Disseminated Simian Varicella Virus Infection in an Irradiated Rhesus Macaque (Macaca mulatta)

Abstract

We describe correlative clinicopathological/virological findings from a simian varicella virus (SVV)-seronegative monkey that developed disseminated varicella 105 days after gamma-irradiation. Twelve other monkeys in the colony were also irradiated, none of which developed varicella. Before irradiation, sera from the monkey that developed disseminated infection and one asymptomatic monkey were available. Analysis indicated that subclinical reactivation of latent SVV from an asymptomatic irradiated monkey likely led to disseminated varicella in the seronegative irradiated monkey. These findings parallel those from humans with disseminated varicella infection and support the usefulness of SVV infection as a model for human varicella-zoster virus infection, particularly virus reactivation after gamma-irradiation.

Simian varicella virus (SVV) causes chicken pox (varicella) in primates. Outbreaks have been recorded to occur in multiple animal facilities (5). Serious, often fatal SVV infection was described previously for Chlorocebus aethiops (4). In 1967 to 1968, 48 cases occurred in Erythrocebus patas (11). Milder outbreaks were reported to occur in Macaca nemestrina (3), M. fascicularis (3, 13), and M. fuscata (3) at the Washington National Primate Research Center, Seattle, Washington. Closely related viruses isolated from different outbreaks, such as deltaherpesvirus (1), Medical Lake macaque virus (16), and Liverpool vervet virus (4), were classified by geographical location and/or monkey species.

Clinical and pathological features of SVV and human varicella-zoster virus (VZV) infection are similar. Also, SVV and VZV are related antigenically, share DNA homology, and become latent in ganglionic neurons (6, 7). VZV reactivates in irradiated (2, 14) and immunocompromised humans, and dissemination has previously been described to occur in a bone marrow transplant recipient 265 days after total-body irradiation (14) and in 12/27 children who received total-body irradiation after stem cell transplantation (8).

Our report herein is necessarily restricted to a single monkey, but the descriptive information provided by these first correlative clinicopathological and virological parameters from a macaque that developed disseminated SVV infection after whole-body irradiation is relevant to our understanding of the pathogenesis of varicella infection.

Case report.

Thirteen male macaques (12 M. mulatta macaques and one M. fascicularis macaque) (Table 1), aged 5 to 7 years, were housed in individual cages in one room. All monkeys were seronegative for Chlorocebus herpesvirus 1, simian immunodeficiency virus, simian T-lymphotrophic virus, and type D simian retrovirus. Except for monkey no. 2, which was born in Vietnam and had a history of antibody to measles virus, the monkeys were born and bred domestically. There is no history of spontaneous SVV infection in any monkeys housed in the University of Maryland School of Medicine (UMSOM) animal facility. All animals were part of a study of stem cell reconstitution after exposure to gamma-irradiation, performed in accordance with U.S. Department of Agriculture Animal Welfare Act regulations and the Guide for Care and Use of Laboratory Animals and with approval of the Institutional Animal Care and Use Committee at the UMSOM.

TABLE 1.

Serology results for irradiated monkeys

Animal no.a	SVV antibody titerb before irradiation	Amt (cGy) gamma-radiation	Result after irradiation
			Presence/absence of:		SVV antibody titerb
			SVV IgGc	SVV IgMc
1	<1:25	600	+	+	1:160
2	1:160	850	+	+	>1:800
3	NAd	875	+	+	1:100
4	NA	600	−	−	NDe
5	NA	875	−	−	ND
6	NA	600	−	−	ND
7	NA	600	−	−	ND
8	NA	850	−	−	ND
9	NA	600	−	−	ND
10	NA	600	−	−	ND
11	NA	850	−	−	ND
12	NA	850	−	−	ND
13	NA	850	−	−	ND

^aAll animals were M. mulatta macaques, except animal no. 2, which was an M. fascicularis macaque.

^bAnti-SVV antibody titers are expressed as the serum dilution that neutralized 80% or more of the SVV plaques compared to control cultures.

