Article Figures & Data
Figures
- FIG. 1.
Measuring the mutation rate during the growth of a single plaque. Plaque formation was performed in confluent monolayers of MDCK cells infected with A and B viruses. A well-isolated parent plaque of approximately 2 mm in diameter was picked at an appropriate time postinfection. To clone selected individual descendant viruses in the parent plaque, a second plaque formation was performed using the virus yield directly from the parent plaque. All plaque viruses were eluted from the agar plug into 300 μl of minimal essential medium containing 0.2% bovine serum albumin, made into aliquots, and stored in a deep freezer (−80°C) until used. The harvested viruses were used without further modification for sequential analysis of their NS genes, which reveals the sequence of the major virus in the plaque. The magnified views of the plaques show the viruses that exist within them. Star, viruses carrying NS gene sequences identical to that of the major virus in the parent plaque; triangle, diamond, spade, club, heart, and square, viruses carrying NS gene sequences different from that of the major virus in the parent plaque. The two triangles in the parent plaque represent a clone line. One of the descendant plaques is descended from a mutant of this type.
- FIG. 2.
Comparison of the generation times of A and B viruses in MDCK cells. Confluent monolayers of MDCK cells (5 × 105 cells) were infected with A/Aichi/1/87 and B/Aichi/29/99 at a multiplicity of infection of 8, kept at room temperature for 1 h, washed three times with minimal essential medium, and then incubated in 500 μl of overlay medium (Table 1, footnote a) excluding agar and DEAE-dextran, at 34 or 37°C. At 0 and 5 h postinfection and at each subsequent hour, a sample of the supernatant was removed and analyzed by plaque titration in MDCK cells. The PFU per 500 μl are shown versus the hours postinfection. (Inset) The generation times were determined as the point of maximum release of PFU per hour in each one-step growth experiment (1, 7). This is generally equivalent to the point at which the PFU reaches 50% of its final value, estimated here by averaging the total PFU at 11, 12, and 14 h. The width of the distribution about the mean was likewise estimated by noting the points at which the PFU reached 16 and 84% of its final value, corresponding to plus and minus 1 standard deviation (1). The change in temperature from 34 to 37°C, tested for A/1/87 only, appeared to have no significant effect on the mean generation time.
Tables
- TABLE 1.
Analysis of mutation rate for NS genes of human influenza viruses
Virus type Expt no. Virusa PFU of parent plaque No. of descendant plaques analyzed Total no. of nucleotides analyzedb No. of nucleotide changesc Mutation frequency/sited Mutation rate Per site per cyclee Per site per yearf B 1 B/Aichi/29/99 6.4 × 106 143 147,576g 1 0.7 × 10−5 1.0 × 10−6 0.8 × 10−3 2 B/Aichi/44/01 2.0 × 106 146 150,672g 0 0 0 0 3 B/Aichi/44/01 4.8 × 106 158 163,056g 1 0.6 × 10−5 0.9 × 10−6 0.8 × 10−3 Avg 0.4 ± 0.3 × 10−5 0.6 ± 0.4 × 10−6 0.5 ± 0.4 × 10−3 A 4 A/Aichi/12/92 3.0 × 106 149 124,117h 1 0.8 × 10−5 1.2 × 10−6 1.5 × 10−3 5 A/Aichi/1/87 2.4 × 106 136 113,288h 2 1.8 × 10−5 2.6 × 10−6 3.2 × 10−3 6 A/Aichi/1/87i 1.2 × 106 138 114,954h 2 1.7 × 10−5 2.5 × 10−6 3.2 × 10−3 Avg 1.4 ± 0.6 × 10−5 2.0 ± 0.9 × 10−6 2.6 ± 1.2 × 10−3 ↵ a To avoid a change in character of the viruses by adaptation to MDCK cells, we limited the number of virus amplifications in MDCK cells, prior to the experiment, to five. The overlay for plaquing consisted of minimal essential medium with 0.6% agar (Difco), 0.25% bovine serum albumin, 0.01% DEAE-dextran, 0.1% glucose, and 2.0 μg of acetyl trypsin per milliliter. Viruses were plaque purified twice in MDCK cells before preparing parent plaques. Plaque formation was performed in MDCK cells at 34°C unless otherwise noted. Parent plaques were grown for 48 h for A viruses and 71 h for B viruses.
