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Studies on Cell Cultures Persistently Infected with Bovine Herpes Mammillitis Virus. The Possible Role of Deionised Water in Inducing A Carrier Status
B r. vet.
J.
(1972), 128,61 I
STUDIES ON CELL CULTURES PERSISTENTLY INFECTED WITH BOVINE HERPES MAMMILLITIS VIRUS. THE POSSIBLE ROLE OF DEIONISED WATER IN INDUCING A CARRIER STATUS By M. M. R WEYEMAMU*, R. H.JOH NSONt AND E. P.J. GIBBS! Department of Veterinary Medicine, Langford H ouse, Langford, near Bristol
SUMMARY The problem of in vitro latency of bovine herpes mammillitis virus associated with irregular cytopathic effect is described. One such persistently infected bovine kidney culture was maintained for three months showing several cycles of cytopathic effect (regression and regeneration) with corresponding cycles of extracellular virus yield. This in vitro carrier status could not be associated with interferon, antibody or serum inhibitors, variations in techniques, media or virus strains. It was overcome empirically by substituting double glass-distilled deionised water for deionised water in all media. I NTROD UCTION Bovine herpes mammillitis (BHM) virus normally grows rapidly and to high titre in tissue culture in which it produces large syncytia (Rweyem amu & Johnson, 1967) . Between D ecember and April 1967/68 and again 1968/69 only a limited cytopathic effect (CPE) followed the inoculation of susceptible cells and the routine assay of this virus in tissue culture becam e impossible. One feature of this poor CPE was the establishment of a carrier state in the inoculated monolayers. Although the cause of this poor CPE and latent infection was not found, the problem was overcome empirically by the substitution of double glass distilled dionised water for deionised water in all tissue culture media and for the final rinses during the washing of glassware. This paper reports the series of experiments that led to this observation. MATERIALS AN D METHODS The TV virus strain of BHM was used in this study (Rweyemamu & Johnson 1967). Methods for cell culture, the cell systems used and techniques for virus assay were as d escribed by Rweyemamu & Johnson (1967). Virus tit res were calculated by the technique of Reed & Muench (1938) and are expressed as tissue culture infective doses 50 per cent (TCID 5 o ) per ml. Present addresses: * Central Veterinary Laboratory, Temeke, Dar-es-Salaam, Tanzania; t Department of Tropica l Veterinary Science, James Cook University, Townsville, Queensland, 4810, Australia ; and t Animal Virus Research Institute, Pirbright, Woking, Surrey, England .
612
BRITISH VETERINARY JOURNAL,
128, 12
For continuity in the script further details of techniques are given with the results. RESULTS
Features of CPE and carner status The features observed may best be summarized from studies made on one culture of bovine kidney (BK) cells which was persistently infected with BHM virus over a period of three months. A secondary BK monolayer was cultured in a Roux flask to a full sheet which showed good pH conversion over 4 days. This monolayer was inoculated with 20 ml of virus at the 19th laboratory passage level in BK cells, at a titre of 10 4 • 3 TCID so /ml. Syncytial foci developed at 24 hours, enlarged by lateral extension over the next 24 hours but then showed degeneration and break up without development of secondary foci. CPE gradually regressed to apparent total disappearance. The culture was left incubated at 37 °C for a month without change of medium being required, by which time the monolayer had regenerated to a full sheet. The medium was then changed and the culture re-incubated for a further 8 days without showing any obvious signs of CPE; there was no detectable virus in the supernatant fluid. The monolayer was passaged to a Roux flask. The culture established itself, showed initial foci of CPE on the third day after passaging and CPE extension by the fourth day. Over the next 10 days, CPE became static and regressed with monolayer recovery and regrowth; the medium had been changed on the 4th, 8th and 9th days. On the 10th day, new foci of CPE were evident. The culture was subsequently passaged three times and maintained up to three months, during which time it demonstrated several cycles of CPE regeneration and regression alternating with cellular growth. Cycles of CPE regeneration corresponded roughly with rise in extracellular virus (Fig. I). Attempts to "cure" cultures by means of antibody were not successful. CPE was less marked in cultures maintained on media containing antibody but the cultures were not completely devoid of CPE foci, especially on passage. Occasionally subcultures from monolayers showed no epE. These cells were susceptible to superinfection with BHM virus, showing a variable degree of CPE following such superinfection. An investigation into some factors which might have been responsible for the development of the carrier state Factors associated with the virus. Latent infection was observed in BK monolayers by using BHM virus at the 3rd, loth, 14th and 19th tissue culture passage levels of the same strain, and by inoculating vesicular fluids from natural cases of BHM. All these samples had previously given good progressive CPE. The possibility of a virus contaminant either in the virus inoculum or in control cell cultures was investigated by (i) repeated passaging of control cultures and staining of representative coverslips; (ii) mixing control cultures from tests which had resulted in latent infection with new batches of cells and allowing them to form a monolayer together and staining coverslips at varying
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intervals; (iii) haemadsorption tests on monolayers; (iv) inoculating different cell cultures with BHM virus which had been neutralized with natural bovine antiserum; (v) examination of cell cultures by electron microscopy (Almeida, Waterson & Plowright, 1967); (vi) immunofluorescent examination for the presence of feline panleucopaenia virus (this virus is non-cytopathic in BK cells and had previously been used extensively in this laboratory-Johnson, 19 6 7) . No contaminant virus was detected.
