A Molecular Genetic Approach to Improved Animal Health

BOVINE RESPIRATORY DISEASE UPDATE

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A MOLECULAR GENETIC APPROACH TO IMPROVED ANIMAL HEALTH The Effect of Interferon Genotype on the Severity of Experimental Bovine Herpesvirus-l Infection Anne M. Ryan, DVM, PhD, and James E. Womack, PhD

Over the past decade, biotechnology has brought large-animal veterinary medicine an increased understanding of the effects of genetic background on disease resistance and production traits. The Human Genome Project has produced a genetic "map" as a tool to study the role of specific genes in development, aging, cancer, reproduction, immunology, physiology, and hereditary diseases. The technological advances of the Human Genome Project have been incorporated into the developing domestic animal gene maps; the human map has also acted as a blueprint to identify "candidate" genes for specific animal diseases. Some examples of genetic diseases in domestic animals that have been identified following the identification of the specific defect in man include: • Leukocyte adhesion deficiency (Holstein cattle) due to a point mutation in the f3 integrin CD18 This work was supported by USDA 87-CRSR-2-3126, USDA 90-CSRS-34116, Binational Agricultural Research and Development Fund US-1687-89, the Texas Advanced Technology Program and the Texas A&M University Regents' Fellowship Program.

From the Department of Veterinary Pathobiology and the Center for Animal Genetics, Institute of Biosciences and Technology, Texas A&M University, College Station, Texas

VETERINARY CLINICS OF NORTH AMERICA: FOOD ANIMAL PRACTICE VOLUME 13 • NUMBER 3 • NOVEMBER 1997

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• Hyperkalemic periodic paralysis (Quarter horses) caused by a point mutation in the skeletal muscle sodium channel a subunit gene • Malignant hyperthermia (swine) due to point mutations in the ryanodine receptor Even in the absence of a homologous human disease, the developing bovine and ovine gene maps have been used to identify genetic markers that are associated with increased production or enhanced disease resistance and can be manipulated in selective breeding programs. Some examples include progressive degenerative myeloencephalopathy (Weaver syndrome) in Brown Swiss (which was inadvertently selected in some herds because it is associated with increased milk production), the fecundity gene in Boorola sheep (controls ovulation rate and litter size), and the callipyge gene, which causes muscular hypertrophy of the hindquarters and associated leanness and feed efficiency in sheep. By far, the greatest successes in domestic animal molecular genetics have been the identification of mutations in individual genes that are responsible for a specific genetic disease. Enhanced meat, milk, and fiber production, reproductive performance, specific carcass traits, and disease resistance are more complex phenotypes, and are generally thought to be influenced by the effects of many genes acting in concert. Such polygenic traits are studied, and the specific genes identified, through either linkage or association studies. Linkage studies are basically a "within family" design in which the relationship between a genetic marker or candidate gene and phenotype is examined in affected and unaffected members of a family. In contrast, association studies have a more epidemiological approach, as they are based in a population of unrelated individuals and are designed to detect an increased frequency of a particular genetic marker among affected individuals. Resistance/ susceptibility to viral diseases is considered a polygenic trait, influenced by the genetic composition of both the virus and host.

THE TYPE I INTERFERON GENE FAMILY IN CATTLE

Interferons (lFNs) are low-molecular-weight proteins with wellcharacterized, potent antiviral, antineoplastic, and immune-modulating properties. As part of the innate immune response, IFNs are critical in protecting the host early in the course of viral infection, before activation of antigen-dependent cellular and humoral immune responses. 25 The type I IFN gene complex, localized to bovine chromosome 8qlS, is composed of IFN-a (IFNA), IFN-f3 (lFNB), IFN-w (IFNW), and trophoblast IFN (lFNT) gene families, each consisting of multiple intronless genes in cattle. 43,44 Although all four families have significant antiviral properties, IFNT is also the major secretory product of preimplantation bovine and ovine embryos, and plays a major role in the maintenance of pregnancy. 31, 32

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The physical organization of the type I IFN gene families on bovine chromosome 8, the number of genes in each family, and the genetic polymorphism among the type I IFNs in both cattle and sheep have been determined. 45, 46 There are approximately 10 IFNA, 6 IFNB, 10 IFNW, and 6 IFNT genes in cattle; similar numbers of IFNA and IFNW genes have been reported in humans. In contrast, IFNT has not been identified to date in any nonruminant species, and only a single IFNB gene is present in man. The high level of genetic polymorphisms identified in 11 bovine type 1 IFN genes (> 70% of the maximum heterozygosity values46) makes them useful genetic markers in linkage analysis or association studies to identify targets for selective breeding studies. IFN AND BOVINE HERPESVIRUS-1 INFECTION IN CATTLE

