Br. J. exp. Path. (1976) 57, 152
THE PATHOGENESIS AND PATHOLOGY OF EXPERIMENTAL QUARANFIL VIRUS INFECTION A. BASKERVILLE AND G. LLOYD From the Microbiological Research Establishment, Porton, Salisbury Received for publication September 16, 1975
Summary.-Mice were infected intranasally with Quaranfil arbovirus, and killed at intervals from 1 day to 2 months later. The infection produced clinical signs of neurological disturbance and a high mortality. The virus could be isolated from the lungs on Days 1-9 and from the brain on Days 1-11 of the infection. Meningoencephalitis developed by Day 5 in the olfactory lobes and spread progressively caudally, involving all regions of the brain by Day 7. The principal features of the inflammatory process were perivascular cuffing, necrosis of neurones, and, in the later stages, spongiform degeneration and marked astrocytic and microglial activity. In the lungs after a short and mild exudative phase interstitial pneumonia developed. This was characterized by proliferation of connective tissue cells in interalveolar septa and later by fibrosis.
QUARANFIL virus is an unclassified arbovirus, which was first isolated from ticks in Egypt by Taylor et al. (1966). The virus has been recovered and antibodies to it have been detected from a small number of human patients, including some with febrile illness, and from several species of birds and ticks (Argas species) in Egypt and Afghanistan (Taylor et al., 1966; Williams, Abdel-Wahab and Hoogstraal, 1970). No direct causal relationship has been established, however between the virus and encephalitis or other disease in man in these areas (Abdel-Wahab, 1970), although it has been shown to cause death after experimental inoculation into mice, chicks, hamsters and guinea-pigs (Taylor et al., 1966). The purpose of this communication is to describe the changes which develop in various organs and the distribution of virus after intranasal infection of mice with Quaranfil virus. MATERIALS AND METHODS
The Egar 1095 strain of Quiaranfil virus was used in all the experiments. The virus was passaged 3 times in suckling mice by intracerebral inoculation (i.e.), harvested on
Day 6, and was later given 16 passages intracerebrally in baby hamsters. The virus was then designated QM3H 16. For experimental infection of mice a 10% suspension of QM3H16 hamster brain in borate-buffered saline having a pH of 8-0 and containing antibiotics and 0 75% bovine albumin was used. This produced a cytopathic effect (CPE) in LLC-MK2 tissue culture cells (TC) and had a titre of 10 9- 3median tissue culture infective doses (TCID50) per ml. The mice were 6 week old female Porton specific-pathogen-free animals. They were lightly anaesthetized with ether and each was given 0 03 ml of v,irus suspension intranasally. An experiment with 100 mice was carried out to study the mortality after this dose of virus, and the time after infection at which deaths occurred was recorded. For sequential pathological examination and for virus isolation studies a further 1000 mice were infected. Ten mice were killed at each of the following stages: daily from Days 1-9 after infection, at Day 11, 12 and 14, and 5 mice at 3, 6 and 8 weeks. For histopathology .5 inice at each stage were used. The trachea and lungs were removed en bloc and these and the brain, liver, spleen and kidneys were fixed in 10% formol saline. The nose was skinned and separated from the cranium at the junction of the nasal and frontal bones and was also fixed in formol saline. The nasal structures were subsequently decalcified in modified Gooding and Stewart's fluid and transverse blocks from them were embedded in paraffin wax by standard methods. Paraffin sections
PATHOLOGY OF EXPERIMENTAL QUARANFIL VIRUS INFECTION
of all tissues were stained with haematoxylin and eosin (H. and E.) and selected tissues and sections were also stained by Cajal's method for astrocytes, by Gordon and Sweet's method for reticulin fibres, and by the Van Gieson technique. For virus isolation, 5 mice were taken at the above stages up to Day 14, and from each mouse the brain, lungs and spleen were dissected out, and 20% homogenates were prepared in tissue culture media by grinding in sterile sand followed by centrifugation. Of the supernatant, 0-1 ml was inoculated into LLC-MK2 tissue culture cells initially sown at 0 5 X 105 viable cells per ml. The cells were maintained in Parker's 199 medium, supplemented with 2% foetal calf serum, 0 025% NaHCO3 and 1% crystamycin. Identification of Quaranfil virus was by neutralization test in TC using sheep anti-Quaranfil serum prepared in the laboratory. For estimation of virus titre the respective tissues homogenates were inoculated i.c. into 3 week old mice at dilutions in the range 10-1-10- 9. As control animals 75 anaesthetized mice were inoculated intranasally with 0 03 ml of a 10% suspension of normal hamster brain in PBS containing antibiotic. Groups of 5 mice were killed at intervals from Day 1 to 1 month after inoculation and from each mouse the trachea, lungs and brain were taken for histopathology. The lungs, brain and spleen from the remaining 25 mice were taken periodically as controls for virological isolation.
