Monkey viral pathology in the Sukhum colony and modeling human viral infections.

The data characterizing spontaneous infections of Old World monkeys: measles, poliomyelitis, hepatitis A (HPA), encephalomyocarditis, coronavirus infection, simian hemorrhagic fever (SHF), are presented. The experimental infections were reproduced with the isolated pathogens. On these models, pathogenesis and epidemiology of these diseases were studied. The efficiency of poliomyelitis, measles and HPA vaccines is shown. The priority of data on the discovery of earlier unknown disease-SHF and "Sukhumi" virus-are emphasized. Several important pathogenic mechanisms common for various hemorrhagic fevers were studied on experimental SHF of macaques. This model is uniquely safe and adequate for the assessment of therapy of hemorrhagic fevers dangerous for humans.

INTRODUCTION

In this article, results of long‐term comparative research of primate viral infections of monkeys and humans are presented. These data are interesting for the characterization of monkeys as an object in medical‐biological research. Also, they are important for preventing epizooty and infection of personnel contacting with monkeys in a monkey colony. The characterization of spontaneous infections in monkeys is very important for further use of monkeys as models of analogous human infections. The similar to human manifestations of the most part of these infections can be reproduced in monkeys that is explained by evolutionary established similarity of main physiological systems of monkeys and humans. The evolutional similarity of infectious agents, seemingly duplicates of human viruses, should be also taken into account. The contribution made by monkeys to research of human virus pathology is invaluable. The use of these laboratory primates helped to prove etiology of the most part of viral infections, to study their pathogenesis and epidemiology, and to develop the preventive care.

The care and use of the monkeys are carried out in accordance with the rules of the humane care of animals in Russia (and in USSR) and with approved experimental protocols.

Studies of viral pathology were started at Sukhum Medical‐Biological Station of the USSR Academy of Medical Sciences in 1950s. Research of spontaneous and experimental poliomyelitis was performed under the guidance of academicians M.P. Chumakov and M.K. Voroshilova, measles—under the leadership of academician P.G. Sergiev.

Continuous virological research began at the Sukhum Institute in 1960s. Serological screening of 7 monkey species (more than 1000 animals) revealed antibodies to wide spectrum of viruses: measles, flu, parainfluenza, adeno‐, rota‐, entero‐, corona‐, and herpes. Infection with these viruses usually was inapparent. However, in a number of cases, activation of latent infections and also epizooties were observed. It happened because of weakening immunodefense (capture, acclimatization stress, radiation exposure).

SPONTANEOUS INFECTIONS

Short information on spontaneous viral infections of various monkey species studied in the Sukhum colony is presented in Table 1. Each infection proceeded with corresponding clinical and pathological signs. Infectious agents were identified. Some of them were identical to human viruses; others were simian viruses.

Table 1

Spontaneous viral infections of monkeys

Nosological diagnosis	Years	Monkey species	Isolated viruses	Family
Measles1, 2	1954,1962	Rhesus macaques, hamadryas baboons, Assam macaques	Human measles virus	Paramyxoviridae
Poliomyelitis2, 3	1957	Rhesus macaques	Human poliovirus	Picornaviridae
Simian hemorrhagic fever4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15	1964,1967,1974	Rhesus macaques, pig‐tailed macaques, patas monkeys	SHV strain “Sukhumi‐64”	Arteriviridae
Encephalomyocarditis16, 17, 18	1970,1978	Rhesus macaques, hamadryas baboons	Encephalomyocarditis virus, strain EMC‐70	Picornaviridae
Conjunctivitis pneumonia19	1972,1973	Rhesus macaques, hamadryas baboons	Monkey adenovirus SV‐14	Adenoviridae
Ulcerative stomatitis20	1974	Rhesus macaques	Monkey herpes B virus	Herpesviridae
Polio‐like enteroviral disease21	1981‐1982	Rhesus macaques	Monkey enterovirus SV‐49	Picornaviridae
Pneumonia and/or enterocolitis22	1982‐1986	Rhesus macaques, hamadryas baboons	Monkey coronaviruses, strains CVMR and CVPH	Coronaviridae
Hepatitis A23, 24, 25	1985‐1992	Cynomolgus monkeys, bear macaques, rhesus macaques, green monkeys, hamadryas baboons	Hepatitis A virus, strains VHA‐MR, VHA‐GM, VHA‐PH	Picornaviridae