IFN secretion has been induced in cattle with a number of viruses,34 and has resulted in partial protection against natural and experimental infections with several bovine pathogens. ll Distinction between the direct antiviral and immunomodulating properties of IFN can be made when bovine herpesvirus-l (BHV-l) is used as the challenge virus, as the effects of BHV-Ion immune function have been characterized in vivo and in vitro.6,8 BHV-1 characteristically produces a persistent latent infection similar to other herpesvirus infections in man. 1 Both bovine and human herpesvirus infections respond clinically to homologous (and in cattle, heterologous) recombinant IFN.2, 29, 41 The role of IFN in inhibiting viral replication has not been established for most bovine pathogens, with the exception of BHV-l. Aerosolized BHV-1 results in localized IFN production by the nasal mucosa which provides partial protection against subsequent infection with BHV-1 or another virus. 17-19 Prior corticosteroid treatment enhances IFN production upon exposure to BHV-l via corticosteroid-induced immunosuppression, which facilitates unrestricted viral replication. 16 This suggests that BHV-l is a strong IFN inducer. Peak nasal IFN production is accompanied by a three- to fourfold decrease in viral titer.6 The influence of an animal's genetic composition on viral resistance has been suspected to play a role in some infections.ll In the following study, the relationship between IFN genotype and severity of clinical infection following experimental BHV-1 infection is examined. THE EFFECT OF IFN GENOTYPE ON THE SEVERITY OF BHV-1 INFECTION

BHV-1 is an important agent in the pathogenesis of bovine respiratory disease complex (BRDC), as the resulting immunosuppression predisposes cattle to secondary bacterial pneumonia. 14, 33, 42 BHV-1 infection impairs neutrophil, lymphocyte, and macrophage function. 6, 8, 13, 27 Pre-

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treatment with recombinant bovine IFNA prevented BHV-1-induced immunosuppression2, 7, 35; although not eliminating clinical disease entirely, IFNA significantly decreased morbidity and mortality in calves subsequently challenged with Pasturella haemolytica. 2-4, 20 Nasal excretion of BHV-1 was unchanged in treated and control calves, 2 suggesting that reduced severity of clinical disease was the result of immunomodulation rather than antiviral effects of IFNA. In vitro and in vivo studies have characterized the immunomodulatory effects of bovine IFNA on leukocyte function in detai1. 2, 5, 7, 9, 10 Exploiting genetic differences in endogenous biological response modifiers, such as IFN, may decrease or prevent the immunosuppression associated with BHV-1 infection and lessen the economic losses attributable to BRDC. Current molecular genetic techniques have greatly enhanced the power of genetic and epidemiologic methods to identify specific genes which may modulate disease resistance. Taking a "candidate gene" approach, the authors retrospectively examined health records and genomic DNAs from 98 unrelated, mixed-breed cattle which had been experimentally infected with BHV-1 to identify associations between type I IFN alleles and the severity of clinical disease. Unrelated, mixed-breed cattle were obtained from the Texas Agricultural Experiment Station, Amarillo wheat pastures, and auction barns in Austin, Texas and Tennessee. Following a 7- to 10-day acclimation period, 98 animals seronegative for BHV-1 (titer < 1:4) were selected; these animals were primarily steers (94 steers versus 4 females). The Cooper strain of BHV-1, supplied in 1 mL ampules of 2.7 X 107 plaque forming units (PFU) virus, was used. One ampule was thawed prior to each experiment, diluted 1:100 in Eagle's minimum essential medium, and inoculated intranasally. All animals in the study demonstrated seroconversion to BHV-1 (titer> 1:512) by day 14. At the end of the study, blood was collected from each animal for DNA analysis. Following BHV-1 challenge (and independent of the DNA analysis), cattle were retrospectively classified into 2 groups on the basis of rectal temperature, feed intake and weight gain during postinoculation (PI) days 4 to 8, the period when most animals became febrile (rectal temperature > 102.5°F). Animals classified as mildly affected (MA) had an average rectal temperature of 104°F or less during this period (range 101.6-104.0°F) whereas severely affected (SA) cattle exceeded 104°F (range 104.1-106.1°F). Daily feed intake and weight gain during PI days 4 to 8 also differed significantly between the two groups. Temperature profiles for the two groups were similar, but both the magnitude and duration of the febrile response was more pronounced in SA cattle. Sex, source, and ambient conditions did not appear to represent confounding variables; consequently, data from individual experiments were pooled prior to analysis. Allelic frequencies for the 16 type I IFN loci examined were compared in MA and SA cattle. For most loci, allelic frequencies in MA animals approximate those from a larger population of cattle derived from the same sources. 45 ,46 The distribution of IFN alleles between MA

