THE JOURNAL OF INFECTIOUS DISEASES. VOL. 135, NO.4. APRIL 1977 © 1977 by the University of Chicago. All rights reserved.
Clinical and Pathogenetic Studies of Medical Lake Macaque Virus Infections in Cynomolgus Monkeys (Simian Varicella) From the Children's Mercy Hospital and the University of Missouri (Kansas City) School of Medicine, Kansas City, Missouri
Herbert A. Wenner, David Abel, Shloe Barrick, and P. Seshumurty
stock was formed in an attempt to raise the fraction of clinical infections. Inoculation. Four separate studies were completed, each separated by intervals of four to six months. Inoculated monkeys were caged individually in an isolation unit. In one of these tests, two sentinel monkeys .were introduced into the unit on the day their companions were inoculated. MLM virus was administered by the intradermal (id), iv, intracardiac (ih), intratracheal (it), and pharyngeal routes. Fifteen monkeys were tested by two routes of inoculation of virus (table 2). Eight id blebs were raised on the anterior thorax; the total volume injected was -- 1.0 ml. One milliliter of virus, mixed with an equal volume of 0.25% ionagar, was delivered directly into the trachea (by surgical catheterization). For pharyngeal inoculation, the mucous membranes of the nose and throat were rubbed with swabs saturated with MLM virus. Volumes delivered by the remaining routes were 0.8-1.2 ml. Between day 2 and day 4 after inoculation, oil of mustard was rubbed onto the skin of the thorax of monkeys inoculated id, it, or via the pharynx. Clinical and pathologic studies. The collection of blood, sera, and tissues for virus detection, serologic studies, histopathology, and fluorescent antibody (FA) labeling have been described [2]. Vero cells were used for the detection of MLM virus. The CF test was used to follow antibody development; a fraction of the sera was studied by indirect immunofluorescence [3].
In 1973, Blakely et al. [1] described a mild vesicular disease of monkeys and associated the infection with a varicella-like herpesvirus. Infected monkeys developed vesicular eruptions in association with antibodies cross-reacting with Herpesvirus varicellae and in lesser degree with H erpesvirus hominis. In this study we confirm the findings of Blakely et al. that the Medical Lake macaque (MLM) virus is closely related to varicella-zoster virus. The features of experimental infection, particularly those relating to pathogenesis, represent major points in this presentation. Materials and Methods
Monkeys. Young, mature cynomolgus monkeys weighing 1.5-2.7 kg were used. Their general health was satisfactory, and all were tuberculin-negative. Samples of blood were procured before inoculation for the studies described. Virus. l\ILM virus was obtained from the Center for Disease Control, Atlanta, Ga. Passage histories and concentrations of cell-associated virus in the inocula are shown in table I. Since low-passage virus produced simian varicella in only 12 of 23 monkeys, the high-passage virus Received for publication June 28, 1976, and in revised form September 22,1976. This study was supported by research grant no. AIl0252 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland. Please address requests for reprints to Dr. H. A. Wenner, The Children's Mercy Hospital, 24th and Gillham, Kansas City, Missouri 64108.
611
Downloaded from http://jid.oxfordjournals.org/ at University of Birmingham on August 28, 2015
The Medical Lake macaque (MLM) virus produced varicelliform eruptions in cynomolgus monkeys. Not all experimentally infected monkeys developed overt disease; viremia was found, and specific antibodies were detected. Specific lesions were found in skin, lymph nodes, and spleen. Focal inflammatory lesions were present in liver, pancreas, and lung (after intratracheal instillation of virus). MLM virus was recovered from these and other organs. The temporal movements of MLM virus in and out of primary and secondary target organs remained partially unsolved. MLM virus is related to the Wu strain of varicella virus.
612
Wenner et al.
Tissues were fixed, blocked, cut, and stained with hematoxylin and eosin. Occasionally, other staining procedures were used as noted. A serum sample from a monkey hyperimmunized with MLM virus was labeled with fluorescein isothiocyanate and used for FA studies [4].
