Virulence of Avian Influenza A Viruses for Squirrel Monkeys

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1 INFECTION AND IMMUNITY, Sept. 1982, P /82/ $02.00/0 Copyright C 1982, Anierican Society for Microbiology Vol. 37, No. 3 Virulence of Avian Influenza A Viruses for Squirrel Monkeys BRIAN R. MURPHY,'* VIRGINIA S. HINSHAW,2 D. LEWIS SLY,3 WILLIAM T. LONDON,4 NANETTE T. HOSIER,I FRANK T. WOOD,1 ROBERT G. WEBSTER,2 AND ROBERT M. CHANOCK1 Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Disease', and National Institute of Neurological and Communicative Disorders and Stroke, Bethesda, Maryland 20205,4 Division of Virology, St. Jude Children's Hospital, Memphis, Tennessee ; and Meloy Laboratories, Rockville, Maryland Received 16 February 1982/Accepted 27 April 1982 Ten serologically distinct avian influenza A viruses were administered to squirrel monkeys and hamsters to compare their replication and virulence with those of human influenza A virus, A/Udorn/307/72 (H3N2). In squirrel monkeys, the 10 avian influenza A viruses exhibited a spectrum of replication and virulence. The levels of virus replication and clinical response were closely correlated. Two viruses, A/Mallard/NY/6874/78 (H3N2) and A/Pintail/Alb/121/79 (H7N8), resembled the human virus in their level and duration of replication and in their virulence. At the other end of the spectrum, five avian viruses were restricted by 100- to 10,000-fold in replication in the upper and lower respiratory tract and were clearly attenuated compared with the human influenza virus. In hamsters, the 10 viruses exhibited a spectrum of replication in the nasal turbinates, ranging from viruses that replicated as efficiently as the human virus to those that were 8,000- fold restricted. Since several avian viruses were closely related serologically to human influenza viruses, studies were done to confirm the avian nature of these isolates. Each of the avian viruses plaqued efficiently at 42 C, a restrictive temperature for replication of human influenza A viruses. Avian strains that had replicated either very efficiently or very poorly in squirrel monkeys still grew to high titer in the intestinal tracts of ducks, a tropism characteristic of avian, but not mammalian, influenza viruses. These observations indicate that some avian influenza A viruses grow well and cause disease in a primate host, whereas other avian viruses are very restricted in this host. These findings also provide a basis for determining the gene or genes involved in the restriction of replication that is observed with the attenuated avian viruses. Application of such information may allow the preparation of reassortant viruses derived from a virulent human influenza virus and an attenuated avian virus for possible use in a live attenuated vaccine for prevention of influenza in humans. Influenza A viruses are widely distributed in nature, with humans, horses, pigs, seals, and a large number of species of birds serving as hosts (4; V. S. Hinshaw and R. G. Webster in Recent Influenza Research and Progress Toward Epidemiological Control, in press). Birds have been the most abundant source of different antigenic variants of influenza A virus. Thus, each of the 12 serologically distinct hemagglutinins and 9 distinct neuraminidases have been detected on viruses recovered from birds (Hinshaw and Webster, in press). It has been postulated that new pandemic human influenza A viruses arise by gene exchange between human and nonhuman influenza A viruses (35). In 1957, the new pandemic H2N2 Asian viruses possessed hemagglutinin, neuraminidase, and two polymerase genes unrelated to those of the then circulating HlNl viruses (26). In 1968, the new H3N2 Hong Kong pandemic virus possessed all of the genes of the previous H2N2 human virus except for the one coding for hemagglutinin (26). Avian influenza A viruses have been considered as a possible source of genes that could be exchanged with those of a human virus during mixed infection, yielding reassortant pandemic viruses such as those that appeared in 1957 and 1968 Human influenza A viruses can replicate to a limited extent in the respiratory tract of birds, and this could be a possible site of mixed infection (Hinshaw and Webster, in press). However, little information is available concerning the ability of avian influenza A viruses to replicate in mammals and humans. Recent studies have shown that currently circulating avian strains can infect several experimentally inoculated mammals, i.e., pigs, ferrets, cats, mink, and 1119