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and SA populations was compared in 2 X 2 contingency tables. Alleles at 3 IFN loci (IFNBl, IFNW4, IFNW8) were significantly associated with the severity of clinical disease following BHV-l challenge. At the IFNBI locus, animals with allele C were four times more likely to be severely affected than cattle without this allele. Allele C was present in 17% of SA cattle compared to 5% of MA cattle (Fig. 1). Seventeen cattle had allele C at this locus; 13 of 17 (76%) were classified as SA whereas 4 (24 0/0) were MA. Four CC homozygotes, all SA, were also identified. There was no apparent heterogeneity in the data from individual experiments, supporting the decision to pool data. In addition, allele B from this same locus was positively associated with the MA rather than SA phenotype. Allele B was present in 20% of the MA population compared to 8% of the SA population. BB homozygotes were both classified as MA. Of the 23 cattle with allele B, 15 (65%) were MA in contrast to 8 (35%) SA. The distribution of allele C was skewed between the MA and SA populations at frequencies of 0.05 and 0.17, respectively. The frequency of allele C in the SA population also differed from that of a larger population of cattle derived from the same source. In addition, frequencies for allele B were different in MA (0.20) and SA cattle (0.08), and the frequency in the MA population was different from the larger population. In the IFNW4 contingency table (Fig. 2), the more severe clinical phenotype was twice as frequent in cattle with allele 2 than cattle with allele 1. Thirty percent of the SA population had allele 2 at this locus compared to 16% of the MA population. No 22 homozygotes were observed, but of the 45 cattle heterozygous for allele 2, 32 (71 0/0) were from the SA population in contrast to 13 (29%) from MA group. The frequency of allele 2 differed between SA and MA populations (0.31

Allele C Other Total

SA 17 85 102

MA 4 82 86

Total 21 167 188

{psi}=4.1 (95% CI=3.1-5.4); Fisher's Exact Test P=O.OI

Allele B Other Total

MA 17 69 86

SA 8 94 102

Total 25 163 188

{psi}=2.9 (95% CI=2.2-3.8); Yates'

x2 =4.8, P
Figure 1. The IFNB1 allele C was 4.1 times more frequent in SA cattle (P = 0.01), whereas allele B was 2.9 times more prevalent in cattle with the milder clinical phenotype (P < 0.05). The distribution of alleles Band C was skewed between MA and SA cattle. Allele frequencies also differed significantly from that of a larger population derived from the same source (allele B: P < 0.01; allele C: P < 0.001). CI = confidence interval.

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Allele SA 2 32

1

72

Total 104

MA 13 67 80

Total 45 139 184

{psi}=2.3 (95% CI=1.8-2.9); Yates' x2 =4.4, P<0.05 Figure 2. The IFNW4 allele 2 was 2.3 times more frequent in cattle classified as SA (P < 0.05). The distribution of allele 2 was skewed between MA and SA cattle, and allele frequencies differed significantly (P < 0.005) from those observed in a larger population of caUle obtained from the same sources.

versus 0.17), and the frequency in SA cattle was increased relative to the general cattle population. The distribution of IFNW8 alleles A and B between groups is shown in Fig. 3. Cattle with allele A at this locus were more than twice as likely to be severely affected following viral challenge. Fifty-seven percent (18/28) of cattle with allele A were classified as SA. The frequency of allele A was skewed between SA and MA cattle (0.56 versus 0.35), and also differed significantly between SA cattle and the general population. Disease association studies are generally based on the assumption of a single major locus which predisposes to disease 15; however, complete concordancy between BHV-1 clinical phenotype and IFN genotype was not observed in this study. Possible explanations for this lack of concordancy include the presence of additional environmental factors, genetic recombination between the IFN and disease loci, or polygenic inheritance influencing penetrance. Because of the inherent limitations of retrospective studies, the authors' results should be confirmed in other cattle popUlations by prospective studies before embarking on a selective breeding program. Genetic control of IFN induction in mice22-24,52 and of IFN production in human leukocytes40 has been documented. Primary genetic defects in IFN production have not been described, but impaired production has been reported for certain diseases, including herpes simplex in man and mouse,3S,51 and may be a consequence of indirect effects of the disease on leukocytes. 47 Although the genetic mechanism is unknown,

