CHAPTER 19

The Pathogenesis of Torovirus Infections in Animals and Humans

MARION KOOPMANS AND MARIAN C. HORZINEK

I. INTRODUCTION

In 1992, the new genus torovirus was added to the family Coronaviridae (Pringle, 1992; Cavanagh and Horzinek, 1993), ending aperiod of controversy about the assignment of this taxonomic cluster. Toroviruses are enveloped, positive-stranded RNA virus es that may cause enteric infections in animals and humans. The first descriptions mentioned superficial morphological re­ semblances with coronaviruses, both being 80-120 nm, enveloped, peplomer­ bearing particles with a pleomorphic appearance as seen by electron micros­ copy (EM) (Weiss et a1., 1983; Woode et a1., 1982). However, further studies revealed morphological and antigenic differences, sparking discussions ab out the place toroviruses should occupy in taxonomy (Horzinek et a1., 1984, 1985, 1986,1987; Horzinek and Weiss, 1984; Koopmans et a1., 1986; Weiss et a1., 1984; Weiss and Horzinek, 1986, 1987; Woode et a1., 1982, 1985). The matter was settled when studies of the replication mechanism and genomic sequence showed fundamental similarities between toro- and coronaviruses (reviewed by Snijder and Horzinek, 1993; Bredenbeek et a1., 1990; den Boon et a1., 1991; Snijder et a1., 1988, 1989, 1990 a-c; see also Chapter 11, this volume). The torovirus prototype Beme virus (BEV) was isolated in Beme, Switzer-

MARION KOOPMANS • Virology Seetion, National Institute of Public Health and Environ· mental Protection, 3720 BA Bilthoven, The Netherlands. MARIAN C. HORZINEK • Virol­ ogy Division, Department of Infectious Diseases and Immunology, University of Utrecht, 3584 CL Utrecht, The Netherlands. The Coronaviridae, edited by Stuart G. Siddell, Plenum Press, New York, 1995.

403 404 MARION KOOPMANS AND MARIAN C. HORZINEK land from a rectal swab taken from a horse with severe gastroenteritis 1 week hefore it died (Weiss et a1., 1983). Salmonella lilIe, isolated from the same swab, was considered to be the cause of the disease in this animal, and BEV has remained a virus in search of a disease. However, since it grows in cell culture and has therefore been extensively studied by the Utrecht group, BEV has been designated the torovirus prototype. The situation is quite different for another torovirus that had been de­ scribed 1 year before; it had been found by EM in feces from calves in a dairy herd in Breda, Iowa, that had severe diarrhea (Woode et a1., 1982). Breda virus (BRV) does not replicate in cell or tissue culture, hut has been identified as a pathogen causing gastroenteritis in calves and possibly in older cattle (Koopmans et a1., 1990, 1991c; SaH et a1., 1981; Woode et a1., 1982, 1985). In the years following the discoveries of BEV and BRV, serological evidence of torovirus infection has been obtained in all ungulates that were tested using a BEV neutralization test (horses, cattle, sheep, goats, pigs), and in rats, rabbits, and some species of feral mice (Weiss et a1., 1984). Also, toroviruslike particles have been detected in stool specimens from pigs (Penrith and Gerdes, 1992; Durharn et a1., 1989; Scott et a1., 1987; L. SaH, personal communication), humans (Beards et a1., 1984, 1986; Brown et a1., 1987; Koopmans et a1., 1991a, 1993b), cats (Muir et a1., 1990), and dogs (Hill and Yang, 1984). There is little doubt that solitary torovirions have been seen by electron microscopists, but their pleomorphism precluded their identification as virus es since confirmatory testing was unavailable (Koopmans et a1., 1991a, 1993b; Liebler et a1., 1992). No antibodies have been found in the sera of cats and humans (Brown et a1., 1988; Weiss et a1., 1984). The purpose of this chapter is to highlight epidemiological and clinical studies with an emphasis on the pathogenesis of toroviruses. We will focus on bovine toroviruses since most information on the infection in vivo has been obtained from studies in cattle. For an update on the structural and morphologi­ cal properties of the virus, on its replication strategy, and on diagnosis of torovirus infections in animals and humans, the reader is referred to other recent reviews (Koopmans and Horzinek, 1994; Snijder and Horzinek, 1993) and to Chapter 11, this volume.

