4th National Symposium on Biosafety

Strategies of Managing Macaque Monkeys and Herpes virus simiae (B-virus)

Julia Hilliard, PhD
Southwest Foundation for Biomedical Research
PO Box 28147
San Antonio, TX 78228-0147
210-6674-1410

I am going to provide you with a better understanding of SPF monkeys and review some concerns about Herpes B, a zoonotic disease.

Sometimes, everything breaks down because of the animal sources we use. We usually buy SPF animals from our friends and colleagues. We trust that the label depicts a true representation of what the animal has gone through in terms of testing and achieving status as an SPF animal. There are some basic virology and serology techniques, and some basic diagnostics that will assist us in assessing what kinds of animals we have, be they rats, rabbits, mice, monkeys, or whatever.

Concerns about B virus began to pick up a lot of momentum in 1987 when we saw what we thought was a resurgence of a zoonotic disease. Since then, I've had the opportunity and privilege of studying many different cases including a fairly new one in Oregon, an older case in California, and an antibody-positive case with no reported disease in New Mexico. I've classified these into deaths, virus-positive, non-fatal cases, antibody positive cases, and antibody cases with encephalitic disease. There's been adeath in Texas and six seropositive cases, all at one institution. Another case has developed in Illinois; one in Michigan; an antibody-positive Vietnam veteran; several deaths in the State of Florida; an antibody-positive one in the Washington area; and another antibody-positive case in Massachusetts. We've seen a cluster of people becoming infected, then a lull, and now another burst of activity. In fact, I'm not sure we had a lull, but that's another discussion altogether.

For those of you unfamiliar with this disease, a monkey infected with B virus does not have to demonstrate any kind of active lesion. The monkey can have conjunctivitis, but can have perfectly healthy eyes, mucosal membranes, and not be problematic at all. We tend to disregard injuries sustained from "healthy" animals or traumatic injuries, thinking that they are certainly not a source of virus to worry about. In fact, it is a nice wet mucosal membrane, and it's good for growing virus. Sharps (metal objects, syringes, wiring, latches, etc.) are frequently found on cages, so these are all points of contact where we or the animals can sustain injuries.

Let me stress that we have guidelines, wisdom, and a lot of information. We also have a lot of information with respect to how viruses get into other species. With many viruses, we believe that for the virus to cross into another species, there has to be a significant virus load or a severe enough injury so that we can inoculate the virus into the other species. In fact, in the laboratory we sometimes barely scratch the surface of the skin to infect a mouse when doing a standard scarification procedure. We've found that the severity of the injury does not really matter, which is a bit surprising to many people.

We've had active cases of B virus infections in humans following as little as a scratched finger. We can have a bite that heals, and gives no sign or signal that it needs more immediate attention. In fact, we shouldn't look to the monkey or to the injury to try and determine whether we've had a transmission of this virus. When we do have a transmission of infection, we go through a period when we have a wide open window to do a great deal of information gathering. The first 5 or 6 symptoms that one encounters are simply cold or flu-like symptoms, so it is very hard to differentiate at this time whether you should get serious or not. As soon as the central nervous system becomes involved, the window starts closing very rapidly. Unfortunately, if we do get to this stage, there is very little that anti-virals can do to interrupt this disease. This window has been characterized in the literature as about a 3 week time course. In fact, it can be six weeks, 4 months, a year, 2 years, or 10 years, so time is really irrelevant.

The type of injury is irrelevant also. We don't have enough information about viral load, but certainly with this time course, we have a lot of latitude with certain cases. Comparing a normal healthy brain with a brain that is infected with B virus, we see first that the entire brain stem is hemorrhagic, and then it collapses. Between C1 and C5 there is complete liquefaction of the spinal cord. As the disease progresses, the entire cord gradually becomes nothing but a pustular mass.

Monkeys are the resource with which we can do the most with in terms of disease prevention, and it doesn't matter what kind of macaque monkey we talk about. All of them can be equally hospitable hosts for Herpes B, and they have done quite well living with B virus. It causesvery little to no problem in them, just as HSV causes few major problems for humans. You can't look at the monkey and remember that. You can't tell if a monkey is healthy or sick. In light of this information, about 4 or 5 years ago the NIH funded a number of centers to produce B virus-free macaque breeding colonies. NIH is extending the scope of this project to breeding colonies of pigtails, as well, and some centers are attempting to establish similar cynomologous breeding colonies. These centers are rather geographically-distributed, and we have worked with each to try different test strategies for producing SPF animals.