Allele A B Total

SA 26 20 46

MA 14 26 40

Total 40 46

86

{psi} =2.4 (95% CI=1.8-3.3); Yates' x2 =3.2, P-0.06 Figure 3. The IFNW8 allele A was 2.4 times more frequent in cattle classified as SA (P = 0.06). The distribution of allele A was skewed between MA and SA cattle; allele frequencies differed significantly (P < 0.001) from those of a larger population of cattle from the same sources.

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allelic differences at specific IFN loci may identify cattle which are unable to counteract the immunosuppressive effects of BHV-I, either by increased neutrophil recruitment and activity or IFN production. Alternatively, these alleles may serve as markers for enhanced susceptibility to the proinflammatory effects of IFN, with cachexia and fever resulting from IFN's role in the acute phase response to BHV-l infection. 12 In vitro studies on the production of IFN and other cytokines and neutrophil function in BHV-I-affected cattle with· specific IFNB and IFNW genotypes are needed. Although previous studies have examined the effects of exogenous bovine IFNA on leukocyte function during BHV-1 infection, IFNB has greater antiviral and antiproliferative properties than bovine IFNA in vitro2I; this suggests that other type I IFNs may also be important in the progression of BHV-1. Both quantitative and qualitative increases in animal production efficiency can be gained by curbing disease-related losses. 48 In cattle, a number of studies have identified associations between the major histocompatibility complex (MHC) and subclinical bovine leukemia virus,36, 37 mastitis incidence,39 and severity of tick infestation. 49 The bovine genome initiative has provided a genetic approach to improve production traits and select for disease resistance by identifying candidate genes for economically relevant traits. 26,30 In addition to the immunomodulatory effects of IFN during BHV-l infection, IFN has direct antiviral effects against many other viruses implicated in the pathogenesis of BRDC.20, 21, 28, 50 Therefore, further studies on the association between IFN genotype and respiratory disease in cattle are warranted to determine if selective breeding programs could modify the severity of disease and benefit cattle producers. References 1. Andrew C, Pererira HG, Wildy P: The herpesviruses. In Viruses of Vertebrates, ed 4. London, Clowes and Sons, 1978, pp 312-356 2. Babiuk LA, Bielefeldt-Ohmann H, Gifford G, et al: Effect of bovine ex I interferon on bovine herpesvirus type-I-induced respiratory disease. J Gen Virol 66:2383-2394, 1985 3. Babiuk LA, Lawman MJP, Gifford GA: Use of recombinant bovine ex I interferon in reducing respiratory disease induced by bovine herpesvirus type 1. Antimicrob Agents Chemother 31:752-757, 1987 4. Babiuk LA, Lawman MJP, Gifford GA: Bovine respiratory disease: Pathogenesis and control by interferon. In A Seminar in Bovine Immunology. Presented at the Western Veterinary Conference, February 16, 1987, Veterinary Learning Systems, sine loco 1987, pp 12-24 5. Bielefeldt-Ohmann H, Babiuk LA: Effects of bovine recombinant ex I interferon on inflammatory responses of bovine phagocytes. J Interferon Res 4:249-263, 1984 6. Bielefeldt-Ohmann H, Babiuk LA: Viral-bacterial pneumonia in calves: Effect of Bovine herpesvirus-Ion immunologic functions. J Infect Dis 151:937-947, 1985 7. Bielefeldt-Ohmann H, Babiuk LA: In vitro and systematic effects of recombinant bovine interferons on natural cell-mediated cytotoxicity in healthy and bovine herpesvirus-linfected cattle. J Interferon Res 5:551-564, 1985 8. Bielefeldt-Ohmann H, Babiuk LA: Alteration of alveolar macrophage function after aerosol infection with bovine herpesvirus-I. Infect Immun 51:344-347, 1986 9. Bielefeldt-Ohmann, Babiuk LA: Alteration of some leukocyte functions following in vitro exposure to recombinant bovine ex and 'Y interferon. J Interferon Res 6:123-136, 1986