Measles

In 1954, measles outbreak occurred in the Sukhumi colony. Infection spread among monkeys kept in the same building. In rhesus macaques and hamadryas baboons, the clinically apparent disease progressed. It was characterized by catarrhal signs and rash abundant. In cynomolgus and green monkeys, infection was inapparent.1 The second measles outbreak took place in 1962 among Assam macaques, which were in experiment with small doses of gamma rays during a year. The disease was severe, with characteristic complications and death.2

Poliomyelitis

In 1957, in the Sukhumi colony in rhesus macaques polio outbreak spontaneously arose. Clinical manifestations and pathomorphological changes specific for polio were registered.3 The main manifestations were paralyses of lower and upper extremities and respiratory muscles. A morphological study indicated a massive loss of anterior horn cells of the spinal cord in cervical and lumbar enlargements. Type 2 poliovirus was isolated from the brain of perished monkeys.

Simian hemorrhagic fever (SHF)

In 1964, an enzooty of new unknown disease occurred. This disease was called by us SHF. In a month, 55 rhesus macaques imported from India died. Main manifestations of the infection were hemorrhagic diathesis and damage of neurological status.4, 5, 6 Clinical signs were also fever, apathy, anorexia, tremor, ataxia, limb weakness (rarely, pareses). There was an increase in tendon reflexes and muscular tone. Most animals had petechial skin rash, more rarely ‐ nosebleed and intestinal bleeding. In the terminal period, temperature and blood pressure drastically decreased. Monkeys died in 8‐12 days. Disorder of blood circulation in the microcirculatory bloodstream, hemorrhagic diathesis in the central nervous system, diffuse encephalomyelitis, widespread destruction of lymphoid elements and focal coagulative necroses in parenchymatous organs were histologically observed. From the blood and brain, a filterable agent was isolated.6, 7 The virus differed from known causative agents of hemorrhagic fevers. He was apathogenic for small laboratory animals, did not possess hemagglutinating properties, and did not reproduce itself in any of 39 cell cultures. Only rhesus macaque embryonic kidney cells (PEMR) were permissive. Immunotyping showed that it was the new virus different from ones known earlier.6, 7 Virus strain discovered by Z.V. Shevtsova in 1964 and designated as “Sukhumi‐64” was registered at the State collection of viruses of the USSR Ministry of Public Health. The certificate was signed by Dr. V.M. Zhdanov, the member of International committee on taxonomy and classification of viruses. Two years later after our first communications,4, 5 American scientists reported that an enzooty of similar disease took place in the NIH rhesus macaque colony in Bethesda at the end of 1964.8 The isolated virus strain was designated as Bethesda 64.9 After exchange of virus strains between USSR and United States, it was found that viruses were antigenically similar but not identical.10, 11 Another 2 outbreaks were observed in the Sukhumi colony in 1967 and 1974. The infection source was African patas monkeys carrying latent infection and located in the same room as Asian macaques. SHF was the serious problem of primatological centers: 17 enzooties were registered by 1996.12 In some colonies, a number of died macaques was from 200 to 500.13 Later, SHV‐related variants were isolated from African monkeys of various species from wild animals as well as from those habitating in National parks. Today, 12 isolates are studied in detail by American and Russian virologists. All of them are arteriviruses, but differ by their genome structures and homology. The strain “Sukhumi‐64” was included in genus Arteriviruses as Sukhumi simarterivirus species called simian hemorrhagic encephalitis virus (SHEV). GenBank, USA, assigned own number to SHEV.14

Encephalomyocarditis

Group illness of encephalomyocarditis was registered twice in the Sukhum colony: among macaques in 1970 and hamadryas baboon in 1978. The disease proceeded with brain and myocardium damage and was lethal in a number of cases.15, 16 The encephalomyocarditis virus isolated from dead monkeys and designated as EMC‐70 was identical to the prototypical strain EMC‐Paris.16 This infection is still the serious problem of colonies and zoos because of many fatal outbreaks from 1980 to 2005.17, 18 Human illness is known only from outbreaks among recruits.

