Roger H. Miller

Mutation rate of the hepadnavirus genome

An essential factor for charting the evolution of hepadnaviruses is an estimation of the mutation rate of the virus genome during replication in the host. In order to determine the mutation rate of the hepadnavirus genome under defined experimental conditions, we transfected 10 neonatal woodchucks with an infectious molecular clone of woodchuck hepatitis virus (WHV). By 4 months post-transfection, all 10 animals showed serological evidence for WHV infection. Subsequently, 1 animal became chronically infected and was used for further study. At 16 months post-transfection WHV DNA from serum virions was cloned and the nucleotide sequence of three independent progeny genomes compared directly with that of the input recombinant DNA. Although the consensus nucleotide sequence remained unchanged, we found three differences in individual progeny genomes when compared to the parental genome sequence. Thus, we estimate the mutation rate of the WHV genome to be less than or equal to 2 X 10(-4) base substitutions/site/year. This figure is one to two orders of magnitude lower than the mutation rates previously calculated for the positive- and negative-strand RNA viruses, but is similar to the mutation rate of the gag gene which is the most slowly evolving gene of retroviruses. Therefore, we find that the hepadnavirus genome is relatively stable during replication in host tissues when compared to other viruses that lack polymerase-associated proofreading functions.

Detection of hepatitis B virus DNA in serum by polymerase chain reaction

Abstract Standard methods of virus DNA detection using the polymerase chain reaction can be time consuming and can involve multiple steps in which contamination with exogenous DNA can occur. Therefore, we developed a simplified method for detecting hepatitis B virus DNA in serum. The main advantages of this method are that it can be performed rapidly, consists of only several steps, and has a false positive rate of

Challenge Pools of Hepatitis C Virus Genotypes 1–6 Prototype Strains: Replication Fitness and Pathogenicity in Chimpanzees and Human Liver-Chimeric Mouse Models

Chimpanzees represent the only animal model for studies of the natural history of hepatitis C virus (HCV). To generate virus stocks of important HCV variants, we infected chimpanzees with HCV strains of genotypes 1-6 and determined the infectivity titer of acute-phase plasma pools in additional animals. The courses of first- and second-passage infections were similar, with early appearance of viremia, HCV RNA titers of >10(4.7) IU/mL, and development of acute hepatitis; the chronicity rate was 56%. The challenge pools had titers of 10(3)-10(5) chimpanzee infectious doses/mL. Human liver-chimeric mice developed high-titer infections after inoculation with the challenge viruses of genotypes 1-6. Inoculation studies with different doses of the genotype 1b pool suggested that a relatively high virus dose is required to consistently infect chimeric mice. The challenge pools represent a unique resource for studies of HCV molecular virology and for studies of pathogenesis, protective immunity, and vaccine efficacy in vivo.

Preparation and storage of competent Escherichia coli cells

Publisher Summary The uptake of foreign DNA by Escherichia coli can be induced either through electroporation, which involves discharging an electrical voltage across bacterial cell membranes, or by making bacteria competent through chemical methods. Two widely used chemical methods involve treating bacteria with calcium chloride or hexamine cobalt and subjecting the cells to a heat shock. The protocol developed for the preparation of competent E. coli was designed to be rapid, convenient, and inexpensive, yet suitable to achieve transformation efficiencies sufficient for routine cloning experiments. Although transformation efficiencies obtained with the protocol are comparable to those of other commonly used methods to prepare competent bacteria, the technique is advantageous in that it is simple and can be completed in a short time once the bacteria are grown. Although the conditions necessary to achieve maximum numbers of transformants are specified, there is flexibility in the assay.

