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Molecular pathogenesis of equine coital exanthema: Temperature-sensitive function(s) in cells infected with equine herpesviruses
Veterinary Microbiology, 11 (1986) 221--237 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
221
MOLECULAR PATHOGENESIS OF EQUINE COITAL EXANTHEMA: TEMPERATURE-SENSITIVE FUNCTION(S) IN CELLS INFECTED WITH EQUINE HERPESVIRUSES
ROBERT J. JACOB
Department of Medical Microbiology and Immunology, A.B. Chandler Medical Center, University o f Kentucky, Lexington, K Y 40536-0084 (U.S.A.) (Accepted for publication 12 August 1985)
ABSTRACT Jacob, R.J., 1986. Molecular pathogenesis of equine coital exanthema: temperaturesensitive function(s) in cells infected with equine herpesviruses. Vet. Microbiol., 11: 221--237. Preliminary experiments have revealed that several laboratory and wild-type strains of the equine herpesvirus (EHV) triad were temperature-sensitive for growth when assayed at 39 ° C. The efficiencies o f plating (EOP) observed were 10 -2 for both EHV 1 and 2, and 1 × 10 -6 for EHV 3. The EOPs were determined by plaque assays which compared titrations at 34°C and 39°C on equine fetal dermal fibroblast cells. Growth yield experiments, assayed at 34 ° C, reflected these EOP's, but did not indicate any difference in yields when infected cultures were incubated at 34°C and 37°C. Temperature shift experiments with EHV 3-infected cultures revealed that a temperature-sensitive function(s) responsible for the reduction in titer appeared to be a late function(s). All strains examined appeared to incorporate H3-thymidine into viral-density DNA at the non-permissive temperature of 39° C. Electron microscopy of EHV 3-infected cell cultures, incubated continuously at the non-permissive temperature and examined at 18 h after infection, revealed structures consistent with the accumulation of nucleocapsids within the nucleus. The evidence presented is consistent with the hypothesis that in equine dermal cells infected with a plaque-purified wild-type strain of EHV 3 (l118LP), a function needed for the egress of nucleocapsids from the nucleus is absent at 39°C. The significance o f these findings relative to the pathogenicity of the disease (equine coital exanthema) caused by this virus is discussed.
INTRODUCTION T h e f a m i l y H e r p e s v i r i d a e is c h a r a c t e r i z e d b y s e v e r a l b i o l o g i c a l p r o p e r t i e s that often jeopardize human and animal health. Two significant properties o f t h i s g r o u p o f v i r u s e s a r e t h e i r a b i l i t y t o r e m a i n l a t e n t in t h e h o s t a n d t h e i r a b i l i t y t o t r a n s f o r m c e l l s ( m a l i g n a n t - o n c o g e n i c ) in c u l t u r e . T h e m o l e c u l a r events that lead to the establishment of latently-infected and transformed cells are considered abortive events because they do not allow completion of the productive cycle. A model detailing the role of wild-type temperature-
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© 1986 Elsevier Science Publishers B.V.
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sensitive functions involved in the establishment of latent or persistent viral infections has been proposed by Preble and Youngner (1975). Therefore, it is reasonable to investigate any naturally-occurring viral function that could be suspected of producing the abortive state in cells susceptible to infection, particularly when such a viral function is consistent with the pathogenesis of infection in the natural host. This report presents evidence that such a function m a y exist for the equine herpesviruses. For several years it has been known that some herpesviruses demonstrate restricted growth at elevated supraoptimal temperatures (Armstrong, 1942; Thompson and Coates, 1942; Hoggan and Roizman, 1959; Rafferty, 1964; Stevens, 1966; Stevens and Jackson, 1967; Aurelian, 1968; Spring and Roizman, 1968; Carmichael and Barnes, 1969; Schwartz and Roizman, 1969; Rapp and Jerkofsky, 1973; Golias et al., 1977; Carmichael et al., 1978; Letchworth et al., 1982). Evidence reported as early as 1942 by T h o m p s o n and Coates demonstrated that herpes simplex virus (HSV) grew to maximal titer in chick e m b r y o cells at 34°C and grew very poorly at 39°C. The most dramatic growth restriction has been seen for pseudorabies virus (Golias et al., 1977) and canine herpesviruses when infected cultures are incubated at temperatures only slightly above normal host b o d y temperatures (Carmichael and Barnes, 1969; Carmichael et al., 1978). Other reports (Hoggan and Roizman, 1959) have shown that HSV-infected cells release more virus into the media when incubated at 34°C, than at 39°C: it appears that the viral particles remain more cell-associated at 39--40 ° C. In addition, evidence indicated that cultures incubated at 37°C and infected with fresh isolates of HSV had a lower percentage of the nucleocapsid population mature into virions by envelopment than did cultures infected with laboratory strains (Schwarz and Roizman, 1969). Several other reports (Esparza et al., 1976; Knipe et al., 1981) have been published regarding the temperature sensitivity of fresh isolates of HSV. One of these reports (Knipe et al., 1981) has localized the region of the viral chromosome regulating temperature sensitivity of a particular isolate o f HSV-1 (strain F). In this manuscript, evidence is presented that the members of the equine herpesvirus triad (EHV 1, 2, 3) are restricted in their growth at 39°C. EHV 3 demonstrates the most dramatic temperature restriction and this restriction appears to be a late function, involved in the egress of capsids from the nucleus of infected equine cells. Such a temperature restriction, defined at this level of maturation, has not been shown for any recent isolate or wild-type laboratory strain of herpesvirus. MATERIALS AND METHODS Cells and virus growth
Monolayer cultures of a diploid strain of horse e m b r y o dermal fibroblasts (KYED) cells and an African Green m o n k e y kidney (AGMK) cell line were used in these studies. The cells were grown in Eagle's minimal essential me-
223 dium supplemented with 5% (v/v) fetal bovine serum and 50 pg of kanamycin m1-1 (Allen and Bryans, 1977). The viruses used were EHV 1 strains Army 183 and T2, EHV 2 strain T328, and EHV 3 strain 1118. The EHV-1 strain Army 183 is a s u b t y p e 1 isolated from the respiratory tract of a horse in Virginia, U.S.A. in 1941 and can cause abortion when inoculated intranasally into pregnant mares. The EHV-1 strain T2 is a s u b t y p e 2 isolated from the respiratory tract of a horse in Kentucky, U.S.A. in 1979, which has not been tested for its ability to cause abortion in pregnant mares (Sabine et al., 1981; Turtinen et al., 1981). The large plaque strain (1118) of EHV 3 has been twice cloned by plaque purification (G. Allen, personal communication, 1980). Conditions for propagation of these EHV strains in KYED cells and preparation of stocks by low-multiplicity (5 × 10 _4 plaque-forming units (pfu) cell- 1) infections has been described elsewhere (Allen and Randall, 1979).
Assay o f virus The assay of virus by serial titrations on monolayer cultures o f KYED cells has been reported previously (Perdue et al., 1974). Briefly, 25-cm 2 flasks of cells were infected for 2 h by rotary shaking at 39°C, overlayed with methylcellulose and incubated at either the permissive (34°C) or nonpermissive temperature (39°C). Assays on virus produced at 34°C, 37°C and 39°C were carried out at 34°C (Table I) because of previous findings (Schwarz and Roizman, 1969) indicating that some herpesvirus give a lower percentage o f mature and infectious virions at 37°C. EHV 1- and EHV 2-infected cultures were incubated for 72 h and EHV 3 cultures for 45 h. Forma Scientific model 3029 incubators, equipped with a chart recorder were used to monitor temperatures to within +0.1°C. Temperature shift experiments were undertaken with high titer virus stock, 3.6 × 109 pfu ml- 1 that was serially diluted and assayed on equine dermal cells which were incubated for 72 h at different temperatures. In 'shift-up' experiments, the infected cells were first incubated at 34°C for 1, 3, 6, 8, 10 or 12 h post-infection (pi), then raised to 39°C for the remainder of the 72 h and compared with cultures continuously incubated at 34°C (0 time). In 'shift-down' experiments, the infected cultures were first incubated at 39°C for 1, 3, 6, 12 or 18 h then lowered to 34°C for the remainder of the 72 h and compared with cultures continuously incubated at 39°C (0 time). All plaques and cytopathic effects were determined by staining monolayers with 0.5% crystal violet in ethanol. All assays were done in duplicate and efficiencies of plating (EOP) were determined by comparing the number of plaques on cultures incubated at 39°C relative to those at 34°C.
