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BAcrmowGIcwm REVIEWS, Sept. 1972, p. 291-310Copyright 0 1972 American Society for Microbiology

Vol. 36, No. 3Printed in U.S.A.

Mechanisms of Virus PathogenicityH. SMITH

Department of Microbiology, University of Birmingham, Birmingham, England

INTRODUCTION ............................................................ 291QUANTITATIVE COMPARISONS OF VIRULENCE: PROPERTIES OF

VIRULENT AND ATTENUATED STRAINS ................ .......... 292ENTRY OF THE HOST: SURVIVAL ON AND PENETRATION OF

MUCOUS MEMBRANES ............. ................................. 292REPLICATION IN VIVO .................................................... 294COUNTERACTING HOST DEFENSE MECHANISMS ...... ................. 295HOST DAMAGE............................................................ 298HOST AND TISSUE SPECIFICITY .......... ............................... 301LITERATURE CITED........................................................ 305

INTRODUCTION

This review stems from a previous report(143) in which the biochemical mechanisms ofbacterial pathogenicity formed the main sub-ject and were discussed in a manner whichprompted the following questions regardingthe pathogenicity of other, less-studied mi-crobes including viruses. Can differences invirulence be detected and measured in vivo?Are reasonably stable virulent and avirulentstrains available so that virulence markers anddeterminants can be recognized (i) by com-paring the behavior of the strains in vitro andin vivo and (ii) by observing the effects of theproducts of the virulent strain on the behaviorof the avirulent strain? How far can avirulencebe due to any inherent inability to grow inhost tissues (as distinct from an inability tocombat host defense mechanisms)? What hostdefense mechanisms act against the microbeand what aggressions inhibit them? Are thepathological effects of the disease due to pro-duction of toxins (acting intracellularly or sys-temically, or both), depletion of nutrients,mechanical blockage of vital tissues, or evoca-tion of hypersensitivity or auto-allergicreactions? Can host and tissue specificities beexplained either by differential distribution ofmicrobial inhibitors or differential suitabilityof tissues for microbial growth?At the time, only incomplete answers to

these questions could be provided for virusesdue to lack of space and knowledge. Now moreinformation has accumulated (148), and theobject of the present review is a more compre-hensive discussion of these and other questionsrelating to the pathogenicity of viruses. Thereview is written from the standpoint of onewho has entered the field of virus pathoge-

nicity from studies of bacterial pathogenicity,and whose predominant impression is that thebroad aspects of pathogenicity of the two mi-crobial types are similar despite the obligateparasitism of viruses. In particular, viral-likebacterial pathogenicity is not determinedsolely by biochemical ability to replicate inhost tissues; virulent and attenuated strains ofviruses replicate in host cells in vitro, yet theydiffer fundamentally in behavior in vivo, pre-sumably-as for bacteria-due to differentcapacities to counteract host defense mecha-nisms and to damage tissues. Also, the factthat the viral factors responsible for virulencemechanisms are induced within host cells doesnot confer uniqueness on viral pathogenicity.Although replicating by different processes,many pathogenic microbes (including bacteria)are intracellular parasites, and often bacterialvirulence factors that are produced extracellu-larly enter and act within host cells, e.g., theinterference with protein synthesis by thetoxin of Corynebacterium diphtheriae. Eventhe latency of viruses has its parallel in thecarrier state of bacteriology. Finally, animalexperiments with viruses are as essential asthose with bacteria, for many aspects of patho-genic behavior in vivo are missing in tissueculture experiments (142).

It is hoped the reader will use the previousreview (143) as a basis for what follows. Theterms "pathogenicity" and "virulence" arenearly synonymous and mean the capacity toproduce disease; as suggested by Miles (93),virulence is used here mainly with respect tocomparisons of the disease-producing capaci-ties of strains within a species. Tumor viruseshave been largely excluded from the reviewsince their pathogenicity is a special case re-quiring separate treatment. Also, latency has

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not been specifically discussed, because thephenomenon has been described many times,there is little real knowledge of the mecha-nisms involved, and the speculations on theirnature are well known (98).

QUANTITATIVE COMPARISONS OFVIRULENCE: PROPERTIES OFVIRULENT AND ATTENUATED

STRAINSAlthough mechanisms of pathogenicity can

be investigated by using a single strain of vi-rus, the existence of stable strains of differingvirulence greatly increases the experimentalscope in the search for virulence markers anddeterminants. Different virus strains exist, andadvances in viral genetics (48) will increase thenumber available, but quantitative comparisonof their virulence is difficult because of the lowefficiency of viable counts. The effects in ani-mals (LDO, lesion size, or mean death time forthe "same" dose) must be related to amountsof virus particles indicated by plaque counts oregg infection. The latter detects only a smallproportion of the total virus particles andtherefore may not measure all the particles(which could vary for different strains) capableof multiplying in experimental animals. Forexample, plaque counts on chick embryo fibro-blasts detected less infectious particles ofSemliki Forest virus than infection of sucklingmice (20), and in this system the proportion oftotal virus particles detected by the plaquecounts was fairly high (approximately 1 in 10)compared with many other virus systems.Thus, for this and other reasons, comparisonsof the virulence of virus strains are often im-precise. Burrows (24), for example, has de-scribed the imprecision of virulence compari-sons between strains of foot-and-mouth diseasevirus and the lack of correlation with behaviorin the field.Only virus strains for which conventional

tests have indicated large differences in viru-lence should be compared to recognize viru-lence markers and determinants. Comparisonsof such well-tested and well-separated strainshave been rare (14, 164) but informative. At-tenuated strains of poliovirus had less affinitythan virulent strains for the cell receptors ofprimate central nervous system (65). Virulentstrains of ectromelia virus had a greater ca-pacity to infect mouse macrophages than at-tenuated strains, although they had equalability to infect hepatic cells (95). Well-testedstrains of Semliki Forest virus of widely dif-ferent virulence (20) have been examined inmice (121). Surprisingly, in view of the wide

difference in virulence, and in contrast withstudies on less well-defined strains of JapaneseB encephalitis virus (70), muscle replicationand systemic infection of the attenuated strainwas at least as high, if not higher, than that ofthe virulent strain; only in the brain was virusreplication higher for the virulent strain thanfor the attenuated strain (121). Because of theease of virulence comparisons in chickens (164),well-established virulent and attenuatedstrains of Newcastle disease virus are avail-able. Early work showed virulent strains tohave a greater capacity to replicate in chickenbrain and penetrate the tissue of eggs (14, 164).Recently, the virulence of strains was asso-ciated with an increased capacity to producecell-fusion effects and plaques in chicken em-bryo fibroblasts (125); there were no major dif-ferences between strains in the kinetics of rep-lication, either in timing or amount of virusreleased (127), but virulent strains producedmore cell-associated hemagglutinin and neur-aminidase (1, 126).