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10. Bielefeldt-Ohmann H, Davis WC, Babiuk LA: Surface antigen expression by bovine alveolar macrophages: Functional correlation and influence of interferon in vitro and in vivo. Immunobiology 171:125-142, 1986 11. Bielefeldt-Ohmann H, Lawman MJP, Babiuk LA: Bovine interferon: Its biology and application in veterinary medicine. Antiviral Res 7:187-210, 1987 12. Bielefeldt-Ohmann H, Martinod SR: Interferon immunomodulation in domestic food animals. In Blecha F, Charley B (eds): Immunomodulation in Domestic Food Animals. San Diego, Academic Press, 1990, pp 215-223 13. Briggs RE, Kehrli M, Frank GH: Effects of infection with parainfluenza-3 virus and infectious bovine rhinotracheitis virus on neutrophil functions in calves. Am J Vet Res 49:682-686, 1988 14. Confer AW, Panciera RJ, Mosier DA: Bovine pneumonic pasturellosis: Immunity to Pasturella haemolytica. J Am Vet Med Assoc 193:1308-1316, 1988 15. Cooper DN, Clayton JF: DNA polymorphisms and the study of disease associations. Hum Genet 78:299-312, 1988 16. Cummins JM, Rosenquist BD: Leukocyte changes and interferon production in calves injected with hydrocortisone and infected with infectious bovine rhino tracheitis virus. Am J Vet Res 40:238-240, 1979 17. Cummins JM, Rosenquist BD: Protection of calves against rhinovirus infection by nasal secretions of interferon induced by infectious bovine rhinotracheitis virus. Am J Vet Res 41:161-165, 1980 18. Cummins JM, Rosenquist BD: Partial protection of calves against parainfluenza-3 virus infection by nasal-secretion interferon induced by infectious bovine rhinotracheitis virus. Am J Vet Res 43:1334-1338, 1982 19. Cummins JM, Rosenquist BD: Temporary protection of calves against adenovirus infection by nasal-secretion interferon induced by infectious bovine rhinotracheitis virus. Am J Vet Res 43:955-959, 1982 20. Czamiecki CW, Anderson KP, Fennie EB, et al: Bovine interferon-a 11 is an effective inhibitor of bovine herpesvirus-1 induced respiratory disease. Antiviral Res 1(suppl):209-215, 1985 21. Czarniecki CW, Hamilton EB, Fennie CW, et al: In vitro biological activities of Escherichia coli-derived bovine interferons a-, (3- and 'Y. J Interferon Res 6:29-37, 1986 22. Dandoy F, DeMaeyer-Guignard J, Bailey D, et al: Mouse genes influence antiviral action of interferon in vivo. Infect Immun 38:89-93, 1982 23. DeMaeyer E, DeMaeyer-Guignard J: Considerations on mouse genes influencing interferon production and action. In Gresser I (ed): Interferon 1. New York, Academic Press, 1979, pp 75-100 24. DeMaeyer E, DeMaeyer-Guignard J: Host genotype influences immunomodulation by interferon. Nature 284:173-175, 1980 25. DeMaeyer E, DeMaeyer-Guignard J: Interferon and Other Regulatory Cytokines. New York, John Wiley and Sons, 1988 26. Feldmann H: Screening and cloning of genes for application in animal genetics and animal breeding. In Geldermann H, Ellendorf F (eds): Genome Analysis in Domestic Animals. New York, VCH, 1990, pp 35-47 27. Filion LG, McGuire RL, Babiuk LA: Nonspecific suppressive effects of Bovine Herpesvirus type 1 on bovine leukocyte functions. Infect Immun 42:106-112, 1981 28. Fulton RW, Downing MM, Cummings JM: Antiviral effects of bovine interferons on bovine respiratory tract viruses. J Clin Microbiol 19:492-497, 1984 29. Fulton RW, Burge LJ, McCracken JS: Effect of recombinant DNA-derived bovine and human interferons on replication of bovine herpesvirus-I, parainfluenza-3 and respiratory synctial viruses. Am J Vet Res 47:751-753, 1986 30. Geldermann H: Application of genome analYSis in animal breeding. In Geldermann H, Ellendorff F (eds): Genome Analysis in Domestic Animals. New York, VCH, 1990, pp 291-323 31. Imakawa K, Anthony RV, Kazemi M, et al: Interferon-like sequences of ovine trophoblast protein secreted by embryonic trophectoderm. Nature 330:377-379, 1987 32. Imakawa K, Hansen TR, Malathy PV, et al: Molecular cloning and characterization of complementary deoxyribonucleic acids corresponding to bovine trophoblast protein-