Electron microscopy Cells cultured as monolayers were infected at a multiplicity o f 5 pfu cell -~ and harvested by scraping into phosphate buffer (0.1 M NaH3PO4, 0.5 mM
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CaC12, pH 7.5). Cell suspensions were centrifuged at 2000 × g for 5 min and resuspended into phosphate-buffered glutaraldehyde (3% glutaraldehyde, 0.1 M NaH3PO4, 0.5 mM CaCl~, pH 7.1). The fixed cells were embedded (Palade, 1952) in an Epon mixture (MaUenhauer, 1964) and, after curing, thin sectioned by ultra-microtome. Thin sections were positively stained b y saturated uranyl acetate in 50% ethanol (Trump, 1961) and 1% lead citrate (Reynolds, 1963). Sections prepared in this manner were examined in the bright field m o d e of a Phillips 400 electron microscope.
Equilibrium density centrifugation The total DNA was extracted from infected KYED cells at 18 h pi, by incubation at 39°C for 3 h in pronase (1 mg ml-1), sodium dodecyl sulphate (SDS: 1.0%), sarkasyol (2.0%), 0.2 M NaC1, 10 mM EDTA, and 0.01 M TrisHC1, pH 7.4. The mixture was made isodense with CsC1 at 1.710 g cc -1. The self-generating gradients were centrifuged at 20°C, 150 000 × g for 24 h in a Beckman VTI 50 rotor. Radioactivity was measured b y total acid precipitable counts in a 750-~1 fraction, dripped from the b o t t o m of the tube. RESULTS
Restricted growth o f equine herpesvirus at 39 ° C The first experiment in this series determined that viral growth yields were different at 34°C, 37°C and 39°C for each equine herpesvirus. In this experiment virally-infected cells were incubated at the different temperatures indicated until cytopathic effect (CPE) was considered to be advanced. From these infected cultures stocks were harvested, prepared in identical volumes and titrated at 34°C before storage. The results are recorded in Table I. A TABLE I G r o w t h yields o f e q u i n e d e r m a l cells i n f e c t e d w i t h equine herpesvirus i n c u b a t e d at different temperatures Type
Temperature of incubation (o C)
Titer a t o t a l cell s o n i c a t e
EHV 1
34 37 39 34 37 39 34 37 39
3.4 × 3.3 x <10 4.8 × 4.8 × 5.8 × 1.3 × 1.8 x <10
EHV 2
EHV 3
a T i t r a t i o n s carried o u t at 34 ° C.
107 107 10 ~ 10 ~ 106 108 108
225 TABLE II Efficiency of plating (EOP) of equine herpesvirus Virus type
Titers on KYED a cells 34°C
EHV1(Army183) EHV 1 (T2) EHV 2 (T328) EHV3(lll8LP) HSV-1 (STH2HFEM) HSV-I (MP) HSV-I (F)
1.9× 1.5 × 3.2 x 1.9× 1.5 x
EOP
39°C 10 s 107 108 109 109
1.9x 3.4 × 5.0 × <10 s 1.5 ×
Titers on AGMKb cells 34°C
EOP
39°C
106 104 106
1.0 × 10 -2 2.0 x 1 0 - ' 1.6 x 10 -5 < i . 0 x 10-6 109 1.0 2.5 × 10 8 1.0 × 10 s 0.4 1.4 × 10 9 2.8 × i 0 ~ 0.2 1 . 2 × 10 9 4 . 0 x 10 8 0.3 4.0 × 10 9 < 1 0 4 < i . 0 × I 0 -s 1.2 × 10 9 < 1 0 4 < 1 . 0 × 10 -s
aKYED is a diploid line of equine dermal cells. bAGMK is a line of African Green Monkey cells. EOP = the ratio of the number of plaques at 34°C over those at 39°C. c o m p a r i s o n o f yields at 3 4 ° C a n d 3 7 ° C f o r e a c h e q u i n e h e r p e s v i r u s revealed little d i f f e r e n c e in g r o w t h yields at these t e m p e r a t u r e s . H o w e v e r , yields o f i n f e c t i o u s virus f r o m c u l t u r e s i n c u b a t e d at 3 9 ° C w e r e d r a m a t i c a l l y r e d u c e d f o r t h o s e i n f e c t e d with E H V 1 and 3 and less so in c u l t u r e s i n f e c t e d with E H V 2. In t h e s e c o n d e x p e r i m e n t in this series, p l a q u e t i t r a t i o n s o f equine herpesviruses w e r e m a d e o n K Y E D cells and t h e EOPs w e r e c o m p a r e d . T a b l e II reveals t h a t t h e E O P s f o r E H V 1 ( A r m y 183 a n d T 2 ) and 2 ( T 3 2 8 ) were 1 0 - ' - 10 -2 a n d t h a t f o r E H V 3 ( l l l 8 L P ) was a p p r o x i m a t e l y 10 -6. Several differe n t s t o c k s o f t h e s e viruses, all p r e p a r e d at 34°C, w e r e e x a m i n e d a n d s h o w n t o h a v e EOPs t h a t were c o n s i s t e n t w i t h t h o s e r e p o r t e d in T a b l e II. T h e t a b l e also allows a c o m p a r i s o n o f t h e s e EOPs with values d e t e r m i n e d f o r t h r e e hum a n herpesviruses t i t r a t e d o n b o t h K Y E D a n d A G M K cells. All t h r e e strains gave d i f f e r e n t results. Results w i t h t h e HSV-1 strains S T H 2 ( H F E M ) and MP, c o n s i d e r e d t o b e strains a d a p t e d to t h e l a b o r a t o r y , i n d i c a t e u n r e s t r i c t e d g r o w t h f o r S T H 2 a n d a m i n o r r e s t r i c t i o n ( ~ 10 -1) f o r t h e MP strain. H o w ever, t h e HSV-1 strain F, a m o r e r e c e n t field isolate, gave an E O P o f 10 -s, i n d i c a t i n g a s t r o n g g r o w t h r e s t r i c t i o n at 39°C. This r e s t r i c t i o n f o r HSV-1 strain F has b e e n i n d i c a t e d in a r e c e n t r e p o r t ( K n i p e et al., 1 9 8 1 ) w h i c h s t a t e d t h a t HSV-1 (F) c o n t a i n e d a t e m p e r a t u r e r e s t r i c t i o n in its a ( i m m e d i ately early) f u n c t i o n s . In t h e p r e s e n t s t u d y , t e m p e r a t u r e sensitivity in t h e h u m a n strains e x a m i n e d a p p e a r e d to be i n d e p e n d e n t o f t h e t w o cell t y p e s used. E q u i n e herpesvirus, in p a r t i c u l a r E H V 3, has a m o r e r e s t r i c t e d h o s t range a n d t h e r e b y l i m i t e d t h e p l a q u e assays t o cells o f e q u i n e origin.