Table 1. Passage histories of Medical Lake macaque (MLM) virus.
Results
Low 3
Table 2. virus.
High 1 2
Passage history"
Viral titer']
RhT (12)/Vero (19) RhT (12)/Vero (19)
2.9 X 10 4 5.0 X 10 4 3.0 X 10 7 5.0 X 10 6
Vero (4) Vero (7)
4:1:
NOTE. Viral titers were determined in Vero cell cultures. The low-passage tissue culture virus was concentrated by negative-pressure dialysis [2]. *Type of cell (number of passages). RhT = rhesus monkey thyroid. tNumber of cell-associated viral particles/mi. :l:Virus recovered from the vesicular fluid of monkey no. M-39.
cession on about day 10 after inoculation (range, six to 12 days). Most vesicles were less than 1 ml in diameter, and clusters of larger vesicles (2-3 mm) were observed occasionally. Crusting occurred within a few days, and healing occurred by day 14 after inoculation. Demonstrable clinical signs of varicella were not seen in 19 monkeys; all, however, developed antibodies to MLM viral antigens. One of two sentinel monkeys housed among the infected animals developed a varicelliform eruption 24 days later, approximately 17 days after the onset of the eruption in inoculated monkeys. The remaining sentinel monkey (no. M-51) failed to develop evidence of infection;
Clinical and subclinical evidence of infection in 34 monkeys inoculated with Medical Lake macaque
Route of inoculation Pharyngeal Intratracheal Intravenous Intracardiac Intradermal Combined§
No. of monkeys developing rash/no. of monkeys tested * 1/2 0/2 3/9 2/5 1/1 8/15
Rash
13 9-12 9-12 8 6-10
Concurrent events
Duration (days) of
Day(s) of onset
Vesiculation
«
2 (mild)t
"'3 (mild) ~4
5 2-6 (mild in one)
Crusting NS NS 3 5 5
Viremia 0/2+ 0/2 2/5 1/2 0/1 8/15
Antibody titer rises
I/U 2/2 5/5 3/3 1/1 15/15
NOTE. Six of the 15 monkeys with rash were sacrificed during the 12 days after inoculation; thus the duration of the eruption is a general approximation based on observation of nine monkeys. NS = not seen. *Rash refers to vesiculation of trunk and extremities; local vesiculation after intradermal inoculation was not included. t An evanescent eruption that could have been easily missed and was verified histologically. +Number of monkeys with the event/number tested. § Of these 15 monkeys, 13 received intradermal inoculation, seven in combination with intracardiac inoculation, and six in combination with intravenous inoculation. The two remaining monkeys received virus both via the pharynx and via the trachea.
Downloaded from http://jid.oxfordjournals.org/ at University of Birmingham on August 28, 2015
Clinical evidence of infection. Some of the features of infection could be related to the portals of inoculation. The id inocula resulted in development of local papules, followed by vesiculation and subsequent crusting. Vesicle fluids yielded infectious virus on subculture in Vero cells. These vesicles persisted for 24-36 hr; encrustation lasted for as long as eight days and up to the time of generalized eruption, if it developed. Vesiculation was enhanced at sites of skin irritation by oil of mustard. Three of four monkeys developed pneumonia after it inoculation, and none developed an exanthem between days 8 and 13 after inoculation. One of the four monkeys, in wh ich virus was rubbed onto the pharynx, also developed viremia. One of the two monkeys that received virus on the pharynx developed an evanescent rash and specific antibodies. Five of 14 monkeys inoculated intravascularly (iv and ih) developed cutaneous lesions. Data on 34 monkeys (table 2) indicate that 15 developed a demonstrable varicelliform eruption. Papules and vesicles developed in rapid sue-
Passage designate, stock no.