11. INFECTION IN CATTLE

A. Enteric Infections

Three strains of BRV have been used to study the course of infection in its natural host. Besides the original "isolate," two additional strains of bovine enteric toroviruses have been reported; the three strains were assigned to two groups (BRVI and BRV2) based on antigenie comparisons: BRVI refers to the first Iowa strain, and BRV2 comprises the two strains that had been detected in feces from a 5-month-old diarrheal calf in Ohio and from a 2-day-old calf in Iowa (SaH et a1., 1981; Woode et a1., 1983, 1985). All three strains are pathogenic for newborn gnotohiotic and nonimmune conventional calves, aged 1 hour to 10 weeks (Woode et a1., 1985). PATHOGENESIS OF TOROVIRUS INFECTIONS 405

Although the respiratory system may be sporadically involved (see Seetion II.C), BRV infections are usually limited to the gut. Between 1 and 3 days after oral inoculation, calves typically develop a watery diarrhea that persists for 4 to 5 days (Woode et a1.,1982, 1983, 1985). The most severe symptoms occurwithin 2 days after onset of the diarrhea, with dehydration, weakness, and depression (Woode, 1987). In the presence of a normal intestinal flora (but in the absence of maternal antibodies), diarrhea generally is more severe than in gnotobiotic calves. Both crypt and villus epithelial cells are infected from the midjejunum through to the large intestine (Woode et a1., 1982; Woode, 1987). Infections with BRV seem to be ubiquitous, as evidence of infection has been obtained in every country where serological and/or virological studies were done: Belgium, Great Britain, France, Germany, India, Italy, The Nether­ lands, Switzerland, and the United States (Vanopdenbosch et a1., 1992a; Brown et a1., 1987, 1988; Lamouliatte et a1., 1987; Liebler et a1., 1992; Koopmans et a1., 1989; Weiss et a1., 1984; Woode et a1., 1985). In addition, unconfirmed torovirus­ like particles have been found in cattle from South Africa (Penrith and Gerdes, 1992) and New Zealand (Horner, personal communication). The infections are quite common in dairy cattle; by 1 year of age, 85-95 % of the animals have antibodies to BRV (Koopmans et a1., 1989; Weiss et a1., 1984; Woode et a1., 1985). In a study among dairy cattle in The Netherlands, most seroconversions took place between 6 and 12 months of age, after the waning of maternal immunity (Koopmans et a1., 1986). The presence of maternal antibodies in calves (in 95% of the animals) did not prevent infection, but may have modified its outcome, since diarrhea generally was mild in BRV-excreting calves (Koopmans et a1., 1990, 1991c). BRV infections accounted for 4 % of cases of diarrhea in calves in this study. BRV­ associated diarrhea under farm conditions was clinically indistinguishable from that caused by the most common enteropathogens (rota- or coronavirus), although it las ted slightly longer (average 9.2,6.8, and 6.8 days, respectively, for the three viruses) and affected slightly older calves (average 12.7, 7.7, and 8.3 days, respectively) (Koopmans et a1., 1991c). Calves 3-4 months of age showed very mild diarrhea O! no diarrhea at all in association with torovirus shedding (Koopmans et a1., 1991c). Besides its association with gastroenteritis in young calves, BRV is a pos­ sible cause of diarrhea in adult dairy cows (Koopmans et a1., 1991c).

B. Pathogenesis of Enteric Torovirus Infections

The exact pathogenic pathways that typify torovirus infections in cattle are not known. However, results from experimental infections of calves suggest a mechanism similar to that of bovine coronaviruses (BCV) (Clark, 1993). Calves may be infected with BRV by the oral route and possibly by the respiratory route (Woode et a1., 1982, 1985; Vanopdenbosch et a1., 1991). The epithelial cells lining the smaH and large intestine become infected, with progression from areas of the midjejunum down through the ileum and colon (Fagerland et a1., 1986). Within the smaH intestine, not only epithelial ceHs at the top of intestinal villi 406 MARION KOOPMANS AND MARIAN C. HORZINEK