Temporal nature o f temperature-restricted growth of EHV 3 in dermal cells Since E H V 3 d e m o n s t r a t e d t h e s t r o n g e s t g r o w t h r e s t r i c t i o n at 3 9 ° C , t h e f o l l o w i n g e x p e r i m e n t was designed to see if t h e t e m p e r a t u r e - s e n s i t i v e g r o w t h
226
restriction is in a function expressed at early (0--6 h) or late times (6--12 h) in the synthesis of EHV 3 virus. 'Shift-up' experiments (change from 34°C to 39°C) were designed to define the stage of the block. The results are presented in Table III. The number of plaques formed in these cultures was not affected by incubation at 34°C during the early times, but was markedly affected b y incubation at 34°C during later times in the synthesis of virus. This observation is consistent with a function which is late in the cycle (12--14 h) of EHV 3 viral replication (Allen and Bryans, 1977). To test the indications that temperature restriction was in a function at late times (6--12 h) and determine the reversibility of the block, a temperature 'shift-down' experiment (change from 39°C to 34°C) was designed. The results are presented in Table IV. When compared to cultures continuously incubated at 34°C (0 time), the number of plaques formed in these cultures was relatively independent of incubation at the restrictive temperature, 39°C, for up to 6 h pi; at best, a slight reduction in the number of plaques T A B L E III T i t r a t i o n o f E H V 3 o n e q u i n e d e r m a l cells f o l l o w i n g t e m p e r a t u r e a l t e r a t i o n H o u r s (pi) i n c u b a t e d at 34°C p r i o r t o 39°C
Plaque titers a f t e r 72 h
Percentage r e d u c t i o n at 39°C
(X lO s) 0a
1 3 6 8 10 12
0
--
4.3 2.0 3.9 4.0 21.0 30.0
88 94 89 88 32 17
a T i t e r o f s t o c k is 3.6 X 109 at 34°C. pi = p o s t - i n f e c t i o n .
TABLE IV T i t r a t i o n o f E H V 3 o n e q u i n e d e r m a l cells f o l l o w i n g t e m p e r a t u r e a l t e r a t i o n H o u r s (pi) i n c u b a t e d at 3 9 ° C prior to 34°C 0a
1 3 6 12 18
Titers a f t e r 72 h
(× 10 9)
Percentage r e d u c t i o n at 39°C
3.6
0
4.6 3.5 2.8 1.9 0.6
-3 22 47 98
a T i t e r o f s t o c k is 3.6 × 109 at 34°C. pi = p o s t - i n f e c t i o n .
227
m a y be seen at 6 h pi. Incubation of cultures at 39°C for longer than 6 h caused a dramatic reduction in titer. Again, these times (6--12 h) are in the late phase (end of the first replication cycle) o f EHV 3 synthesis.
Fig. 1. Electron micrograph o f KYED cells at 18 h after infection with equine herpesvirus 3. Cells were infected at multiplicity o f 5 pfu cell -1 and incubated at 34°C. Specimens were prepared by lead citrate-urynal acetate staining (see Materials and Methods). Arrows indicate enveloped virus in the cytoplasm and intercellular spaces. B indicates budding virus. The bar represents 1 ~m.
228
Examination o f equine cells infected with E H V 3 at the restricted temperature, 39°C Electron micrographs o f EHV 3-infected equine dermal cells incubated for 18 h at either 34°C or 39°C are shown in Figs. 1--6. Figure 1 illustrates an
Fig. 2. Electron micrograph of the perinuclear region in a K Y E D cell infected with E H V 3. Conditions are as indicated in Fig. 1. Arrows indicate budding virus, nuclear m e m brane, I and H (B) forms of nucleocapsids. T h e bar represents 0.1 urn.
229
infected cell incubated at 34°C. It can be seen (arrows) that both the cytoplasm and intercellular spaces are filled with mature virions {enveloped) that are typical o f herpesviruses. In addition, budding through the nuclear membrane can be seen, indicated b y arrows B. Figure 2 is an enlargement showing the nuclear membrane (arrows B, Fig. 1) o f a cell infected at 34°C for 18 h. It illustrates capsid envelopment, clearly showing envelope, tegument and nucleocapsid-like structure. In addition, this micrograph illustrates capsidlike structures, reminiscent of B or H and I capsids, seen within the nucleus of infected cells (Carmichael and Barnes, 1969; Gibson and Roizman, 1972; Perdue et al., 1975). The electron-dense cores contain nucleic acid. Figure 3 illustrates the enveloped virions seen in the cytoplasm of these cells.
Fig. 3. Electron micrograph o f the cytoplasm o f KYED cells i n f e c t e d with EHV 3. Conditions are as indicated in Fig. 1. Arrows are u s e d to indicate enveloped virion in the cytoplasm. T h e bar represents 0.1 ~m.
230
In contrast to these cells, Figs. 4--6 are electron micrographs of infected equine dermal cells, prepared in an identical manner as above, but incubated for 18 h pi at 39°C. Figure 4 illustrates a multinuclear giant cell containing
Fig. 4. Electron micrograph of KYED cells infected with EHV 3 at a multiplicity o f 5 and incubated at 39°C for 18 h. All conditions for infection and specimen preparation are as indicated in Fig. 1. Nuclei (N), cytoplasm (C), rough endoplasmic reticulum (er), chromatin (chr) and membrane reduplications (mRD). The bar represents 1.0 um.
231
Fig. 5. E l e c t r o n m i c r o g r a p h o f t h e n u c l e a r m e m b r a n e b o u n d a r y o f a t y p i c a l K Y E D cell at 18 h a f t e r i n f e c t i o n w i t h E H V 3 i n c u b a t e d at 39°C. All n o m e n c l a t u r e o f arrows are as i n d i c a t e d in Fig. 4 w i t h t h e a d d i t i o n o f i n d i c a t i o n s o f n u c l e o c a p s i d f o r m s ( N c ) at t h e n u c l e a r m e m b r a n e . T h e b a r r e p r e s e n t s 1.0 urn.
232
cytoplasm active in protein synthesis which contains rough endoplasmic reticulum (er). However, evidence of viral-like structures in the cytoplasm, or at the plasma membrane, is missing. The nuclei present evidence of membrane reduplication (arrow mRD) and margination of chromatin {arrow chr).
Fig. 6. E l e c t r o n m i c r o g r a p h o f t h e n u c l e u s o f a t y p i c a l K Y E D cell at 18 h a f t e r i n f e c t i o n w i t h E H V 3 i n c u b a t e d at 39 ° C. All n o m e n c l a t u r e s o f a r r o w s are as i n d i c a t e d in Figs. 4 a n d 5. T h e b a r r e p r e s e n t s 1.0 urn.
233
Figure 5 is an enlargement of a perinuclear region o f this cell and illustrates cytoplasm abundant in endoplasmic reticulum and the absence of virion-like structures. However, nucleocapsid-like structures are seen at the inner lameUa of the nuclear membrane (arrow Nc). Membrane reduplication and chromatin margination are also seen. Figure 6 illustrates the relative abundance of these nucleocapsid structures in the nucleus of infected cells incubated for 18 h at a temperature A
B o
_
_
q
. . . . . .
0
'~
5-
C
*t
I I
!
"
'1
3"
o a
!
2-
I ! I
1
I
'b
1'0
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Fract io. Fig. 7. CsC1 equilibrium gradients o f D N A isolated f r o m K Y E D cells i n f e c t e d w i t h equine herpesviruses at a m u l t i p l i c i t y o f 2 0 p f u cell -~ and i n c u b a t e d for 18 h pi. In all cases the cells were labeled w i t h H 3 - m e t h y l t h y m i d i n e ( 2 0 pCi p1-1) for t o t a l time. Panel A represents D N A e x t r a c t e d from E H V 1 - i n f e c t e d cells i n c u b a t e d at 3 4 ° C. Panels B and C represent D N A e x t r a c t e d f r o m cells i n c u b a t e d at 3 9 ° C and i n f e c t e d w i t h E H V 1 or E H V 3, respectively. T h e marker D N A is e x t r a c t e d f r o m P32-1abeled E H V 3 virions.
234 (39°C) restrictive for viral growth. Envelopment (budding) of these capsids, and virion forms in the cytoplasm, was not seen. More than 75 of 100 cells examined were of this kind.