2 1120 MURPHY ET AL. mice (1, 9, 15, 19, 31, 33, 39). The detection of an avian virus in seals with viral pneumonia (13, 34) suggests that avian viruses infect mammals in nature. Studies by Beare (personal communication) indicated that avian viruses had little, if any, ability to infect humans, and there have been only two reports of human infection with an avian virus (3, 35). The isolation of influenza A/Seal/Mass/1/80 (H7N7) virus from the eye of an acutely infected laboratory worker shows that some avian-like influenza viruses have the potential for replication in humans (32). There is no serological evidence that suggests transmission of avian influenza A viruses to humans (4, 10). One goal of the present study was to evaluate the ability of avian viruses to replicate in the upper and lower respiratory tracts of a nonhuman primate. Squirrel monkeys were chosen to determine the extent to which a variety of avian viruses could replicate in primate cells, since this readily available primate develops illness when infected with each of three human influenza A viruses, and viral replication in these animals can be readily quantitated (19). The replication of avian viruses in hamsters and ferrets was also examined to compare viral replication in other mammals. A second goal was to identify an avian influenza virus that is restricted in replication in primate cells. Such an attenuated virus could then possibly serve as a donor of attenuating genes to produce a live reassortant human vaccine virus that is restricted in its replication in humans. MATERIALS AND METHODS Viruses. The human influenza A virus, A/ Udorn/307/72 (H3N2), used in these studies was a cloned virus shown to be virulent in humans and squirrel monkeys (18, 23). The virus suspension used in the present study was grown in the allantoic cavity of specific pathogen-free (SPF) eggs (SPAFAS, Storrs, Conn.) and titered % tissue culture infective dose (TCID50) per ml on canine kidney (MDCK) cell monolayer culture. The isolation and characterization of the other human influenza A viruses (See Table 1) have been described (16, 17, 22; Murphy et al., unpublished data). These viruses were grown in the allantoic cavity of 10-day-old eggs. The initial isolation of the avian influenza A viruses used in this study has been described previously (9). Briefly, these viruses were isolated in 10- to 11-day-old embryonated eggs from cloacal swabs of healthy feral ducks or from a sick domestic turkey. The 10 avian viruses included in this study were chosen because the'y produced plaques on monolayer in primary chick kidney (CK) cell culture and on a continuous line of MDCK cells and therefore could be readily quantitated in vitro. The first or second egg passage of these viruses was grown in 10- to 11-day-old SPF embryonated chicken eggs and then was cloned by plaque-toplaque passage in primary CK cultures prepared from 1- to 4-day-old SPF chicks. A suspension of each INFECT. IMMUN. strain was then prepared for use in this study by growing the plaque-purified virus in the allantoic cavity of 10-to to 11-day-old embryonated SPF eggs. To ensure that these stocks did not contain retrovirus, each stock was assayed for reverse transcription as described previously (29), and none was found. Animals. Squirrel monkeys (Saimiri sciureus) were feral adults which had been quarantined and conditioned in the laboratory for at least 6 months before inoculation. Each monkey selected for the study had a serum hemagglutination-inhibiting (HI) antibody titer < 1:4 for the virus administered. Compatible pairs of monkeys were housed in airflow-controlled isolation cages, and routine feeding and care were performed as previously described (7, 18). In an 0.5 ml inoculum, 1070 TCID50 of virus was administered transtracheally to monkeys lightly anesthetized with ketamine hydrochloride (40 mg/kg) administered intramuscularly. Control animals received normal allantoic fluid. The clinical observations were made blind, the observer did not know whether the animal had received infectious or control fluid. The animals were clinically evaluated and sampled as described previously (18) except that, in the present study, each animal also had a tracheal lavage on