are infected but also cells in the upper third of the crypts (Fagerland et a1., 1986; Pohlenz et a1., 1984). This has important consequences for the outcome of the infection as can be deduced from the physiology of the gastrointestinal tract: in an uninfected gut there is a well-attuned balance between the sequestration of cells at the top of the intestinal villi and the production of new enterocytes in the crypts. Crypt epithelial cells rapidly divide and are pushed up toward the top of the intestinal villus. During this process the new cells mature into absorbing cells by the development of fingerlike (brush border) projections that increase the cell surface many times and by the production of brush border­ associated enzymes. Following a toroviral infection, the turnover rate of epithe­ lial cells increases because infected cells die and detach (Fagerland et a1., 1986; Pohlenz et a1., 1984), leading to an efficient spread of the infection. In fecal preparations from experimentally BRV infected calves, hemagglutination (HA) titers up to 3 x 107 units/ml have been measured (Woode et a1., 1983), corre­ sponding to virus titers of 10 11 to 1012 (Zanoni et a1., 1986). With such high titers the infection will spread rapidly. The lost epithelium is replaced by immature cells that do not yet possess a mature brush border, thereby presenting insuffi­ cient absorptive surface and digestive enzymes (15-65% reduction in D-xylose resorption in BRV-infected calves) (Woode et a1., 1982, 1985). Since BRV infects both crypt cells and the villus epithelium, restoration of the normal structure and function may be slower than after infections that affect only the villous epithelium (e.g., by rotaviruses and BCV). A longer duration of diarrhea can therefore be expected (Koopmans et a1., 1991c) with more severe complications. The decrease in digestive and absorptive capacities leads to accumulation of lactose in the gut lumen, which in turn results in water and electrolyte reten­ tion leading to diarrhea and occasionally to dehydration, acidosis, hypoglyce­ mia, and death.

C. Respiratory Infection

Recently, Vanopdenbosch et a1. (1992a,b) reported the isolation of a toro­ virus from the respiratory tract of calves with pneumonia [bovine respiratory torovirus (BRTV)]. If confirmed, this finding would suggest that bovine toro­ viruses are both entero- and pneumotropic, as has been clearly demonstrated for BCV (Reynolds, 1983; Reynolds et a1., 1985; Saif et a1., 1986). Most likely different strains are involved, because respiratory tract symptoms were rarely observed in calves after experimental BRV infection that suffered from diarrhea, whereas in the calves described by Vanopdenbosch et a1. (1992a,b) respiratory symptoms were predominant (Koopmans et a1., 1990; Woode et a1., 1982, 1985). Also, when testing RNA extracts from cells infected with the BRTV isolate in a diagnostic polymerase chain reaction assay with BEV /BRV consensus primers, no detectable amplification product was obtained (M. Koopmans, unpublished results). The "respiratory" isolate is the only bovine torovirus that has been adapted to cell culture, but its presence still needs to be confirmed. Using infected cells in an immunofluorescence test, Vanopdenbosch et a1. (1992b) found high levels of antibody in all calves (n = 50) that persisted for 5 months. PATHOGENESIS OF TOROVIRUS INFECTIONS 407

These authors also detected antibodies in 20 batches of fetal bovine serum, in contrast with earlier findings (Koopmans et a1., 1990; Woode et a1., 1982).

D. Infection of Other Organ Systems

The unexpected finding of antibodies in all commercially available batches of fetal bovine serum and in 7 of 13 precolostral calf sera (Vanopdenbosch et a1., 1992b) indicated the possibility of transplacental torovirus infections in cattle; this concept was underscored by the demonstration of toroviral antigen in placental cotyledons of spontaneously ahorted calves using an immunofluores­ cence assay. The same authors suggested a possible role in central nervous disturbances, sudden death, and in a syndrome resembling mucosal disease (Vanopdenbosch et a1., 1992a).