ENTRY OF THE HOST: SURVIVAL ONAND PENETRATION OF MUCOUS

MEMBRANESSome virus diseases are more communicable

than others. Communicability depends on thefactors (24) determining virus release from onehost, survival in the intermediate environ-ment, and entry into another host. Only thelatter process is discussed here. Some virusesenter host tissues directly by trauma or insectbite, but most infections start on the mucousmembranes of the respiratory and alimentarytracts. To initiate infection, virus particlesmust first survive on these mucus-coveredmembranes in the presence of viral and non-viral commensals. Subsequently, to replicate,the virus must enter host cells either in themucous membrane itself or in tissues fartherafield after penetration through the surfacemembrane. Replication in mucous membranecells can produce the disease effects directly asin respiratory diseases, but sometimes it pro-vides a staging post for subsequent damagingreplication in another site, e.g., poliovirus rep-licates first in the alimentary tract cells andultimately in anterior horn cells. Detailedknowledge of the factors influencing the earlystages of virus disease is almost completelylacking, mainly owing to lack of techniques forobserving the behavior of a few, highly dis-persed virus particles on mucous surfaces withtheir indigenous microbial populations. Respir-atory-tract infections provide most of ourpresent information, and they are the main

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examples in the following discussion of earlyvirus attack. Descriptions of the mucus-pro-ducing cells and the clearance action of themucociliary movement have been provided byothers (42, 80, 118, 161) and need not be re-peated here.What factors determine the initial site of

virus lodgement? In the respiratory tract, thesize of the droplet to which the virus is at-tached is the prime factor (42), but some dif-ferences in deposition occur in different an-imal species due to different size, anatomy,and posture (24). Within one species, smalldifferences in anatomy and breathing patterncan influence deposition of virus (134). Apartfrom these mechanical factors, chemical fac-tors which might determine tissue specificity(16, 17) may play a role in primary lodgementif present in the pathway of the incomingvirus. The site of virus deposition can influ-ence the subsequent pathogenesis of the dis-ease. A recent reinvestigation (24) of the path-ogenesis of foot-and-mouth disease followingthe discovery that the disease can be aerosol-transmitted showed differences in disease de-velopment according to whether initial infec-tion occurred through the natural site, the pha-ryngeal region, or through tongue epitheliumformerly thought to be the natural site.How do viruses penetrate the moving mucus

blanket which sweeps particles towards thepharynx? Mucus depth and speed of flow areobviously important in this process. Little isknown of the depth and nature of mucus indifferent parts of the respiratory tract, al-though it appears that the cilia beat in a wa-tery layer underlying a stiffer particle trans-porting layer, and in the lower respiratorytract mucus thickness decreases and the com-position changes (37, 80, 118). Mucus flowrates are better understood. There is a velocitygradient from small to large airways (4) andindividual rates can vary widely, for example,4-fold in chickens and 10-fold in humans (11,122). Obstructions in the tract such asbronchial junctions and protrusions can pro-duce local changes in flow rate (42). Many fac-tors affect mucous secretion and ciliary action(11, 118, 166), including ion concentration inthe air, temperature, and humidity; the lasttwo affect nasal epithelium rather than lowerparts of the tract where the air has been pre-heated and humidified. Clearly, the thinnerthe mucus and the slower the rate of flow, themore likely the occurrence of epithelial infec-tion or penetration. This has been demon-strated experimentally. Reduction of nasomu-cociliary activity of chicks by exposure to low

temperature or by injections of cocaine or pilo-carpine increased infection rates with astandard exposure of Newcastle disease virus(11, 13). What happens in natural infection isstill a matter of conjecture. Viral infectionshould occur more easily in the lower than inthe upper respiratory tract because of a slowermovement of a thinner protective film. Mucusflow might be almost stationary at respiratorytract obstructions, thus providing foci for virusattack (42); or gaps in the mucus coat mightoccur, exposing the underlying tissue (118).However, in epidemics, infection occurs in toomany individuals to implicate simultaneousimpairment of mucociliary action. The pene-tration mechanisms operating in many virusinfections may have little connection with var-iations of the mucous blanket and may residein the properties of the virus itself, such as anextra affinity for the surface receptors or phag-ocytic action of susceptible mucosal cellsagainst which they brush during the mucocil-iary movement. Better techniques for ob-serving the behavior of small numbers of virusparticles on mucous surfaces will be neededbefore these possibilities can be investigated.

If mucus contains virus inhibitors, how dovirulent viruses counteract them? There islittle doubt that mucus can inhibit or kill someviruses. The slightly alkaline pH inactivatessome viruses, such as foot-and-mouth diseasevirus (24), and nonspecific inhibitory sub-stances have been found in mucus and homo-genates of mucosae (123). Perhaps the majorantiviral activity in respiratory and alimentarytract mucus resides in specific immunoglobu-lins (largely immunoglobulin A) arising fromprevious infections or immunization (74, 159).How virulent viruses overcome these nonspe-cific and specific virus inhibitors is unknown.Local concentration of virus particles at onesite might saturate the inhibitory materials,allowing active virus to penetrate the mucus.But this is pure speculation. Comparisons ofvirulent and avirulent strains as regards resist-ance to mucus inhibitors might be revealing.

Clearly, some viruses, such as influenza vi-rus, attack the mucosal cells, but do otherspass through the mucosa without establishinginfection in the membrane itself? Simple pen-etration appears to occur in infections withAfrican swine fever and rinderpest wherethere are rapid recoveries of virus from thelymph nodes draining the respiratory tract (24).The mechanisms of penetration are not clear,but carriage by nondestructive macrophagesmay occur as for ectromelia virus by mousealveolar macrophages (98).

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How are any inhibitory effects of commen-sals overcome? Although tissue culture andanimal experiments show that different typesof commensals could, and probably do, inter-fere with early virus attack by such mecha-nisms as interferon production or usage of es-sential metabolites (like arginine by myco-plasmas [157]), there is no real proof that suchinterference occurs in natural infection or thatviruses counteract it. Some experiments aresuggestive; for example, reduction of com-mensal bacteria might have accounted for theincreased susceptibility of oxytetracycline-treated pigeons to Venezuelan equine encepha-litis virus (94).When mucosal infection occurs, which sur-

face cells are attacked? Differential suscepti-bility of the upper and lower respiratory tracthas been detected by applying the same doseof virus to the nasal epithelium and adminis-tering it as a small-particle aerosol. The upperrespiratory tract of man was more susceptibleto infection with coxsackievirus A21 and rhi-novirus NIH 1734, the lower to adenovirustype 4, and both tracts were equally infectedwith influenza virus (34, 35, 81). But the initialtarget cells were not recognized. Histologicaland immunofluorescent studies have showninfluenza virus in ciliated intermediate, basal,and possibly goblet cells of human nasal epi-thelium (43), but again the initial site of attackwas not clear. In Newcastle disease of chick-ens, however, the acinous mucous cells ap-peared to be the target cells (13). Organ cul-tures of relevant parts of the respiratory tractmay allow a closer study of early events, forexample, the close adhesion between influenzavirus and the cilia and microvilli of nasal epi-thelium that precedes virus replication (41, 56).

REPLICATION IN VIVOAlthough ability to replicate in host tissues

is not the only factor in virus virulence, it isessential, and the more rapid the rate of repli-cation the more likely the success of the virusin producing its disease syndrome. At present,a method for measuring the absolute rate ofvirus replication in vivo comparable to that inbacteriology (92) does not exist. However, se-quential determinations of virus contents oftissues, the resultants of virus replication, de-struction, and removal, indicate in some in-stances a more rapid replication of virulentstrains than avirulent strains either in the tis-sues generally or in a vital site (14, 121). Also,some support for a connection between thereplication rate and virulence comes fromtissue culture studies. For strains of some (65)

but not all viruses (138), there is a relationbetween plaque size and virulence, and, al-though the size of the plaque can be deter-mined by cytotoxic factors (3, 125, 127) andnot by rate of replication, in some cases thelatter seems the dominant factor (132, 164).