Sequence comparison of woodchuck hepatitis virus replicative forms shows conservation of the genome

The complete nucleotide sequence of virion DNA from two isolates of woodchuck hepatitis virus (WHV 7 and WHV 59) was determined along with the sequence of supercoiled DNA from one of those isolates (WHV 7). The sequences of the two WHV isolates were compared with the previously published sequences of two other isolates (WHV 1 and WHV 8). The range of nucleotide sequence variation of the four isolates (1 to 3.5%) was similar to those of HBVs of the same subtype (1.5 to 2%), but less than those of HBVs of different subtypes (8 to 10%). When amino acid sequences from the four WHV isolates were aligned, a consensus sequence could not be obtained at 13 sites. At 11 of 13 sites (7 in polymerase, 2 in presurface, 1 in surface, and 1 in the X region) the viruses could be grouped into pairs, so that WHV 1 and WHV 59 shared one amino acid and/or WHV 7 and WHV 8 shared a different amino acid. WHV 7 was passaged twice in woodchucks and supercoiled DNA was isolated. The nucleotide sequence of WHV 7 supercoiled DNA (derived from liver) was compared to that of WHV 7 virion DNA (derived from serum). Sequence comparison of virion and free supercoiled DNA from WHV 7 showed no nucleotide changes, except for a single nucleotide deletion thought to represent an error during molecular cloning or a defective viral genome. Thus, the nucleotide sequence of two different replicative forms of WHV DNA, separated by two in vivo passages, were virtually identical.

Characterization of primers for optimal amplification of hepatitis B virus DNA in the polymerase chain reaction assay

While the polymerase chain reaction assay has been shown to be effective in detecting serum hepatitis B virus (HBV) DNA, a systematic evaluation of the characteristics of optimal primer pairs has not yet been reported. Several factors related to the selection of primer pairs were examined and the findings are summarized as follows: (1) primers specific for the single- or double-stranded region of the HBV genome are equally effective in amplifying serum DNA; (2) maximum amplification is seen using primers, separated by no more than 500 nucleotides; and, (3) primers with up to 14% mismatch are effective at amplifying HBV DNA.

Genetic and Biological Characterization of Two Hepatitis B Virus Variants: A Precore Mutant Implicated in Fulminant Hepatitis and a Surface Mutant Resistant to Immunoprophylaxis

Two hepatitis B virus (HBV) variants were studied in chimpanzees to determine their genetic and biological characteristics. The first variant, mutant HBV strain HT, had an in-frame stop codon at the 28th position of the precore gene and caused fulminant hepatitis in two individuals. Strain HT had an infectivity of ≥107 chimpanzee infectious doses per ml and caused moderately severe hepatitis in all three infected animals. Analysis of the complete nucleotide and deduced amino acid sequence of strain HT showed significant heterogeneity when compared to 33 published HBV sequences. The most notable differences were found within a region that spans the 3′ half of the X gene, through the precore/ core gene, to the 5′ end of the polymerase gene. Thus, strain HT is highly infectious and pathogenic, and possesses a unique genome sequence. The second variant, mutant HBV strain AS, emerged in a vaccinated infant and had an Arg-substitution at the 145th codon of the surface gene. Strain AS had an infectivity titer of 106 chimpanzee infectious doses per ml and caused hepatitis in seronegative chimpanzees. Analysis indicated that wild-type virus was also present. A polymerase chain reaction-based nucleotide assay showed that an inoculum at the end point of infectivity consisted exclusively of the mutant virus. This was confirmed by detection of only the mutant in the serum of a chimpanzee infected with the end point (10−6) dilution of the reference serum. Thus, strain AS is infectious and pathogenic. To test whether licensed recombinant HB vaccines could protect against challenge with strain AS, we infected 4 chimpanzees that had received one of two commercially available vaccines. All chimpanzees were protected during the 6-month duration of the study. Therefore, strain AS may not pose a threat to properly immunized individuals.

Nucleotide sequence of the avian influenza A/Mallard/NY/6750/78 virus polymerase genes

The avian influenza A/Mallard/NY/6750/78 virus is currently being evaluated as a donor of attenuating genes in the construction of live avian-human influenza A reassortant virus vaccines for use in humans. We determined the nucleotide sequences of the three polymerase gene segments of this virus. This completes the nucleotide sequence of the six transferrable genes of the avian donor virus. Comparison of the nucleotide and deduced amino acid sequences of the non-glycoprotein genes of the avian A/Mallard/78 virus with representative avian and human influenza A viruses suggests that the PB1 gene of H2N2 subtype human influenza A viruses may have been derived from a non-human, possibly avian influenza A virus by genetic reassortment. In addition, several regions of conserved amino acids with potential functional significance were identified in the deduced amino acid sequences of the polymerase proteins.

