FR2581655A2 - Papillomavirus probes and method for the in vitro diagnosis of papillomavirus infections. - Google Patents

Abstract

The invention relates to papillomaviruses, in particular HPV DNAs obtained from these papillomaviruses whose physical structures are derived from figure 10, or alternatively to probes containing these HPV DNAs or to fragments obtained from the latter. It also relates to boxes or "kits" containing distinct groups of probes containing one of these HPV DNAs or HPV DNA fragments as well as to a method for the detection and identification of papillomaviruses using these different probes.

Description

Papillomayirus probes and method for in vitro diagnosis of papillomavirus infections
The invention relates to papillomavirus DNAs, and more particularly to probes derived from these papillo viruses. as well as methods implementing them for the in vitro diagnosis of papillomavirus infections The expression "papillomavirus" covers a large number of viruses having in common the fact that they are held responsible for several forms of viral infections. etagant between skin warts or relatively benign mucous membranes and hyperplasias likely to degenerate into intraepithelial neoplasias and skin cancers. Among papillomavirus infections. mention will also be made more particularly of epidermodysplasia verruciformis.

which will sometimes be referred to below as "EV"
A number of types of papillomavirus have already been described. In the main patent, numerous new types and subtypes of papillomavirus have been described which have been isolated from warts or disseminated macular lesions, which may give rise to the early development of skin cancer in humans. 'large proportions of patients affected by it.

 Recent studies have revealed the importance of immune factors and the role of various types of human papillomavirus (HPV), these factors adding to the role already described in the literature of various genetic factors and actinic radiation in the pathogenesis of papillomavirus infections.

 The invention of the main patent was based on observations which had been made as to the relative behavior of a large number of newly isolated papillomaviruses. the essential genomic characteristics of which have been defined below.

 A significant number of new Ilomavirus papi types and subtypes have been described in the main patent. The same was true of the use of the DNAs of these new papillomaviruses, taken alone or in combination, with each other and / or with previously known HPV DNAs, in more refined in vitro diagnostic techniques.

 Generally speaking. it was noted in the main patent that the; papillomavirus. although very different from each other, had sizes in the range of 7000-8000 base pairs. In addition, their genomes may nevertheless present certain degrees of homologation, evaluated as percentages of homologies between types; and papillomavirus subtypes, in hybridization tests carried out under so-called non-stringent or non-strict conditions or even under stringent or strict hybridization conditions.

 It has been said that the papillomaviruses which present percentages of homology lower than 50% under strict or stringent conditions belong to different types. The viruses for which we observe. under these strict or stringent conditions, percentages of homology greater than 50% are considered to be apparent.

belonging to the same type. They can form different subtypes within this same type
heidization tests under non-strict or non-stringent conditions involve the mutual contacting of DNAs from two virus isolates under the following conditions described by HEILMA.N CA et al, 1980. J. Virol., 38, 385-407. and CROISSANT et al. 1382.

C.R. Acad. Sc. Paris, 234. 581-585 (heteroduplex molecules).

 Hybridization tests under strict or stringent conditions involve the mutual contacting of DNAs coming from two virus isolates under the conditions described by KREMSDORF, D. et al. ((1982).

a, Virol. 43: 436-447 and 1383, s. Virol. 48: 340-351) and
DAVIS RW et al., 1971, Methods Enzymol., 21, 413-428 (heteroduplex molecules)
Based on these considerations, several new viruses have been described in the main patent. Likewise, genetic recombinants have been described comprising all or part of the genomes (called
DNA-HPVs) of these viruses. The invention of the main patent therefore concerned each of the DNA-HPVs chosen from all of the DNAs which have sizes which range between 7000 and 8000 base pairs and are characterized by the maps of restriction which appear in the drawings also reproduced in this application, with regard more particularly to the
DNA-HPVs obtained from papillomaviruses and which correspond to the designations HPV-2d, HPV-10b, HPV-14a,
HPV-14b, HPV-15, HPV-17a, HPV-17b, HPV-19, HPV-20, HPV-21 HPV-22, HPV-23, HPV-24, HPV-28, HPV-29, HPV-31 and HPV-32.