^cAnti-SVV IgG and IgM antibodies were analyzed by indirect immunofluorescence. +, presence; −, absence.

^dNA, not available.

^eND, not done.

Monkey no. 1 developed a generalized maculopapulovesicular rash (Fig. 1A and B) 105 days after receiving 600 cGy of total-body irradiation. This monkey and its cage were immediately removed from the room. A plaque reduction assay, performed (12) on plasma obtained before irradiation from the affected monkey, did not reveal any anti-SVV antibody (Table 1). After irradiation, platelets, total white blood cells (WBCs), and blood mononuclear cells (MNCs) were reduced from preirradiation levels of 300,000, 10,000, and 6,000 to 4,000, 7,000, and 3,000 per μl, respectively. Furthermore, serum creatine phosphokinase (2,182 U/liter) and aspartate aminotransferase (131 U/liter) were elevated, while total protein (4.8 g/dl) and albumin (2.9 g/dl) were mildly decreased after irradiation.

FIG. 1.

Gross pathological abnormalities of SVV-infected monkey. Erythematous maculopapular lesions were present on face (A) and trunk (B). Note white raised plaques and ulcers affecting the gingiva, hard palate, and tongue (C), diffuse petechial hemorrhage and necrosis in liver (D), and plaques and consolidation in lung (E).

Preirradiation plasma from one other monkey (no. 2) was available and revealed antibody to SVV at a dilution of 1:160. Of 13 total monkeys that were irradiated, 3 (no. 1, no. 2, and no. 3) were positive, after irradiation, for anti-SVV immunoglobulin G (IgG) and IgM antibodies by indirect immunofluorescence (Table 1). Seropositivity of these three samples was confirmed by SVV plaque reduction assay, which detected anti-SVV antibodies at dilutions of 1:160, >1:800, and 1:100, respectively. The plaque assay revealed that the antibody titer increased from <1:25 before irradiation to 1:160 after irradiation in monkey no. 1 with disseminated disease. In monkey no. 2, the antibody titer increased from 1:160 before irradiation to >1:800 after irradiation, supporting the possibility that subclinical reactivation was the likely source of SVV that infected irradiated monkey no. 1.

After irradiation, in monkey no. 2, WBCs and MNCs increased from 10,000 and 4,000 per μl to 11,000 and 5,000 per μl, respectively, and platelets decreased from 340,000 to 300,000 per μl. The increase in WBCs and MNCs in monkey 2 months after irradiation most likely reflected immunological recovery. In monkey no. 3, platelets and red blood cells increased from 1,000 to 3,000 per μl and 2,000 to 4,000 per μl, respectively, after irradiation.

Because of concern for an infectious (or zoonotic) disease outbreak, monkey no. 1 was euthanized 6 h later and necropsied under biosafety conditions to prevent contamination of personnel or environment. Organs were examined histologically and virologically. Paraformaldehyde-fixed, paraffin-embedded sections were analyzed immunohistochemically by use of a 1:500 dilution of rabbit polyclonal anti-SVV antibody as described previously (10).

Gross pathology.

Severe lesions were seen on the gingiva, hard palate, and tongue of monkey no. 1 (Fig. 1C). The liver was friable and contained petechial hemorrhages and multifocal white plaques (Fig. 1D). The lungs were pale and appeared consolidated (Fig. 1E), with numerous discrete, 1- to 4-mm, firm, elevated plaques.

Histology.

Skin revealed melanosis and epidermal hyperplasia with increased keratin and some cells with poorly defined basophilic intranuclear inclusions and necrosis (Fig. 2A). Petechial hemorrhages were present on the mucosa of the stomach, jejunum, and ileum with Cowdry A inclusions, a hallmark of herpesvirus infection (Fig. 2B). Areas of mucosal sloughing in the large intestine were seen (Fig. 2C). The liver had several small areas of necrosis-containing neutrophils (Fig. 2D). In the lung, hemorrhage and fibrin were seen in some alveoli along with scattered small areas of inflammation, primarily neutrophilic (Fig. 2E and F). The spleen had a cloudy appearance with generalized petechial hemorrhage. Mesenteric lymph nodes were reactive with eosinophils and macrophages (data not shown). The adrenal glands were hemorrhagic (Fig. 2G). Cowdry A inclusion bodies were found in multiple organs (Fig. 2A, B, D, F, and G).