Close evolutionary relatedness of the hepatitis B virus and murine leukemia virus polymerase gene sequences

Previous work indicates that hepatitis B virus (HBV) and retroviruses utilize a unique mechanism for genome replication by reverse transcription of RNA and share homology in biologically important nucleotide and protein sequences. The data presented here extend previous findings of sequence homology among the genomes of the members of these virus families. HBV was found to possess sequences homologous to the retrovirus protease and reverse transcriptase gene sequences. Homology was not found to the retrovirus integrase sequence consistent with the observation that hepadnaviruses do not integrate into cellular DNA as a necessary step in their replication cycle. Overall, the homology of the hepadnavirus polymerase gene was strongest with that of the murine leukemia viruses (MLVs). Also, the hepadnavirus polymerase shares organizational similarities to the MLV polymerase sequence. Analysis suggests that the ancestor of both hepadnaviruses and retroviruses possessed an overlapping long open reading frame in the polymerase gene sequence. In addition, low stringency blot hybridization using hepadnavirus DNA probes indicates that HBV is more closely related to MLV sequences than the sequences of MLV-related viruses and endogenous retrovirus-like genetic elements. Taken together, the data indicate that the polymerase gene sequence of the hepadnavirus and MLV genomes are organized in a similar fashion which suggests that these viruses evolved from a common ancestor.

Evidence for a bidirectional promoter complex within the X gene of woodchuck hepatitis virus

The genetic organization of hepadnaviruses is unusual in that all cis-acting regulatory sequences are located within genes. Thus, in the mammalian hepadnavirus genome, the presurface, surface, and X transcript promoters reside within the polymerase gene while the pregenome transcript promoter is located within the X gene. In this study we have identified two additional promoters within the woodchuck hepatitis virus (WHV) X gene that stimulate production of transcripts in vitro. First, we cloned regions of the WHV X gene into a promoterless expression vector (pGL2) to examine their ability to promote expression of firefly luciferase and mapped a previously unidentified promoter to positions 1475-1625 of the WHV8 genome. Deletion analysis revealed that the essential domain of this promoter, termed the ORF5/deltaX transcript promoter, mapped to nucleotides 1525-1625. Analysis revealed that this transcript initiated at nucleotide 1572 in both human (HuH-7) and woodchuck (WLC-3) hepatoma cell lines. Consistent with this finding, DNA footprinting analysis revealed protection of nucleotides 1567-1578 on the positive strand of the WHV8 genome. The function of this transcript in vivo is unclear, however, it may be used to produce a truncated form of the X protein that initiates at an AUG codon at position 1743-1745 on the WHV8 genome. Next, a second promoter was identified at positions 1625-1975 that was responsible for production of an antisense transcript. The activity of this promoter was comparable to that of the previously characterized surface transcript promoter of WHV in the absence of an enhancer. The antisense transcript promoter resides immediately upstream of open reading frame (ORF) 6, a previously identified ORF on the strand opposite of the known WHV protein-encoding sequences, that is thought to represent a vestigial gene. Analysis indicates that the antisense transcript had multiple start sites: nucleotides 1683 and 1762 on the WHV8 genome when assayed in HuH-7 cells, and nucleotide 1786 when assayed in WLC-3 cells. These data are consistent with footprinting analysis of supercoiled WHV DNA that revealed that the regions encompassing nucleotides 1696-1685, 1781-1766, and 1801-1787 on the negative sense DNA strand were protected from nuclease degradation. It is possible that such a transcript was once used in protein expression in an ancestral virus and may now be used for genetic control of WHV replication and/or gene expression. Overall, these data are consistent with the presence of a bidirectional promoter complex within the WHV X gene.