Simian Varicella
613
40
Viremia. Viremia was detected in four of 16 monkeys given high-passage virus. With the lowpassage virus stock, viremia was detected in seven of 11 monkeys (table 3). The temporal aspects of viremia are brought into sharper perspective in a study involving 12 monkeys inoculated either id or ih. Two monkeys were sacrificed daily after day 2 after inoculation for detection of virus not only in the blood, but also in visceral organs. The results relating to viremia are summarized in table 4. These data provide evidence that viremia revealed as early as the second day persisted and was still detectable on day 6 after inoculation (monkey no. 1\1-63). Viremia in the monkeys inoculated id began later and may have ended earlier than in monkeys inoculated ih, Localization of MLM virus in selected tissues and body fluids. Ten of the 12 monkeys mentioned in table 4 were sacrificed at intervals for localization of virus in body tissues and fluids prior to anticipated development of skin lesions. Additional data were procured for three monkeys during the period immediately after the eruptive phase of infection, when viremia is not usually present and antibodies are already measurable. These data are summarized in table 5. Viremia developed by day 3 after id inoculation. At that time the only organ yielding virus was the testis. Presumably, viremia was initiated at the dermal sites of viral replication, and
OBSERVED SENTINAL
+
0
C 39
Figure 1. Illustrative course of infection with Medical Lake macaque (MLM) virus in cynomolgus monkey no. M-51, which was housed amid experimentally infected monkeys from April 11, 1975 to June 3, 1975. Clinical disease failed to develop, and antibody conversion did not occur. The monkey was challenged with MLM virus intradermally and iv on June 3, '1975. For skin eruption, the plus signs indicate vesiculation at sites of intradermal inoculation. WBC = white blood cell.
I I INOCULATED
I
I
I
I
I
I
I
I
I
I
I
FOR M 51
613/ 76
/0-0-0
0" 0-
o
..........
0_
38f-
0_
0
37,. I
I
I
I
SKIN ERUPTION
+
VIREMIA CF TITER
+
+
+
+
+
0
c8
cB
~ GENERALIZED VESICULATION-ENCRUSTATION-!
0
0
0
0 256
128
256
~ _e", LEUKOCYTES e" e__ • ;::::::::::::::-t LYMPHOCYTES ../ ""'.. e-e-1~"
10000,.
wac
ABSOLUTE COUNTS
",-..
0_0_0
10001-
/ -3
•
Clinical and Pathogenetic Studies of Medical Lake Macaque Virus Infections in Cynomolgus Monkeys (Simian Varicella) From the Children's Mercy Hospital and the University of Missouri (Kansas City) School of Medicine, Kansas City, Missouri
Herbert A. Wenner, David Abel, Shloe Barrick, and P. Seshumurty
stock was formed in an attempt to raise the fraction of clinical infections. Inoculation. Four separate studies were completed, each separated by intervals of four to six months. Inoculated monkeys were caged individually in an isolation unit. In one of these tests, two sentinel monkeys .were introduced into the unit on the day their companions were inoculated. MLM virus was administered by the intradermal (id), iv, intracardiac (ih), intratracheal (it), and pharyngeal routes. Fifteen monkeys were tested by two routes of inoculation of virus (table 2). Eight id blebs were raised on the anterior thorax; the total volume injected was -- 1.0 ml. One milliliter of virus, mixed with an equal volume of 0.25% ionagar, was delivered directly into the trachea (by surgical catheterization). For pharyngeal inoculation, the mucous membranes of the nose and throat were rubbed with swabs saturated with MLM virus. Volumes delivered by the remaining routes were 0.8-1.2 ml. Between day 2 and day 4 after inoculation, oil of mustard was rubbed onto the skin of the thorax of monkeys inoculated id, it, or via the pharynx. Clinical and pathologic studies. The collection of blood, sera, and tissues for virus detection, serologic studies, histopathology, and fluorescent antibody (FA) labeling have been described [2]. Vero cells were used for the detection of MLM virus. The CF test was used to follow antibody development; a fraction of the sera was studied by indirect immunofluorescence [3].