What is the status of these colonies? What's been done to these colonies? What is an SPF colony? How are they produced and are they all the same? Are they all comparable?

In 1990, when we actually began looking for animals to include in this "club", 12.1% of 984 potential entrants were found to be seropositive. We were going after the very best monkeys that we could find, and still 12% were seropositive for anti B virus antibodies. From 1990 to 1994, we rigorously tested many of these colonies; we did ELISA, western blots, confirmatory assays, and repeated testing sometimes every 8 weeks on these animals. You'd think that would clean it up for sure, in spite of the fact I was trained in a school believing that only c-section derived animals could be this clean. The problem with c-section derived animals is that we don't end up with hardy breeders.

In 1994, we were up to 2,174 breeding animals, and we had a 5.8% history of one or more non-negative tests. Now, a non-negative test is or can be a very confusing term. Of those 5.8%, only 0.4% were seropositive. Let me explain the problems with non-negative animals.

We need to form an idea about what to do with non-negative animals, particularly if we're die-hard advocates of having a specific pathogen free monkey. In summary, we started in late 1989 with 6 institutions doing frequent serologic testing and monitoring the first year. Animals were sampled every 4 to 6 weeks in some colonies, and multiple tests were run. This is a very critical point. Multiple tests need to be run because you increase the sensitivity and specificity of your tests if you run serial or complementary tests. That's great when you're beginning a colony like this, but may not be so helpful later. Most institutions employed single cage housing during the first year. Some institutions housed serologically negative animals in pairs or harems when they felt confident that they had been through a two year testing period and were reasonably assured that they were clean animals. During the second, third and fourth years we moved serologically negative animals together and began to set up larger SPF groups. Different places use different strategies, but some went to group housing much earlier than others.

As colonies approach SPF status, it's very hard to apply the same testing paradigm that you have been previously using. The prevalence of the disease has now dropped to a very low state, and it's very hard to apply diagnostics to a condition that is very rare. So now with the SPF colonies, we're in the same position as we are with human testing. That is, seronegative latent (false-negative) and non-cross reactive (false-positive) animals emerge.

Only 0.4% of the animals were seropositive in the fifth year. That's 9 out of 2,000 breeders. One animal was negative for 9 months and then indeterminate for a period of 25 months. The patten of seropositivity can decline, it can increase, and it can cycle. We've had an animal that stayed clean through 1990, 1991, and 1992. In 1992, it showed a positive blip on one test but not the other 2 tests, then returned back to negative. So, it's easy at this point to say " I think that's a false-positive". "We'll leave that animal within the group." But then, in the middle of 1992, the animal again showed a positive blip and a month later he's got positives on all tests. What that means is that we don't understand enough about the pathogenesis of latency and reactivation. We have had other examples. As you might imagine, you get every possible example with 2,000 breeders and their offspring.

One animal stayed negative until a blip occurred in 1990, but through 1993 all tests were negative, so do we consider that a false positive in 1990? Increasing patterns of sero-reactivity are a little bit easier and a little bit more straightforward. In another case in 1990, we went through negative tests, and in 1992 we started to see some spiking of activity. Again, it wasn't confirmable as a seropositive animal, but by 1993 and 1994, we were clearly working with a positive animal. Where did that animal get the virus? He's been in the corral group since 1989, he's tested clean, and all of a sudden, he springs forth as a positive animal.

We tried a series of tests to see if we could look at those low level virus shedders and determine if there were definitive tests we could do to identify those animals sooner and get them out of the colonies. In fact, we're still quite a ways from that. One animal was followed by immunofluorescence assay, by competition ELISA, by western blot, and by titration ELISA. The results went up and down, and sooner or later, he developed more "significant" banding patterns on the western blot, even as his IFA, (which is not a very sensitive test), becomes positive.