Adenovirus

From 1972 to 1973 year, there have been cases of acute conjunctivitis sometimes accompanied by pneumonia. From the conjunctive content of diseased animals was isolated a virus cytopathic for a number of cell cultures.19 Biological, physico‐chemical, and electron‐microscopic analyses showed that it was simian adenovirus SV‐37.

Herpesvirus

The group of rhesus macaques underwent radiation sickness and lapsed into ulcerative stomatitis. The simian herpes virus B was isolated from diseased sites.20

Enterovirus

In 1982, the infection was spread among rhesus macaques. It was clinically characterized by the disorder of lower limb locomotor activity and the development of atrophy and contractures. Histologically observed myositis with lysis of limb muscle fibers and inflammation of brown fat were found. The loss of neurons was detected in lumbar segments of the spinal cord. From diseased monkeys, the SV‐49 virus was isolated.21

Coronavirus

We also described spontaneous coronavirus infection in rhesus macaques and hamadryas baboons. It was persistent with periodical recrudescences including pneumonia and (or) enterocolitis.22 A morphological study revealed diffuse lymphocytic and macrophage infiltration of the intestine mucosa. In the lungs, changes in characteristic of pneumonia (the presence of giant cells, signs of carnification) were determined. Coronaviruses were found in bowel, pancreas, and lungs of perished monkeys. Strains were designated as CVMR and CVPH and registered at the State collection of viruses.

Hepatitis A

In contrary to existent opinion, we found the natural sensitivity of Old World monkeys to hepatitis A virus (HAV). Epizooties of this infection in 4 monkey species imported from natural habitats were studied and described by us.23, 24 Strains isolated from diseased monkeys were not antigenically different from human HAV.25

EXPERIMENTAL INFECTIONS AND MODEL DEVELOPMENT

The second part of our article is dedicated to modeling 6 above‐described infections in monkeys (Table 2).

Table 2

Models of viral infections

Infection	Used virus	Monkey species	Studied
Poliomyelitis26	Human poliovirus	Rhesus macaques, cynomolgus monkeys, green monkeys, hamadryas baboons	Pathogenesis, epidemiology, testing vaccine strains
Measles27	Human measles virus	Rhesus macaques, hamadryas baboons	Pathogenesis, vaccinal process, testing vaccine strains
Coronavirus infection28	Coronavirus of rhesus macaques	Rhesus macaques	Clinical and pathomorphological characterization
Encephalomyocarditis29, 30	Encephalomyocarditis virus EMC‐70	Rhesus macaques, hamadryas baboons, green monkeys, patas monkeys	Clinical and pathomorphological characterization
Hepatitis A31, 32	Human hepatitis A virus	Rhesus macaques	Clinical and pathomorphological characterization, testing vaccine
Simian hemorrhagic fever13, 33, 34	SHV “Sukhumi‐ 64”	Rhesus macaques	Clinical and pathomorphological characterization, thanatogenesis

Experimental poliomyelitis was induced in 4 monkey species. In animals infected by human poliovirus, paralyses of lower limbs and characteristic pathomorphological changes in cells of ventral horns of the spinal cord were observed.26 Problems of polio pathogenesis and epidemiology were studied with these models, and immunogenicity of live‐attenuated virus was shown. Obtained data allowed to develop successful prophylaxis of poliomyelitis.

Inoculation of 3 monkey species by material from children suffering from measles played an important role in study and prophylaxis of measles. Virological and histological research revealed main pathogenic mechanisms and the role of systemic change of reticuloendothelium and lymphoid tissue. Comparative research performed in monkeys gave data on pathogenesis of experimental measles and vaccinal process developed after introduction of live‐attenuated vaccine L‐4 (Leningrad‐4) used in measles preventive care.27

Experimental coronavirus infection of rhesus macaques and hamadryas baboons was performed using the strains isolated from diseased monkeys and antigenically related to human coronavirus OS‐43. Infection similar to spontaneous was persistent with the latent course and periodical recrudescences including pneumonia and (or) enterocolitis.28 Actuality of our model is defined by an increasing role of coronaviruses in human pathology, especially by the appearance of strain causing severe acute respiratory syndrome (SARS).