days 2, 4, and 6 postinoculation. The lavage was performed by injecting 1.5 ml of sterile phosphate-buffered saline through a no. 8 French endotracheal tube, positioned at midtrachea, and aspirating the washings. The endotracheal tubes were fashioned from disposable urethral catheters (Sovereign B, sterile single-use feeding tube and urethral catheter, Monoject, St. Louis, Mo.) by trimming either end to approximately 10 cm in length. Fifteen-week-old Golden Syrian female hamsters (Charles River, Lakeview, Newfield, N.J.) were anesthetized with pentobarbital sodium and inoculated intranasally with TCID50 of virus in a 0.1-ml inoculum. Six hamsters from each group were sacrificed daily on days 1, 2, 4, and 7 (see experiment 1, Table 4) and on days 1, 2, 3, and 4 (see experiment 2, Table 4). Homogenates of the individual lungs and nasal turbinates were prepared as described previously (28). The titer of virus in individual lung or nasal turbinate specimens was determined, and the daily mean log10 titer was expressed as TCID5J/gm of tissue. Ferrets and ducks (pekin white, 2 to 4 months old) were used after serological tests and attempts at virus isolation failed to document prior or current infection with an influenza A virus. The ferrets were inoculated intranasally with % egg infectious dose (EID50) of virus and ducks were inoculated with 107 to 108 EID50 of virus. The animals were examined daily for clinical signs of disease. Cloacal and tracheal swabs and feces were taken daily from the birds and treated with antibiotics before assaying for infectious virus as previously described (10). Nasal washes from the ferrets were collected as described (21), except that the animals were anesthetized with ketamine (Bristol Laboratories, Syracuse, N.Y.) before sampling to facilitate handling and to increase mucosal secretions. Nasal washes were titrated in embryonated chicken eggs to determine the EID50mM of wash. Sera from the ferrets at 14 days after virus administration were examined for HI antibody. Criteria for designation of illness. Squirrel monkeys were observed twice daily for 10 days and also on days 14 and 21 for signs of systemic illness by noting feed

3 VOL. 37, 1982 TABLE 1. nfludmiisered VIRULENCE OF AVIAN INFLUENZA A VIRUSES 1121 Efficiency of plaque formation of human and avian influenza A viruses in primary CK or MDCK cells at 37, 41, and 42 C subtyr Antigenic Tissue used (LoglO) PFU/ml at: A/Mallard/Alb/88/76 H3N8 (Hav7Neq2) CK A/Pintail/Alb/286/78 H4N8 (Hav4Neq2) CK A/Mallard/Alb/573/78 HlNl (HswlNl) CK A/Mallard/Alb/827/78 H8N4 (Hav8Nav4) CK A/Mallard/NY/6874/78 H3N2 (Hav7N2) CK A/Turkey/MN/5M9 H1ON7 (Hav2Neql) CK A/Pintail/Alb/119/79 H4N6 (Hav4Navl) CK A/Pintail/Alb/121M9 H7N8 (HavlNeq2) CK A/Mallard/NY/6750/78 H2N2 (H2N2) CK A/Mallard/NY/6750/78 H2N2 (H2N2) MDCK A/Pintail/Alb/358/79 H3N6 (Hav7Navl) CK A/Pintail/Alb/358n9 H3N6 (Hav7Navl) MDCK A/Udorn/307n2b H3N2 (H3N2) CK <3.7 A/Udom/307/72b H3N2 (H3N2) MDCK <3.7 A/Californialom78b HlNl (HlNl) CK 6.0 <2.7 <2.7 A/California/10/78b HlNl (HlNl) MDCK <2.7 A/Hong Kong/123/77b HlNl (HlNl) MDCK 5.5 <0.7 <0.7 A/Georgia/lol/n4b H3N2 (H3N2) MDCK <0.7 A/Alaska/6n77b H3N2(H3N2) MDCK 6.5 <0.7 <1.7 A/Washington/897/80b H3N2 (H3N2) MDCK <1.7 a These subtype designations are from the current nomenclature (38). The designations in parentheses are from the previous nomenclature (37). b Human influenza A virus. consumption and general level of activity. Respiratory signs of dyspnea, coughing, and sneezing were also noted during a 10-min observation period. Lateral and dorsoventral thoracic radiographs were also taken before virus inoculation and on days 2, 6, and 14 after virus inoculation. Signs of upper respiratory illness were scored daily when swabs for virus isolation were taken. A score of 1 was given to any animal with a dry discharge around the nares. A score of 2 was given if the nasal swab contained purulent discharge when removed from the nostril, and a score of 3 was recorded if rhinorrhea was present before swabbing. These upper respiratory illness scores were used to calculate the severity of illness (see Table 3). The total score achieved over the 10-day observation period was added for each animal, and the mean score ± the standard error was determined for the group. Tissue culture and virus assay. Primary CK cultures were prepared from 1- to 4-day-old SPF chicks as described (14). When the virus was plaque purified, 0.25,ug of trypsin per ml was incorporated in the overlay. Cloned virus was found to plaque efficiently in CK monolayers in the absence of trypsin. Efficiency of plaque formation at various temperatures was assayed by plaque titration in primary CK monolayers or MDCK monolayers on six-well plastic plates (Costar, Cambridge, Mass.) with Liebowitz Medium 15 (M.A. Bioproducts, Walkersville, Md.), and 1 p.g of tolylsulfonyl phenylalanyl chloromethyl ketone trypsin per ml (Worthington Diagnostics, Freehold, N.J.) with an 0.8% agarose overlay. Infectivities of viral inocula, tissue homogenates, nasopharyngeal swabs, and tracheal lavages were determined by endpoint titration cytopathic effect on MDCK monolayers in 24-well plates (Costar) with Eagle no. 2 medium supplemented with antibiotics and 1,ug of tolylsulfonyl phenylalanyl chloromethyl ketone trypsin per ml as previously described (30). The EID50 was determined as described previously (10). HI tests. The HI tests were performed as described (20). RESULTS Efficiency of plaque formation of human and avian influenza A viruses. Avian influenza A viruses are able to replicate at 42 C, a temperature restrictive for efficient replication of human influenza A viruses (6). The efficiencies of plaque formation of the 10 avian influenza A viruses were determined (Table 1) in CK or MDCK tissues or both and compared with those of recent human influenza A viruses belonging to the HlNl and H3N2 subtypes that were known to produce influenza illness in volunteers (16, 17, 19; Murphy et al., unpublished data). Some of the human influenza A viruses did not plaque efficiently in CK tissue, and their efficiencies of plaque formation were therefore evaluated inmdck tissue. Each of the 10 avian viruses produced plaques with high efficiency at 42 C, whereas each of the human influenza A viruses failed to produce plaques at this temperature. The restriction in replication of human influenza virus at 42 C was observed in both CK and MDCK tissue. This biological test confirms the avian heritage of each of the 10 avian influen-

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5 VOL. 37, 1982 VIRULENCE OF AVIAN INFLUENZA A VIRUSES 1123 TABLE 3. Correlation of viral replication with severity of illness in squirrel monkeys' Duration of Severity of illness viral replication in nasopharynx High Low Long 5 0 Short 0 6 aa long duration of viral shedding is >4.0 days (Table 2) and a high level of severity of illness is a score of >5.0. The Kendal coefficient of association is 1.00; P < 0.01, Fisher exact test. za A viruses, some of which are closely related antigenically to the human strains (Table 1). Evaluation of the avian influenza A viruses in squirrel monkeys. Each monkey received TCID50 of avian influenza A virus transtracheally, and the duration and magnitude of virus replication and the clinical response were assessed and compared with those of a human influenza A virus (Table 2). There was a correlation between the level of virus replication and clinical response, which indicates that the illness induced by the virus was a function of its ability to replicate in the monkey (Table 3). The A/Mallard/NY/6750/78 virus was completely attenuated in squirrel monkeys. Conversely, the A/Pintail/Alb/121/79 and A/Mallard/NY/6874/78 viruses replicated efficiently in squirrel monkeys and induced illness of a severity and duration comparable to that of the human influenza A virus. Radiographic evidence of pneumonia was seen in 1 of the 4 animals infected with A/Mallard/NY/6874/78 virus, in 2 of the 4 animals infected with A/Pintail/Alb/121/79 virus, and in 2 of the 11 animals infected with A/Udorn/72 human influenza A virus. Signs of systemic illness were also observed in monkeys infected with each of these three viruses with the same frequency as radiographically detected illness. In general, the avian viruses did not induce a high titer of serum HI antibody, although some of the avian viruses grew moderately well. This finding is similar to primary infection of ducks with avian influenza A viruses in which immunity is induced despite the development of a low level of serum HI antibody (8, 12). Evaluation of the avian influenza A viruses in hamsters. Hamsters were inoculated intranasally with approximately 105 TCID50 of virus, and 0 the amount of virus in the lungs and