We have become strong believers in testing animals frequently by using a variety of testing strategies. We don't want to miss those animals or call them indeterminate when, in fact, they have detectable antibody. When we're talking about undetectable antibody in some of these animals, it is unlikely that they have zero antibody. We're talking about making the test more and more sensitive in order to identify these low level antibodies. The bad news about these animals, when they pop up, is that they have been in harem groups. They've been part of the program now since 1990. I'd like to point out that an animal will occasionally leave the harem for hospitalization or for some other procedure. It will return to the harem and within a few months it will suddenly be sero-positive. We don't know what happens in these circumstances. We know it's very important for each of the breeding facilities to very carefully monitor every move of the animal, because that animal may come back positive and can clearly infect other animals in the group. If you know that in some cases it takes up to 25 months to detect antibodies, then you clearly have a problem of having compromised all of your hard work for the last 4 or 5 years.

Achieving and maintaining these colonies means differentiation of false and true sero-conversions. That's a very difficult task that cannot be done easily. Containing seronegative latent infections is very important in maintaining the integrity of the colonies. But if you are a die-hard about having a seronegative colony or an SPF colony, these are the kinds of considerations that you have to take into account. You have to be able to integrate case history and housing records to identify contacts.

When you work with an institution from which you derive an SPF animal, make certain that they have very tight housing records-i.e., when animals may have gone into the hospital; when they may have been treated; or when they may have been around other animals. Evaluate the serologic status of the colony as a whole, rather than as a single animal. A single animal without the restof the colony information is rather meaningless and can get you in trouble. The reason I point this out is that after all of the years and all of the talks, we still get calls from people who have sustained injuries from macaques who say, "But we don't need to test because we're working with an SPF animal". Presently there are very few, if any, SPF animals. We may be approaching it in this NIH project, but we're not far enough into this project to guarantee that we have seronegative B virus-free animals. We're almost at that point, yet we're almost at that point of jeopardizing this whole production because of financial constraints and NIH limitations. But if you do want to use these animals, then you want to play a dynamic role in maintaining the population that has been developed over the last 6 to 7 years. The recommendations at this point (from what we have learned since 1989) are that these colonies essentially should be closed to immigration. We shouldn't put new animals into these colonies. Only the progeny animals should be contributing to the population increase. We shouldn't trade animals back and forth. When I send you a negative animal, I essentially lose my ability to monitor that animal and not just serologically. Of course, you can send me serum and swabs to test, but I lose track of all of the places that animal can go to become exposed.

Maintaining a housing database is essential. This is extremely important for facilities and institutions that work with a large number of these animals. Many of you only handle a small number and you just want to make sure that they're clean during the short interval of time that you have them on an experimental protocol. Test negative groups semi-annually. Don't mix in new animals. Once you get to the point where most of the animals are clean, as they are now, test often, because you will have false positives and false negatives in any kind of tests. Use a more cautious approach, a consolidated approach to testing, so that you can use a constellation of tests and put all that information together.

Isolate positive animals and contacts immediately; especially watch those contacts. When you are in the second year of your protocol and discover that you had an animal break, you really want to return to frequently testing the other animals that have been co-housed. If you have sero-negative animals that you are going to necropsy, please consider sending us the trigeminal and any of the dorsal root ganglia, preferably all of them, so that we can begin to get a handle on the limits of sensitivity of our diagnostic abilities.

To show you the strategy and how complex it is, most of the institutions that are actively participating go through a program of testing the animal on SPF-ELISA. If it is negative, it certainly goes into the SPF group. But, there are other ways it can get into the SPF group that can be a bit more nefarious in the end. Serum can be tested by a western blot and have an indeterminate result if it was positive on ELISA. But if it is indeterminate here, and then it goes into the competition ELISA, which is a bit more rigorous, it can be positive and go in this way. Or, if it is clearly positive on the western blot or the competition ELISA, it then gets culled out of the group. So, you can have a cycle of animals that are constantly tested that constantly fall into different categories.

During the first year 12.1% of the animals were culled because of B virus. This dropped to 1.3% in the second year, and 0.5% thereafter. Repeat testing demonstrated that most colonies have remained at 0.5% through years 4 and 5. We're not seeing an increase at this point. But we are seeing the sporadic individual animal break. We don't know what the long term implications of that are going to be. The take-home message must be the risks of one-shot testing.