Ability to proliferate in vivo depends on aninherent ability to replicate in the biochemicalconditions of the host tissues, coupled with acapacity to resist or not to stimulate host de-fense mechanisms which would otherwise killor remove them. Distinguishing between theseeffects is not easy for any type of microbe (147),but it is particularly difficult for viruses be-cause of the absolute parasitism involved. Thecellular factors required for virus replicationare complex (104), and it is almost impossible,with present techniques, to distinguish clearlythe influence of their absence in a particularsituation from the influence of host factors (de-fense mechanisms) which destroy virus or in-terfere with replication. Nevertheless, the dis-tinction has been made occasionally, for exam-ple, when lack of replication has been shown tobe due to the host defense mechanism, inter-feron (15, 31). And further attempts to distin-guish "replication factors" (147) from host de-fense factors seem worthwhile, since they maylead to the recognition of virus-induced prod-ucts which inactivate or resist host defensefactors (e.g., interferon antagonists). Ability toreplicate and interference with host defensefactors are discussed in this and the followingsections.The ability of a virus to replicate in a partic-

ular cell depends on inherent features of thecell (104) as well as the virus. These featurescan be involved in one or more stages of repli-cation: attachment, penetration, uncoating,provision of energy and precursors of low-mo-lecular-weight, nucleic acid and protein syn-thesis, assembly, and release (104). Cell cul-ture experiments have shown that "replicationfactors" vary from cell type to cell type. Thus,by comparison with cells supporting full virusreplication, certain cell types appeared to lacksuch factors at the receptor stage for poliomye-litis virus (65), at penetration for feline virus(155), at uncoating for mouse hepatitis virus(136), at viral nucleic acid synthesis and matu-ration for some PK-negative mutants of rabbitpoxvirus (48), and at maturation or envelop-ment for KB-negative mutants of adenovirustype 12, some DK mutants of herpes simplexvirus, and Sendai virus (48). Also, it is abun-dantly clear from experiments in culture thatvirus replication is influenced by changes inthe environment of the cell. These include

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temperature, pH (65), and small molecularmaterials such as arginine (18), leucine (50),yeast extract materials (27), and some fattyacids (82). Furthermore, replication in somecell types can be promoted by extracts ofothers (139).

In animals, virus pathogenicity will be af-fected by variation of the availability of repli-cation factors in particular hosts or tissues andunder different environmental conditions; andattenuated viruses may have a decreased ca-pacity to use the factors. Experiments compa-rable in depth to those conducted in tissue cul-ture have not yet been accomplished in ani-mals or organ culture. Nevertheless, there aresigns of the influence of replication factors inthe following studies. With regard to receptors,the ability of homogenates of some primatetissues to bind poliomyelitis virus paralleledtheir susceptibilities to virus replication anddamage in infection; also, virulent strains ad-hered to susceptible nerve tissues morestrongly than avirulent strains (65). The effectof temperature on virus virulence (14, 150)probably reflects temperature sensitivity ofprocesses needed for virus production ratherthan an influence on host defense mechanisms,and the protective effect of fever on virusinfection may result from a similar mecha-nism. Leucine enhanced vaccinia infection ofmice as well as that of cell cultures (50), butsimilar animal experiments with arginine andappropriate viruses have not been done. Lackof the complete set of replication factors mayresult in production of defective virus particleswhich can influence virus infection by in-ducing host defenses against infective particles(69). The spread of a virus in a host may beprevented-by a layer of insusceptible cells (i.e.,those lacking replication factors), barring entryto a target organ; thus, a blood-borne virusunable to grow in vascular endothelium maynot enter the brain, placenta, or skin tissues(98).

In organ cultures, the defense mechanisms ofthe reticuloendothelial system and the inflam-matory response are largely absent. Hence, theinfluence of replication factors on virus growthcan be studied in a system removed to someextent from the influence of host defense fac-tors without complete destruction of the invivo character of experiments. Infection pat-terns in organ culture may parallel those inwhole animals, for example, in the replicationof rhinovirus in different human tissues (67),infectious bovine rhinotracheitis virus in dif-ferent bovine tissues (137), and influenza virusin different ferret tissues (16, 17). In these

cases, host defense mechanisms (other thanthose present or induced in infected cells) maynot be as important in infection as replicationfactors. On the other hand, when the patternof infection is not repeated in organ culture,for example, in growth of trachoma-inclusionconjunctivitis agents in baboon and guinea pigconjunctiva (115), host defense mechanismsmay be more important in pathogenicity invivo. Comparisons of virulent and attenuatedvirus strains in organ cultures might revealsome aspects of the influence of replicationfactors on pathogenicity. Thus, in bovine pha-ryngeal epithelium, virulent strains of foot-and-mouth virus multiplied more rapidly andto a higher titer than attenuated strains (24).Such investigations are rare. They should beextended to strains of other viruses andpressed deeper by using modifications of tissueculture methods, for example, one-step growthcurves (56, 68).

COUNTERACTING HOST DEFENSEMECHANISMS

Although much has been written on hostdefense mechanisms against viruses (2, 15, 57,65, 95, 98, 164), little concrete information ex-ists on the ability of viruses to counteractthem (98, 105, 164). In particular, there seemto be few studies of the early stages of virusinfection (95), where comparisons of the be-havior of virulent and attenuated strains mightreveal viral invasive mechanisms as they havedone in bacteriology (143). In this section, hostdefense mechanisms are summarized to form abackground against which virus counteractionis described, and possible determinants of itare discussed.

Nonspecific defense against virus infectionsis stronger in adult than young hosts (14, 20,63, 102, 128), is reduced by treatment with cor-tisone or X-rays (106, 150), and contains bothhumoral and cellular factors. Humoral factorsinclude the low pH of inflammatory exudates(65, 95, 164) and nonspecific virus inhibitors intissues and serum. These inhibitors may bepresent before infection (103, 150) or be in-duced by it (156, 162). Cellular factors includethose present or induced in any tissue whichthe virus attacks, such as host nucleases ca-pable of destroying virus nucleic acid (104) orinterferon (15, 98). There are probably also fac-tors present in the phagocytic and other cellsof the reticuloendothelial system (95) whichcan prevent the spread of viruses into suscep-tible tissues such as influenza virus into liverparenchyma (95) or neuroviruses into the brain(75). Macrophages ingest and destroy some

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viruses (57, 63, 73, 95, 105, 130, 152), and anti-macrophage serum has enhanced infectionswith yellow fever virus, vesicular stomatitisvirus, and herpes simplex virus (62, 114, 170).The role of polymorphonuclear leukocytes indefense against virus infections is not as clearas that of macrophages and has not been in-vestigated thoroughly (57, 95, 98, 105). Al-though present, neutrophils do not figure soprominently in early inflammatory lesions asthey do in bacterial infections. Nevertheless,they may play some part in aborting virusinfections since, in the few available studies,they phagocytosed virus particles and eitherdestroyed them or inhibited replication (60, 98,113, 130, 167).In a preimmunized host, or a few days after

primary infection, a virus must contend withthe specific defenses of the host. Here, neutral-izing antibodies supplement the nonspecificinhibitors in body fluids, and cellular viricidalmechanisms are strengthened by influence ofthese antibodies and immune lymphocytes(119). Antibody may combine with surfacecomponents of the virus essential for host-cellpenetration or opsonize the virus leading tophagocytosis and destruction by the macro-phages (57, 95). The virus-antibody combina-tion can be reversed, and, although comple-ment potentiates some combinations, it doesnot necessarily have a viricidal action (98). Inprimary infection, immunoglobulin M may bemore important than immunoglobulin G be-cause of its earlier appearance. As regards cel-lular mechanisms, there is no clear evidenceyet that macrophages from immune animalsare more viricidal than normal macrophages(98). But there are indications from the influ-ence of antilymphocyte serum and from otherexperiments (72, 170) that immune lympho-cytes are important in defense against somebut not all virus infections; they may reactwith viral antigens and stimulate the infiltra-tion and activity of macrophages. In somevirus infections, such as those with cytomega-lovirus and herpes simplex virus, there appearsto be a dynamic equilibrium between virusreplication and destruction by specific defen-ses, because immunosuppression can result inclinical disease (36, 101).