 The invention of the main patent also related to mixtures or "cocktails" of DNA-HPVs isolated from new or previously known papillomaviruses. 9 the date of filing of the main patent, or hybridization probes containing them likely to be implemented more effectively for the diagnosis of various categories of infections, or even of the levels of risk which the discovery in a patient of The number of papillomavirus probes described in the main patent, to which were added those constituted from the genomic DNAs of papillomavirus previously isolated and their associations in determined mixtures, therefore makes it possible to carry out refined diagnoses, in particular great discrimination between the various categories of infections attributable to the various types of papillomavirus or likely to develop as a result of the latter types and, within a given category of infections, to better predict the degree of risk than these the latter turn into more formidable diseases. In particular. the invention of the main patent provided means making it possible, in the case of infections manifested by epidermodysplasia verruciformis, to better appreciate the degree of risk that these latter evolve towards skin cancers.

As such, the invention of the main patent relates more particularly to diagnostic compositions; a group of preferred compositions was defined as containing 1) at least the DNA of HPV2d, 2) at least one of the DNAs of HPV10b, 28 and 29, 3) at least one of the DNAs of HPV17, 24 , 4) at least one of the DNAs of HPV14, 15, 17, 19, 20. 21, 22
and 23, 5) at least one of the DNAs of HPV15 and 17, 6) the DNA of HPV24, 7) the DNA of HPV14, 32, 8) the DNA of HPV31, 9) the DNA of HPV32 , it being understood that the DNAs of the nine groups were chosen so as to be in all circumstances different from each other.

In Figures ta 9 of the accompanying drawings, the physical restriction maps of the
DNA-HPVs concerned.

 The physical maps give the position of sites of cleavage by various restriction endonucleases.

The origin of the cards is generally made up of a single clipping site. Distances to origin are expressed as a percentage of genome length.

 The present invention relates to newly isolated papillomaviruses, genomic DNAs which can be extracted therefrom or fragments of these genomic DNAs, as well as new hybridization probes which can be formed from these DNA-HPVs or DNA fragments. HPVs.

 The invention also relates to novel "cocktails" of probes containing them or their use in "CLCKtails" of probes already described in the main patent.

pal, however supplemented with one or other of the DNA-HPVs which are the subject of the present application and. therefore, more sophisticated diagnostic kits or "kits" are available.

The DNA-HPVs according to the present invention are characterized by the restriction maps making the omits in FIG. 10 and respectively called HPV-IP2 and
HPV-IP4.

 A hydridation probe constructed from the genome of HPV-IP2 is particularly useful for diagnosis and / or in vitro screening, under the conditions which have been described in the main patent and which will be described further. far, types of papillomavirus which conxtitj a risk for the development of genital neoplasms and. in particular, cervical cancer.

A hybridization probe constructed from
HPV-IP4 is particularly useful for the diagnosis and / or the screening in vitro of the types of papilloms virus constituting a risk for the development of precancerous or cancerous leaions of the skin,
Hybridization probe cases will advantageously be incorporated into the diagnostic mixtures of affections of the same type to which it will be discussed later.

The invention also relates to fragments of
DNA HPVs previous or able to hybridize with these. especially under strict conditions. Likewise, it relates to the recombinant DNAs containing all or part of each of the DNA-HPVs mentioned above. and more particularly recombinant DNAs containing fragments corresponding to the genes El, E6-E7, L1 and L2 respectively or alternatively fragments containing sequences corresponding to the intergenic regions of said DNA-MPvs. Finally, it relates to the probes which can be constituted from these respective DNA-HPVs or from the corresponding fragments and the in vltro diagnostic methods involving said probes.
The preparations of viral DNA or :: were extracted according to the techniques which will be described below in Examples 1 and 2.

In what follows, the conditions will be described more precisely; in which viruses have been isolated. then the conditions, in which the DNA-HPVs were obtained from these viruses
Molecular cloning and characterization of @@ new type of HPV associated with neoplasias and enenital cancers (HPV IP2).