Synthesis o f EHV 3 viral-density DNA at the restrictive temperature, 39°C The following experiment was designed to determine if viral DNA was synthesized at the restrictive temperature and further supports the interpretation that the nucleocapsid forms seen in the electron micrograph contain viral DNA in KYED Cells infected with EHV 3. Figure 7 shows the results of CsC1 equilibrium density gradients of total DNA isolated from equine dermal cells infected with EHV 1 (Fig. 7A) and incubated at the permissive temperature (34°C) for 18 h pi. Gradients of DNA isolated from cells infected with EHV 1 (Fig. 7B) and EHV 3 {Fig. 7C) and incubated for 18 h at the restrictive temperature (39°C) axe also shown. The marker DNA has been isolated from purified virions of EHV 3 and has a density of 1.726 g cc -1 which is higher than the density o f EHV 1, 1.716 g cc -~. DISCUSSION
The results presented in this report indicate that, at least for those strains of the equine herpesvirus triad examined (Army 183, T2, T328, l l l 8 L P ) , there appears to be restricted growth at 39°C. These strains included both the subtype 1, Army 183, and subtype 2, T2, of EHV 1. The reason why EHV 1 and EHV 3 gave poorer yields than EHV 2 at 39°C, has not yet been determined. Possibly, the products made at the restrictive temperature of 39°C in cells infected by EHV 1 or 3 are highly cell-associated at this temperature and much less able (as expected of nucleocapsids) to spread the infection than those products of the EHV 2 infections. It is n o t clear if the identification of EHV 2 as an equine cytomegalovirus, and the highly cellassociated nature of these viruses, is significant. It is possible that EHV 2 is more adapted to a cell-mediated dissemination of infection. When the temperature restriction in EHV 3 was examined by temperature shift experiments the block in maturation was limited to late in the replication cycle and did not require early incubation at the non-permissive temperature (39°C) to be effective. The evidence that DNA of viral density was synthesized at the non-permissive temperature and electron microscopic demonstration of DNA-containing nucleocapsids building up in the nuclei of infected cells (Carmichael and Barnes, 1969; Gibson and Roizman, 1972; Perdue et al., 1975) are consistent with this finding. However, enveloped virions did not appear in the cytoplasm of the EHV 3-infected cells incubated at the non-permissive temperature. Parenthetically, DNA with a density greater than viral DNA, characteristic of defective viral particles in a population, was not seen in the CsC1 gradients.
235 EHV 3 demonstrates the strongest growth restriction seen in the equine herpesvirus triad. Examination of more recent field isolates of EHV 3 from geographically unrelated areas supports the findings on strain l 1 1 8 L P (J. Evermann, personal communication, 1985). The growth restriction in EHV 3-infected equine dermal cells appears to be a block in a required late function that cannot be overcome b y incubation at the permissive temperature (34°C) early in viral replication. This is in contrast to t w o previous reports on temperature sensitivity with other herpesviruses. In the first, Stevens and Jackson (1967) indicated that a temperature-sensitive restriction in bovine herpesvirus 1 (BHV 1) occurred early in the growth cycle and blocked viral DNA synthesis. It should be noted that this temperature sensitive restriction was effective at 40--42°C and could be overcome by incubation at permissive temperatures early in the replication cycle. In the second, it was reported (Knipe et al., 1981) that HSV-1 strain F was restricted at 39°C. HSV-1 (F) produces a temperature-sensitive ~ (immediately early) gene product at the non-permissive temperature (39°C) that maps between 0.83-0.87 and 0.97--1.0 map units and that infected cell polypeptide patterns are largely ~ (immediately early). These patterns are reminiscent of ts mutants in complementation group 1--2 (P.J. Godowski and D.M. Knipe, unpublished data, 1981). These findings would indicate that nucleocapsids are n o t formed at 39°C during HSV-1 (F) infections. However, the findings on EHV 3 infections presented here support a more recent finding (Letchworth et al., 1982) that a 39°C temperature growth restriction in bovine herpesvirus 2 (BHV 2) replication is a block in a late function. In addition, preliminary results from experimental analysis of EHV 3-infected cell proteins (M. Steiner, personal communication, 1985) support the conclusion that certain 7 (late) proteins, some of which appear to be glycosylated, are not being synthesized at the restrictive temperature (39°C). The significance of these findings is that the restrictive temperature reported (39°C) is consistent with b o d y temperatures c o m m o n l y found in the equine host, though the temperature of superficial tissue in the equine m a y be lower. This is o f importance since most clinical presentations (Bryans and Allen, 1973; Studdert, 1974; Bryans, 1980) of EHV 3 infections, e.g. coital exanthema, appear as lesions on superficial tissues that typically progress to a mild, self-limiting exanthematic disease. The clinical presentation of EHV 3 infection is often reviewed as an infectious, b u t innocuous, genital disease (O'Callaghan et al., 1982). The prognosis does not indicate a progression of the disease that would involve the internal organs or a fetus of a pregnant mare (Bryans and Allen, 1973; Studdert, 1974; Bryans, 1980; O'Callaghan et al., 1982), as do those infections with EHV 1. The finding in this report and clinical presentation of the disease caused by EHV 3 are consistent (Bryans, 1980). Further investigations into the molecular pathogenesis of this viral disease could reveal w h y this herpesvirus appears to be relatively innocuous in its natural host.
236 ACKNOWLEDGEMENTS
The author would like to acknowledge the technical assistance of Ms. J e n n y Borchelt, Mr. R. Geissler (Pathology) and Dr. D. Powell for use of their electron microscope. The assistance of Mr. Bradley Moore in the preparation of this manuscript was appreciated. In addition, I would like to thank Drs. Allen and Bryans, Department of Veterinary Sciences, for many of the viral strains used in this study. The work was supported by Public Health Service Grant AI 17620 from the National Institute of Allergy and Infectious Diseases.
REFERENCES Allen, G. and Bryans, J.T., 1977. Replication of equine herpesvirus type 3: kinetics of infectious particle formation and virus nucleic acid synthesis. J. Gen. Virol., 34: 421--430. Allen, G.P. and Randall, C.C., 1979. Proteins of equine herpesvirus type 3. I. Polypeptides of the purified virion. Virology, 92: 252--254. Armstrong, C., 1942. Some recent research in the field of neurotropic viruses with special reference to lymphocytic choriomeningitis and herpes simplex. Mil. Surg., 91 : 129--145. Aurelian, L., 1968. Effect of environmental conditions on formation and release of canine herpesvirus in canine kidney cells. Am. J. Vet. Res., 29: 1945--1952. Bryans, J.T., 1980. Herpesvirus diseases affecting reproduction in the horse. Vet. Clin. North Am.: Large Anita. Pract., 2: 303--310. Bryans, J.T. and Allen, G.P., 1973. In vitro and in vivo studies of equine coital exanthema. In: J.T. Bryans and H. Gerber (Editors), Proc. 3rd Int. Conf. Equine Dis. S. Karger, Basel, pp. 322--336. Carmichael, L.E. and Barnes, F.D., 1969. Effect of temperature on growth of canine herpesvirus in canine kidney cells and macrophage cultures. J. Infect. Dis., 120: 664--668. Carmichael, L.E., Barnes, F.D. and Percy, D.H., 1978. Temperature as a factor of resistance in young puppies to canine herpesvirus. J. Infect. Dis., 120: 669--678. Esparza, J., Purifoy, D., Schaffer, P. and Benyesh-Melnick, M., 1976. Isolation, complementation and preliminary phenotypic characterization of temperature-sensitive mutants of herpes simplex virus type 2. Virology, 57: 554--574. Gibson, W. and Roizman, B., 1972. Proteins specified by herpes simplex virus: VIII. Characterization and composition of multiple capsid forms of subtype 1 and 2. J. Virol., 10: 1044--1052. Golias, F., Sabo, A. and Svobodovam, J., 1977. Susceptibility of various cell lines to virulent and attenuated strains of pseudorabies virus at supraoptimal temperature. Acta Virol., 21: 25--30. Hoggan, M.D. and Roizman, B., 1959. The effect of the temperature of incubation on the formation and release of herpes simplex virus in infected FL cells. Virology, 8: 508--524. Knipe, D.M., Batterson, W., Nosal, C., Roizman, B. and Buchan, A., 1981. Molecular genetics of herpes simplex virus: VI. Characterization of a temperature sensitive mutant defective in the expression of all early gene products. J. Virol., 38: 539--547. Letchworth, C.J.III, Carmichael, L.E. and Greisen, H.A., 1982. Sensitivity of bovid herpesvirus 2 replication to temperatures found in natural host. Arch. Virol., 73: 273--286.