Viruses break through host defenses to causedisease and, as for bacteria, this process de-pends not only on the strength of the defensesand the microbe's capacity to counteract them,but also on the number of invaders. A suffi-ciently large infecting dose can overwhelm theinitial defenses of a susceptible host and causeirreparable damage before the induced de-

fenses can be mounted. Thus, morbidity andmortality in Newcastle disease of chickens isdirectly related to dosage (164), and RiftValley fever virus will kill mice in only 6 hr ifa high dose is given intravenously (98). How-ever, in natural disease and most laboratoryexperiments, a small infecting dose is in-volved. This must be built up to a populationsufficiently large to cause damage against theactivities of the host defense mechanisms thatinitially are heavily weighted against the fewinvading microbes. In the nonimmune host,defense mechanisms act at three overlappingstages. First there are those preexisting in thetissues. Then there are those induced fairlyquickly and nonspecifically, such as interferonproduction and the inflammatory response.And finally there are the specific responses.Since many virus diseases are self-limiting (asregards pathological effects but not necessarilyas regards virus elimination), it appears thateven virulent strains of these viruses cannotwithstand the specific responses which prob-ably determine recovery from disease. Hence,in acute virus disease, the virulence mecha-nisms are first those which interfere with thetwo primary stages of host defense and thenthose which delay and possibly reduce the spe-cific response to which viruses appear espe-cially vulnerable.

Little is known about the resistance of vi-ruses to the nonspecific viricidins in bodyfluids. Virulent strains of influenza virus ap-peared to resist these factors in serum morethan avirulent strains (150), but possible rea-sons for the differential resistance, such assubtle differences in the envelope proteins ofthe strains, were not investigated. Similarly,alimentary tract viruses such as enteroviruseswithstand a low pH, but the biochemical basisfor this resistance is unknown.Virus species and strains within species

differ both in the amount of interferon theyinduce and in their susceptibility to it. Most ofthe work has been done in tissue culture. Inmany cases, virulent strains of viruses induceless interferon or are more resistant to it thanattenuated strains, but there are instanceswhen this is not so (14, 15, 25, 65, 89, 100, 120).However, virus virulence is almost certainlydetermined by more than one mechanism.These mechanisms may be additive in theircontributions to virulence rather than interde-pendent (cf., bacteria [140]). Thus, a strict cor-relation between virulence and one factor suchas induction of or resistance to interferonwould not be expected. The role of interferonin virus infection is still not clearly estab-

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lished, owing to the difficulty of dissociatingits action from more specific processes. Never-theless, interferon is produced in vivo, and it isreasonable to assume that a capacity to reduceits production or resist its action would be anadvantage to an invading virus. How could avirus achieve these ends? Strains which induceearly inhibition of host cell ribonucleic acid(RNA) and protein synthesis would depressinterferon production, and a blocker of inter-feron production- in vitro has been reported(31, 71). Also, there are persistent reports ofviruses producing in vitro antagonists of in-terferon-termed variously as stimulators,enhancers, and anti-interferons (29, 53, 61,78, 135, 160). The chemical nature of thesesubstances is unknown, and whether they areproduced in infection and play any role invirus invasion has yet to be assessed. Never-theless, these interferon antagonists have beenrecognized, and some of them may prove to beviral counterparts to bacterial "aggressins"(143). Similarly, if virus internal proteins canbe proved to prevent the destruction of viralnucleic acids by host nucleases, as speculatedby Newton (104), they might also qualify asviral "aggressins."Some viruses are killed after ingestion by

macrophages and others are not (57, 95, 98).Ability to resist the killing mechanisms ofmonocytes and possibly to replicate withinthem appears to be one of the main virulencemechanisms of some viruses. The Hampsteadmouse strain of ectromelia has an increasedability to grow in mouse macrophages com-pared with the Hampstead egg strain, and it iscorrespondingly more virulent for mice.Within macrophages, virus is protected fromextracellular inhibitors such as antibody, andthus wandering macrophages can spread infec-tion through the blood, lymph, and tissues,while fixed macrophages can provide an initialfocus of infection in larger organs such as theliver (98). Some viruses are phagocytosedpoorly or not at all by macrophages (95); thisis a virulence mechanism for viruses which aredestroyed by macrophages. On the other hand,for a virus which survives and replicates withinmacrophages, ability to be ingested is an es-sential virulence mechanism. The WE3 strainof lymphocytic choriomeningitis virus heavilyinfects the liver and spleen of mice because, incontrast to the Armstrong strain, it is ingestedby the macrophages of these organs.At present, we are ignorant of the viral prod-

ucts or mechanisms which determine virusingestion, survival, or replication within mac-rophages. This is not surprising. Only re-

cently have we learned a little regarding intra-cellular survival of bacteria, despite manyyears of work on their biology and chemistry(143). Perhaps the first point to note is thatmacrophages do not appear to provide a goodenvironment for replication of any virus. Manymacrophages in an inoculated population donot become infected, often viruses survive butdo not multiply within macrophages, and,even when replication occurs, yields of infec-tious virus are small with much incompletevirus (98). Ingestion will probably be affectedby the nature of the virus envelope, and sero-logical and biochemical examination of theenvelope proteins of strains differing in ease ofingestion might yield interesting results.Within the macrophage, the virus envelopemay also play a role in virus survival by di-rectly inhibiting viricidins, but it is equallypossible that virus survival may be due to anoverall inhibition of macrophage function by acytotoxic action of the virus. Clearly some vi-ruses such as myxoviruses, vaccinia virus, andmeasles virus exert cytotoxic effects on macro-phages and inhibit phagocytic activity towardsbacteria (57, 105, 130). These cytotoxic effectsmay be a result of virus replication and thuscome under the heading of damage to the hostwhich could aid or hinder, according to thefunction of the macrophage, further invasionby the same virus or another pathogen. Butthey would have little relevance to the survivaland replication of the initial infecting virusparticles. On the other hand, the constituentsof these initial particles might themselves in-hibit macrophage function, including viricidalactivity sufficient to allow their limited repli-cation. Until we know more about the bio-chemical basis of virus cytotoxicity, we cannotdecide between these possibilities and recog-nize the basis of virus survival and replicationwithin macrophages. Similarly, we are igno-rant of the reasons for the inhibition of phago-cytic activity towards bacteria of polymor-phonuclear leukocytes after treatment withviruses such as mumps virus, influenza virus,and coxsackievirus (105, 130). If neutrophilscontribute to defense against virus infection,such interference with their function by virusesmay be a virulence mechanism.

Viruses could delay or reduce the protectiveeffect of antibody by being present in suchlarge amounts that any local antibody isswamped, by being "bad" antigens for in-ducing antibody, by antigenic variation, andby infecting and inhibiting the function of an-tibody-forming cells. Virus strains vary in theirability to evoke antibody (164), and "slow vi-

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ruses," such as the scrapie agent, do not ap-pear to induce antibody (169). The reasonswhy some antigens are "good" and others"bad" are unknown (144). The fact that virusesoften have host-cell-membrane constituents intheir envelope proteins provides the possibilityof virus antigens being more "host-like" andtherefore "bad" antigens, but this has not yetbeen proven. Furthermore, I am unaware ofany comparison between virulent and atten-uated strains of virus which has shown virulentstrains to be the less immunogenic; it isusually the other way round (164). Antigenicvariation occurs in influenza virus, rhinovi-ruses, foot-and-mouth disease virus, and otherviruses infecting the respiratory and alimen-tary tracts, and this must contribute to theability of these viruses to attack fresh hostswhich have neutralizing antibodies onlyagainst previous variants. But we do not haveevidence that antigenic variation during thecourse of infection contributes to virulence asfor example in protozoal diseases (147). Somevirus infections depress antibody synthesis,but it is not usually completely prevented. Insome infections it is increased, for example, inVenezuelan encephalitis of mice and guineapigs (105). Like depression of macrophage ac-tivity, the way in which antibody-forming cellsare inhibited is unknown. Also, in all experi-ments so far, the methods for detecting changein antibody production have not involved theinfecting virus (98). Hence we do not knowwhether response to the latter was suppressed,the crucial point as regards counteraction ofhost defenses in primary virus infection.

Cellular immunity as judged by graft rejec-tion or delayed hypersensitivity reactions isdepressed in most virus infections (105).Again, cellular immunity against the infectingvirus has not been examined. Some virusesgrow in lymphocytes and produce immunosup-pression with or without cytotoxic damage (98).The mechanisms of this intracellular growthare obscure but, like macrophage infection,lymphocyte infection provides a ready vehiclefor spread of virus infection in some diseasessuch as those caused by ectromelia virus, dis-temper virus, tick-borne encephalitis virus,and lymphochoriomeningitis virus (98).

HOST DAMAGEIn attempts to understand the mechanisms

responsible for the pathological effects of virusdisease, four broad questions arise. Whichpathological effects are specific to virus attackrather than nonspecific responses to generalinjury (153)? Which cells are damaged by virus

replication? Does this damage explain the spe-cific pathological effects? And how is thedamage produced?

In some cases, specific pathological effectsare easy to detect, for example, paralysis inpoliomyelitis resulting from damaged anteriorhorn tissue. On the other hand, the biochem-ical and pathological manifestations of shockthat occur in poxvirus infections (149) areprobably nonspecific responses to injury com-parable to those found in anthrax (141) andmalaria (51). When such blanketing nonspe-cific responses occur, identifying the triggermechanisms in virus diseases will prove moredifficult than for bacterial diseases (45). Unlikesome bacterial toxins, the virus products re-sponsible for the triggering effects have notbeen isolated (see below). Hence, infectedanimals or tissues must be used in all investi-gations. It is therefore difficult to distinguishthe effects of virus replication (e.g., cellularamino acid changes due to virus-coded proteinsynthesis) from the results of damage to hosttissue (e.g., cellular amino acid changes ac-companying lysis), and there are also technicalhardships in dealing with heavily infectedanimals. In bacteriology, the pathological syn-drome has been studied successfully withoutinterference from bacterial growth by re-moving bacteria with antibiotic treatment at astage of the disease when the host was about tosuccumb (41). This method might be adaptedfor virus work if replication but not the diseasesyndrome in animals or organ cultures couldbe stopped by methods comparable to thoseused in tissue culture (6).

Cellular damage of animal tissues by virusattack has been recognized for many years bythe classical methods of histopathology; forexample, anterior horn cells are damaged bypoliovirus, respiratory epithelium by influenzavirus, and brain cells by Newcastle diseasevirus. Now that these methods have been sup-plemented by electron microscopy and immu-nofluorescent techniques, it is apparent thatviral re"plication occurs in cells without signifi-cant damage. In seeking the important celldamage in virus disease, it would be unwise toassume that lack of morphological damagemeans absence of relevant biochemical dam-age, in view of the profound effect virus repli-cation has on cell biochemistry and the experi-ence from other fields, such as pharmacology,that small biochemical lesions can have strik-ing pathological effects, especially if they oc-cur in the nervous or vascular systems. Anycell type showing evidence of virus replicationor presence should be considered a candidate

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for the primary site of damage, although thoseovertly damaged should probably receiveattention first.A direct connection between cell damage

and specific pathological effects is perhapsstrongest for those viruses which damage cellsof the nervous system, for example, poliovirusand the encephalitis-producing viruses. Respi-ratory tract viruses such as rhinoviruses andinfluenza virus damage the epithelial cells ofthe respiratory tract with resultant local symp-toms, and the destruction of some respiratorytract cells by influenza virus will ease the wayfor its well-known secondary invaders, pneu-mococci and staphylococci. But it is difficultto believe that the unpleasant systemic andsometimes fatal effects of influenza are merelydue to damage of respiratory epithelium. Ei-ther the virus grows in and damages other sitesor virus components (or host cell breakdownproducts) are liberated from the damaged res-piratory epithelium, and, like bacterial toxins,have a toxic action elsewhere. In this connec-tion, it is interesting that large doses of influ-enza virus are toxic (95, 149, 154). Similarly,the systemic effects of viruses which producerashes and skin pocks are possibly not directlyconnected with the skin cell damage but withdamage elsewhere. If damage of host cells iswidespread, then, as in malaria (112), deathmay follow from the nonspecific pathologicaleffects of pharmacologically active materialsliberated from the damaged host cells. Thismay be the explanation for the fatal shocksyndrome seen in some poxvirus infections(149).As suggested by Ginsberg (54), virus-induced

cell damage may result from a passive role ofthe virus-a simple repercussion of the processof replication, such as the depletion of cellularcomponents essential for cell life or mechan-ical harm due to excessive production of virusor its components. Nevertheless, there is in-creasing evidence that two more positive proc-esses of cell damage occur, namely, virus cyto-toxic activity (6, 54) and immunological reac-tion of the host against virus-infected cells(119, 129, 165).There are two levels at which pathologically

important cytotoxic activity can operate: bio-chemical damage without morphologicaldamage and that occurring with morphologicaldamage such as cell lysis, fusion, or death (6).The latter (called here morphological damage)is what is usually meant by cytotoxic (or cyto-pathic) effect. But both processes must be con-sidered, since the former (called here biochem-ical damage) could cause the decisive patho-

logical damage, for example in nerve cells,even when there is subsequent or accompa-nying morphological damage in the same orother tissues. In attempts to elucidate thesecytotoxic effects, the first question is whetherthey can be divorced from the process of virusreplication and be connected with virus-in-duced compounds which may or may not becomponents of the virion. Then we wish toknow if the processes of morphological damagecan be separated from aspects of biochemicaldamage. Finally, we need to know the natureand mode of action of the virus-induced com-pounds responsible for the cytotoxic effects.Some progress has been made in answeringthese questions for a few viruses, but only intissue culture experiments. How far the find-ings can be extended to other viruses and tothe pathology of animal infections remains tobe seen.Morphological damage can occur in tissue

culture without production of infectious virus.Thus influenza virus, Newcastle disease virus,fowl plague virus, a murine picornavirus, andmengovirus damaged cells which were eitherincapable or poorly able to support virus repli-cation (6). Cells were also damaged by polio-virus, vaccinia virus, and rabbit poxvirus inthe presence of chemical inhibitors of virusreplication, such as p-fluorophenylalanine, andalso by ultraviolet light-inactivated vacciniavirus, rabbit poxvirus, and reovirus (6). Inthese experiments, high multiplicities of virusinfection were used. Further support for thefact that virus replication and morphologicaldamage need not be closely linked is providedby the observations that virulent strains ofsome viruses such as Newcastle disease virus(124, 125) have greater damaging effects in re-lation to replication rate than avirulentstrains; and the damaging effects of the samevirus, such as reovirus, in the same cell linecan vary with different cultural conditionswhich provide similar yields of virus (45). Withregard to biochemical damage, the cut-offphenomenon (90) or inhibition of host-cellmacromolecular synthesis can occur in theabsence of the production of infectious virus.Thus, RNA and protein synthesis were de-pressed in poliovirus-infected HeLa cellstreated with guanidine, which prevented repli-cation (64), and in cells treated with vesicularstomatitis virus after inactivation with ultravi-olet light (163). Finally, some pathologicaldamage occurs in animals in the absence ofnew infectious virus (131).Some preformed virion components seem to

exert cytotoxic effects. Sendai virus, New-

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castle disease virus, measles virus, and simianvirus 5 produced rapid polykaryocytosis in cellcultures, but only when high virus multiplici-ties were used. This indicated that preformedproducts were responsible for the fusion effectsand, in the experiments with simian virus 5,puromycin and actinomycin D were added tostop de novo protein synthesis (26, 66, 83, 107).Components of herpesvirus also seem to pro-duce syncytia (158). The penton of adenoviruscauses cell rounding and cell detachment fromglass (46, 55). A double-stranded RNA frombovine enterovirus caused rapid death, withoutthe production of infectious virus, of cells sus-ceptible and insusceptible to enterovirus infec-tions (33). Whether the RNA itself was cyto-toxic or incomplete replication occurred givingrise to cytotoxic proteins is a matter of specu-lation (6). Biochemical damage, more specifi-cally interference with host-cell macromolec-ular synthesis, has been achieved with thefiber antigen of the adenovirus capsid whichinhibited with RNA, deoxyribonucleic acid(DNA), and protein synthesis (88) and mayhave been achieved with a double-strandedRNA from poliovirus which interfered withprotein synthesis in lysates of rabbit reticulo-cytes (44). Finally, it is interesting to mentionhere that large quantities of some viruses, suchas influenza virus and poxviruses, cause rapidtoxic effects in animals (97, 149, 154).

