We used these two MCF viruses with different biological phenotypes to distinguish the effect of specific viral genes and genetic determinants on thymotropism and leukemogenicity. Our goal was to identify the viral sequences that distinguish thymotropic, nonleukemogenic viruses such as MCF 30-2 from thymotropic, leukemogenic viruses such as MCF 247. We cloned MCF 30-2, compared the genetic hallmarks of MCF 30-2 with those of MCF 247, constructed a series of recombinants, and tested the ability of recombinant viruses to replicate in the thymus and to induce leukemia. The results established that (i) MCF 30-2 and MCF 247 differ in the numbers of copies of the enhancer sequences in the long terminal repeats. (ii) The thymotropic phenotype of both viruses is independent of the number of copies of the enhancer sequences. (iii) The oncogenic phenotype of MCF 247 is correlated with the presence in the virus of duplicated enhancer sequences or with the presence of an enhancer with a specific sequence. These results show that the pathogenic phenotypes of MCF viruses are dissociable from the thymotropic phenotype and depend, at least in part, upon the enhancer sequences. On the basis of these results, we suggest that the molecular mechanisms by which the enhancer sequences determine thymotropism are different from those that determine oncogenicity.     

Comparison of Different Cell Substrates on the Measurement of Human Influenza Virus Neutralizing Antibodies

Eight cell lines were systematically compared for their permissivity to primary infection, replication, and spread of seven human influenza viruses. Cell lines were of human origin (Caco-2, A549, HEp-2, and NCI-H292), monkey (Vero, LLC-MK2), mink (Mv1 Lu), and canine (MDCK). The influenza viruses included seasonal types and subtypes and a pandemic virus. The MDCK, Caco-2, and Mv1 Lu cells were subsequently compared for their capacity to report neutralization titers at day one, three and six post-infection. A gradient of sensitivity to primary infection across the eight cell lines was observed. Relative to MDCK cells, Mv1 Lu reported higher titers and the remaining six cell lines reported lower titers. The replication and spread of the seven influenza viruses in the eight cell substrates was determined using hemagglutinin expression, cytopathic effect, and neuraminidase activity. Virus growth was generally concordant with primary infection, with a gradient in virus replication and spread. However, Mv1 Lu cells poorly supported virus growth, despite a higher sensitivity to primary infection. Comparison of MDCK, Caco-2, and Mv1 Lu in neutralization assays using defined animal antiserum confirmed MDCK cells as the preferred cell substrate for influenza virus testing. The results observed for neutralization at one day post-infection showed MDCK cells were similar (<1 log₂ lower) or superior (>1 log₂ higher) for all seven viruses. Relative to Caco-2 and Mv1 Lu cells, MDCK generally reported the highest titers at three and six days post-infection for the type A viruses and lower titers for the type B viruses and the pandemic H9N2 virus. The reduction in B virus titer was attributed to the complete growth of type B viruses in MDCK cells before day three post-infection, resulting in the systematic underestimation of neutralization titers. This phenomenon was also observed with Caco-2 cells.     

Effect of the addition of oligosaccharides on the biological activities and antigenicity of influenza A/H3N2 virus hemagglutinin

Influenza A/H3N2 viruses have developed an increased number of glycosylation sites on the globular head of the hemagglutinin (HA) protein since their appearance in 1968. Here, the effect of addition of oligosaccharide chains to the HA of A/H3N2 viruses on its biological activities was investigated. We constructed seven mutant HAs of A/Aichi/2/68 virus with one to six glycosylation sites on the globular head, as found in natural isolates, by site-directed mutagenesis and analyzed their intracellular transport, receptor binding, and cell fusion activities. The glycosylation sites of mutant HAs correspond to representative A/H3N2 isolates (A/Victoria/3/75, A/Memphis/6/86, or A/Sydney/5/97). The results showed that all the mutant HAs were transported to the cell surface as efficiently as wild-type HA. Although mutant HAs containing three to six glycosylation sites decreased receptor binding activity, their cell fusion activity was not affected. The reactivity of mutant HAs having four to six glycosylation sites with human sera collected in 1976 was much lower than that of wild-type HA. Thus, the addition of new oligosaccharides to the globular head of the HA of A/H3N2 viruses may have provided the virus with an ability to evade antibody pressures by changing antigenicity without an unacceptable defect in biological activity.     

Virological events leading to spontaneous AKR thymomas.

The spontaneous leukemias of AKR mice are caused by mink cell focus-forming (MCF) viruses. These viruses are generated by recombination between several endogenous murine retroviruses. The virological events leading to the generation of the leukemogenic agent were investigated by using an oligonucleotide specific for the U3 region of the leukemogenic virus and env-reactive oligonucleotide probes specific for the different classes of endogenous murine leukemia virus. It was shown that (i) the leukemogenic MCF virus is formed by recombination between at least three different endogenous sequences; (ii) the U3 donor for the leukemogenic virus is the inducible xenotropic virus Bxv-1; (iii) all spontaneous tumors contain viruses with duplicated enhancer regions in their long terminal repeats; (iv) enhancer duplication is a somatic event, since Bxv-1 contains only one copy; (v) the first recombinant virus detectable in mass populations of thymocytes by Southern hybridization analysis contains all structural features of the ultimate leukemogenic virus; and (vi) the multiple novel viruses in a given tumor represent progeny of the same unique recombination events. On the basis of these results, an analysis of the virological events leading to AKR thymomas is presented.     

The cytoplasmic tail of influenza A virus neuraminidase (NA) affects NA incorporation into virions, virion morphology, and virulence in mice but is not essential for virus replication.

In this study, we investigated the role of the conserved neuraminidase (NA) cytoplasmic tail residues in influenza virus replication. Mutants of influenza A virus (A/WSN/33 [H1N1]) with deletions of the NA cytoplasmic tail region were generated by reverse genetics. The resulting viruses, designated NOTAIL, contain only the initiating methionine of the conserved six amino-terminal residues. The mutant viruses grew much less readily and produced smaller plaques than did the wild-type virus. Despite similar levels of NA cell surface expression by the NOTAIL mutants and wild-type virus, incorporation of mutant NA molecules into virions was decreased by 86%. This reduction resulted in less NA activity per virion, leading to the formation of large aggregates of progeny mutant virions on the surface of infected cells. A NOTAIL virus containing an additional mutation (Ser-12 to Pro) in the transmembrane domain incorporated three times more NA molecules into virions than did the NOTAIL parent but approximately half of the amount incorporated by the wild-type virus. However, aggregation of the progeny virions still occurred at the cell surface. All NOTAIL viruses were attenuated in mice. We conclude that the cytoplasmic tail of NA is not absolutely essential for virus replication but exerts important effects on the incorporation of NA into virions and thus on the aggregation and virulence of progeny virus. In addition, the relative abundance of long filamentous particles formed by the NOTAIL mutants, compared with the largely spherical wild-type particles, indicates a role for the NA cytoplasmic tail in virion morphogenesis.