In 1973, Blakely et al. [1] described a mild vesicular disease of monkeys and associated the infection with a varicella-like herpesvirus. Infected monkeys developed vesicular eruptions in association with antibodies cross-reacting with Herpesvirus varicellae and in lesser degree with H erpesvirus hominis. In this study we confirm the findings of Blakely et al. that the Medical Lake macaque (MLM) virus is closely related to varicella-zoster virus. The features of experimental infection, particularly those relating to pathogenesis, represent major points in this presentation. Materials and Methods
Monkeys. Young, mature cynomolgus monkeys weighing 1.5-2.7 kg were used. Their general health was satisfactory, and all were tuberculin-negative. Samples of blood were procured before inoculation for the studies described. Virus. l\ILM virus was obtained from the Center for Disease Control, Atlanta, Ga. Passage histories and concentrations of cell-associated virus in the inocula are shown in table I. Since low-passage virus produced simian varicella in only 12 of 23 monkeys, the high-passage virus Received for publication June 28, 1976, and in revised form September 22,1976. This study was supported by research grant no. AIl0252 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland. Please address requests for reprints to Dr. H. A. Wenner, The Children's Mercy Hospital, 24th and Gillham, Kansas City, Missouri 64108.
611
Downloaded from http://jid.oxfordjournals.org/ at University of Birmingham on August 28, 2015
The Medical Lake macaque (MLM) virus produced varicelliform eruptions in cynomolgus monkeys. Not all experimentally infected monkeys developed overt disease; viremia was found, and specific antibodies were detected. Specific lesions were found in skin, lymph nodes, and spleen. Focal inflammatory lesions were present in liver, pancreas, and lung (after intratracheal instillation of virus). MLM virus was recovered from these and other organs. The temporal movements of MLM virus in and out of primary and secondary target organs remained partially unsolved. MLM virus is related to the Wu strain of varicella virus.
612
Wenner et al.
Tissues were fixed, blocked, cut, and stained with hematoxylin and eosin. Occasionally, other staining procedures were used as noted. A serum sample from a monkey hyperimmunized with MLM virus was labeled with fluorescein isothiocyanate and used for FA studies [4].
Table 1. Passage histories of Medical Lake macaque (MLM) virus.
Results
Low 3
Table 2. virus.
High 1 2
Passage history"
Viral titer']
RhT (12)/Vero (19) RhT (12)/Vero (19)
2.9 X 10 4 5.0 X 10 4 3.0 X 10 7 5.0 X 10 6
Vero (4) Vero (7)
4:1:
NOTE. Viral titers were determined in Vero cell cultures. The low-passage tissue culture virus was concentrated by negative-pressure dialysis [2]. *Type of cell (number of passages). RhT = rhesus monkey thyroid. tNumber of cell-associated viral particles/mi. :l:Virus recovered from the vesicular fluid of monkey no. M-39.
cession on about day 10 after inoculation (range, six to 12 days). Most vesicles were less than 1 ml in diameter, and clusters of larger vesicles (2-3 mm) were observed occasionally. Crusting occurred within a few days, and healing occurred by day 14 after inoculation. Demonstrable clinical signs of varicella were not seen in 19 monkeys; all, however, developed antibodies to MLM viral antigens. One of two sentinel monkeys housed among the infected animals developed a varicelliform eruption 24 days later, approximately 17 days after the onset of the eruption in inoculated monkeys. The remaining sentinel monkey (no. M-51) failed to develop evidence of infection;
Clinical and subclinical evidence of infection in 34 monkeys inoculated with Medical Lake macaque
Route of inoculation Pharyngeal Intratracheal Intravenous Intracardiac Intradermal Combined§
No. of monkeys developing rash/no. of monkeys tested * 1/2 0/2 3/9 2/5 1/1 8/15
Rash
13 9-12 9-12 8 6-10
Concurrent events
Duration (days) of
Day(s) of onset
Vesiculation
«
2 (mild)t
"'3 (mild) ~4
5 2-6 (mild in one)
Crusting NS NS 3 5 5
Viremia 0/2+ 0/2 2/5 1/2 0/1 8/15
Antibody titer rises
I/U 2/2 5/5 3/3 1/1 15/15
NOTE. Six of the 15 monkeys with rash were sacrificed during the 12 days after inoculation; thus the duration of the eruption is a general approximation based on observation of nine monkeys. NS = not seen. *Rash refers to vesiculation of trunk and extremities; local vesiculation after intradermal inoculation was not included. t An evanescent eruption that could have been easily missed and was verified histologically. +Number of monkeys with the event/number tested. § Of these 15 monkeys, 13 received intradermal inoculation, seven in combination with intracardiac inoculation, and six in combination with intravenous inoculation. The two remaining monkeys received virus both via the pharynx and via the trachea.