Factors associated with the cell system (a) Age of monolayer. Young cultures were more susceptible to BHM virus infection than old cultures during the period when persistent infection became a problem. This variation in susceptibility associated with age of monolayer is not a significant factor during periods when standard ePE is obtained (Rweyemamu & Johnson, 1967). (b) Cell variation. During this period of poor ePE with BHM virus in BK cells it was observed that the problem of latent BHM virus infection became common to all cell systems used (Rweyemamu & Johnson, 1967). On one occasion all cell cultures were destroyed, and the virology laboratory was fumigated with formalin vapour. When seed virus was inoculated into new cell stocks the problem remained.
BRITISH VETERINARY JOURNAL,
128, 12
(c) Cell surfacefactors. Poste (1970) has suggested that following infection ofa cell with a syncytia forming virus the cell coat thickness must be approximately 35 Angstrom units before cells gain the capacity to fuse. In conjunction with Dr Poste it was shown that the system of cell surface changes that occur in polykaryocytosis developed in cell cultures exhibiting latent infection with BHM virus, even though extensive ePE did not develop (T able I ). TABLE " THE EFFECT OF CELL COAT THICKNESS ON BHM VIRUS
CYTOPATHOGENICITY
Mean cell coat thickness Number of observations
Bifore virus infection
After virus injection
Bovine kidney sample A
10
SO'2Ao
30'7A o
Bovine kidney sample B
10
38 '7Ao
27'4Ao
Bovine skin (fibroblastic)
8
42'3A o
30 '2Ao
Bovine skin (epithelial)
8
6S'SAo
60'IAo
Cell type
•+
Fusion *
CPE*
+* + +
+* + +
= present ; - = absent,
Bovine skin monolayers often exhibit a mixed population of fibroblastic and epithelial type cells. Under normal conditions, inoculation of these cultures with BHM virus results in a rapid and progressive ePE in the fibroblastic cell population with rarely any ePE in the epithelial cells. During the period when the ePE of BHM virus was poor in BK cells the epithelial type cells were refractory to infection and the fibroblastic type cells showed only limited ePE. Cell coat thicknesses of each type of cell at this time are given in Table 1. The variation in cell coat thickness between the two types of cells further suggests that this was not a factor in inducing the in vitro latency of BHM virus in BK monolayers.
Factors associated with viral inhibition. (a) Interferon. The possibility that interferon might be responsible for the chronic in vitro infection with BHM virus was investigated by using techniques that are said to by-pass the action of interferon, vi;:;. trypsin treatment of carrier cultures, maintenance of cultures at lowered temperature, frequent changes of maintenance medium and incorporation of hydrocortisone in the medium. No experiment revealed any evidence of interferon. (b) Serum inhibitors. The possible presence of inhibitors to viral growth, either specific or non-specific, was investigated by using different batches of antibody-free calf sera including fo etal calf serum, in a neutralization test (Rweyemamu & Johnson, 1968) . No experiment demonstrated an inhibitory effect that could be attributed to serum; even stock serum samples which previously had been associated with progressive CPE gave poor results.
BOVINE HERPES MAMMILLITIS VIRUS
6r5
(c) pH of tissue culture medium. Maintenance medium used at pH 7·5, 8·5 and g·o did not improve the CPE of BHM virus. (d) Mycoplasma and y east contamination. After the previous observation by Rweyemamu & Johnson (1967) that mycoplasma and low grade yeast contamination of cell cultures was associated with poor virus assay results, mycostatin * and kanamycint antibiotics were routinely incorporated in all the media. It was conceivable that either of these antibiotics were not completely effective against low grade contamination or were themselves inhibitory to virus cytopathogenicity. No difference in the type ofCPE followed the inoculation of cells maintained in antibiotic-free and antibiotic-containing media. Control monolayers which were subcultured thrice in the absence of these antibiotics were examined without success for the presence of mycoplasma and fungal contamination at each of the three passage levels. Miscellaneous factors. (a) Temperature of incubation. At the time of onset of the irregular CPE of
BHM virus, it was found that there was a temperature variation within and between incubators of ± 2°C. However no improvement in the poor CPE resulted from titrating virus at different temperatures ranging between 22 °C and 42 °C. (b) Effect of using double glass distilled deionised water in the media and for final rinsing of all glassware. The work to date had not yielded an answer to the problem. At this time an opacity of cell monolayers of all types was noticed; this became more obvious when versene was added. The cell monolayers were relatively resistant to the action of versene requiring several changes before complete stripping, as the versene rapidly turned acid on contact with the monolayers. This suggested the presence of high levels of metallic ions in/on the cell membranes and, in consequence, led to the empirical use of double glass distilled deionised water for preparing all media and for the final rinses of washed glassware; this was in preference to the previous use of deionised water. An immediate and marked improvement was obvious in the susceptibility of cell monolayers to the action of versene and more importantly in the susceptibility to BHM virus infection. Using this system, the titre of BHM virus in standard titrations using a tenfold dilution rate (Rweyemanu & Johnson, 1967) was usually 3 Log lo units higher than comparative titrations in cells grown in media containing deionised water. Chemical analyses of mains, deionised and double glass distilled deionised water were made on numerous occasions during February- March 1969 by the research laboratories of Bristol Waterworks and the manufacturers of the deionising equipment. Except for the silicate concentration all results were within normally acceptable limits. The silicate concentration in mains water in the Langford area rises in late winter and early spring following the breakdown of algae in the reservoirs. Si0 2 was detectable in the mains water-
* Mycostatin (50 units/ml) E. R. Squibb &
Sons Inc. , New York.
t Kanamycin Sulphate B.P.C. (o·rmg/ml ) Bayer Products Co., Surbiton-upon-Thames.