Bablanian, Tamm, and their colleagues haveshown that morphological damage of cells in-fected with poliovirus or vaccinia virus is dueto de novo synthesis and accumulation ofvirus-induced proteins; this was achieved bycareful time-sequence examinations of the ef-fects on morphological damage of adding andremoving compounds which either interferedwith the production of infectious virus such asguanidine or with protein synthesis such asstreptovitacin A, cycloheximide, and puro-mycin (6-8). Similar conclusions that de novoprotein synthesis is needed for morphologicaldamage have been made for mengovirus (3, 21,52, 59), influenza virus (133), Molluscum con-tagiosum virus (85), and Newcastle diseasevirus (124). Also, in some instances, inhibitionof host-cell macromolecular synthesis appearsto be due to virus-induced protein synthesis (6,90).Although virus-induced inhibition of host-

cell macromolecular synthesis could producedecisive biochemical effects in animals (seeabove) and in time will kill cells, in severalinstances in tissue culture it appears that therapid morphological damage of cells is notdependent on the "cut-off" phenomenon.

First, noninfected cells with drug-inhibitedmacromolecular synthesis were not as dam-aged as infected cells. For example, L cellswith RNA synthesis inhibited by actinomycinD to an extent comparable to that seen inmengovirus infection, and LLC-MK2 cellswith protein synthesis inhibited with puromy-cin, cycloheximide, and streptovitacin A,comparable to that seen in vaccinia infection,did not suffer the rapid morphological damageseen in virus infection (6, 9). Second, sequen-tial observations of virus-infected cells some-times coupled with treatment with compoundsinhibiting RNA and protein synthesis showeda lack of parallelism between the appearanceof morphological damage and the occurrence ofthe "cut-off" phenomenon for poliovirus (6),mengovirus (3, 59), reovirus (45), influenzavirus (133), simian virus (66), and herpesvirus(47). Third, in the case of adenovirus, differentparts of the capsid have different activities,the penton affecting morphology and the fiberantigen macromolecular synthesis (47, 88).

In investigating the nature and mode of ac-tion of the virus-induced compounds that areresponsible for cytotoxic effects, preformed vi-rion components (see above) are the easiertarget. By fractionating split virions, it shouldbe possible to identify cytotoxic componentssuch as the penton and fiber antigens of ade-novirus and possibly the double-stranded RNAfrom bovine enterovirus. But the majority ofcytotoxic compounds are probably extravirioncompounds found in infected cells. Hence thetask of identification is more difficult. Stephenand Birkbeck (151) advocated a direct ap-proach comparable to that used in bacteriol-ogy, namely, to isolate the virus-induced com-ponents from infected cells, to free them fromintact virus, and to attemptto produce thetoxic effects in uninfected cells. This approachrequired the design of techniques for the diffi-cult process of introducing potentially cyto-toxic viral products into fresh cells. Usingmagnesium sulfate solutions to increase mem-brane permeability, Stephen and his colleagues(19) have obtained evidence of cytotoxic fac-tors induced by vaccinia virus in HeLa cells.Furthermore, by using appropriate immuno-sorbents, they (168) have provided evidencethat the infected cells contain both virus-spe-cific and host-specific cytotoxic factors. Thus,there is increasing evidence that viruses pro-duce cytotoxins; but how do they act? Inhibi-tion of host-cell macromolecular synthesis orother interference with the functions of the cellcould be produced directly by the virus prod-uct, as diphtheria toxin interferes with protein

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synthesis. On the other hand, the virus-in-duced product might release autolytic enzymesfrom the cell's own lysosomes (58), and theresults of Stephen and his colleagues are inter-esting in this respect. Obviously we are begin-ning to know something of the mechanisms ofvirus cytotoxicity, but much remains to bedone in tissue culture and even more in re-lating the results of such experiments to path-ological effects in animals.Immunopathology is responsible for host

damage in bacterial and other microbial dis-eases (143, 147), but it is more likely to occurin virus diseases, because the obligate para-sitism involved increases the chances of cell-bound antigens occurring and also the exist-ence of autoimmune phenomena. It is nowclearly established that some viruses incorpo-rate host-cell constituents, especially mem-brane constituents, into their structure. Henceantibodies against these virus-host complexescould reactwith the membrane constituents ofinfected and normal cells. Also, virus infectionmay change the host-cell membrane constitu-ents and form neoantigens, the antibodiesagainst which could react with infected andnormal cells.Host damage could result from any of the

four types of allergic reactions described byCoombs and Gell (32): type I, reaginic anti-body-mediated anaphylactic reaction; type II,antibody and often complement-mediated cy-totoxic reaction against cell-bound antigens;type III, antibody-antigen complex Arthus-type reaction; and type IV, reaction of activelyallergized cells without antibody. In additionto cell destruction, cell proliferation mightalso occur as a result of type II reactions (165).It appears that one or more of these four typesof allergic reactions may be involved, in somecases of damage, in a number of virus diseasessuch as encephalitis in measles, poxvirusrashes, pneumonia from respiratory syncytialvirus, yellow fever, hemorrhagic dengue,mumps, coxsackie B virus infections, canineinfective hepatitis, Kyasamur Forest disease,blue tongue of sheep, hog cholera, and Aleu-tian disease of mink (96, 165). In these dis-eases, the evidence for immunopathology ismostly suggestive. But for lymphochoriomen-ingitis in mice, we have the viral counter-part of tuberculosis and streptococcal nephritiswhere sufficient solid experimental evidencehas accumulated for us to be reasonably surethat immunopathology (glomerulonephritisfrom immune complex, type III reaction inchronic disease, and cytotoxic type II reactioninvolving antibody and complement in acute

disease) plays a major role in the observeddamage. This evidence has been so well de-scribed and reviewed that it need not be re-peated here (40, 99, 108-111, 165).Immunopathology is such an attractive ex-

planation for virus damage that it is receivingmuch current attention (111, 129, 165). Per-haps a few words of warning against too easyassumptions of its complicity in cases of virusdamage may not be out of place. Firstly, meredemonstration that an infected host is immu-nologically sensitive to virus products by adiagnostic test such as a skin test is no proofof the implication of the sensitivity in themain pathological effects of disease. More ex-tensive investigations are needed; the mainsystemic and local effects of the disease mustbe simulated by immunological reactions in-voked in a sensitized host by products of theappropriate virus or in an infected host by an-tibody or immune cells from an immunizedhost. Such evidence is not easily obtained, par-ticularly for human diseases lacking good an-imal models. Secondly, the lack of knowledgeof the mechanisms of direct virus cytotoxicityadds to the difficulty of distinguishing suchmechanisms from immunopathological ones.Thirdly, it should be remembered that, al-though interesting, the number of immuno-pathological cases is probably small comparedwith those due to direct virus cytotoxicity(165).