A new type of HPV has been detected in DNA extracted from cervical cancer from 1 uteras. by hy @ ridation. under non-striated conditions with a radioactive probe specific for HPV type 16. No cross-hybridization is detectable when the hybridization was carried out under strict hybridization conditions. this study of the sensitivity of the DNA of this HPV to several restriction enzymes showed that the enzyme Bglll cut the viral DNA once. After digestion of the DNA extracted from the tumor with the Bgl11 endonuclease. the fraction containing 8kb DNA molecules (size of a papillomavirus genome) were purified by centrifugation in a sucrose gradient. The Skb molecules were inserted via the Bgill site. in a vector constitutes plasmid PL15.5 (containing a single site for cleavage by
BglII and by BamHI) inserted by its BamHI site. in the DNA of bacteriophage lambda L47.1. After packaging of the recombinant DNA and infection of host bacteria (Escherichia coli, strain LA101), the lysis plaques corresponding to recombinant phages were detected by hybridization of replicates of the infected bacterial cultures, with a radioactive HPV16 DNA, in non-strict conditions. Several recombinant bacteriophages, containing all the viral sequences. have been isolated the cut of phage DNA by the insertion enzyme
BglII generates an Ekb fragment hybridizing with the HPV16 probe under non-strict conditions; cutting the DNA of the recombinant phages and the DNA of the original tumor by mixing the enzymes BglII and PstI generates the same 5 fragments in the sum of the molecular weights is equal to the size of a genome of the papillomaviruses.

The DNA of the new HPV was excised from the DNA of the recombinant bacteriophages, purified by electroelution, and recloned in the plasmid PL15.5. A restriction map of viral DNA was established from the sensitivity of this DNA to 18 restriction endonucleases, which made it possible to locate 21 cleavage sites (FIG. 9). The map thus established is different from the map of the genomes of the HPVs identified to date. The sequence homology between the DNA of the new HPV and the DNA of the HPVs identified with this Bour was analyzed by replica molecular hybridization experiments carried out under strict conditions. The homology detected was always less than 5 7, the greatest homology being detected with the genome of HPV16.

The new virus characterized from cervical cancer therefore constitutes a new type of HPV, provisionally known as HPVIP2.

 Analysis, using an electron microscope. of hereroduplex molecules formed, under different conditions, between the DNA of HPVIP2 and the DNA of HPVI made it possible to align the physical maps of these 2 genomes and to define the theoretical position of the different genes carried by the DNA of the HPVIP2.

PUTATIVE LOCATION OF THE MAIN GENES AND THE INTRAGENIC REGION OF HPV-IP2 ON THE MAP OF THIS GENOME
End coordinates
5 '3'
E6-E7 62 71.5
El 71 95
E2 95.5 11.5
L2 11 30.5 L1 31.5 52
Intergenic region 52 63.5
The use of radioactive probes prepared from the DNA of the purified HPVIP2 made it possible to determine the pathogenic power of these viruses. HPVIP2 DNA was detected in one case of bowenoid papules of the external genitalia out of the 14 studied. in 2 invasive cervical cancers out of the 51 studied and in 1 case of intraepithelial cervical neoplasia out of the 28 studied. HPVIP2 therefore constitutes a type of HPV with genital tropism presenting an oncogenic potential. whose frequency is slightly lower than that of HPV'a, and much lower than that of HPV16. It is necessary to incorporate it into any mixture of HPV DNA intended for the preparation of molecular probes, with a view to the diagnosis or screening of the types of HPV constituting a risk for the development of genital neoplasias. in particular, cervical cancer,
Molecular clonaae and characterization of a new HPV tvoe associated with Cancerous skin lesions (HPV
IP4).

 A new type of HPV has been detected in DNA extracted from a biopsy of actinic keratosis. a precancerous skin lesion, by molecular hybridization.

under strict conditions, with a mixture with radioactive probes specific for HPV type 5, 8 and 14.

No cross-hybridization was detected when the hybridization was carried out with specific probes of the 1,2,3,7,10,13,16,18,28, IP1 types (previously called HPV31
IP2, and IP3 (previously called HPV32).

A study of the DNA sensitivity of this HPV to several restriction enzymes has shown that the enzyme
EcoRI cuts viral DNA once. After digestion of the DNA extracted from the biopsy by the endonuclease EeoRI. friction containing skb DNA molecules (pap genome size; llomavirus) was purified by centrifugation in a sucrose gradient. The 8kb mosecules have card inserted. by the EcoRI site, in the DNA of bacteriophage ss gt wes. B. After packaging of the recombinant DNA and infection of host bacteria (Escherichia col, strain LA101), the lysis plaques corresponding to recombinant phages were detected by hybridization of replicates of the bacterial cultures by radioactive mixture of the DNA of HPVs , 8. and 14. under non-strict conditions. Several recombinant bacteriophages. containing all the viral sequences have been isolated: the cleavage of phage DNA by the insertion enzyme EcoR 1 generates a fragment of 9kb hybridizing with the specific probe of HPV5, S and 14 under non-strict conditions: the cleavage of 1 DNA from recombinant phages and DNA from the original lesion by mixing the enzymes EcoRI and PstI generates the same 6 fragments whose sum of molecular polds is equal to the size of a genome of paplliomavlrus. The DNA of the new HPV was excised from the DNA of a recombinant bacteriophage, purified by electroelution. and recloned into the plasmid P @ P65. A restriction map of viral DNA was established on the basis of the sensitivity of this DNA to 15 restriction endonotlases, which made it possible to locate 23 cleavage sites (FIG. 10). The map thus established is different from the map of HPV gnomes identified to date.