nasal turbinates was determined (Table 4). Replication of AIMallard/NY/6750/78 virus was 1,000-fold reduced in the upper and lower respiratory tracts compared to the human wild-type virus. At the other end of the spectrum were the A/Pintail/Alb/121/79 and A/Mallard/Ny/6874/78 viruses which were only slightly reduced in replication. In general, replication of the avian viruses was more restricted in nasal turbinates than in lungs. There was a correlation between the replication levels of avian influenza A viruses in the upper respiratory tracts of hamsters and squirrel monkeys (Table 5). TABLE 4. Replication of avian influenza viruses in the nasal turbinates and lungs of hamstersa Difference in level of replication Maximum Maximum between human and avian virus Influenza A virus level of level of (maximum level of replication of administered replication in nasal replication human virus - maximum level of in lungs replication of avian virus) Nasal turbinates Lungs Expt 1 A/Mallard/NY/6750/ ± ± A/Mallard/Alb/827n8 3.3 ± ± A/Pintail/Alb/119/ ± ± A/Pintail/Alb/286/ ± ± A/Mallard/Alb/88n6 4.7 ± ± A/Udorn/307n2 6.1 ± ± 0.24 Expt 2 A/Mallard/Alb/573n8 4.3 ± ± A/Turkey/MN/5M9 6.2 ± ± A/Pintail/358n9 4.0 ± ± A/Pintail/Alb/121M9 6.2 ± ± AlMallard/NY/6874/ ± ± A/Udorn/307/ ± ± 0.20 a Each hamster received approximately 1050 TCID50 of virus intranasally. Eight hamsters from each group were sacrificed on days 1, 2, 4, and 7 (experiment 1) and on days 1, 2, 3, and 4 (experiment 2). The values listed represent the mean log1o titer ± the standard error expressed as TCID50/gm of tissue and is the highest mean titer reached on one of the four days of sacrifice.

6 1124 MURPHY ET AL. TABLE 5. Correlation of the replication of influenza A viruses in the upper respiratory tracts of hamsters and squirrel monkeys' Level of Duration of replication in replication in squirrel monkeys hamsters Long Short High 5 1 Low 0 5 aa high level of replication in hamsters is defined as a mean titer of greater than 4.0 TCID50/gm (log10) of virus in nasal turbinates (Table 4) and a long duration of replication in squirrel monkeys is defined as greater than 4.0 days of virus shedding. The Kendal coefficient of association is 0.83; P < 0.02, Fisher exact test. Evaluation of selected avian viruses in ducks and ferrets. Two viruses (A/Mallard/NY/6750/78 and A/PintailIAlb/358/79) that were attenuated in squirrel monkeys and two (A/Turkey/Minn/5/79 and A/Mallard/NY/6874/78) that were not were evaluated in ducks and ferrets for their levels of replication and virulence (Table 6). Each of the avian viruses produced an asymptomatic infection in the ducks and replicated in both the respiratory and gastrointestinal tracts. The human virus did not infect the ducks. In ferrets, each of the avian strains replicated but failed to produce signs of disease, whereas the human virus induced fever and nasal discharge in these animals. The A/Mallard/NY/6750/80 virus reached a lower peak titer than the other avian viruses or the human virus. Compared with the human virus, the avian viruses stimulated a low level of serum HI antibody, although the level and duration of virus replication resembled that observed for the human virus. DISCUSSION The present study demonstrates that the 10 serologically distinct avian influenza A viruses exhibit a spectrum of virulence for squirrel monkeys, a primate that has been shown to develop illness after infection with human influenza A viruses (18). At one end of the spectrum was the INFECT. IMMUN. A/Mallard/NY/6750/78 virus which infected squirrel monkeys but did not induce illness. This virus also showed restricted replication in hamsters and ferrets. Although this virus is closely related serologically to the 1957 H2N2 influenza virus, its avian character was demonstrated by its ability to replicate at high temperatures (42 C) and by its enterotropism for ducks. At the other end of the spectrum were several viruses that grew moderately well and induced illness comparable to that produced by the human virus. These latter observations have several implications for the generation of new influenza A virus pandemics in the human population. First, it is clear that some avian viruses are as virulent for primates (2 of the 10 viruses tested) as currently circulating human influenza A viruses, and by inference such avian viruses could be virulent for humans. A