HOST AND TISSUE SPECIFICITYThe occurrence of host and tissue specificity

in virus infections is so well documented (12,65, 79, 149, 154) that descriptions of the manyexamples will not be repeated here. On theother hand, studies of the biochemical bases ofthese phenomena in virus infections are evenmore in their infancy than similar studies inbacteriology (12, 79, 115, 149). The two phe-nomena are considered together, becausebroadly they can be explained on similar prin-ciples, although most of the examples dealwith tissue specificity. It should be empha-sized that the two specificities are not all-or-none phenomena (limited virus replicationmay well occur in the nonsusceptible tissue orhost, especially if the infecting dose is high),and they often vary with the age of the host.Thus, coxsackievirus infects muscle cells ofyoung but not old mice (76), and the MHV(PRI) strain of mouse hepatitis virus infectsliver macrophages of young but not old C3Hmice (12).The real difficulty in studies of host and

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tissue specificity is not lack of ideas of possibleexplanations for the phenomena but the designof experimental systems to investigate them ina manner relevant to natural infection.Clearly, the availability of "replication fac-tors" in host cells and their surrounding fluidsand their variation under different environ-mental conditions probably determine manycases of host and tissue specificities (146, 147).Similarly, other cases will depend on variationin levels of antiviral substances from host tohost and tissue to tissue, or differential induc-tions of interferon and immune mechanismseither in level or in time (31, 65, 87). Also, theroute of infection may play some role in tissuespecificity. One susceptible tissue may beeasily accessible to the incoming virus andbecome infected, whereas another equally sus-ceptible tissue may be protected by a barrierof cells which either do not support virus repli-cation or destroy virus. For example, in micethe KUpffer cells of the liver seem to protectthe parenchyma cells from infection withblood-borne influenza and myxoma viruses,and infection via the bile duct circumvents thebarrier (95). A blood-brain barrier appears toprevent infection of the brain by certain blood-borne viruses which attack neurons if inocu-lated directly into the central nervous system(14). This barrier may be insusceptible vas-cular endothelial cells, but general reductionof the viremia by the cells of the whole reticu-loendothelial system may be a major factor inthe "blood-brain barrier" (75). However, thelatter does not seem to operate in some cases.Thus, in mice, an avirulent strain of SemlikiForest virus produced a viremia at least ashigh and probably higher than a virulent strain,yet its attack on the brain cells was abortivecompared with the virulent strain whose lethaleffect depended on rapid invasion and replica-tion in the brain (121). Despite the clear impli-cation of route of infection in some cases oftissue specificity, variations of "replicationfactors" and host defense mechanisms arealmost certainly more important in both tissueand host specificity, and methods of investi-gating them outside the animal host are dis-cussed below.The first essential is that the specificities of

animal infection should be retained in theexperimental system. Since virus susceptibil-ities change when cells differentiate in normaltissue cultures (12, 68, 77, 142), the lattercannot be used directly to investigate host andtissue specificity in natural infection. Never-theless, studies of differing cell susceptibilitiesin such cultures (48, 104), might serve as mod-

els for adaptation to the systems describedbelow.

Infection in the chick embryo has been rec-ommended as a system in which tissue speci-ficities characteristic of human and fowl infec-tions can be reproduced for a number of vi-ruses such as fowl pox, vaccinia, herpes, pseu-dorabies, influenza, Rous sarcoma, and New-castle disease viruses (12, 22, 23). However,this system does not appear to have been usedextensively to investigate mechanisms of spec-ificity, although experiments with whole em-bryos, primary chick cell cultures, and organcultures could be conducted along the samelines as those described below. Thus, the dif-ference in susceptibility between chicken lungcells and fibroblasts to Sendai virus appearedto be due to more efficient maturation of virusparticles in the lung cells (38).

Short-term studies with primary cell cul-tures or suspensions of relevant tissues haveyielded most of our available information ontissue and host specificity. Using such prepara-tions and membrane fractions from them,Bang (12) and Holland (65) provided good evi-dence that the presence or absence of surfacereceptors for poliovirus determined suscepti-bility or resistance to infection of differentprimate tissues and of primate and nonpri-mate tissues; in particular, cell resistance toinfection disappeared when the receptors wereby-passed by using virion RNA as the in-fecting material. In other cases, experimentswith primary cell cultures have shown that thecell receptors determining initial adsorptionare probably not the important factors in sus-ceptibility. The MHV (PRI) strain of mousehepatitis virus infects PRI mice but not C3Hmice, and this difference in host specificity isreflected in the susceptibility of liver macro-phages (12). Yet adsorption of MHV (PRI)virus to resistant mouse (C 3H) macrophageswas similar to that occurring with susceptiblemouse (PRI) macrophages; penetration of theresistant macrophages seems to have occurred,but there appeared to be no uncoating (136).Similarly, work with primary cell culturesshowed initial adsorption, but absence of pene-tration occurred with insusceptible cells forRous sarcoma virus (117) and feline herpes-virus (155). Also, experiments with primarycell cultures of human fetal tissue, placenta,and leukocytes with Sendai and rubella virusesshowed that interferon production varied withcell type and virus (10).

In primary cell cultures, there have beenattempts to confer susceptibility on resistantcells by extracts of or products from suscep-

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tible cells. As for treatment of conventionaltissue culture cells, susceptibilities of primarycell cultures have been affected by externallyapplied materials such as serum and tissueextracts. Bang and his colleagues reported thatmouse (C 3H) liver macrophages resistant tomouse hepatitis virus were rendered suscep-tible by treatment with extracts of susceptiblemouse (PRI) liver macrophages, but in theseexperiments there may have been a change invirus rather than a change in cell susceptibility(12). The source (horse, calf, or mouse) andconcentration of the serum used to suspendmouse liver macrophages influenced their sus-ceptibility to hepatitis virus, possibly by af-fecting intracellular events (86). RecentlyMathews (91) reported that encephalomy-ocarditis virus promoted protein synthesis incell-free systems of certain mammalian andavian cells, such as Krebs II ascites cells, butnot in extracts of rabbit reticulocytes, untilcell sap from ascites cells was added. The ex-periments indicated that a tissue-specific fac-tor, possibly transfer RNA (5), was required fortranslation of the viral RNA by the rabbit retic-ulocyte extract. If cases are found where re-sistance or susceptibility of primary cell cul-tures can be changed by treatment with cellextracts of susceptible or resistant cells, thenappropriate fractionation might provide thechemical basis for susceptibility or resistance.For example, as far as I am aware, no one hastried to extend the work of Holland and hiscolleagues on the importance of receptors forpoliovirus infection by attempting to confersusceptibility on resistant cells by treatmentwith receptor extracts from susceptible cells.Such experiments would be comparable to theenhancement of infection of monkey kidneycultures by echovirus by treatment with re-ceptor-like substances from red blood cells(139). In this connection, recent work (28, 30,84) has shown the uncoating and release ofnucleoprotein from polivirus by protein frac-tions from membranes of susceptible but notunsusceptible cell lines and similarly from in-fluenza virus by chick embryo cell membranes.In the future, experiments with organ cul-

tures coupled with those in animals may proveof equal importance to work with primary cellcultures in studies of host and tissue speci-ficity. Unlike tissue cultures, organ culturesusually retain their parent specificities of nat-ural infection (12, 68) and, as suggested earlier,when this is not so, it indicates that the spe-cific or nonspecific host defense mechanismspresent in animals but absent in organ cul-tures may play an important role in the tissue

or host specificity. As an indication of thecourse research might take when this situationoccurs in studies of virus specificity, the readeris referred to descriptions of work on the hostspecificities of trachoma conjunctivitis agents(115) which, although not viruses, are handledtechnically as if they were. The followingsummary of recent attempts to identify thebasis of tissue specificity of influenza virus (16,17, 56; S. Rosztoczy, G. L. Toms, and H.Smith, unpublished data) illustrates the stagesof research when specificities are reproducedin organ culture.

Influenza virus infection in the ferretseemed an appropriate system for study, sincein ferrets influenza appeared to take the samecourse as in man, with a definite localizationin the upper respiratory tract (49). First, aquantitative survey of ferret organs for virusfollowing inoculation intranasally or into thebloodstream was conducted to identify notonly highly susceptible tissues but, for compar-ison, insusceptible ones and possibly those ofintermediate susceptibility. One to five daysafter intranasal inoculation, of 16 different tis-sues examined, only nasal mucosa, lungs, tra-chea, and esophagus contained detectablevirus. The nasal mucosa appeared the mostsusceptible tissue: the average total amountsof infective virus in lungs, trachea, and esoph-agus were approximately 2%, 0.001%, and0.1%, respectively, of that in nasal mucosa.When virus was inoculated into the blood-stream, only nasal mucosa became infectedand to a considerable degree. The high suscep-tibility of nasal mucosa to influenza virus wasthus established; other workers reached similarconclusions (116).