237 Mallenhauer, S., 1964. Plastic embedding mixtures for use in electron microscopy. Stain Technol., 39: 111--114. O'Callaghan, D.J., Gentry, G.A. and Randall, C.C., 1982. The equine herpesvirus. In: H. Fraenkel-Conrat and R. Wagner (Series Editors), The Viruses: Vol. 2, The Herpesviruses, B. Roizman (Editor). Plenum Press, New York, pp. 215--305. Palade, G.E., 1952. A study o f fixation for electron microscopy. J. Exp. Med., 95: 285--298. Perdue, M.L., Kemp, M.C., Randall, C.C. and O'Callaghan, D.J., 1974. Studies on the molecular anatomy o f the L-M strain of equine herpes virus t y p e 1: proteins of the nucleocapsid and intact virion. Virology, 59: 201--216. Perdue, M.L., Cohen, J.C., Kemp, M.C., Randall; C.C. and O'Callaghan, D.J., 1975. Characterization o f three species of nucleocapsids o f equine herpesvirus type 1 (EHV1). Virology, 64: 187--204. Preble, O.T. and Youngner, J.S., 1975. Temperature sensitive viruses and the etiology of chronic and inapparent infections. J. Infect. Dis., 131: 407--473. Rafferty, K.A., Jr., 1964. Kidney tumors o f the leopard frog: a review. Cancer Res., 24 : 169--200. Rapp, F. and Jerkofsky, M.A., 1973. Persistent and latent infections. In: A.S. Kaplan (Editor), The Herpesvirus. Academic Press, New York, pp. 271--287. Reynolds, E.S., 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol., 17 : 208--215. Sabine, M., Robertson, G. and Walley, J., 1981. Different subtypes of equine herpesvirus 1 b y restriction endonuclease analysis. Aust. Vet. J., 57: 148--149. Schwartz, J. and Roizman, B., 1969. Similarities and differences in the development of laboratory strains and freshly isolated strains of herpes simplex virus in HEp-2 cells: electron microscopy. J. Virol., 4: 879--889. Spring, S.B. and Roizman, B., 1968. Herpes simplex virus products in productive and abortive infection. III. Differentiation of infectious virus derived from nucleus and cytoplasm with respect to stability, size and adhesion to cells. J. Virol., 2: 979--985. Stevens, J.G., 1966. Selective inhibition o f herpesvirus DNA synthesis at elevated temperatures. Virology, 29: 570--579. Stevens, J.G. and Jackson, N.L., 1967. Studies on a temperature sensitive step essential to herpesvirus DNA replication. Virology, 32: 654--661. Studdert, M.J., 1974. Comparative aspects o f equine herpesvirus. Cornell Vet., 64: 94-104. Thompson, R.L. and Coates, M.S., 1942. The effect of temperature upon the growth and survival of myxoma, herpes, and vaccinia viruses in tissue culture. J. Infect. Dis., 71: 83--85. Trump, B.F., 1961. A method for staining epoxy sections for light microscopy. J. Ultrastruct. Res., 5: 343--348. Turtinen, L., Allen, G., Darlington, R. and Bryans, J., 1981. Serology and molecular comparisons o f several EHV-1 strains. Am. J. Vet. Res., 42: 2099--2104.
221
MOLECULAR PATHOGENESIS OF EQUINE COITAL EXANTHEMA: TEMPERATURE-SENSITIVE FUNCTION(S) IN CELLS INFECTED WITH EQUINE HERPESVIRUSES
ROBERT J. JACOB
Department of Medical Microbiology and Immunology, A.B. Chandler Medical Center, University o f Kentucky, Lexington, K Y 40536-0084 (U.S.A.) (Accepted for publication 12 August 1985)
ABSTRACT Jacob, R.J., 1986. Molecular pathogenesis of equine coital exanthema: temperaturesensitive function(s) in cells infected with equine herpesviruses. Vet. Microbiol., 11: 221--237. Preliminary experiments have revealed that several laboratory and wild-type strains of the equine herpesvirus (EHV) triad were temperature-sensitive for growth when assayed at 39 ° C. The efficiencies o f plating (EOP) observed were 10 -2 for both EHV 1 and 2, and 1 × 10 -6 for EHV 3. The EOPs were determined by plaque assays which compared titrations at 34°C and 39°C on equine fetal dermal fibroblast cells. Growth yield experiments, assayed at 34 ° C, reflected these EOP's, but did not indicate any difference in yields when infected cultures were incubated at 34°C and 37°C. Temperature shift experiments with EHV 3-infected cultures revealed that a temperature-sensitive function(s) responsible for the reduction in titer appeared to be a late function(s). All strains examined appeared to incorporate H3-thymidine into viral-density DNA at the non-permissive temperature of 39° C. Electron microscopy of EHV 3-infected cell cultures, incubated continuously at the non-permissive temperature and examined at 18 h after infection, revealed structures consistent with the accumulation of nucleocapsids within the nucleus. The evidence presented is consistent with the hypothesis that in equine dermal cells infected with a plaque-purified wild-type strain of EHV 3 (l118LP), a function needed for the egress of nucleocapsids from the nucleus is absent at 39°C. The significance o f these findings relative to the pathogenicity of the disease (equine coital exanthema) caused by this virus is discussed.
INTRODUCTION T h e f a m i l y H e r p e s v i r i d a e is c h a r a c t e r i z e d b y s e v e r a l b i o l o g i c a l p r o p e r t i e s that often jeopardize human and animal health. Two significant properties o f t h i s g r o u p o f v i r u s e s a r e t h e i r a b i l i t y t o r e m a i n l a t e n t in t h e h o s t a n d t h e i r a b i l i t y t o t r a n s f o r m c e l l s ( m a l i g n a n t - o n c o g e n i c ) in c u l t u r e . T h e m o l e c u l a r events that lead to the establishment of latently-infected and transformed cells are considered abortive events because they do not allow completion of the productive cycle. A model detailing the role of wild-type temperature-
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sensitive functions involved in the establishment of latent or persistent viral infections has been proposed by Preble and Youngner (1975). Therefore, it is reasonable to investigate any naturally-occurring viral function that could be suspected of producing the abortive state in cells susceptible to infection, particularly when such a viral function is consistent with the pathogenesis of infection in the natural host. This report presents evidence that such a function m a y exist for the equine herpesviruses. For several years it has been known that some herpesviruses demonstrate restricted growth at elevated supraoptimal temperatures (Armstrong, 1942; Thompson and Coates, 1942; Hoggan and Roizman, 1959; Rafferty, 1964; Stevens, 1966; Stevens and Jackson, 1967; Aurelian, 1968; Spring and Roizman, 1968; Carmichael and Barnes, 1969; Schwartz and Roizman, 1969; Rapp and Jerkofsky, 1973; Golias et al., 1977; Carmichael et al., 1978; Letchworth et al., 1982). Evidence reported as early as 1942 by T h o m p s o n and Coates demonstrated that herpes simplex virus (HSV) grew to maximal titer in chick e m b r y o cells at 34°C and grew very poorly at 39°C. The most dramatic growth restriction has been seen for pseudorabies virus (Golias et al., 1977) and canine herpesviruses when infected cultures are incubated at temperatures only slightly above normal host b o d y temperatures (Carmichael and Barnes, 1969; Carmichael et al., 1978). Other reports (Hoggan and Roizman, 1959) have shown that HSV-infected cells release more virus into the media when incubated at 34°C, than at 39°C: it appears that the viral particles remain more cell-associated at 39--40 ° C. In addition, evidence indicated that cultures incubated at 37°C and infected with fresh isolates of HSV had a lower percentage of the nucleocapsid population mature into virions by envelopment than did cultures infected with laboratory strains (Schwarz and Roizman, 1969). Several other reports (Esparza et al., 1976; Knipe et al., 1981) have been published regarding the temperature sensitivity of fresh isolates of HSV. One of these reports (Knipe et al., 1981) has localized the region of the viral chromosome regulating temperature sensitivity of a particular isolate o f HSV-1 (strain F). In this manuscript, evidence is presented that the members of the equine herpesvirus triad (EHV 1, 2, 3) are restricted in their growth at 39°C. EHV 3 demonstrates the most dramatic temperature restriction and this restriction appears to be a late function, involved in the egress of capsids from the nucleus of infected equine cells. Such a temperature restriction, defined at this level of maturation, has not been shown for any recent isolate or wild-type laboratory strain of herpesvirus. MATERIALS AND METHODS Cells and virus growth
Monolayer cultures of a diploid strain of horse e m b r y o dermal fibroblasts (KYED) cells and an African Green m o n k e y kidney (AGMK) cell line were used in these studies. The cells were grown in Eagle's minimal essential me-
223 dium supplemented with 5% (v/v) fetal bovine serum and 50 pg of kanamycin m1-1 (Allen and Bryans, 1977). The viruses used were EHV 1 strains Army 183 and T2, EHV 2 strain T328, and EHV 3 strain 1118. The EHV-1 strain Army 183 is a s u b t y p e 1 isolated from the respiratory tract of a horse in Virginia, U.S.A. in 1941 and can cause abortion when inoculated intranasally into pregnant mares. The EHV-1 strain T2 is a s u b t y p e 2 isolated from the respiratory tract of a horse in Kentucky, U.S.A. in 1979, which has not been tested for its ability to cause abortion in pregnant mares (Sabine et al., 1981; Turtinen et al., 1981). The large plaque strain (1118) of EHV 3 has been twice cloned by plaque purification (G. Allen, personal communication, 1980). Conditions for propagation of these EHV strains in KYED cells and preparation of stocks by low-multiplicity (5 × 10 _4 plaque-forming units (pfu) cell- 1) infections has been described elsewhere (Allen and Randall, 1979).