BRITISH VETERINARY JOURNAL,
128, 12
approximately 3 p.p.m. compared with less than I p.p.m. in summer-in the deionised water at concentrations between 0'2 and 0·8 p .p.m., but in the double glass distilled deionised water at less than 0'1 p.p.m. InJune 1969, by which time the problem of poor CPE with BHM virus had ceased, virus was titrated in BK cells grown in media prepared with double glass distilled deionised water to which had been added 12 p. p.m. of silicate as Na 2SiO. The CPE of the virus was normal in the test and control titrations. DIS CUSS ION
The various factors which have been associated with in vitro persistent infections with animal viruses are summarized in Table II. The investigations in these TABLE II FACTORS ASSOCIATED WITH
Factor
in vitro
CARRIER STATE OF ANIMAL VIRUSES
Riferences
Virus
I.
Antibody
Wheeler & Canby Hoggan & Roizman Wheeler Hinze & Walker Szanto
( 1959 ) (1959) (1960) (196 1) ( 196 3)
H erpes simplex Hrepes simplex Herpes simplex H erpes simplex H erpes simplex
2.
Interferon
Ho & Enders Chany Glasgow & Habel Glasgow & Habel Henle Isaacs Aurelian & R oizman Walker Baron Taniguchi Vilcek
(1959 ) ( 196 1) (1962 ) (1963) (1963) (1963) (1964) (1964) (1066 ) (1966) (1969)
Poliovirus Parainfluenza-3 Vaccinia H erpes simplex (Review) (R eview) H erpes simplex (Review) (R eview) H erpes simplex (Review)
3. Temperature of incubation
Morgan Wheeler & Canby Coleman & Jawetz Lwoff Stevens Stevens & J ackson
(1959) ( 1960) (1961) (1962 ) (1966 ) ( 1967)
(R eview) H erpes simplex Herpes simplex Poliovirus IBR IBR
4. Genetic resistance
Ginsberg Aurelian & Roizman Walker Hampar & Copeland Roizman
(1958 ) (19 64) (1964) (1965) (1966)
(Review) H erpes simplex (R eview) H erpes simplex H erpes simplex
5. R egulated infections
Walker
(1964)
(R eview)
6. Metabolic inhibitors
Tamm & Eggers
(1965)
(R eview)
7. Lack of nutrients
Morgan
(1959)
(Review)
IBR = Infectious bovine rhino tracheitis
BOVINE HERPES MAMMILLITIS VIRUS
laboratories into the in vitro carrier state observed with BHM virus could not identify which of these or other factor (s) were responsible. The deionised water appeared to be, at least partly, responsible for the problem. It is conceivable that the marked improvement in the assay of BHM virus obtained by making all media in double glass distilled deionised water related to the removal, or inactivation, of a residual substance inhibitory to BHM virus growth. Future work on this aspect will have to take into account the seasonal and regional variations in the water constituents and the effici ency of deionising and distillation equipment. More sensitive systems of biochemical and chemical analyses of water are required than were available at the time of this work. Other virus laboratories in the United Kingdom have experienced similar problems; more specifically Dr W. B. Martin ( Ig68, personal communication) has noticed in vitro latent infection with his own strain ofBHM virus in Scotland and also while working in K enya with strains of Allerton virus, a serologically related virus (Martin, Hay, Crawford, Le Bouvier & Crawford, Ig66). ACKNOWLEDGEMENTS
We are grateful to Miss R. Laurillard for technical assistance, to Dr G. Poste for the measurements of cell coat thickness by the technique of ellipsometry, Mr Bays of Bristol Waterworks for the analysis of water samples and to Professor C. S. Grunsell for criticism. The work reported here is part of a project supported by the Agricultural Research Council. Most of the data in this paper are given in greater detail in a thesis on bovine herpes mammillitis submitted in Ig68 by M. M. Rweyemamu to the University of Bristol for the degree of Doctor of Philosophy. REFERENCES
ALMEIDA,J. D., WATERSON, A. P. & PLOWRIGHT, W. (1967). Arch. ges. Virusforsch. 20, 392. AURELIAN, L. & ROIZMAN, B. (1964). Virology, 22,452. BARON, S. (1966). In Interferons, ed. N. B. Finter, p. 268. Amsterdam : North Holland Publishing Co. CHANEY, C. (1961). Virology, I3, 485. COLEMAN, V. &JAWETZ, E. (1961). Virology, I3, 375· GINSBERG, H. ( 1958). Progr. med. Virol. 1,36. GLASGOW, L. A. & HABEL, K. (1962). J. expo med. u5, 503· GLASGOW, L. A. & HABEL, K . (1963) . Virology, I9, 328. HAMPAR, B. & COPELAND, M. L. (1965). J. Bact. 90, 205· HENLE, W. (1963) . J. I mmun. 9 1, 145· HINZE, H . C. & WALKER, D. L . ( 1961 ). J. B act. 82, 498. Ho, M. & ENDERS,J. F. (1959). Virology, 9, 446. HOGGAN, M. D. & ROIZMAN, B. (1959). Am. J. Hyg. 70, 208. ISAACS, A. (1963) . Adu. Virus R es. IO, I. JOHNSON, R. H. (1967). Res. uet. Sci. 8, 256. LWOFF, A. (1962). Cold Spring HaTh . Symp. quant. B ioi. 27, 159. MARTIN, W. B., HAY, D., CRAWFORD, L. V., LE BOUVIER, G. L. & CRAWFORD, E. M . (1966). J. gen. Microbiol. 45, 32 5. MORGAN, H . R. (1959). Prohl. Virol.4, I. POSTE, G. ( 1970) . Adu. Virus Res. I6, 303. REED, L. J . & MUENCH, H. (1938) . Am. J. Hyg. 27, 493.