In organ cultures of ferret tissues, influenzavirus infection followed the pattern in vivo fornasal mucosa, lung, trachea, esophagus, andaorta. After receiving the same inoculum, virusreplicated significantly in nasal mucosa, lung,and trachea, and particularly well in nasalmucosa, but significant replication was notdetected in the esophagus and aorta. Unex-pectedly, organ cultures of bladder and ovi-duct (including the upper end of the uterus),which were not infected in the original experi-ments in vivo after intranasal or intracardialinoculation, supported virus growth. However,direct inoculation of ferrets into the bladderand upper end of the uterus resulted in localinfection. The conclusions from the work thusfar are as follows. First, influenza virus wasmore ubiquitous in infecting tissues than wasfirst thought; the urogenital tract infectionrecalled reports of isolation of virus from urine

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in some cases of human influenza (39). Second,route of infection plays a most important partin localization of influenza virus in ferrets andprobably man, since the incoming virus meetsa highly susceptible tissue first, and other sus-ceptible tissues are protected by a blood-tissuebarrier which may be a reduction of viremia bythe whole reticuloendothelial system, as sug-gested for the blood-brain barrier by Johnsonand Mims (75), or possibly an adsorption andinactivation by blood cells (S. Rosztoczy, G. L.Toms, and H. Smith, unpublished data).Third, some parallel had been established be-tween infection in vivo and virus growth pat-terns in organ cultures, suggesting that "repli-cation factors" rather than host defense mech-anisms of the inflammatory and immune re-sponse play some role in tissue specificity.Fourth, highly susceptible, moderately suscep-tible, and poorly susceptible tissues had beenindicated for future detailed comparison oftheir behavior when infected with influenzavirus. Finally, factors which could vary in vivoand might be responsible for specificity differ-ences, such as variations in temperature, pH,small molecular materials in the environment,and microbial flora, would not appear to play adecisive role in this example of tissue speci-ficity. All these factors are standardized orcontrolled in the antibiotic-containing organculture, the infection pattern of which paral-leled the susceptibilities in vivo.Deeper investigations on the differing sus-

ceptibilities of the organ cultures could bebased on the methods and approaches whichhave had some success in studies of the sus-ceptibility of tissue culture cells (48, 104) andprimary cell cultures (see above). Single-cyclegrowth experiments with varying virus inoculaand coupled with treatment with antiserum atdifferent stages could be attempted to investi-gate more closely the stages of virus replica-tion in poorly, moderately, and highly suscep-tible tissues. Investigations of receptors couldbe conducted by the methods used by Holland(65) for poliovirus, including infection experi-ments with viral nucleic acid and destructionof receptors by the Vibrio cholerae enzyme(neuraminidase). Attempts could be made totransfer susceptibility or resistance by isolatedmembranes or extracts from the various organcultures or by feeder techniques using suscep-tible and poorly susceptible tissues in closeproximity to one another. It is also possiblethat different strains of virus with a differentenvelope protein or neuraminidase may beblocked in infecting the normally highly sus-ceptible nasal mucosa and thus provide some

clue as to the important step in susceptibility.All these types of experiments are possible,but for their operation and interpretation theorgan culture system has two serious disadvan-tages. First, in organ culture, cell types differand only a relatively small proportion of themmay be susceptible to infection; in fact, differ-ences in susceptibility of organ cultures (andthe parent tissue in vivo) may be merely a re-flection of the relative number of susceptiblecells rather than differences in inherent sus-ceptibility of the cells. Second, organ cultureshave cut edges and under-surfaces which willexpose previously unexposed cells to virus at-tack, at least at the start of infection in vivo;virus attachment to or replication in thesecells may confuse an issue. Some method, suchas immunofluorescence or electron microscopy,of checking where virus attachment and repli-cation is occurring in the different cell types oforgan culture would seem an essential adjunctto the experimental approaches describedabove. Overall, it seems to me and my col-leagues (S. Rosztoczy, G. L. Toms, and H.Smith, unpublished data) that only if suscepti-bility or resistance is determined by differen-tial numbers of susceptible cells in the tissueor at early stages in replication (such as ab-sorption, penetration, and possibly uncoating)is the organ culture system capable of pro-viding the answers at its present stage of so-phistication.

In a first attempt at such deeper investiga-tions (56), infection of ferret nasal mucosa andesophagus were compared in experimentsbased on single-cycle growth studies. Virusadsorbed with equal efficiency to both tissues.Electron microscopy showed virus directlyadsorbed to the cilia and cell surfaces of therespiratory tissue. However, in contrast to thenasal mucosa, virus adsorbed to esophagusappeared to be mainly associated with anamorphous substance covering the epithelialcells and rarely in direct contact with the cellmembrane. Subsequently, virus penetrated thecells of the nasal mucosa, and large amounts ofnewly synthesized virus were recovered within8 hr of infection. In contrast, with the smallinoculum used in these experiments, virusfailed to penetrate the esophageal surface inquantities sufficient to promote the produc-tion of significant amounts of new virus. It ispossible that larger inocula or removal of thesurface covering would allow some virus pene-tration and replication, but a strong adsorp-tion to the thick surface covering, preventingclose adhesion of most virus particles to thecells of the surface, appears to be the main

304 SMITH


reason for the relative insusceptibility ofesophagus to influenza infection. On the otherhand, the reason for the high susceptibility ofnasal mucosa is not clear. A strong adherenceto the cilia and microvilli was observed in thisand other work (41). Obviously the presenceof cilia and microvilli is not essential for ad-sorption and penetration, since nonciliatedcells of ferret bladder were susceptible to in-fection both in vivo and in vitro.

In conclusion, this review will have achievedits purpose if it encourages some virologistswell versed in the elegant methods of tissueculture to turn their attention and adapt theirmethods to less well-defined organ culture andwhole animal systems. In this way we maylearn more about virus disease, the raisond'6tre for our subject.

LITERATURE CITE1. Alexander, D. J., P. Reeve, and W. H. Allen.

1970. Characterisation and biological proper-ties of the neuraminidase strains of New-castle disease virus which differ in virulence.Microbios 6:155-165.

2. Allison, A. C. 1967. Cell-mediated immune re-sponses to virus infections and virus-inducedtumors. Brit. Med. Bull. 23:60-65.

3. Amako, K., and S. Dales. 1967. Cytopathologyof meningovirus infection. I. Relationshipbetween cellular disintegration and virulence.Virology32:184-200.

4. Asmundsson, T., and K. H. Kilburn. 1970.Mucociliary clearance rates at various levelsin dog lungs. Amer. Rev. Resp. Dis. 102:388-397.

5. Aviv, H., I. Boime, and P. Leder. 1971. Proteinsynthesis directed by encephalomyocarditisvirus RNA: properties of a transfer RNA-dependent system. Proc. Nat. Acad. Sci.U.S.A.68:2303-2307.

6. Bablanian, R. 1972. Mechanisms of virus cyto-pathic effects. Symp. Soc. Gen. Microbiol.22:359-381.

7. Bablanian, R., H. J. Eggers, and I. Tamm.1965. Studies on the mechanisms of polio-virus-induced cell-damage. I. The relationbetween poliovirus-induced metabolic andmorphological alterations in cultured cells.Virology 26:100-113.

8. Bablanian, R., H. J. Eggers, and I. Tamm.1965. Studies on the mechanism of polio-virus-induced cell damage. II. The relationbetween poliovirus growth and virus-inducedmorphological changes in cells. Virology 26:114-121.

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