Experimental encephalomyocarditis was reproduced in 25 monkeys of 4 species.29 We showed that the virus strain EMC‐70 causes changes in monkey brain and myocard. At the acute stage of illness, ECG was similar to cardiogram of infarction patients. Encephalomyocarditis is a serious problem for many primatological organizations. The experimental primate model is actual and used for trials of vaccines and preparations.30

Hepatitis A (HPA) was reproduced in 4 monkey species (rhesus macaques, cynomolgus monkeys, green monkeys, and hamadryas baboons) inoculated with strains isolated from monkeys and 1 strain isolated from a patient.31 The most significant and studied HPA model is rhesus macaque infected by human HAV. This model is cheaper and more available than its previous analogues in chimpanzees and marmosets. We obtained results on infection chronicity and HAV persistence.32 Our data brought new knowledge about HPA epidemiology: HAV long‐term conservation in immune organisms of sensitive primates allows to consider primates as animal reservoir in intra‐epidemic period. Cultural inactivated HPA vaccine offered by German scientists F. Deinhardt and B. Fleming was tested in the rhesus macaque model.32 Its good protective properties were revealed. In the last years, using vaccines manufactured with similar technology showed their high efficiency in human immunization.

Simian hemorrhagic fever

Hemorrhagic fever similar to spontaneous 1 was reproduced by us in macaques with the strain “Sukhumi‐64”.33 Problems of infection pathogenesis were studied. We showed the virus tropism to cells of the reticuloendothelial system—monocyte‐macrophage system. Death of these cells leads to release of tissue procoagulant and blood coagulation increase. The first stage of hypercoagulation is followed by hypocoagulation. The coagulation profiles are similar to profiles at disseminated intravascular coagulation syndrome.34 Analogous data were obtained by a number of authors during Ebola hemorrhagic fever research: virus tropism to endothelial cells and to macrophages, and coagulation damage is a result of virus tropism to cells of the monocyte‐macrophage system.35 Comparison of our results with literature data showed that general pathophysiological mechanisms underlie the pathogenesis of various hemorrhagic fevers. Only primates can be considered as an adequate model because in primates disease proceeds in a similar to human way. The strain “Sukhumi‐64” is not pathogenic for human, so experimental SHF reproduced with this virus in macaques is proposed by us as the model of human hemorrhagic fevers.13, 34

CONCLUSION

Today, an interest to the virus and to the model has been increased that is explained by growing epidemic potential of the most dangerous hemorrhagic human fevers and the possibility of their introduction to non‐endemic territories.36 Hemorrhagic fever viruses were added to the list of bioterrorism agents. The situation with Ebola virus causes much trouble. From 1976, when this infection firstly appeared in Sudan, Zaire and other African countries, outbreaks occur every 2‐4 years, and lethality is 25%‐90%. The largest outbreak, announced by WHO to be Public Health Emergency, took place in West Africa in 2014. Human illness fit with multiple lethal cases of gorillas and chimpanzees in nearest forests. There is the danger of sharp decreasing anthropoid apes in nature.37 It is established that ill and dead monkeys are the infection source for humans.

Therapeutic and preventive treatments of human hemorrhagic fevers are absent. For testing the developed preparations, the experimental model is necessary. Monkeys are the only animals in which these diseases proceed in a similar to human way, but the direct work with infection agents is very dangerous. There were human lethal cases after laboratory infection.38 In literature, our model of experimental hemorrhagic fever is discussed as the unique safe and adequate model for the assessment of new preparations. Research in this field is acknowledged to be very important.39

Our data show that studies conducted in monkeys of the Sukhum colony are the significant part of great contribution in research of human viral infections and their prophylaxis made by primates.