618
BRITISH VETERINARY JOURNAL, 128, 12
R OIZMAN, B. (1966). In Comparative Leukaemia, Res. Proc. Intern. Wenner-Gram Symp. Stockholm, 1965, p. 73. Oxford & New York: Pergamon Press. R WEYEMAMU, M. M. & JOHNSON, R. H. (1 967). Br. vet. J. 123, 482 . RWEYEMAMU, M. M. (1968). Ph.D. Thesis University of Bristol. STEVENS,j. G. (1966). Virology, 29, 570. STEVENS, j. G. & JACKSON, N. L. (1967). Virology, 32, 654. SZANTO, j. C. (1963). Acta Virol. 7, 392. T AMM, I. & EGGERS, H. j . (1965). In Viral and Rickettsial Infections of Man, ed. F. L. Horsfall & I. Tamm, 4th edn., p . 305. London: Pitman. TANIGUCHI, S. (1966). Virology, 30 , 333. VILCEK, j . (1969) Interferon, Virology Monographs, No.6. Vienna: Springer. WALKER, D. L. (1964). Prog. med. Virol.6, 1 I I. WHEELER, C. E. (1960). J. Immun. 84> 394. WHEELER, C. E . & CANBY, C . M. (1959). Arch. Derm. Chicago, 79, 86. WHEELER, C. E. & CANBY, C. M. (1960). J. Bact. 79, 657. (Acceptedjor publication 20 July 1972)
Etudes de cultures cellulaires infectees de fa~on pennanente par Ie virus bovin herpetique IDaDUIlelonnaire. Le role possible de I'eau desionisee dans I' induction d ' une categorie de porteurs de gennes (RweyeD1aD1u et al.) ResUD1e. Le probU:me de la latence in vitro de virus bovin herpetique mammelonnaire associe a un effet cytologique irregulier est decrit. Une telle culture de rein de bovide infectee de fac;:on permanente fut maintenue pendant trois mois montrant plusieurs cycles d'effet cytologique, regression et regeneration avec des cycles correspondant a l'extension extra cellulaire d u virus. Cette categorie de germes in vitro ne pouvait etre associee avec des interferons, anticorps ou inhibiteurs seriques, par des variations techniques, des milieux ou des souches virales. Ceci fut surmonte empiriquement en substituant de l'eau desionisee doublement distillee a l'eau desionisee dans tous les milieux. Versuche DJit Zellkulturen, die fortdauernd DJit Virus des bovinenherpes D1lLDlIIlilitis infiziert w aren. Moglicher Einfiuss von de-ionisierteD1 Wasser bei der Verursachung von tibertrigern (RweyeD1aD1u et al. ) ZusaD1D1enfass ung. Beschreibung des Problems der in vitro auftretenden Latenz des Virus des bovinen Herpes mamrnilitis assoziiert mit unregelmassigem zytopathischem Effekt. Eine solche fortdauernd infizierte Kultur bovinen Nierengewebes wurde drei Monate lang unterhalten und zeigte verschiedene Zyklen von zytopathischem Effekt, R egressionen und Regeneration mit entsprechenden Zyklen von extrazellularer Virusproduktion. Dieser in vitro beobachtete Obertragerzustand konnte nicht auf Interferon oder Antikorper- oder Serumhemmer, oder auf Variationen in Methodik, Medien oder Virusstamrnen zuriickgeftihrt werden. Es gelang durch rein empirisches Vorgehen, dieser Erscheinung Herr zu werden, indem in allen Medien das de-ionisierte Wasser durch doppeltdestilliertes glassdestilliertes de-ionisiertes Wasser ersetzt wurde. Estudios en cultivos de celulas infectados con virus de la D1aDJ.ilitis del herpes bovino. Posible papel del agoa desionizada en inducir un estado de propagacion (Rwey eD1aD1u et al.) Res UD1en. Se describe el problema de la latencia in vitro del virus de la marnilitis herpetica bovina asociado a un efecto citopatico irregular. Uno de tales cultivos de rilion bovino infectado persistentemente se mantuvo durante tres meses mostrando diversos ciclos de efectos citopaticos, de regresion y regeneraci6n con los ciclos correspondientes de producci6n extracelular del virus. Este estado de propagacion in vitro no se pudo asociar con el interferon inhibidores de anticuerpos 0 del suero, variaciones en la tecnica, en los medios 0 las razas de virus. Se pudo evitar empiricamente sustituyendo el agua desionizada de doble destilacion en vid rio por agua desionizada en todos los medios.
J.
(1972), 128,61 I
STUDIES ON CELL CULTURES PERSISTENTLY INFECTED WITH BOVINE HERPES MAMMILLITIS VIRUS. THE POSSIBLE ROLE OF DEIONISED WATER IN INDUCING A CARRIER STATUS By M. M. R WEYEMAMU*, R. H.JOH NSONt AND E. P.J. GIBBS! Department of Veterinary Medicine, Langford H ouse, Langford, near Bristol
SUMMARY The problem of in vitro latency of bovine herpes mammillitis virus associated with irregular cytopathic effect is described. One such persistently infected bovine kidney culture was maintained for three months showing several cycles of cytopathic effect (regression and regeneration) with corresponding cycles of extracellular virus yield. This in vitro carrier status could not be associated with interferon, antibody or serum inhibitors, variations in techniques, media or virus strains. It was overcome empirically by substituting double glass-distilled deionised water for deionised water in all media. I NTROD UCTION Bovine herpes mammillitis (BHM) virus normally grows rapidly and to high titre in tissue culture in which it produces large syncytia (Rweyem amu & Johnson, 1967) . Between D ecember and April 1967/68 and again 1968/69 only a limited cytopathic effect (CPE) followed the inoculation of susceptible cells and the routine assay of this virus in tissue culture becam e impossible. One feature of this poor CPE was the establishment of a carrier state in the inoculated monolayers. Although the cause of this poor CPE and latent infection was not found, the problem was overcome empirically by the substitution of double glass distilled dionised water for deionised water in all tissue culture media and for the final rinses during the washing of glassware. This paper reports the series of experiments that led to this observation. MATERIALS AN D METHODS The TV virus strain of BHM was used in this study (Rweyemamu & Johnson 1967). Methods for cell culture, the cell systems used and techniques for virus assay were as d escribed by Rweyemamu & Johnson (1967). Virus tit res were calculated by the technique of Reed & Muench (1938) and are expressed as tissue culture infective doses 50 per cent (TCID 5 o ) per ml. Present addresses: * Central Veterinary Laboratory, Temeke, Dar-es-Salaam, Tanzania; t Department of Tropica l Veterinary Science, James Cook University, Townsville, Queensland, 4810, Australia ; and t Animal Virus Research Institute, Pirbright, Woking, Surrey, England .
612
BRITISH VETERINARY JOURNAL,
128, 12
For continuity in the script further details of techniques are given with the results. RESULTS
Features of CPE and carner status The features observed may best be summarized from studies made on one culture of bovine kidney (BK) cells which was persistently infected with BHM virus over a period of three months. A secondary BK monolayer was cultured in a Roux flask to a full sheet which showed good pH conversion over 4 days. This monolayer was inoculated with 20 ml of virus at the 19th laboratory passage level in BK cells, at a titre of 10 4 • 3 TCID so /ml. Syncytial foci developed at 24 hours, enlarged by lateral extension over the next 24 hours but then showed degeneration and break up without development of secondary foci. CPE gradually regressed to apparent total disappearance. The culture was left incubated at 37 °C for a month without change of medium being required, by which time the monolayer had regenerated to a full sheet. The medium was then changed and the culture re-incubated for a further 8 days without showing any obvious signs of CPE; there was no detectable virus in the supernatant fluid. The monolayer was passaged to a Roux flask. The culture established itself, showed initial foci of CPE on the third day after passaging and CPE extension by the fourth day. Over the next 10 days, CPE became static and regressed with monolayer recovery and regrowth; the medium had been changed on the 4th, 8th and 9th days. On the 10th day, new foci of CPE were evident. The culture was subsequently passaged three times and maintained up to three months, during which time it demonstrated several cycles of CPE regeneration and regression alternating with cellular growth. Cycles of CPE regeneration corresponded roughly with rise in extracellular virus (Fig. I). Attempts to "cure" cultures by means of antibody were not successful. CPE was less marked in cultures maintained on media containing antibody but the cultures were not completely devoid of CPE foci, especially on passage. Occasionally subcultures from monolayers showed no epE. These cells were susceptible to superinfection with BHM virus, showing a variable degree of CPE following such superinfection. An investigation into some factors which might have been responsible for the development of the carrier state Factors associated with the virus. Latent infection was observed in BK monolayers by using BHM virus at the 3rd, loth, 14th and 19th tissue culture passage levels of the same strain, and by inoculating vesicular fluids from natural cases of BHM. All these samples had previously given good progressive CPE. The possibility of a virus contaminant either in the virus inoculum or in control cell cultures was investigated by (i) repeated passaging of control cultures and staining of representative coverslips; (ii) mixing control cultures from tests which had resulted in latent infection with new batches of cells and allowing them to form a monolayer together and staining coverslips at varying
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intervals; (iii) haemadsorption tests on monolayers; (iv) inoculating different cell cultures with BHM virus which had been neutralized with natural bovine antiserum; (v) examination of cell cultures by electron microscopy (Almeida, Waterson & Plowright, 1967); (vi) immunofluorescent examination for the presence of feline panleucopaenia virus (this virus is non-cytopathic in BK cells and had previously been used extensively in this laboratory-Johnson, 19 6 7) . No contaminant virus was detected.