E. BRV Infection and the Immune System

In addition to crypt and villus epithelial cells of the small and large intes­ tine, BRV also infects the dome epithelium overlying Peyer's patches. The dome epithelial cells, including the M cells, show the same cytopathic changes that occur in the absorptive villous cells (Woode et a1., 1984; Pohlenz et a1., 1984). In addition, the germinal centers in Peyer's patches of BRV-infected calves were found depleted of lymphocytes and occasionally showed fresh hemorrhage (Woode et a1., 1982). The M cells are specialized gut epithelial cells that endo­ cytose macromolecules and microorganisms from the gut lumen and present them to underlying lymphoid tissue; they playa key role in the development of an immune response against enteric pathogens. Direct or IgA-mediated adher­ ence to M cells has been observed for other viruses that infect them (bovine astrovirus) or use their trans epithelial transport capacity to cross the epithe­ lium and to invade neural and lymphoid tissues (reovirus and poliovirus) (Pear­ son et a1., 1978; Sicinski et a1., 1990; Weltzin et a1., 1989; Wolf et a1., 1981). Infection of M cells by BRV and bovine astrovirus results in a cytopathic effect, while poliovirus and reovirus infections are noncytopathic. It is unknown whether BRV (or astroviruses) pass the epitheliallining via the M cell pathway; no such evidence was obtained in clinical and pathological studies (Fagerland et a1., 1986; Woode et a1., 1982, 1985). The tropism of BRV for dome cells may have consequences for the (mu­ cosal) immune response. Development of BRV-specific IgM antibodies inter­ estingly occurred very late after primary infection of sentinel calves « 1 month of age), and no memory response was seen in them after a second BRV infection at 10 months of age (Koopmans et a1., 1990). An alternative explanation for these findings may be the suppressive effect of maternal antibodies on infection: In experimental BeV infections the immune response was delayed in calves that had been fed colostrum with high titers of specific antibody compared with the response in calves fed low-titered colostrum (Heckert et a1., 1991). 408 MARION KOOPMANS AND MARIAN C. HORZINEK

F. Chronic Infection

Chronic torovirus infections also may occur, as data from arecent epidemi­ ological study suggest. In a closed herd of 10 dairy calves repeated torovirus shedding was observed in several animals, with intervals of several weeks to 4 months. When the calves were introduced into a herd of adult cows, they all had an episode of diarrhea in association with BRV shedding, followed by serocon­ version. The adult cows showed no evidence of active BRV infection in the 2 weeks preceding the arrival of the calves, but they all had high levels of preexist­ ing antibodies (Koopmans et a1., 1990). These observations indicate the pres­ ence of carriers that shed low levels of virus or undergo recurrent subclinical infections. Virus persistence and shedding may be an important source of virus in epizootics of neonatal torovirus infections. Infections with BCV may serve as a model for understanding the situation in bovine torovirus infections, given the elose resemblance in other aspects of the pathogenesis of these viruses. Evidence of coronavirus shedding has been found in up to 75% of elinically normal cows (Collins et a1., 1987j Crouch and Acres, 1984), hut only 5% were shedding free virus that could be detected in regular enzyme-linked immuno­ sorbent assay (ELISA)j the remaining 70% were identified using an ELISA for the detection of immune complexes (Crouch and Acres, 1984). In a follow-up study, free virus could be detected intermittently in some animals, but immune­ complexed virus was present in the feces throughout the 12 weeks of the study (Crouch et a1., 1985). Chronic shedders can be a source of infection as shown by Bulgin et a1. (1989): calves from carrier cows had a 60% chance of developing clinical illness, whereas those from noncarriers had only a 22 % chance. With the development of highly sensitive, polymerase chain reaction­ based detection methods for toroviruses, the role of carriers in the epidemiology and pathogenesis of torovirus infections can now he addressed (Koopmans et a1., 1993a).