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1: A comparison with ovine trophoblast protein-1 and bovine interferon-a II. Mol Endocrinol 3:127-139, 1989 Jericho KWF, Langford EV: Pneumonia in calves produced with aerosols of bovine herpesvirus-1 and Pasturella haemolytica. Can J Comp Med 42:269-277, 1978 Knight DJ, Ashton RJ: Induction of circulating interferon in calves with 3 different types of double stranded ribonucleic acid. Res Vet Sci 28:386-388, 1980 Lawman MJP, Gifford G, Gyongyossy-Issa M, et al: Activity of polymorphonuclear (PMN) leukocytes during bovine herpesvirus-1 induced respiratory disease: Effects of recombinant bovine interferon a P. Antiviral Res 8:225-238, 1987 Lewin HA, Bemoco D: Evidence for BoLA-linked resistance and susceptibility to subclinical progression of bovine leukemia virus infection. Anim Genet 17:197-207, 1986 Lewin HA, Wu MC, Stewart JA, et al: Association between BoLA and subclinical bovine leukemia virus infection in a herd of Holstein-Fresian cows. Immunogenetics 27:338-344, 1988 Linnavuori K: History of recurrent mucocutaneous herpes correlates with relatively low interferon production by herpes simplex virus-exposed cultured monocytes. J Med Virol 25:61-68, 1988 Lunden A, Sigurdardottin S, Andersson L: Analysis of the MHC genotypes of bulls and disease frequency in their progeny [abstract]. Anim Genet 20 (suppl1):31, 1989 Pitkaranta A, Linnavouri K, Hovi T: Virus-induced interferon production in human leukocytes: A low responder to one virus can be a high responder to another virus. J Interferon Res 11:17-23, 1991 Roney CS, Rossi CR, Smith PC, et al: Effect of human leukocyte A interferon on the prevention of infectious bovine rhinotracheitis virus infection in cattle. Am J Vet Res 46:1251-1255, 1985 Roth JA: Immunosuppression and immunomodulation in bovine respiratory disease. In Loan RW (ed): Bovine Respiratory Disease. College Station, Texas A&M University Press, 1984, pp 143-192 Ryan AM, Gallagher DS, Womack JE: Syntenic mapping and chromosomal localization of bovine a and J3 interferon genes. Mamm Genome 3:575-578, 1992 Ryan AM, Gallagher DS, Womack JE: Somatic cell mapping of omega and trophoblast interferon genes to bovine syntenic group U18 and in situ localization to chromosome 8. Cytogenet Cell Genet 63:6-10, 1993 Ryan AM, Womack JE: Hybridization profiles and restriction fragment length polymorphisms for bovine and ovine interferon genes. Animal Biotechnology 4:11-30, 1993 Ryan AM, Womack JE: Type I interferon genes in cattle: Restriction fragment length polymorphisms, gene numbers and physical organization on bovine chromosome 8. Anim Genetics 24:9-16, 1993 Samuel CE: Mechanisms of the antiviral actions of interferon. Prog Nucleic Acid Res Mol BioI 35:27-72, 1988 Schook GE: Selection for disease resistance. J Dairy Sci 73:1349-1362, 1989 Stear MJ, Newman MJ, Nicholas FW, et al: Tick resistance and the major histocompatibility system. Aust J Exp BioI Med Sci 62:47-52, 1984 Trueblood MS, Manjara J: Response of bovine viruses to interferon. Cornell Vet 62:3-12, 1972 Zawatzky R, Hilfenhaus J, Marcucci F, et al: Experimental infection of inbred mice with herpes simplex virus type 1. I: Investigations of humoral and cellular immunity of interferon induction. J Gen Virol 53:31-38, 1981 Zawatzky R, Kirchner H, DeMaeyer-Guignard J, et al: An X-linked locus influences the amount of circulating interferon induced in the mouse by herpes Simplex virus type 1. J Gen Virol 63:325-332, 1982

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