If you're going to get a B virus-free animal, make sure you understand that there's a big difference between a B virus free animal and an antibody-free animal. False negatives, seronegative-latent hosts, and immunologcally unreactive hosts are always a possibility in early stage disease, a time when we have no markers to detect false positives or unidentified cross reactors. We have not found any definitive leads on what things may interfere with our tests after testing thousands of potentially cross-reactive viruses, etc. The benefits of comprehensive testing, the benefits of using a battery of tests along with case histories, and the epidemiologic analysis, is a real imperative when we handle our macaques, particularly our specific pathogen free macaques. Most importantly, whenever an injury occurs and there's been a potential exposure of a human, we should test an animal, whether it is SPF-derived or not.

Strategies of Managing Macaque Monkeys and Herpes virus simiae (B-virus) Breakout Session
Rapporteur: Louisa Chapman, MD Medical Epidemiologist Centers for Disease Control and Prevention 1600 Clifton Road Atlanta, GA 30333 404-639-1028

Dr. Hilliard outlined important lessons emphasized through the human cases of B virus infection she has reviewed over the past decade:

The best opportunity to intervene effectively to prevent human B virus infections is with the monkey. NIH has funded efforts to produce macaque colonies free of B virus. Specific operating protocols have varied between these centers, but in general all colonies housed all macaques in single cages for the first year and performed frequent serologic testing. During years 2-4 macaques that tested repeatedly negatively were moved to grouped housing. The predictive value of any test results is always a function of the prevalence of disease in the tested population. Therefore, as the prevalence of B virus infection in the colonies decreases, the value of serologic testing also decreases because the difficulty of interpreting results of serologic testing increases because the rate of false-positives tests due to cross-reactions and of sero-indeterminent results increases. (This is not unique to B virus serology; this is true in all instances in which diagnostic testing is used for screening of clinically healthy individuals.) Some monkeys have tested positive after several years of residence in so-called B virus free colonies.

It is uncertain at present how to interpret these. Some clearly represent false positive testing results. Some are monkeys who were removed from the colony because of illness or other reasons and became infected on the outside before returning to the colony. Some are probably monkeys who themselves were infected within the confines of the so-called B virus free colony by monkeys that had been removed, infected, and then re-introduced from the outside. It is unclear whether or to what extent some of these may represent monkeys who had long periods of clinical and serologic latency between the time of infection and seroconversion, or whether some may be animals who, although latently infected, reverted to a seronegative status, then appeared to sero-convert following reactivation disease. Our knowledge of the pathogenicity and immunology of B virus latency in these macaques is too incomplete. However, what is clear is that although SPF colonies can produce macaques that are less likely to be infected with B virus, there are at present no colonies within the United States that can be guaranteed to produce B virus free monkeys without fail. Therefore, caution must still be exercised with macaques from any source in the United States. In addition, serial testing of any macaqueshould be performed before it is assumed to be B virus negative. Dr. Hilliard's lab would like to receive the trigeminal and sacral ganglia from necropsied macaques that had non-negative test results (indeterminant results) for PCR studies aimed at improving the understanding of how indeterminent test results should be interpreted.

Breakout discussions:

The guidelines for the prevention and treatment of B virus infection in exposed humans published in Clinical Infectious Diseases in February 1995 were a composite document intended to describe the current state of clinical understanding of human B virus infection and provide guidance to the physician on the spot to evaluation and treatment of exposed persons, and to people charged with developing institutional occupational health policies. Universal agreement does not exist even among the named authors on all points in the document, and this document can assist but cannot replace the clinical decision making necessary on evaluation and treatment of human infections.