Assay o f virus The assay of virus by serial titrations on monolayer cultures o f KYED cells has been reported previously (Perdue et al., 1974). Briefly, 25-cm 2 flasks of cells were infected for 2 h by rotary shaking at 39°C, overlayed with methylcellulose and incubated at either the permissive (34°C) or nonpermissive temperature (39°C). Assays on virus produced at 34°C, 37°C and 39°C were carried out at 34°C (Table I) because of previous findings (Schwarz and Roizman, 1969) indicating that some herpesvirus give a lower percentage o f mature and infectious virions at 37°C. EHV 1- and EHV 2-infected cultures were incubated for 72 h and EHV 3 cultures for 45 h. Forma Scientific model 3029 incubators, equipped with a chart recorder were used to monitor temperatures to within +0.1°C. Temperature shift experiments were undertaken with high titer virus stock, 3.6 × 109 pfu ml- 1 that was serially diluted and assayed on equine dermal cells which were incubated for 72 h at different temperatures. In 'shift-up' experiments, the infected cells were first incubated at 34°C for 1, 3, 6, 8, 10 or 12 h post-infection (pi), then raised to 39°C for the remainder of the 72 h and compared with cultures continuously incubated at 34°C (0 time). In 'shift-down' experiments, the infected cultures were first incubated at 39°C for 1, 3, 6, 12 or 18 h then lowered to 34°C for the remainder of the 72 h and compared with cultures continuously incubated at 39°C (0 time). All plaques and cytopathic effects were determined by staining monolayers with 0.5% crystal violet in ethanol. All assays were done in duplicate and efficiencies of plating (EOP) were determined by comparing the number of plaques on cultures incubated at 39°C relative to those at 34°C.
Electron microscopy Cells cultured as monolayers were infected at a multiplicity o f 5 pfu cell -~ and harvested by scraping into phosphate buffer (0.1 M NaH3PO4, 0.5 mM
224
CaC12, pH 7.5). Cell suspensions were centrifuged at 2000 × g for 5 min and resuspended into phosphate-buffered glutaraldehyde (3% glutaraldehyde, 0.1 M NaH3PO4, 0.5 mM CaCl~, pH 7.1). The fixed cells were embedded (Palade, 1952) in an Epon mixture (MaUenhauer, 1964) and, after curing, thin sectioned by ultra-microtome. Thin sections were positively stained b y saturated uranyl acetate in 50% ethanol (Trump, 1961) and 1% lead citrate (Reynolds, 1963). Sections prepared in this manner were examined in the bright field m o d e of a Phillips 400 electron microscope.
Equilibrium density centrifugation The total DNA was extracted from infected KYED cells at 18 h pi, by incubation at 39°C for 3 h in pronase (1 mg ml-1), sodium dodecyl sulphate (SDS: 1.0%), sarkasyol (2.0%), 0.2 M NaC1, 10 mM EDTA, and 0.01 M TrisHC1, pH 7.4. The mixture was made isodense with CsC1 at 1.710 g cc -1. The self-generating gradients were centrifuged at 20°C, 150 000 × g for 24 h in a Beckman VTI 50 rotor. Radioactivity was measured b y total acid precipitable counts in a 750-~1 fraction, dripped from the b o t t o m of the tube. RESULTS
Restricted growth o f equine herpesvirus at 39 ° C The first experiment in this series determined that viral growth yields were different at 34°C, 37°C and 39°C for each equine herpesvirus. In this experiment virally-infected cells were incubated at the different temperatures indicated until cytopathic effect (CPE) was considered to be advanced. From these infected cultures stocks were harvested, prepared in identical volumes and titrated at 34°C before storage. The results are recorded in Table I. A TABLE I G r o w t h yields o f e q u i n e d e r m a l cells i n f e c t e d w i t h equine herpesvirus i n c u b a t e d at different temperatures Type
Temperature of incubation (o C)
Titer a t o t a l cell s o n i c a t e
EHV 1
34 37 39 34 37 39 34 37 39
3.4 × 3.3 x <10 4.8 × 4.8 × 5.8 × 1.3 × 1.8 x <10
EHV 2
EHV 3
a T i t r a t i o n s carried o u t at 34 ° C.
107 107 10 ~ 10 ~ 106 108 108
225 TABLE II Efficiency of plating (EOP) of equine herpesvirus Virus type
Titers on KYED a cells 34°C
EHV1(Army183) EHV 1 (T2) EHV 2 (T328) EHV3(lll8LP) HSV-1 (STH2HFEM) HSV-I (MP) HSV-I (F)
1.9× 1.5 × 3.2 x 1.9× 1.5 x
EOP
39°C 10 s 107 108 109 109
1.9x 3.4 × 5.0 × <10 s 1.5 ×
Titers on AGMKb cells 34°C
EOP
39°C
106 104 106
1.0 × 10 -2 2.0 x 1 0 - ' 1.6 x 10 -5 < i . 0 x 10-6 109 1.0 2.5 × 10 8 1.0 × 10 s 0.4 1.4 × 10 9 2.8 × i 0 ~ 0.2 1 . 2 × 10 9 4 . 0 x 10 8 0.3 4.0 × 10 9 < 1 0 4 < i . 0 × I 0 -s 1.2 × 10 9 < 1 0 4 < 1 . 0 × 10 -s
aKYED is a diploid line of equine dermal cells. bAGMK is a line of African Green Monkey cells. EOP = the ratio of the number of plaques at 34°C over those at 39°C. c o m p a r i s o n o f yields at 3 4 ° C a n d 3 7 ° C f o r e a c h e q u i n e h e r p e s v i r u s revealed little d i f f e r e n c e in g r o w t h yields at these t e m p e r a t u r e s . H o w e v e r , yields o f i n f e c t i o u s virus f r o m c u l t u r e s i n c u b a t e d at 3 9 ° C w e r e d r a m a t i c a l l y r e d u c e d f o r t h o s e i n f e c t e d with E H V 1 and 3 and less so in c u l t u r e s i n f e c t e d with E H V 2. In t h e s e c o n d e x p e r i m e n t in this series, p l a q u e t i t r a t i o n s o f equine herpesviruses w e r e m a d e o n K Y E D cells and t h e EOPs w e r e c o m p a r e d . T a b l e II reveals t h a t t h e E O P s f o r E H V 1 ( A r m y 183 a n d T 2 ) and 2 ( T 3 2 8 ) were 1 0 - ' - 10 -2 a n d t h a t f o r E H V 3 ( l l l 8 L P ) was a p p r o x i m a t e l y 10 -6. Several differe n t s t o c k s o f t h e s e viruses, all p r e p a r e d at 34°C, w e r e e x a m i n e d a n d s h o w n t o h a v e EOPs t h a t were c o n s i s t e n t w i t h t h o s e r e p o r t e d in T a b l e II. T h e t a b l e also allows a c o m p a r i s o n o f t h e s e EOPs with values d e t e r m i n e d f o r t h r e e hum a n herpesviruses t i t r a t e d o n b o t h K Y E D a n d A G M K cells. All t h r e e strains gave d i f f e r e n t results. Results w i t h t h e HSV-1 strains S T H 2 ( H F E M ) and MP, c o n s i d e r e d t o b e strains a d a p t e d to t h e l a b o r a t o r y , i n d i c a t e u n r e s t r i c t e d g r o w t h f o r S T H 2 a n d a m i n o r r e s t r i c t i o n ( ~ 10 -1) f o r t h e MP strain. H o w ever, t h e HSV-1 strain F, a m o r e r e c e n t field isolate, gave an E O P o f 10 -s, i n d i c a t i n g a s t r o n g g r o w t h r e s t r i c t i o n at 39°C. This r e s t r i c t i o n f o r HSV-1 strain F has b e e n i n d i c a t e d in a r e c e n t r e p o r t ( K n i p e et al., 1 9 8 1 ) w h i c h s t a t e d t h a t HSV-1 (F) c o n t a i n e d a t e m p e r a t u r e r e s t r i c t i o n in its a ( i m m e d i ately early) f u n c t i o n s . In t h e p r e s e n t s t u d y , t e m p e r a t u r e sensitivity in t h e h u m a n strains e x a m i n e d a p p e a r e d to be i n d e p e n d e n t o f t h e t w o cell t y p e s used. E q u i n e herpesvirus, in p a r t i c u l a r E H V 3, has a m o r e r e s t r i c t e d h o s t range a n d t h e r e b y l i m i t e d t h e p l a q u e assays t o cells o f e q u i n e origin.