Factors associated with the cell system (a) Age of monolayer. Young cultures were more susceptible to BHM virus infection than old cultures during the period when persistent infection became a problem. This variation in susceptibility associated with age of monolayer is not a significant factor during periods when standard ePE is obtained (Rweyemamu & Johnson, 1967). (b) Cell variation. During this period of poor ePE with BHM virus in BK cells it was observed that the problem of latent BHM virus infection became common to all cell systems used (Rweyemamu & Johnson, 1967). On one occasion all cell cultures were destroyed, and the virology laboratory was fumigated with formalin vapour. When seed virus was inoculated into new cell stocks the problem remained.
BRITISH VETERINARY JOURNAL,
128, 12
(c) Cell surfacefactors. Poste (1970) has suggested that following infection ofa cell with a syncytia forming virus the cell coat thickness must be approximately 35 Angstrom units before cells gain the capacity to fuse. In conjunction with Dr Poste it was shown that the system of cell surface changes that occur in polykaryocytosis developed in cell cultures exhibiting latent infection with BHM virus, even though extensive ePE did not develop (T able I ). TABLE " THE EFFECT OF CELL COAT THICKNESS ON BHM VIRUS
CYTOPATHOGENICITY
Mean cell coat thickness Number of observations
Bifore virus infection
After virus injection
Bovine kidney sample A
10
SO'2Ao
30'7A o
Bovine kidney sample B
10
38 '7Ao
27'4Ao
Bovine skin (fibroblastic)
8
42'3A o
30 '2Ao
Bovine skin (epithelial)
8
6S'SAo
60'IAo
Cell type
•+
Fusion *
CPE*
+* + +
+* + +
= present ; - = absent,
Bovine skin monolayers often exhibit a mixed population of fibroblastic and epithelial type cells. Under normal conditions, inoculation of these cultures with BHM virus results in a rapid and progressive ePE in the fibroblastic cell population with rarely any ePE in the epithelial cells. During the period when the ePE of BHM virus was poor in BK cells the epithelial type cells were refractory to infection and the fibroblastic type cells showed only limited ePE. Cell coat thicknesses of each type of cell at this time are given in Table 1. The variation in cell coat thickness between the two types of cells further suggests that this was not a factor in inducing the in vitro latency of BHM virus in BK monolayers.
Factors associated with viral inhibition. (a) Interferon. The possibility that interferon might be responsible for the chronic in vitro infection with BHM virus was investigated by using techniques that are said to by-pass the action of interferon, vi;:;. trypsin treatment of carrier cultures, maintenance of cultures at lowered temperature, frequent changes of maintenance medium and incorporation of hydrocortisone in the medium. No experiment revealed any evidence of interferon. (b) Serum inhibitors. The possible presence of inhibitors to viral growth, either specific or non-specific, was investigated by using different batches of antibody-free calf sera including fo etal calf serum, in a neutralization test (Rweyemamu & Johnson, 1968) . No experiment demonstrated an inhibitory effect that could be attributed to serum; even stock serum samples which previously had been associated with progressive CPE gave poor results.
BOVINE HERPES MAMMILLITIS VIRUS
6r5
(c) pH of tissue culture medium. Maintenance medium used at pH 7·5, 8·5 and g·o did not improve the CPE of BHM virus. (d) Mycoplasma and y east contamination. After the previous observation by Rweyemamu & Johnson (1967) that mycoplasma and low grade yeast contamination of cell cultures was associated with poor virus assay results, mycostatin * and kanamycint antibiotics were routinely incorporated in all the media. It was conceivable that either of these antibiotics were not completely effective against low grade contamination or were themselves inhibitory to virus cytopathogenicity. No difference in the type ofCPE followed the inoculation of cells maintained in antibiotic-free and antibiotic-containing media. Control monolayers which were subcultured thrice in the absence of these antibiotics were examined without success for the presence of mycoplasma and fungal contamination at each of the three passage levels. Miscellaneous factors. (a) Temperature of incubation. At the time of onset of the irregular CPE of
BHM virus, it was found that there was a temperature variation within and between incubators of ± 2°C. However no improvement in the poor CPE resulted from titrating virus at different temperatures ranging between 22 °C and 42 °C. (b) Effect of using double glass distilled deionised water in the media and for final rinsing of all glassware. The work to date had not yielded an answer to the problem. At this time an opacity of cell monolayers of all types was noticed; this became more obvious when versene was added. The cell monolayers were relatively resistant to the action of versene requiring several changes before complete stripping, as the versene rapidly turned acid on contact with the monolayers. This suggested the presence of high levels of metallic ions in/on the cell membranes and, in consequence, led to the empirical use of double glass distilled deionised water for preparing all media and for the final rinses of washed glassware; this was in preference to the previous use of deionised water. An immediate and marked improvement was obvious in the susceptibility of cell monolayers to the action of versene and more importantly in the susceptibility to BHM virus infection. Using this system, the titre of BHM virus in standard titrations using a tenfold dilution rate (Rweyemanu & Johnson, 1967) was usually 3 Log lo units higher than comparative titrations in cells grown in media containing deionised water. Chemical analyses of mains, deionised and double glass distilled deionised water were made on numerous occasions during February- March 1969 by the research laboratories of Bristol Waterworks and the manufacturers of the deionising equipment. Except for the silicate concentration all results were within normally acceptable limits. The silicate concentration in mains water in the Langford area rises in late winter and early spring following the breakdown of algae in the reservoirs. Si0 2 was detectable in the mains water-
* Mycostatin (50 units/ml) E. R. Squibb &
Sons Inc. , New York.
t Kanamycin Sulphate B.P.C. (o·rmg/ml ) Bayer Products Co., Surbiton-upon-Thames.
BRITISH VETERINARY JOURNAL,
128, 12
approximately 3 p.p.m. compared with less than I p.p.m. in summer-in the deionised water at concentrations between 0'2 and 0·8 p .p.m., but in the double glass distilled deionised water at less than 0'1 p.p.m. InJune 1969, by which time the problem of poor CPE with BHM virus had ceased, virus was titrated in BK cells grown in media prepared with double glass distilled deionised water to which had been added 12 p. p.m. of silicate as Na 2SiO. The CPE of the virus was normal in the test and control titrations. DIS CUSS ION
The various factors which have been associated with in vitro persistent infections with animal viruses are summarized in Table II. The investigations in these TABLE II FACTORS ASSOCIATED WITH
Factor
in vitro
CARRIER STATE OF ANIMAL VIRUSES
Riferences
Virus
I.
Antibody
Wheeler & Canby Hoggan & Roizman Wheeler Hinze & Walker Szanto
( 1959 ) (1959) (1960) (196 1) ( 196 3)
H erpes simplex Hrepes simplex Herpes simplex H erpes simplex H erpes simplex
2.
Interferon
Ho & Enders Chany Glasgow & Habel Glasgow & Habel Henle Isaacs Aurelian & R oizman Walker Baron Taniguchi Vilcek
(1959 ) ( 196 1) (1962 ) (1963) (1963) (1963) (1964) (1964) (1066 ) (1966) (1969)
Poliovirus Parainfluenza-3 Vaccinia H erpes simplex (Review) (R eview) H erpes simplex (Review) (R eview) H erpes simplex (Review)
3. Temperature of incubation
Morgan Wheeler & Canby Coleman & Jawetz Lwoff Stevens Stevens & J ackson
(1959) ( 1960) (1961) (1962 ) (1966 ) ( 1967)
(R eview) H erpes simplex Herpes simplex Poliovirus IBR IBR
4. Genetic resistance
Ginsberg Aurelian & Roizman Walker Hampar & Copeland Roizman
(1958 ) (19 64) (1964) (1965) (1966)
(Review) H erpes simplex (R eview) H erpes simplex H erpes simplex
5. R egulated infections
Walker
(1964)
(R eview)
6. Metabolic inhibitors
Tamm & Eggers
(1965)
(R eview)
7. Lack of nutrients
Morgan
(1959)
(Review)
IBR = Infectious bovine rhino tracheitis
BOVINE HERPES MAMMILLITIS VIRUS
laboratories into the in vitro carrier state observed with BHM virus could not identify which of these or other factor (s) were responsible. The deionised water appeared to be, at least partly, responsible for the problem. It is conceivable that the marked improvement in the assay of BHM virus obtained by making all media in double glass distilled deionised water related to the removal, or inactivation, of a residual substance inhibitory to BHM virus growth. Future work on this aspect will have to take into account the seasonal and regional variations in the water constituents and the effici ency of deionising and distillation equipment. More sensitive systems of biochemical and chemical analyses of water are required than were available at the time of this work. Other virus laboratories in the United Kingdom have experienced similar problems; more specifically Dr W. B. Martin ( Ig68, personal communication) has noticed in vitro latent infection with his own strain ofBHM virus in Scotland and also while working in K enya with strains of Allerton virus, a serologically related virus (Martin, Hay, Crawford, Le Bouvier & Crawford, Ig66). ACKNOWLEDGEMENTS
We are grateful to Miss R. Laurillard for technical assistance, to Dr G. Poste for the measurements of cell coat thickness by the technique of ellipsometry, Mr Bays of Bristol Waterworks for the analysis of water samples and to Professor C. S. Grunsell for criticism. The work reported here is part of a project supported by the Agricultural Research Council. Most of the data in this paper are given in greater detail in a thesis on bovine herpes mammillitis submitted in Ig68 by M. M. Rweyemamu to the University of Bristol for the degree of Doctor of Philosophy. REFERENCES
ALMEIDA,J. D., WATERSON, A. P. & PLOWRIGHT, W. (1967). Arch. ges. Virusforsch. 20, 392. AURELIAN, L. & ROIZMAN, B. (1964). Virology, 22,452. BARON, S. (1966). In Interferons, ed. N. B. Finter, p. 268. Amsterdam : North Holland Publishing Co. CHANEY, C. (1961). Virology, I3, 485. COLEMAN, V. &JAWETZ, E. (1961). Virology, I3, 375· GINSBERG, H. ( 1958). Progr. med. Virol. 1,36. GLASGOW, L. A. & HABEL, K. (1962). J. expo med. u5, 503· GLASGOW, L. A. & HABEL, K . (1963) . Virology, I9, 328. HAMPAR, B. & COPELAND, M. L. (1965). J. Bact. 90, 205· HENLE, W. (1963) . J. I mmun. 9 1, 145· HINZE, H . C. & WALKER, D. L . ( 1961 ). J. B act. 82, 498. Ho, M. & ENDERS,J. F. (1959). Virology, 9, 446. HOGGAN, M. D. & ROIZMAN, B. (1959). Am. J. Hyg. 70, 208. ISAACS, A. (1963) . Adu. Virus R es. IO, I. JOHNSON, R. H. (1967). Res. uet. Sci. 8, 256. LWOFF, A. (1962). Cold Spring HaTh . Symp. quant. B ioi. 27, 159. MARTIN, W. B., HAY, D., CRAWFORD, L. V., LE BOUVIER, G. L. & CRAWFORD, E. M . (1966). J. gen. Microbiol. 45, 32 5. MORGAN, H . R. (1959). Prohl. Virol.4, I. POSTE, G. ( 1970) . Adu. Virus Res. I6, 303. REED, L. J . & MUENCH, H. (1938) . Am. J. Hyg. 27, 493.
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R OIZMAN, B. (1966). In Comparative Leukaemia, Res. Proc. Intern. Wenner-Gram Symp. Stockholm, 1965, p. 73. Oxford & New York: Pergamon Press. R WEYEMAMU, M. M. & JOHNSON, R. H. (1 967). Br. vet. J. 123, 482 . RWEYEMAMU, M. M. (1968). Ph.D. Thesis University of Bristol. STEVENS,j. G. (1966). Virology, 29, 570. STEVENS, j. G. & JACKSON, N. L. (1967). Virology, 32, 654. SZANTO, j. C. (1963). Acta Virol. 7, 392. T AMM, I. & EGGERS, H. j . (1965). In Viral and Rickettsial Infections of Man, ed. F. L. Horsfall & I. Tamm, 4th edn., p . 305. London: Pitman. TANIGUCHI, S. (1966). Virology, 30 , 333. VILCEK, j . (1969) Interferon, Virology Monographs, No.6. Vienna: Springer. WALKER, D. L. (1964). Prog. med. Virol.6, 1 I I. WHEELER, C. E. (1960). J. Immun. 84> 394. WHEELER, C. E . & CANBY, C . M. (1959). Arch. Derm. Chicago, 79, 86. WHEELER, C. E. & CANBY, C. M. (1960). J. Bact. 79, 657. (Acceptedjor publication 20 July 1972)
Etudes de cultures cellulaires infectees de fa~on pennanente par Ie virus bovin herpetique IDaDUIlelonnaire. Le role possible de I'eau desionisee dans I' induction d ' une categorie de porteurs de gennes (RweyeD1aD1u et al.) ResUD1e. Le probU:me de la latence in vitro de virus bovin herpetique mammelonnaire associe a un effet cytologique irregulier est decrit. Une telle culture de rein de bovide infectee de fac;:on permanente fut maintenue pendant trois mois montrant plusieurs cycles d'effet cytologique, regression et regeneration avec des cycles correspondant a l'extension extra cellulaire d u virus. Cette categorie de germes in vitro ne pouvait etre associee avec des interferons, anticorps ou inhibiteurs seriques, par des variations techniques, des milieux ou des souches virales. Ceci fut surmonte empiriquement en substituant de l'eau desionisee doublement distillee a l'eau desionisee dans tous les milieux. Versuche DJit Zellkulturen, die fortdauernd DJit Virus des bovinenherpes D1lLDlIIlilitis infiziert w aren. Moglicher Einfiuss von de-ionisierteD1 Wasser bei der Verursachung von tibertrigern (RweyeD1aD1u et al. ) ZusaD1D1enfass ung. Beschreibung des Problems der in vitro auftretenden Latenz des Virus des bovinen Herpes mamrnilitis assoziiert mit unregelmassigem zytopathischem Effekt. Eine solche fortdauernd infizierte Kultur bovinen Nierengewebes wurde drei Monate lang unterhalten und zeigte verschiedene Zyklen von zytopathischem Effekt, R egressionen und Regeneration mit entsprechenden Zyklen von extrazellularer Virusproduktion. Dieser in vitro beobachtete Obertragerzustand konnte nicht auf Interferon oder Antikorper- oder Serumhemmer, oder auf Variationen in Methodik, Medien oder Virusstamrnen zuriickgeftihrt werden. Es gelang durch rein empirisches Vorgehen, dieser Erscheinung Herr zu werden, indem in allen Medien das de-ionisierte Wasser durch doppeltdestilliertes glassdestilliertes de-ionisiertes Wasser ersetzt wurde. Estudios en cultivos de celulas infectados con virus de la D1aDJ.ilitis del herpes bovino. Posible papel del agoa desionizada en inducir un estado de propagacion (Rwey eD1aD1u et al.) Res UD1en. Se describe el problema de la latencia in vitro del virus de la marnilitis herpetica bovina asociado a un efecto citopatico irregular. Uno de tales cultivos de rilion bovino infectado persistentemente se mantuvo durante tres meses mostrando diversos ciclos de efectos citopaticos, de regresion y regeneraci6n con los ciclos correspondientes de producci6n extracelular del virus. Este estado de propagacion in vitro no se pudo asociar con el interferon inhibidores de anticuerpos 0 del suero, variaciones en la tecnica, en los medios 0 las razas de virus. Se pudo evitar empiricamente sustituyendo el agua desionizada de doble destilacion en vid rio por agua desionizada en todos los medios.