How much risk does macaque blood actually pose? B virus generally is not thought to be viremic. Uncertainty exists as to whether or not there is an early viremic phase just after infection, or whether disseminated disease in immunocompromised animals spreads by blood or along nerves routes. Dr. Hilliard has never documented B virus in blood or serum of macaques either by culture or PCR, and tends to think that viremia, if is occurs at all, is very rare. However, there are reports in the literature of isolation of B virus from blood or serum and human infections from needle stick injuries that she cannot fully explain. In some instances the needle stick may have occurred through gloves contaminated with B virus from saliva or other sources, or the blood may have contained epithelial cells that are the source of the virus. Additionally some people feel that dissemination of B virus into the spleen or liver argues for viremia, although these organs also are innervated and again spread along nerve routes is still thought by most herpes virologists to explain this source of virus in internal organs. This is an area where uncertainty remains, but in general blood is thought to carry little risk of infection.

Virus is in saliva because it is shed from the buccal mucosa. Dr. Hilliard has identified B virus in the stool of infected humans, but not in macaque stool.

The most important specimens for human diagnosis are acute and convalescent sera, preferably 4 weeks or more apart. If you contact Dr. Hilliard's lab they will give you a list of additional specimens that optimally they would like to receive to assist in diagnostics on any human case. In general the virology lab can give more help with interpretation in situations of uncertainty if they have maximum information, including acute and >4 weeks convalescent serum on the patient and the monkey, wound cultures on the patient and conjunctiva, buccal mucosa, and other relevant site cultures on the monkey. However, in addition to what is ideal virologically, we must consider what is ideal mdically, economically, and practically. Occupational health standard procedures that are so cumbersome that workers become reluctant to report minor injuries may backfire in that workers may avoid reporting injuries at all unless they are witnessed or very major. All these considerations must be weighed in deciding on policy for occupational injuries. In general routine biopsies of potential exposures at the time of injury are neither necessary nor desirable, and Dr. Hilliard does not recommend them. However, biopsies of sites with active infection may be valuable in extending our understanding of human infection even days after the injury. In general, Dr. Hilliard will request a biopsy when she feels it will be helpful, generally in instances where infection is proven or highly likely. Dr. Hilliard has had 100% recovery rate of virus from punch biopsies obtained when acute infections have been documented.

How long can virus survive outside the body and remain infectious? Virus is lifeless without being in a cell that can support it's growth. However, in controlled experiments with B virus intentionally inoculated in dried sterile water on a stainless steel plate, virus could be cultured from the site after 7 days. Virus does not pose a threat to humans by its mere presence on a surface in the absence of a portal of entry. However, injuries resulting from cages and other inanimate objects potentially contaminated with B virus by shedding macaques must be considered to carry real risk as sources for human infection.

What is CDC's policy on the biosafety hazard associated with macaques? All macaques are assumed to be B virus positive and treated accordingly. They are not routinely screened, but they are cultured and blood collected for serologic testing in association with potential human exposures. All macaques are kept in biosafety level 2 and all have biohazard signs.

The value of prophylaxis at time of injury is controversial. In most instances the person will not have been exposed since most infected macaques are not shedding at any given point in time. It is doubtful that acyclovir prophylaxis will prevent B virus contamination from progressing to infection, although it may delay seroconversion. In general the recommendation was to be reluctant to prophylaxis but aggressive in treatment of any human disease that may be B virus until it proven not to be. However, the recent MMWR publication of study results suggesting that antiviral prophylaxis of needle stick injuries may decrease risk of HIV infection of health care workers should be reviewed and the decision to prophylax potential B virus exposures considered in light of it.

In general the most important measure for cleansing wounds is through prolonged cleansing as rapidly after injury as possible. Agents used for cleansing are less important than the rapidity and thoroughness of the cleansing. Dr. Hilliard does recommend the use of some type of detergent because it removes the envelope and therefore inactivates the virus.

Laboratorians working with macaque tissue and specimens are at risk as well as monkey handlers. About half of the human infections that Dr. Hilliard has documented have been in laboratory as opposed to animal workers.

Non-macaque monkeys are not likely to be infected with B virus unless they have been housed with macaques. Most frequently infected non-macaque monkeys develop disease similar to human B virus disease.

Reference:

Holmes GP, Chapman LE, Stewart JA, et.al., Guidelines for the Prevention and Treatment of B-Virus Infections in Exposed Persons. Clinical Infectious Diseases, 1995; 20:21-39.

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Strategies of Managing Macaque Monkeys and Herpes virus simiae (B-virus)

Breakout discussions:

Reference: