Proc. Natl. Acad. Sci. USA Vol. 85, pp. 8098-8101, November 1988 Evolution Evolution to predominance of swine influenza virus hemagglutinin mutants of predictable phenotype during single infections of the natural host (host-range mutants/pleiotropism selection/adaptation) EDWIN D. KILBOURNE*t, BERNARD C. EASTERDAYt, AND SANDY MCGREGOR§ *Mount Sinai School of Medicine of the City University of New York, New York, NY 10029; tSchool of Veterinary Medicine, University of Wisconsin- Madison, Madison, WI 53706; and §Bioproducts for Science, 2826 Latham Drive, Madison, WI 53713 Contributed by Edwin D. Kilbourne, August 5, 1988

ABSTRACT L and H2 mutants of the A/NJ/11/76 H1N1 Monoclonal Antibodies. The nature and derivation of two strain of swine influenza virus differ by having either a lysine monoclonal antibodies, Sa-13 and 9C8, and methods for or a glutamic acid at position 153 of the hemagglutinin antigenic analysis of viruses have been described (1). glycoprotein of the virus. In two separate experiments, exper- Experimental Design and Inoculation of Swine. Four- to imental infection of swine with various doses of the H2 mutant 5-week-old weanling pigs negative for hemagglutination in- resulted in the emergence in 11 of 20 animals of virus with the hibiting antibody to swine influenza virus were used in L phenotype. All evidence indicates that the H2-- L mutation, experiments I and II. Groups of four (experiment I) or five selection, and evolution to predominance occurred within the (experiment II) animals were inoculated intranasally with 7-day span ofindividual infections. L and H2 mutations appear various dilutions of virus in a 2-ml volume as indicated in to act as alleles in the adaptation of virus, respectively, to Tables 1 and 2. In experiment I, a single uninoculated animal natural and laboratory hosts. Although the gradual evolution was included in each group as a contact. In both experiments, of mutants during sequential infections is commonplace, the pigs receiving different (i.e., L and H2) viruses were housed present recognition of rapid and predictable evolution of in different rooms, separated by an unoccupied room without mutants of increased replication efficiency and specific pheno- -lateral access. In experiment II, animals inoculated with two type in the natural host, to our knowledge, is unprecedented. different doses ofthe same virus were housed in a single room divided by a 3-ft-high barrier precluding physical contact between groups (1 ft = 0.3048 m). From contemporary strains of swine influenza virus, two Extensive precautions were taken to minimize the oppor- hemagglutinin (HA) phenotypes are readily recovered by tunity for interroom spreading of virus by investigators or immunoselection with either of two HA-specific monoclonal animal caretakers. Street clothes were left in a dressing room antibodies (1) during virus passage in laboratory hosts (2, 3). and gowns or coveralls were worn to the animal-holding L-phenotype mutants replicate poorly in chicken embryos anteroom. The gowns or coveralls were then removed and and Madin-Darby canine kidney (MDCK) cells but are highly fresh coveralls were put on, along with boots, gloves, a infective for swine, in which they appear to predominate in surgical mask, and a hat. Only the supplies (needles, sy- natural infection (4). Virus of the H phenotype is less ringes, swabs, and tubes) that were used for that room were infective for swine by a factor of 50-100 (4) and replicates taken into the room. Upon completion of the sampling efficiently in laboratory hosts. L and H phenotypes are procedure in the room, boots were washed and supplies that defined definitively on the basis of characteristic reactivity had been used were placed in a plastic bag or other suitable patterns with monoclonal antibodies (1, 3). The molecular container and taken into the anteroom. The coveralls were basis of the two pleiotropic phenotypes involves a Lys -* Glu removed; gloves, hats, and masks were discarded; hands substitution at position 153 (L -* H2) and a Gly -+ Glu were washed; and gown or coveralls were put on before going substitution at position 155 (L -- H1) of the H1 HA molecule to the next room. Sample material remained in the container (3). These positions are analogous to positions 156 and 158, in the hall and all materials from all the rooms were removed respectively, of the well-studied H3 HA model (5, 6). from the hall at the same time. The same procedure and During the course of experimental infections of swine, we sequence were used in cleaning and feeding procedures. By discovered that, in swine