The sequence homology between the DNA of the new HPV and the DNA of the HPVs identified to date has been analyzed by replica molecular hybridization experiments carried out under strict conditions. A homology of less than 50% has been detected between the DNA of the new HPV and the DNA of certain types of HPV previously identified in lesions of epidermodysplasia verruciformis @@ PV5.8.12.14. - 19.20, 21 and 25), but no homology was detected with the other types of HPV The new virus characterized from actinic keratosis therefore constitutes a new type of HPV provisionally called HPVIP4.

 The use of a radioactive probe prepared from purified HPVIP4 DNA made it possible to demonstrate HPVIP4 in 42% of the 17 patients with wart-shaped epidermodysplasia studied and in x out of y biopsies of actinic keratosis analyzed. Because of its great frequency in patients suffering from epidermodysplasia verruciformis. a disease characterized by the frequent development of skin cancers, and its association with a fraction of the lesions of actinic keratosis considered as precursors of squamous cell cancer of the skin, HPVIP4 constitutes a type of HPV with tropisome cutaneous presenting a potential It is necessary to incorporate it into any mixture of HPV DNA intended for the preparation of molecular probes for the diagnosis or screening of the types of HPV constituting a risk for the development of precancerous or cancerous lesions of the skin. .

 Given the great diversity of HPVs capable of being isolated from the different forms of worms or other skin or mucosal lesions. it is however preferred to use, for the diagnosis of each type of affection mentioned in the table, angels comprising more than one or two DNA-HPVs, when other DNA-HPVs have and recognized it can also be used in the development of the same type of affection.

The diagnosis of the nature of the infection and its possible evolution will be all the more effective as the number of probes used will be higher. In addition, hybridization tests carried out with different mixtures of probes will allow differential diagnoses having an equally greater degree of probability of the nature of the disease from which the patient suffers.

 The invention therefore relates more particularly still to mixtures or cocktails of different DNAs-HPVs (or probes containing these DNAs-HPVs or of sequences of these latter), capable of being used in combination to carry out global diagnostics of the different forms of papillomavirus infections possibly for the purpose of predicting the possible course of the infection.

Preferred mixtures in accordance with the invention are identified in the table below TASLEAU
CHARACTERISTICS OF HPV DNA MIXTURES FOR USE IN VIROLOGICAL DIAGNOSIS
PAPILLOMAVIRUS INFECTIONS Designation Constitution) Diseases to be diagnosed with mixtures 1 1,2d *, 4 Warts of the skin or thrush
(in particular, common and plantar warts).

Differential diagnosis of epidermodysplasia
verruciform.

2 3.10a, 10b *, 28 *, 29 * Flat or intermediate skin warts or mu
women. Intraepithelial neoplasias and cancers
skin. Differential diagnosis of epidermodys
warty plasia.

3 5.17a *, 24 * Epidermodysplasia verruciformis. Intra neoplasias
epithelial and skin cancer.

4 5,8,12,14a *, 14b *, 19 *, 20 *, 21 *, 22 *, IP4 Epidermodysplasia verruciformis.

5 9.15 *, 17a *, 17b Epidermodysplasia verruciformis.

6 24 * Epidermodysplasia verruciformis.

7 5,8,14b *, 32 *, IP4 Skin cancer of epidermodysplasia verruciformis.

 Intraepithelial neoplasia and skin cancer.

8 13.31 * Oral focal epithelial hyperplasia; Diagnostic
differential of intraepithelial neoplasias
oral.

9 32 *, IP4 Intraepithelial neoplasias and skin cancers.

1) The new types of HPV used in the constitution of probe mixtures are indicated by an asterisk
The above table also indicates the natures of the conditions likely to be more particularly diagnosed by the use of the mixtures appearing on the left part of the table. It is recalled that the restriction maps of the other DNA-HPVs identified in the above table are contained in Figures 1 to 9.

 It should be noted that the HPV-IP2 can be considered as particularly representative of probes usable for the detection of the risks of development of genital neoplasias and in particular. cervical cancer.

The invention therefore relates more particularly to kits or "kits" for diagnosis comprising at least 10 groups appearing in groups numbered from t to 10 in the table under the heading "Designation of mixtures"
In the above. we mainly considered the use, as probes. whole cloned DNA MPVs.

These can however be ubstituted by cloned fragments of these different DNAs. in particular by the El or L1 genes and by the E6-E7 genes.

The basic principle of DNA detection in v-ltro-
HPV will naturally put into play hybridizations operated under strict or less strict conditions. One can operate for example as follows. It was of course understood that the diagnostic tests described cannot be considered as limiting the conditions of use of the probes or mixtures of probes. according to the invention.

 The purpose of examinations using probes prepared from mixtures of cloned I YPVs DNAs is to reveal an HPV and to identify the type of HPV in a biopsy, in cells obtained by scraping lesions, or in fixed biopsy sections by the Carnoy mixture (ethanol. chloroform, acetic acid 6: 3: 1) and included in the paraffin. The examination requires the prior extraction of DNA from the samples according to methods including principle is known and involves the analysis of this DNA by molecular hybridization experiments, carried out under strict or less strict conditions, using rauioactive probes (marked with 32p or 35S) from HPVs DNA mixtures. Each exam requires. in general. the use of several mixtures of probes.

Several hybridization methods can be used. We can. for example. -, be -in work s hybridization method on spot. This method includes. after denaturation of DNA. deposition of an aliquot of DNA on membranes (nitrocellulose or
GenescreenpIus). the hybridization of each membrane. under the usual conditions, with a mixture of probes and the detectian of radioactive hybrids by expasition of the membranes in contact with an x-ray film. We can also use a hybridization method on replica,
This method comprises the electrophoretic separation in agarose gel of the DNA fragments generated after treatment of the DNA with restriction enzymes transfer of the fragments, after alkaline denaturation. on membranes (nitrocellulose, Genesoreenplus) and their hybridization, under usual conditions, with different mixtures of probes, The formation of radioactive hybrids is detected after exposure of the membranes to contact with an x-ray film.

 An in situ hybridization method can also be used. for example on tissue sections fixed by the Carnoy mixture and included in the paraffin.

 The radioactive probes consist either of HPV DNAs labeled by the "nick-translation" method. either by RNAs prepared by transcription of viral DNAs inserted into a vector, for example of the SPE type. The use of radioactive probes has the advantage of great sensitivity. but this does not exclude the use of non-radioactive probes, for example biotinylated and capable of being recognized by antibodies either labeled themselves, or themselves recognized by antibodies carrying an enzymatic, fluorescent marker, etc.

 The invention therefore also relates to kits or "kits" containing a plurality of the above-mentioned probes, and comprise - HPV-IP2 and HPV-IP4 respectively taken in isolation, - either the preceding probes, mixed with others.

in particular those defined above, these "kits" being intended for in vitro diagnostic studies by hybridization between viral preparations obtained from patients and the various groups or mixtures.

As it goes without saying and as it follows moreover from what precedes, the invention is only limited to those of its modes of application and embodiments; have been more specifically considered; on the contrary, it embraces all its variants; in particular the reference in the claims to a DNA-HPV designation followed by a predetermined number, and to which corresponds a DNA-HPV whose restriction map has been provided in the drawings, is understood to mean that these claims cover all of the DNA -HPVs that have in common with this
DNA-HPV particular to be able to be classified in the same type, according to the definition which was given above, and a fortiori to DNA-HPV belonging to the same subtype.

 It will be noted that the recombinant DNAs designated below were deposited on May 3, 1985 at the C.N.C.M.