↵ b For the nucleotide sequence analysis, the viral RNA was directly extracted from 100 μl of each descendant plaque virus (300 μl eluant) using Isogen LS (Nippon Gene, Japan). Half of this viral RNA was used to synthesize cDNA of the NS genes by RT-PCR as described previously (21) using primers ANSF(+) (5′-AGCAAAAGCAGGGTGACAAAGACATAA-3′, positions 1 to 27) and ANSR(−) (5′-AGTAGAAACAAGGGTGTTTTTTATCATTAA-3′, positions 861 to 890) for A viruses and BNSF(+) (5′-AGCAGAAGCAGAGCATTTGTTTAG-3′, positions 1 to 24) and BNSR(−) (5′-AGTAGTAACAAGAGGATTTTTATTTT-3′, positions 1073 to 1098) for B viruses. RT-PCR products were used directly for sequence analysis on an ABI 3100 DNA sequencer operated according to the manufacturer's protocols. For sequence reactions, in addition to the primers indicated above, we used primers 153(−) 5′-GTCTCCCATTCTCATTACTGCTTC-3′, positions 637 to 660) and 196(+) (5′-AAGGGCTTTCACCGAAGAGGGAGCAT-3′, positions 467 to 492) for A viruses and 157(−) (5′-CCAACACTCTCAAGGACAACACAT-3′, positions 586 to 609) and 172(+) (5′-GACATGAACAACAAAGATGCAAG-3′, positions 489 to 511) for B viruses.
↵ c Number of nucleotide differences between descendant and parent NS genes. Details of each mutation are given in Table 2.
↵ d Ratio of number of nucleotide changes detected to number of nucleotides analyzed.
↵ e Following Parvin et al. (23), the mutation rate was determined by dividing the observed frequency of nucleotide mutations per site by the number of infectious cycles. This assumes that all mutants continue to replicate as the plaque grows. If mutant lines die out, the mutation rate obtained in this way may underestimate the true mutation rate. We are assuming seven infectious cycles for A and B viruses.
↵ f Calculated by correcting observed mutation frequencies over 71 h (B viruses) and 48 h (A viruses) to 365 days (8,760 h).
↵ g For each descendant plaque, the 1,032 nucleotides at positions 41 to 1072 of the NS gene were analyzed.
↵ h For each descendant plaque, the 833 nucleotides at positions 28 to 860 of the NS gene were analyzed.
↵ i Parent plaque formed at 37°C.
- TABLE 2.
Mutations observed in NS genes of descendant plaque viruses compared to the parent plaque
Expt no.a Virus Clone no. Mutation (position)b Amino acid change (position)c No. of nucleotide differences between parent and reference virusd 1 B/29/99 3 G→T (238) S→I (NS1, 65) 26 (0.75 × 10−3) 3 B/44/01 8 T→A (678)e Y→N (NS1, 212) 42 (1.1 × 10−3) 4 A/12/92 53 C→A (339) L→I (NS1, 105) 45 (2.25 × 10−3) 5 A/1/87 93 T→C (121) F→S (NS1, 32) 45 (2.8 × 10−3) 136 G→A (849) Silent (NEP, 117) 6 A/1/87 52 A→G (355) K→R (NS1, 110) 125 A→G (665) Silent (NS1, 213) H→R (NEP, 56) ↵ a The numbers of the experiments are according to those in Table 1.
↵ b Nucleotide number is according to A/Aichi/2/68 (M34829) for A viruses and B/Ann Arbor/1/66 (M20225) for B viruses (the reference viruses). The numbers in parentheses are GenBank accession numbers.
↵ c The NS gene codes for two overlapping proteins, NS1 and NEP (nuclear export protein). The amino acids are numbered according to the proteins in the reference viruses listed in footnote b.
↵ d The numbers in parentheses show the evolutionary rates estimated from the differences between the reference virus and parent plaque, expressed in nucleotide differences per site per year. These rates are close to those reported by others (see text).
↵ e B/44/01, like several other B viruses, is missing the noncoding nucleotide at position 40 in B/Ann Arbor/1/66. The remaining nucleotides are numbered as if the omission had not occurred.