Downloaded from http://jid.oxfordjournals.org/ at University of Birmingham on August 28, 2015
Clinical evidence of infection. Some of the features of infection could be related to the portals of inoculation. The id inocula resulted in development of local papules, followed by vesiculation and subsequent crusting. Vesicle fluids yielded infectious virus on subculture in Vero cells. These vesicles persisted for 24-36 hr; encrustation lasted for as long as eight days and up to the time of generalized eruption, if it developed. Vesiculation was enhanced at sites of skin irritation by oil of mustard. Three of four monkeys developed pneumonia after it inoculation, and none developed an exanthem between days 8 and 13 after inoculation. One of the four monkeys, in wh ich virus was rubbed onto the pharynx, also developed viremia. One of the two monkeys that received virus on the pharynx developed an evanescent rash and specific antibodies. Five of 14 monkeys inoculated intravascularly (iv and ih) developed cutaneous lesions. Data on 34 monkeys (table 2) indicate that 15 developed a demonstrable varicelliform eruption. Papules and vesicles developed in rapid sue-
Passage designate, stock no.
Simian Varicella
613
40
Viremia. Viremia was detected in four of 16 monkeys given high-passage virus. With the lowpassage virus stock, viremia was detected in seven of 11 monkeys (table 3). The temporal aspects of viremia are brought into sharper perspective in a study involving 12 monkeys inoculated either id or ih. Two monkeys were sacrificed daily after day 2 after inoculation for detection of virus not only in the blood, but also in visceral organs. The results relating to viremia are summarized in table 4. These data provide evidence that viremia revealed as early as the second day persisted and was still detectable on day 6 after inoculation (monkey no. 1\1-63). Viremia in the monkeys inoculated id began later and may have ended earlier than in monkeys inoculated ih, Localization of MLM virus in selected tissues and body fluids. Ten of the 12 monkeys mentioned in table 4 were sacrificed at intervals for localization of virus in body tissues and fluids prior to anticipated development of skin lesions. Additional data were procured for three monkeys during the period immediately after the eruptive phase of infection, when viremia is not usually present and antibodies are already measurable. These data are summarized in table 5. Viremia developed by day 3 after id inoculation. At that time the only organ yielding virus was the testis. Presumably, viremia was initiated at the dermal sites of viral replication, and
OBSERVED SENTINAL
+
0
C 39
Figure 1. Illustrative course of infection with Medical Lake macaque (MLM) virus in cynomolgus monkey no. M-51, which was housed amid experimentally infected monkeys from April 11, 1975 to June 3, 1975. Clinical disease failed to develop, and antibody conversion did not occur. The monkey was challenged with MLM virus intradermally and iv on June 3, '1975. For skin eruption, the plus signs indicate vesiculation at sites of intradermal inoculation. WBC = white blood cell.
I I INOCULATED
I
I
I
I
I
I
I
I
I
I
I
FOR M 51
613/ 76
/0-0-0
0" 0-
o
..........
0_
38f-
0_
0
37,. I
I
I
I
SKIN ERUPTION
+
VIREMIA CF TITER
+
+
+
+
+
0
c8
cB
~ GENERALIZED VESICULATION-ENCRUSTATION-!
0
0
0
0 256
128
256
~ _e", LEUKOCYTES e" e__ • ;::::::::::::::-t LYMPHOCYTES ../ ""'.. e-e-1~"
10000,.
wac
ABSOLUTE COUNTS
",-..
0_0_0
10001-
/ -3
•