III. INFECTION IN HORSES

A. A Virus in Search of a Disease

Similar to the situation for bovine toroviruses, infection of horses is quite common in the populations that have been examinedj 81 % of the adult horses in Switzerland and The Netherlands possess neutralizing antibodies to BEV (Weiss et a1., 1983, 1984), 35% in Germany, and 38% in India (Liebermann, 1990). Equine toroviruses may cause similar disease pictures as BRV in calves, but epidemiological studies in populations most likely at risk have not been done so far. Diarrhea in young foals is a big problem, and epidemiological studies should be aimed at this age group «1 month of age). Infection experiments with BEV have been very limited: two yearlings that had been injected intravenously with 107 TCIDso of tissue culture grown BEV seroconverted without clinical symp­ toms (Weiss et a1., 1984). Virus shedding was not monitored in these horses. A PATHOGENESIS OF TOROVIRUS INFECTIONS 409

3-day-old gnotobiotic foal was inoculated orally, and again no symptoms were seen, although virus shedding and seroconversion occurred. Attempts to infect a 3-month-old foal were unsuccessful (Dr. F. Scott, Moredun Research Institute, Edinburgh, England, personal communication, 1992). Tissue culture-passaged virus was used in these experiments, which may be of low virulence as a result of the adaptation.

B. The Torovirus Mutant BEV

Irrespective of many attempts (Weiss, unpublished observations), isolation of BEV has been a unique event and could only be repeated with the field sampie from the same horse. This observation suggests that the Berne isolate is a mutant. Most parts of the BEV genome have been sequenced; it would be interesting to obtain sequence information from pathogenic toroviruses and to look for explanations for the difference in pathogenicity. Of special interest is a pre­ sumed pseudogene [open reading frame 4 (ORF 4)1 that has been identified in the BEV genome. The predicted amino acid sequence of the ORF 4 product bears similarities to the C-terminal part of the hemagglutinin esterase (HE) of BCV and of human influenza C virus; however, the 5' two-thirds of the HE gene are missing, and an ORF 4 product has not been identified in BEV virions or in lysates of infected cells (Snijder et al., 1991). The BCV HE (like the influenza C virus HE) uses N-acetyl-9-0-acetylneuraminic acid as a receptor to initiate the infection of cultured cells and probably plays a role in infection of host cells in vivo (Clark, 1993; Schultze and Herrler, 1992). Recently, 2 kb of the 3'-end of BRV has been sequenced; it was demonstrated that BRV has a complete HE gene that is probably functional (L. A. H. M. Cornelissen and R. J. de Groot, unpub­ lished observation; Koopmans et al., 1986).

C. Host Range

Infection with BEV in vitra is limited to cells of equine origin. Neutralizing antibodies have been found in sera from cattle, goats, sheep, pigs, rabbits, and feral mice, indicating a elose antigenie relationship between toroviruses of these species or cross-species infections with one or a few related toroviruses. The latter option is not likely. Mouse immune sera raised against BRV2 show very little cross-reactivity with BRV, except at the level of the peplomers; the sera recognized the polypeptides of the homologous virus and the two highest­ molecular-weight proteins (105 kDa and 85 kDa) of BRV1 in radioimmune precipitation. The same sera inhibited hemagglutination of the heterologous serotype and efficiently neutralized the infectivity of BEV (Koopmans et al., 1986). No evidence of viral replication was obtained in rats, mice, or lambs that had been experimentally infected with BRV (Woode et al., 1982; Woode, 1987). 410 MARlON KOOPMANS AND MARIAN C. HORZINEK

In contrast, the respiratory bovine toroviruses reportedly replicate in cells frorn a wide range of host species (Vanopdenbosch et al., 1992b).

IV. TOROVIRUS INFECTIONS IN OTHER SPECIES

Toroviruslike particles and torovirus antibodies have been found in species other than the cattle and horse, indicating that these viruses rnay infect a broad range of animal hosts (Muir et al., 1990; Scott et al., 1987; Weiss et al., 1984). Although in most cases stool specimens frorn animals with diarrhea were exarnined, the pathogenic role of these toroviruses remains unclear, and epide­ rniological studies are needed to study the causal relationship between virus presence and disease (Durham et al. , 1989; Hill and Yang, 1984; Muir et al. , 1990; Scott et al., 1987). The toroviruslike particles found in hurnans (Beards et al. , 1984, 1986) cross-react antigenically with BRV (Kooprnans et a1., 1993b); preliminary data frorn an ongoing epidemiological study in Brazil indicate an association with diarrhea (Kooprnans and Guerrant, submitted for publication).

V. REFERENCES

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