inoculated with the H2-mutant using these procedures cross-contamination during influenza virus, L-mutant virus predominated at the end of infection. experiments has not been observed by us over a period of We present here evidence that this remarkable phenomenon several years. involves mutation, selection, and evolution to a predictable All pigs were observed daily for signs of disease and nasal viral phenotype during a single infection of its natural host. swabs were collected daily or every other day as indicated. All pigs were bled for serum antibody studies 28 days after MATERIALS AND METHODS infection. Viruses. The viruses employed are mutants or reassortants of swine influenza virus A/NJ/11/76 (HlNl) and are char- RESULTS acterized in Table 3 and elsewhere (3, 7). Viruses were stored In experiment I, a preliminary titration of L and H mutants at - 70'C as chicken embryo allantoic fluid seeds. Virus of A/NJ/11/76 (HlNl) virus was carried out by inoculation isolation was accomplished by inoculation of 10-day-old of equivalent amounts of two chicken embryo (egg) infective chicken embryos or MDCK cells as described (4, 8). doses (EID50) of each virus into swine (Tables 1 and 2). With the large doses employed, all pigs were infected and patterns The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: HA, hemagglutinin. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 8098 Downloaded by guest on September 30, 2021 Evolution: Kilbourne et al. Proc. Natl. Acad. Sci. USA 85 (1988) 8099

Table 1. Virus shedding by swine infected with H2-phenotype Table 2. Virus shedding by swine infected with L phenotype swine influenza virus swine influenza virus Dose, Virus shedding on the following Dose, Virus shedding on the following Pig log days after inoculation H L Pig logse days after inoculation no. EID50 1 2 3 4 5 6 7 9 11 shift no. EID50 1 2 3 4 5 6 7 9 11 13 Experiment I Experiment I 36 7.5 + HL L + - - + 46 7.4 + + + + - -- 37 + H L L - - + 47 + + + + - - - 38 - HH + - - 0 48 + + + + - - - 40 + H HL + - - + 49 + + L + --- 39 - + + L +- - 50* + + + + -- - 31 5.5 + H HL + - - + 41 5.4 + + + - - - - 32 + + H H -- 0 42 + + L + --- 33 -- HL L - - + 43 + + + + - - - 34 + H H L L - + 44 - + L + + - + 35* - - + + + + - 45* - + + + + - - Experiment II Experiment II 111 4.7 - + H H H L LH + 14 4.5 - + + + L L + 113 - - + + + + L + 24 L + + + L L + 115 + H H HL HL LL + 112 - + + LL L + 310 + - H + + + L + 300 - + + L L + + 578 - H HHH L L + 598 + + + LLL + 18 3.7 --- - + 28 3.5 - + - LL L + 19 ---- + H 0 125 - - + + + + + 22 - - -- H + 0 138 - + L L L L L 26 - - - - + + + - 272 - L + L + L + 400 ------+ 487 - - - + + + + L/H' virus was reisolated in chicken embryos and/or MDCK cells 13 2.5 - - - + and demonstrated to be of L or H2 antigenic and growth phenotype. 20 - - - + + + L In mixed isolates (HL or LH) predominant phenotype is indicated by 27 - + + + + + + the first letter. Animals 99, 274, 338, 590, 591, and 274 received a dose 29 - - + + + of 102.7 EID50 of H2 virus; none of the animals shed virus. Animals 30 _ 21, 25, 98, 100, and 589 received a dose of 101.7 EID50; none of the 16 1.5 - - + + + animals shed virus; however, animal 21 died without signs of 271 - - + + LL infection. Symbols for the H-. L shift are as follows: +, H-* L shift; 273 - - + L L 0, no shift; -, isolate not phenotyped. Symbols for virus shedding .. - are as follows: +, swine influenza virus was isolated in chicken 399. .. + embryos from nasal swabbings; HL, H, or L, a mixed isolate, H, or 592. .. ..- + L virus, respectively, was reisolated; -, no virus was isolated. Symbols are as in Table 1. *Uninoculated animal housed with inoculated animals of this group. *Uninoculated animal housed with inoculated animals of this group. of viral shedding were similar. This result was unexpected iting antibody serum titer of all 20 H2-inoculated pigs in because a previous experiment had shown only sporadic experiment I was less by a factor of 2 than that found for shedding of virus in 2 of 5 pigs inoculated with 105 EID50 of L-inoculated pigs. Similarly, in experiment II, comparison of H2 virus (4). However, in the present experiment, when geometric mean titers of 20 L-inoculated pigs with those of excreted virus was typed with Sa-13 and 9C8 monoclonal the 10 H2-inoculated pigs from which virus was recovered antibodies, it was found that initial shedding of H2 virus was demonstrated a lower response by a factor of 2 in the latter. followed by its replacement in 6 of 10 pigs by virus of the L These results were the same when either L or H2 viruses were phenotype (Table 1). In a second experiment (experiment II) used as test antigens. a greater range of viral dilutions was employed to reach the It is probable that the reduced immunologic response in titration end point. In this experiment, all L-inoculated pigs H2-inoculated swine reflects the diminished total virus rep- were infected, even by the smallest dose inoculated (101.5 lication that occurred in these animals despite the later EID50) (Table 2). However, H2 virus was less infective by a emergence in them of the L variant. factor of >100; the interpolated end point of the titration was Was L-Phenotype Virus Selected in Vivo from a Preexisting 10-3.2 EID50. As in experiment I, virus was reisolated from Mixture of L and H2 Phenotypes? We have already presented most positive nasal swabs and phenotyped in hemagglutina- evidence that the H2 virus inoculum was free of contaminat- tion inhibiting tests with monoclonal antibodies. Again, initial ing L virions and that care was taken to prevent cross predominance of the inoculum virus (H2) was followed by its contamination during the experiments. However, examining supplantation by L-phenotype virus by 5-6 days in 5 of 8 this question directly, passage in chicken embryos of the swine initially positive for virus. In those given the lowest cryopreserved H-virus inoculum of experiment I, with and infecting dose of virus (103-7 EID50), 2 swine excreted H2 without Sa-13 (H suppressive) antibody, revealed contami- virus on the sixth or seventh day. In 3 other swine, virus nating L virus in an L/H ratio of 1:33,000. Similar retrospec- could not be reisolated for study. All isolates from L- tive examination of the actual H2 inoculum from experiment inoculated swine were low in titer and, of these, selectively II, by inoculation of both chicken embryos and MDCK cells, sampled isolates manifested no change from L-antigenic demonstrated an EID50 titer of 10 -. Virus at the end-point phenotype (Table 2). dilution was of a H2 phenotype. The titration end point when Antibody Response. Antibody response was concordant viral dilutions were inoculated with a 1:40 dilution of Sa-13 with virus isolation, occurring only in pigs from which virus monoclonal antibody, suppressive to H2 but nonsuppressive was recovered. The geometric mean hemagglutination inhib- to L virus, was <10-1 (i.e., no L virus was demonstrable at Downloaded by guest on September 30, 2021 8100 Evolution: Kilbourne et al. Proc. Natl. Acad. Sci. USA 85 (1988) Phenotypic Characterization of Inoculum and Shed Viruses. The phenotypic characterization of L and H2 inocula and comparisons with the phenotypes of early and late isolates from pigs 115 and 578 and high-yield reassortants of virus 115 (3) are shown in Table 3. The first point to be made is that the mutants isolated from swine were identical in phenotype with either L or H2 A/NJ/11/76 viruses used in initiation of infection. As noted 120- (3), the swine 115 L and H reassortant viruses in which the swine viruses HA genes had been segregated retained their 100 respective antigenic phenotypes (i.e., titer ratios), although

L2. 80 their HA titers increased relatively (as with X-53a) with the incorporation of A/PR/8/34 genes. All HA phenotypes (L, C) 60 H1, and H2) have been encountered in laboratory manipula- a. tions of the wild-type parental virus of the two inocula.

40 DISCUSSION 20 This study of the comparative infectivity of L and H mutants of the A/NJ/11/76 (HlNi) swine influenza virus has con- 3 4 5 6 7 firmed the much greater efficiency of the L mutant in Day Post-inoculation initiating experimental infection in swine. Furthermore, when as few as 30 swine-infective doses of H2 phenotype FIG. 1. Evolution ofthe L mutant virus to predominance in swine virus were inoculated, initial infection with H2 phenotype 115 after inoculation of 104- EID50 of H (H2) virus. Virus was virus and its early predominance were followed later during recovered from nasal swabs by inoculation of MDCK-cell mono- the same emergence layers. Plaque forming units are presented as no. x 10-1. infection by and eventual predominance of virus with the L phenotype in all swine (5/5) so inoculated. a 10- 1 dilution). Titration of virus in the absence of antibody In a separate experiment, an uninoculated contact animal revealed that a decline in infective virus of 1.7 orders of exposed to swine given a larger dose of H virus acquired infection and was found to be shedding L virus. In a total of magnitude (from 107 2 EID50 per 0.1 ml) had occurred during storage and shipment. Even assuming that all virions inacti- 20 animals infected in two experiments with widely ranging amounts H2 L to vated had been the L phenotype, it can be calculated that of virus (Table 1), virus evolved predom- of inance in 11 animals. there had been no more than 50 infective particles of L virus Quantitation and phenotypic characterization ofvirus shed in the original undiluted inoculum. Thus, even the highest from one animal studied in detail demonstrated a pattern of a dose (104 7) this inoculum given represents viral dilution successively of the initial prevalence of H mutants, the later (10-2.5) calculated to preclude chance administration of any appearance of L mutants, the shedding of a mixed population hypothetical contaminating L virus. of L and H mutants, and the final predominance of L virus. Most persuasive ofthe in vivo evolution of L virus from H2 In considering the relative efficacy of L and H2 mutants in virus is a shedding pattern exemplified by a single H2 infection of swine, their natural host, it is evident not only virus-infected pig, swine 115 (Fig. 1). In brief, only virus that the L mutant is more infective but also that the H2 identical in biologic and antigenic phenotype to the inoculum mutant, fully capable of infecting and replicating to a high was isolated on day 3 after inoculation and predominated in titer in swine, is rapidly overtaken and superceded by L high titer on day 4. However, within 2 days, the excretory mutants that arise during the course of infection engendered patterns had reversed, with only L virus recoverable on days by H2 virus. That emerging L mutants can find susceptible 6 and 7. We conclude that both mutation and selection of cells remaining in the swine respiratory tract during the virus occurred within infected pigs. fourth, fifth, and sixth days of infection suggests (i) that H2 Table 3. Phenotypic characterization of L and H mutants and their high-yield reassortants log2 HI titer with the following log2 monoclonal HI hemagglu- antibody ratio,titer tination Virus Virus Sa-13 9C8 Sa-13/9C8 titer phenotype A/NJ/11/76(L) (inoculum) 2 11 0.18 4 L A/NJ/11/76(H2) (inoculum) 7 11 0.64 7 H2 A/NJ/11/76(H) mutant* 10 3 3.33 6 HI Isolate 115 (L) 1 12 0.08 2 L Isolate 115 (H2) 8 11 0.73 6 H2 Isolate 578 (L) 1 12 0.08 4 L Isolate 578 (H2) 6 10 0.6 7 H2 Reassortant 115(L)R 2 11 0.18 10 L Reassortant 115(H2)R 6 11 0.55 12 H2 Reassortant X-53a 9 4 2.25 13 HI The virus phenotype is based on the hemagglutination titer and Sa-13/9C8 antibody titer ratios (L, <0.25; H', >1.0; H2, 0.5-0.8) by the relationship Sa-13 HI titer divided by 9C8 HI titer. HI, hemagglutination inhibiting antibody. *Non-immunoselected egg passage mutant of A/NJ/11/76 (H2) virus. Downloaded by guest on September 30, 2021 Evolution: Kilbourne et al. Proc. NatL. Acad. Sci. USA 85 (1988) 8101

virus replication is intrinsically restricted to a limited number In that instance, strong selection is exerted by an exogenous of susceptible cells, (ii) that L mutants actively interfere agent and host adaptation is not involved. unidirectionally with H2 mutant replication, or (iii) that the It is possible that L and H phenotypes act as alleles in H2 -* L mutation and selection occurs at a high frequency at facilitating replication in natural infection in different hosts. any or all stages of infection. And, of course, any combina- The apparent predominance of H virus in pre-1957 swine tion of these mechanisms might be operative. influenza viruses (14) may simply reflect prolonged labora- The importance of the present observations lies not in the tory passage of these viruses in chicken embryos in compar- reaffirmation that single amino acid substitutions in the ison to more recently isolated strains. Analysis of contem- influenza virus HA gene can markedly influence viral repli- porary (post-1976) swine influenza virus isolates from swine cation (9, 10) or that HA mutants can arise during adaptation (4, 14), humans (7), and turkeys (E.D.K., unpublished data) of virus to an alien host (11). Unprecedented, to our knowl- shows a preponderance of virus with the L phenotype. edge, are the predictability of this evolutionary event and its However, in experimental infection of turkeys, no preferen- occurrence within the span of a single exogenous infection in tial replication ofL viruses is demonstrable and, indeed, field the natural host of the infecting virus. Certainly, the sequen- strain or reassortant H-phenotype viruses have a striking tial HA mutations involved in the antigenic drift of influenza affinity for the nasal mucosa (E.D.K. and B.C.E., unpub- virus are unpredictable, as was most strikingly shown by lished data). However, evidence does not yet fully support Raymond et al. (12) who described two pathways in the the concept of L and H mutants as mammaliotropic and natural evolution of closely similar 1950 and 1977 H1N1 avianotropic alleles, respectively. Neither L nor H variants prototype viruses. More relevant is evidence that adaptation ofwild-type swine influenza viruses isolated from humans are of the H1N1 human influenza virus to eggs was effected by infective for turkeys. Thus, neither L nor H phenotype in the HA mutants belonging to any of three distinct antigenic absence of other viral mutations adapts the virus to turkeys phenotypes (11). In unpublished experiments, we have es- in a single step as is the case with swine-PR8(L) reassortants tablished that L and H phenotypes are not determined by in swine (4). propagation in eggs of MDCK cells as hosts. Indeed, both these laboratory hosts favor the selection of H mutants, The excellent technical assistance of Ms. B. Pokorny and Ms. making it all the more remarkable that the L mutation in Martha Sheerar is gratefully acknowledged. This work was sup- swine is so readily demonstrable. ported by Public Health Service Grant Al 09304. Experimental infection with equine infectious anemia vi- 1. Kilbourne, E. D., Gerhard, W. & Whitaker, C. W. (1983) Proc. rus, a retrovirus, resulted in sequential disease episodes at 4- Natl. Acad. Sci. USA 80, 6399-6402. to 8-week intervals, each associated with the emergence of 2. Kilbourne, E. D., Both, G. W. & Gerhard, W. (1984) in distinct antigenic variants (13). However, this situation only Negative Strand Viruses, eds. Compans, R. M. & Bishop, D. superficially resembles the rapid evolution of L mutants in (Academic, New York), pp. 233-237. swine. Equine infectious anemia virus is a retrovirus that 3. Kilbourne, E. D., Taylor, A. H., Whitaker, C. W., Sahai, R. & causes persistent infection, differing emergent variants ap- Caton, A. J. (1988) Proc. Natl. Acad. Sci. USA 85, 7782-7785. pear after immune response has occurred, and variants 4. Kilbourne, E. D., McGregor, S. & Easterday, B. C. (1979) Infect. Immun. 26, 197-201. appear to be escape mutants from developing host antibody. 5. Both, G. W., Shi, C. H. & Kilbourne, E. D. (1983) Proc. Natl. In contrast, L and H mutants are indistinguishable by swine Acad. Sci. USA 80, 6996-7000. influenza virus polyclonal antibodies and undergo antigenic 6. Wiley, D., Wilson, I. A. & Skehel, J. J. (1981) Nature (London) change not from immunoselection but as a pleiotropic con- 289, 373-378. comitant of selection for replication advantage (7); their 7. Kilbourne, E. D. (1978) Proc. Nati. Acad. Sci. USA 75, 6258- antigenic differences are detectable only with specific mono- 6262. clonal antibodies. This evolution of "lab-adapted" virus to 8. Kilbourne, E. D. (1980) Yale J. Biol. Med. 53, 41-45. flourish in another ecologic niche-the swine respiratory 9. Rogers, G. N., Paulson, J. C., Daniels, R. S., Skehel, J. J., tract-is thus effected by a single-base change resulting in a Wilson, I. A. & Wiley, D. C. (1983) Nature (London) 304, 76- -* 78. Glu Lys amino acid substitution at position 153 (156 in the 10. Deom, C. M., Caton, A. J. & Schultze, I. T. (1986) Proc. Natl. H3 model) of the viral HA (3). Whether or not one views this Acad. Sci. USA 83, 3771-3775. event as a simple reversion to L as the phenotype most 11. Robertson, J. S., Bootman, J. S., Newman, R., Oxford, J. S., commonly isolated from natural infections of swine (4, 14), Daniels, R. S., Webster, R. G. & Schild, G. S. (1987) Virology the predictability ofthe event and the magnitude ofchange in 160, 31-37. viral replication capacity are striking as a model of instant, 12. Raymond, F. L., Caton, A. J., Cox, N. J., Kendal, A. P. & predictable evolutionary adaptation to a changed environ- Brownlee, G. G. (1986) Virology 148, 275-287. ment. Especially noteworthy is the emergence of the L 13. Salinovich, O., Payne, S. L., Montelaro, R. C., Hussain, mutant to predominance during the course of a 7-day infec- K. A., Issel, C. J. & Schnorr, K. L. (1986) J. Virol. 57, 71-80. 14. Kendal, A. P., Noble, G. R. & Dowdle, W. R. (1977) Virology tion. 82, 111-121. Looking more broadly at microbial infections in general, 15. McDermott, W., Muschenheim, C., Hadley, S. J., Bunn, P. A. only the rapid development of bacterial resistance to amino- & Gorman, R. V. (1947) Ann. Inter. Med. 27, 769-822. glycoside antibiotics (15, 16) appears analogous to the H -* 16. Hall, W. H. & Spink, W. W. (1947) Proc. Soc. Exp. Biol. Med. L mutation in occurring within the span of a single infection. 64, 403-406. Downloaded by guest on September 30, 2021