virus such as the A/Pintail/Alb/121/79 (H7N8) virus might be directly introduced into the human population and spread unhindered by immunological constraints because the human population would have no prior experience with viruses sharing either of its surface antigens. These suggestions must be offered with the caveat that the infectious doses and the disease-producing doses of the avian and human influenza A viruses were not examined in the squirrel monkey because of the limited availability of this species. Such a comparison might reveal large differences between the virulent human and avian viruses. Second, since some avian viruses can replicate efficiently in primates, it is possible that humans could be the host in which an exchange of genes between human and avian viruses could occur. Furthermore, the existence of several viruses that replicate efficiently in primate cells suggests that there are a large number of genes present in the total avian influenza A virus population that function efficiently in primate cells. Such genes might function efficiently in the genome of a new, potentially pandemic, reassortant virus. To assess the potential diversity of this gene pool, it will be important to determine the degree of TABLE 6. Evaluation of selected avian influenza A viruses in ducks and ferrets Ducks' Ferrets" Cloaca Trachea Nasal washes Influenza A virus No. of days Reciprocal of virus otiterof virus of virus Peak virus titer serum (day 14) shedding (logl0 EID5s/ml) shedding shedding (log,( EIDo0/ml) HI antibody titer A/Mallard/NY/6750/ A/Pintail/Alb/358/ A/Turkey/Minn/5/ A/Mallard/NY/6879/ A/Udorn/307/ a A minimum of two animals was used for each virus tested and values presented are means.

7 VOL. 37, 1982 nucleic acid homology between the genes present in virulent avian influenza A viruses and those of human influenza A viruses, as well the degree of homology between avian influenza A virus strains. It is important to emphasize, however, that since influenza A viruses were isolated in the early 1930s only two new putative reassortant viruses (the 1957 [H2N2] and 1968 [H3N2] strains) have arisen and spread to man. This implies that proper conditions for the generation, emergence, and spread of pandemic reassortant viruses are only rarely achieved in nature. Many live, attenuated virus vaccines have been generated by passage in cells obtained from a heterologous host (5). Avian influenza A viruses are passaged continuously from bird to bird, presumably largely by a fecal-oral mode (11, 36; V. S. Hinshaw and R. G. Webster, in press). From the vantage point of humans, this represents passage of virus in a heterologous host, and one would expect some of the avian viruses would be attenuated for humans. Also, since the avian viruses replicate in the columnar epithelial cells of the gut (8, 36), multiplication of the avian viruses in the gut also represents passage in a heterologous organ system because the gut is not a known site of replication of human influenza A viruses. Adaptation to grow in this heterologous organ system could lead to genetic changes that restrict replication in the cells of the human respiratory tract. Thus, it was not surprising to identify an avian virus, A/MallardINY/6750/78, that was attenuated for primates and ferrets and showed restricted replication in the respiratory tract of these mammals as well as in that of hamsters. It should be possible to identify the genes in this avian virus that are responsible for attenuation in primates and subsequently to transfer these genes via genetic reassortment to new human influenza A epidemic viruses and thereby produce live attenuated vaccine viruses. In this manner, restriction of replication of the reassortant viruses for humans would be effected by naturally occurring avian influenza A virus genes rather than by mutant genes produced by chemical mutagenesis or selected by passage of virus at a low temperature, in an unnatural host, over a limited period of time. Since many of the influenza genes that have evolved over a long period of time in birds differ significantly from the corresponding genes of human influenza viruses (2, 24, 25, 27), it is possible that the avian genes will retain their attenuated characteristics after limited replication in humans and thereby prove to be phenotypically stable. 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