FIG. 2.

Histopathological abnormalities in hematoxylin and eosin-stained sections of SVV-infected monkey. (A) Skin reveals necrosis immediately below the epidermis (arrow) (magnification, ×220). (B) Stomach reveals Cowdry A inclusion bodies (arrows), also seen in insets for panels A, D, F, and G (magnification, ×660). (C) Intestine with extensive inflammation (thick arrow) (magnification, ×110) and loss of mucosal lining (thin arrows). (D) Liver with necrosis (arrows) (magnification, ×110). (E and F) Lung showing hemorrhagic necrosis (arrow) (magnification, ×110) and inflammation (magnification, ×660), respectively. (G) Adrenal gland with hemorrhage and necrosis (magnification, ×220). (H) Agarose gel electrophoresis after PCR amplification of SVV open reading frame 21 DNA. Lanes 1 and 7, 100-bp size markers; lane 2, no DNA; lane 3, cloned DNA fragment containing SVV open reading frame 21 sequences; lane 4, no DNA; lanes 5 and 6, DNA from whole blood from monkey no. 1 on the day of appearance of rash.

Virology.

PCR (Zoologix, Inc., Chatsworth, Calif.) revealed SVV gene 21-specific sequences in DNA extracted from blood of monkey no. 1 on the day of rash (Fig. 2H). Because the abundance of viral DNA would be high during acute infection, PCR for SVV DNA was not nested. PCR for other viruses (VZV, rhesus cytomegalovirus, and monkeypox virus) and reverse transcription-PCR for measles virus were negative in all monkeys (data not shown).

Immunology.

SVV-specific antigen was found in necrotic skin of monkey no. 1 (Fig. 3A).

FIG. 3.

Immunohistochemical analysis of skin from monkey no. 1, obtained using anti-SVV (A) or normal rabbit serum (B) for staining. (C and D) SVV-infected Vero cells and uninfected cells, respectively, stained with anti-SVV antiserum.

Most disseminated SVV outbreaks have been reported to occur in monkeys in captivity (4, 11, 15, 17). Spontaneous SVV outbreaks have been reported for both species (M. mulatta and M. fascicularis) that were used in this study (5). To our knowledge, only one previous occurrence has been attributed to spontaneous SVV reactivation (15). Our report is the first to document pathological/virological findings from an SVV-seronegative rhesus macaque that developed disseminated varicella after irradiation. Serological analysis before irradiation was available only for monkey no. 1 and no. 2. The detection of anti-SVV antibody in monkey no. 2 before irradiation and the presence of anti-SVV IgG and IgM antibodies along with an increased antibody titer after irradiation is consistent with subclinical varicella reactivation, as described for immunocompromised humans (9). Because serum from monkey no. 3 was not available before irradiation, the significance of detecting anti-SVV IgG and IgM antibody in this monkey after irradiation is unknown. The absence of SVV IgG antibody in monkeys no. 4 to 13 after irradiation indicates that these monkeys were probably seronegative before irradiation and thus not a source of SVV for monkey no. 1; the reason these monkeys did not develop varicella is unknown. Most likely, irradiation of monkey no. 2 and possibly monkey no. 3 led to subclinical reactivation of latent SVV, which in turn led to primary SVV infection in monkey no. 1. Although irradiation seems to be the most likely reason for virus reactivation, we cannot rule out the stress of captivity or related factors which have been predicted earlier to result in virus reactivation (5). Correlative clinicopathological and virological information provided by this first study of even a single monkey is relevant to understanding the pathogenesis of varicella infection. Further, our results suggest the usefulness of irradiation of primates latently infected with SVV as a model for VZV reactivation in humans. Future studies will analyze SVV reactivation in latently infected immunosuppressed monkeys.