(National Collection of Cultures of Micro-Organisms of the INSTITUT PASTEUR of Paris), under the numbers appearing below
PLI55 / IP2 ................ n I-450
pSP65 / IP4 n I-449

Claims (7)

 1 - DNA-HPV having a size of between approximately 7000 and approximately 8000 base pairs or a fragment of this DNA-HPV, this DNA-HPV consisting of a variant of DNA-HPVs defined in the main patent, this DNA-HPV being obtained from one of the papillomaviruses corresponding to the designations HPV-IP2 and HPV-IP4.  2 - DNA fragment according to claim 1, charac terized in that it corresponds to the E1 genes. E6-E7. L1 or L2 or that it still corresponds to a fragment corresponding to an intergenic region of said DNA-HPV.  3 - recombinant DNA containing a DNA-HPV according to claim 1 or a fragment according to claim 2.  4 - DNA, fragment or recombinant DNA according to any one of claims 1, 2 or 3 for use as a probe for detecting DNA-HPVs,  5 - Composition usable for the detection of papillomavirus in a biological medium containing it, characterized in that it contains a DNA-HPV in accordance with claim 1 associated with one or more DNA-HPVs distinct.  5 - Kit or kit comprising a plurality of probes or mixture of distinct probes, characterized by 9 groups of probes; each of which comprises 1) at least the DNA of HPV2d. 2) at least one of the DNAs of HPV 1Ob, 28 and 29. 3) at least one of the DNAs of HPV 17, 24. 4) at least one of the DNAs of HPV 14, 15, 17, 19, 20. 21,  22, 23 and IP4. 5) at least one of the DNAs of HPV 15 and 17, 6) the DNA of HPV 24, 7) the DNA of one of the DNAs of HPV 14, 32 and IP4.  8) the DNA of HPV 31, 9) the DNA of HPV 32, IO) at least one of the DNAs of HPV 16. 18 and IP2. it being understood that the DNAs of the nine groups are chosen so as to be in all circumstances different from each other insofar as each of the nine groups would be reduced to one of the DNAs which compose it.  7 - Method for detecting and identifying papillomaviruses contained in a biological sample comprising carrying out hybridization tests with uo or more -DNA-HPVs, fragments of DNA-HPVs or recombinant DNAs in accordance with any one of the claim 1 to 3 or more separate compositions according to claim 4, and the detection of those of DNA-HPVs, fragments or recombinant DNAs or compositions which give rise to hybridization prefer it with DNA-HPVs previously supported from said biological samples.  8 - Biologically pure purified papillomaviruses. characterized in that they belong to the same type as one of these papillomaviruses identified in claim 1.  BIBLIOGRAPHY  (1) Durst, M. et al., 1983, Proc. Natl. Acad. Scl.  U.S.A., 80: 3812-3815,  (2) Coggin, J.R., Jr. et al., 1979. Cancr Res. 39: 545.  546.  (3) Gissmann. L. et al., 1982, @. Virol. 44: 393-400.  (4) Green. M. et al., 1982, Proc. Natl. Acad. Sci. USA.  79: 4437 4441.  (5) Heilman, C.A. et al., 1980. Virol. 36: 395-407  (6) Jablonska, S. et al., 1972. Cancer Res., 32: 583-583.  (7) Jablonska, S. et al., 1982. Springer Semin. Immuno  pathol. 5: 33-62.  (8) Kremsdorf, D. et al., 1982. J. Virol. 43: 436-447.  (9) Kremsdorf, D. et al., 1983. J. virol. 48: 340-351. (10) Lutzner, M.A. et al., 1978. Bull. Cancer. 65: 169-182. (11) Lutzner, M.A. et al., 1983, Lancet 11: 422-424. (12) Migozzi. M et al., 1965. Bull. Soc. Franc. Derm.  Syph. 72: 747-748. (13) Orth. G. et al .. 1380. Cold Spring Harbor Conf. Cell  Proliferation. 7: 259-292. (14) Orth, G et al. 1981, J. Invest. Dermatol.  76: 97-102. (15) Orth, G. et al. 1979. Cancer Res. 39: 1074-1082. (16) Ostrow, R.S. et al. 1982, Proc. Natl. Acad. Sci.  U.S.A., 79: 1634-1638. (17) Ostrow, R.S. et al., 1983. Ann. Acad, Dermatol.  8: 398-404 (18) Pfster. H. et al., 1983, Cancer Res. 43: 1435-1441. (19) Pfister, H. et al., 1983, J. Virol. 47: 363-366. (20) Pfister. H. et al., 1981, Int. 3. Cancer. 27: 645-650. (21) Rueda, L.A. et aL., 1976, Med. Cut. HE HAS. 2: 113-136. (22) Ruiter, M. et al. 3. Invest. Dermatol. , 47: 247-252. (23) Sutcliffe, J.G., 1978. Nucleic Acids Rees.  5: 2721 - 2728. (24) Tsumori. T. et al. 1983. J. Gen. Virol. 54: 967-969.