Temporal nature o f temperature-restricted growth of EHV 3 in dermal cells Since E H V 3 d e m o n s t r a t e d t h e s t r o n g e s t g r o w t h r e s t r i c t i o n at 3 9 ° C , t h e f o l l o w i n g e x p e r i m e n t was designed to see if t h e t e m p e r a t u r e - s e n s i t i v e g r o w t h
226
restriction is in a function expressed at early (0--6 h) or late times (6--12 h) in the synthesis of EHV 3 virus. 'Shift-up' experiments (change from 34°C to 39°C) were designed to define the stage of the block. The results are presented in Table III. The number of plaques formed in these cultures was not affected by incubation at 34°C during the early times, but was markedly affected b y incubation at 34°C during later times in the synthesis of virus. This observation is consistent with a function which is late in the cycle (12--14 h) of EHV 3 viral replication (Allen and Bryans, 1977). To test the indications that temperature restriction was in a function at late times (6--12 h) and determine the reversibility of the block, a temperature 'shift-down' experiment (change from 39°C to 34°C) was designed. The results are presented in Table IV. When compared to cultures continuously incubated at 34°C (0 time), the number of plaques formed in these cultures was relatively independent of incubation at the restrictive temperature, 39°C, for up to 6 h pi; at best, a slight reduction in the number of plaques T A B L E III T i t r a t i o n o f E H V 3 o n e q u i n e d e r m a l cells f o l l o w i n g t e m p e r a t u r e a l t e r a t i o n H o u r s (pi) i n c u b a t e d at 34°C p r i o r t o 39°C
Plaque titers a f t e r 72 h
Percentage r e d u c t i o n at 39°C
(X lO s) 0a
1 3 6 8 10 12
0
--
4.3 2.0 3.9 4.0 21.0 30.0
88 94 89 88 32 17
a T i t e r o f s t o c k is 3.6 X 109 at 34°C. pi = p o s t - i n f e c t i o n .
TABLE IV T i t r a t i o n o f E H V 3 o n e q u i n e d e r m a l cells f o l l o w i n g t e m p e r a t u r e a l t e r a t i o n H o u r s (pi) i n c u b a t e d at 3 9 ° C prior to 34°C 0a
1 3 6 12 18
Titers a f t e r 72 h
(× 10 9)
Percentage r e d u c t i o n at 39°C
3.6
0
4.6 3.5 2.8 1.9 0.6
-3 22 47 98
a T i t e r o f s t o c k is 3.6 × 109 at 34°C. pi = p o s t - i n f e c t i o n .
227
m a y be seen at 6 h pi. Incubation of cultures at 39°C for longer than 6 h caused a dramatic reduction in titer. Again, these times (6--12 h) are in the late phase (end of the first replication cycle) o f EHV 3 synthesis.
Fig. 1. Electron micrograph o f KYED cells at 18 h after infection with equine herpesvirus 3. Cells were infected at multiplicity o f 5 pfu cell -1 and incubated at 34°C. Specimens were prepared by lead citrate-urynal acetate staining (see Materials and Methods). Arrows indicate enveloped virus in the cytoplasm and intercellular spaces. B indicates budding virus. The bar represents 1 ~m.
228
Examination o f equine cells infected with E H V 3 at the restricted temperature, 39°C Electron micrographs o f EHV 3-infected equine dermal cells incubated for 18 h at either 34°C or 39°C are shown in Figs. 1--6. Figure 1 illustrates an
Fig. 2. Electron micrograph of the perinuclear region in a K Y E D cell infected with E H V 3. Conditions are as indicated in Fig. 1. Arrows indicate budding virus, nuclear m e m brane, I and H (B) forms of nucleocapsids. T h e bar represents 0.1 urn.
229
infected cell incubated at 34°C. It can be seen (arrows) that both the cytoplasm and intercellular spaces are filled with mature virions {enveloped) that are typical o f herpesviruses. In addition, budding through the nuclear membrane can be seen, indicated b y arrows B. Figure 2 is an enlargement showing the nuclear membrane (arrows B, Fig. 1) o f a cell infected at 34°C for 18 h. It illustrates capsid envelopment, clearly showing envelope, tegument and nucleocapsid-like structure. In addition, this micrograph illustrates capsidlike structures, reminiscent of B or H and I capsids, seen within the nucleus of infected cells (Carmichael and Barnes, 1969; Gibson and Roizman, 1972; Perdue et al., 1975). The electron-dense cores contain nucleic acid. Figure 3 illustrates the enveloped virions seen in the cytoplasm of these cells.
Fig. 3. Electron micrograph o f the cytoplasm o f KYED cells i n f e c t e d with EHV 3. Conditions are as indicated in Fig. 1. Arrows are u s e d to indicate enveloped virion in the cytoplasm. T h e bar represents 0.1 ~m.
230
In contrast to these cells, Figs. 4--6 are electron micrographs of infected equine dermal cells, prepared in an identical manner as above, but incubated for 18 h pi at 39°C. Figure 4 illustrates a multinuclear giant cell containing
Fig. 4. Electron micrograph of KYED cells infected with EHV 3 at a multiplicity o f 5 and incubated at 39°C for 18 h. All conditions for infection and specimen preparation are as indicated in Fig. 1. Nuclei (N), cytoplasm (C), rough endoplasmic reticulum (er), chromatin (chr) and membrane reduplications (mRD). The bar represents 1.0 um.
231
Fig. 5. E l e c t r o n m i c r o g r a p h o f t h e n u c l e a r m e m b r a n e b o u n d a r y o f a t y p i c a l K Y E D cell at 18 h a f t e r i n f e c t i o n w i t h E H V 3 i n c u b a t e d at 39°C. All n o m e n c l a t u r e o f arrows are as i n d i c a t e d in Fig. 4 w i t h t h e a d d i t i o n o f i n d i c a t i o n s o f n u c l e o c a p s i d f o r m s ( N c ) at t h e n u c l e a r m e m b r a n e . T h e b a r r e p r e s e n t s 1.0 urn.
232
cytoplasm active in protein synthesis which contains rough endoplasmic reticulum (er). However, evidence of viral-like structures in the cytoplasm, or at the plasma membrane, is missing. The nuclei present evidence of membrane reduplication (arrow mRD) and margination of chromatin {arrow chr).
Fig. 6. E l e c t r o n m i c r o g r a p h o f t h e n u c l e u s o f a t y p i c a l K Y E D cell at 18 h a f t e r i n f e c t i o n w i t h E H V 3 i n c u b a t e d at 39 ° C. All n o m e n c l a t u r e s o f a r r o w s are as i n d i c a t e d in Figs. 4 a n d 5. T h e b a r r e p r e s e n t s 1.0 urn.
233
Figure 5 is an enlargement of a perinuclear region o f this cell and illustrates cytoplasm abundant in endoplasmic reticulum and the absence of virion-like structures. However, nucleocapsid-like structures are seen at the inner lameUa of the nuclear membrane (arrow Nc). Membrane reduplication and chromatin margination are also seen. Figure 6 illustrates the relative abundance of these nucleocapsid structures in the nucleus of infected cells incubated for 18 h at a temperature A
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Fract io. Fig. 7. CsC1 equilibrium gradients o f D N A isolated f r o m K Y E D cells i n f e c t e d w i t h equine herpesviruses at a m u l t i p l i c i t y o f 2 0 p f u cell -~ and i n c u b a t e d for 18 h pi. In all cases the cells were labeled w i t h H 3 - m e t h y l t h y m i d i n e ( 2 0 pCi p1-1) for t o t a l time. Panel A represents D N A e x t r a c t e d from E H V 1 - i n f e c t e d cells i n c u b a t e d at 3 4 ° C. Panels B and C represent D N A e x t r a c t e d f r o m cells i n c u b a t e d at 3 9 ° C and i n f e c t e d w i t h E H V 1 or E H V 3, respectively. T h e marker D N A is e x t r a c t e d f r o m P32-1abeled E H V 3 virions.
234 (39°C) restrictive for viral growth. Envelopment (budding) of these capsids, and virion forms in the cytoplasm, was not seen. More than 75 of 100 cells examined were of this kind.
Synthesis o f EHV 3 viral-density DNA at the restrictive temperature, 39°C The following experiment was designed to determine if viral DNA was synthesized at the restrictive temperature and further supports the interpretation that the nucleocapsid forms seen in the electron micrograph contain viral DNA in KYED Cells infected with EHV 3. Figure 7 shows the results of CsC1 equilibrium density gradients of total DNA isolated from equine dermal cells infected with EHV 1 (Fig. 7A) and incubated at the permissive temperature (34°C) for 18 h pi. Gradients of DNA isolated from cells infected with EHV 1 (Fig. 7B) and EHV 3 {Fig. 7C) and incubated for 18 h at the restrictive temperature (39°C) axe also shown. The marker DNA has been isolated from purified virions of EHV 3 and has a density of 1.726 g cc -1 which is higher than the density o f EHV 1, 1.716 g cc -~. DISCUSSION
The results presented in this report indicate that, at least for those strains of the equine herpesvirus triad examined (Army 183, T2, T328, l l l 8 L P ) , there appears to be restricted growth at 39°C. These strains included both the subtype 1, Army 183, and subtype 2, T2, of EHV 1. The reason why EHV 1 and EHV 3 gave poorer yields than EHV 2 at 39°C, has not yet been determined. Possibly, the products made at the restrictive temperature of 39°C in cells infected by EHV 1 or 3 are highly cell-associated at this temperature and much less able (as expected of nucleocapsids) to spread the infection than those products of the EHV 2 infections. It is n o t clear if the identification of EHV 2 as an equine cytomegalovirus, and the highly cellassociated nature of these viruses, is significant. It is possible that EHV 2 is more adapted to a cell-mediated dissemination of infection. When the temperature restriction in EHV 3 was examined by temperature shift experiments the block in maturation was limited to late in the replication cycle and did not require early incubation at the non-permissive temperature (39°C) to be effective. The evidence that DNA of viral density was synthesized at the non-permissive temperature and electron microscopic demonstration of DNA-containing nucleocapsids building up in the nuclei of infected cells (Carmichael and Barnes, 1969; Gibson and Roizman, 1972; Perdue et al., 1975) are consistent with this finding. However, enveloped virions did not appear in the cytoplasm of the EHV 3-infected cells incubated at the non-permissive temperature. Parenthetically, DNA with a density greater than viral DNA, characteristic of defective viral particles in a population, was not seen in the CsC1 gradients.
235 EHV 3 demonstrates the strongest growth restriction seen in the equine herpesvirus triad. Examination of more recent field isolates of EHV 3 from geographically unrelated areas supports the findings on strain l 1 1 8 L P (J. Evermann, personal communication, 1985). The growth restriction in EHV 3-infected equine dermal cells appears to be a block in a required late function that cannot be overcome b y incubation at the permissive temperature (34°C) early in viral replication. This is in contrast to t w o previous reports on temperature sensitivity with other herpesviruses. In the first, Stevens and Jackson (1967) indicated that a temperature-sensitive restriction in bovine herpesvirus 1 (BHV 1) occurred early in the growth cycle and blocked viral DNA synthesis. It should be noted that this temperature sensitive restriction was effective at 40--42°C and could be overcome by incubation at permissive temperatures early in the replication cycle. In the second, it was reported (Knipe et al., 1981) that HSV-1 strain F was restricted at 39°C. HSV-1 (F) produces a temperature-sensitive ~ (immediately early) gene product at the non-permissive temperature (39°C) that maps between 0.83-0.87 and 0.97--1.0 map units and that infected cell polypeptide patterns are largely ~ (immediately early). These patterns are reminiscent of ts mutants in complementation group 1--2 (P.J. Godowski and D.M. Knipe, unpublished data, 1981). These findings would indicate that nucleocapsids are n o t formed at 39°C during HSV-1 (F) infections. However, the findings on EHV 3 infections presented here support a more recent finding (Letchworth et al., 1982) that a 39°C temperature growth restriction in bovine herpesvirus 2 (BHV 2) replication is a block in a late function. In addition, preliminary results from experimental analysis of EHV 3-infected cell proteins (M. Steiner, personal communication, 1985) support the conclusion that certain 7 (late) proteins, some of which appear to be glycosylated, are not being synthesized at the restrictive temperature (39°C). The significance of these findings is that the restrictive temperature reported (39°C) is consistent with b o d y temperatures c o m m o n l y found in the equine host, though the temperature of superficial tissue in the equine m a y be lower. This is o f importance since most clinical presentations (Bryans and Allen, 1973; Studdert, 1974; Bryans, 1980) of EHV 3 infections, e.g. coital exanthema, appear as lesions on superficial tissues that typically progress to a mild, self-limiting exanthematic disease. The clinical presentation of EHV 3 infection is often reviewed as an infectious, b u t innocuous, genital disease (O'Callaghan et al., 1982). The prognosis does not indicate a progression of the disease that would involve the internal organs or a fetus of a pregnant mare (Bryans and Allen, 1973; Studdert, 1974; Bryans, 1980; O'Callaghan et al., 1982), as do those infections with EHV 1. The finding in this report and clinical presentation of the disease caused by EHV 3 are consistent (Bryans, 1980). Further investigations into the molecular pathogenesis of this viral disease could reveal w h y this herpesvirus appears to be relatively innocuous in its natural host.
236 ACKNOWLEDGEMENTS
The author would like to acknowledge the technical assistance of Ms. J e n n y Borchelt, Mr. R. Geissler (Pathology) and Dr. D. Powell for use of their electron microscope. The assistance of Mr. Bradley Moore in the preparation of this manuscript was appreciated. In addition, I would like to thank Drs. Allen and Bryans, Department of Veterinary Sciences, for many of the viral strains used in this study. The work was supported by Public Health Service Grant AI 17620 from the National Institute of Allergy and Infectious Diseases.
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