RESULTS
Clinical signs.-These are described for the mortality experiment, which was typical of the series as a whole. Clinical signs were first noticed on Day 3, when 8 mice were hunched and disinclined to move and had laboured respiratory movements. By Day 4 more mice were affected in this way, and in addition the eyelids of several were closed and encrusted with exudate. The earliest sign of neurological disturbance was on Day 6, when a small number of mice trailed one or both hind legs behind the body. Two mice died at this stage. On Day 7 20 mice died and a further 20 had paralysis of one or both hind limbs. These legs
had little motor power but responded to a foot-pad pinch with a brisk reflex twitch. Most mice were now weak and either disinclined or unable to move. Convulsions lasting for several minutes could be provoked by touching the body or by a sudden loud noise. A total of 55 animals died on Days 8 and 9. All mice surviving to Day 9 had clinical neurological signs. The hind limb reflexes were absent at this stage and one or both hind limbs were in flaccid paralysis. The 3 animals which survived beyond Day 12 gradually recovered and were able to eat, but 1 month later they had not regained the use of affected limbs.
Mortality The number of deaths and the stages at which they occurred are summarized in Table I. The overall mortality was 97/100 (97%). Virus isolation The CPE had 3 recognizable phases: 1. Changes appeared in discrete and localized areas throughout the monolayer. Affected cells were reduced in size and exhibited varying degrees of pleomorphism and degeneration. 2. The characteristic epithelial type of morphology of the cells was lost. Some cells were refractile and rounded in outline while others appeared stellate. 3. Complete destruction of the monolayer. The period between stages 1 and 3 varied from 6-24 hours. It was noted that the CPE was most marked when the media had a pH value of between 5 and 6 at Stage 1. The time required for primary isolation of Quaranfil virus from experimental tissues was found to vary between 8-14 days. Subsequent passage reduced this interval to 6-12 days after the first passage, and to 2-6 days after the fourth passage. The results of virus isolation from the brain
TABLE I Days after infection Number of mice died
153
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 0 0 0 0 0 2 20 26 29 9 6 5 0 0 0
154
A. BASKERVILLE AND G. LLOYD
TABLE II 1 Days after infection Number of virus fLung 4/5 isolations 1 Brain 0/5
2
3
4
5/5 3/5
4/5 4/5
4/5 4/5
and lungs at different stages are presented in Table II. Since the spleen samples were consistently negative for virus this organ is not included in the Table. The titre of virus in the brain and lungs during the course of the infection is shown in Fig. 1. The maximum titre reached in the lungs was 105 mouse lethal doses (MLD50) on Day 1 and this fell progressively to Day 9. Virus was first detected in the brain in low concentration (
THE PATHOGENESIS AND PATHOLOGY OF EXPERIMENTAL QUARANFIL VIRUS INFECTION A. BASKERVILLE AND G. LLOYD From the Microbiological Research Establishment, Porton, Salisbury Received for publication September 16, 1975
Summary.-Mice were infected intranasally with Quaranfil arbovirus, and killed at intervals from 1 day to 2 months later. The infection produced clinical signs of neurological disturbance and a high mortality. The virus could be isolated from the lungs on Days 1-9 and from the brain on Days 1-11 of the infection. Meningoencephalitis developed by Day 5 in the olfactory lobes and spread progressively caudally, involving all regions of the brain by Day 7. The principal features of the inflammatory process were perivascular cuffing, necrosis of neurones, and, in the later stages, spongiform degeneration and marked astrocytic and microglial activity. In the lungs after a short and mild exudative phase interstitial pneumonia developed. This was characterized by proliferation of connective tissue cells in interalveolar septa and later by fibrosis.
QUARANFIL virus is an unclassified arbovirus, which was first isolated from ticks in Egypt by Taylor et al. (1966). The virus has been recovered and antibodies to it have been detected from a small number of human patients, including some with febrile illness, and from several species of birds and ticks (Argas species) in Egypt and Afghanistan (Taylor et al., 1966; Williams, Abdel-Wahab and Hoogstraal, 1970). No direct causal relationship has been established, however between the virus and encephalitis or other disease in man in these areas (Abdel-Wahab, 1970), although it has been shown to cause death after experimental inoculation into mice, chicks, hamsters and guinea-pigs (Taylor et al., 1966). The purpose of this communication is to describe the changes which develop in various organs and the distribution of virus after intranasal infection of mice with Quaranfil virus. MATERIALS AND METHODS
The Egar 1095 strain of Quiaranfil virus was used in all the experiments. The virus was passaged 3 times in suckling mice by intracerebral inoculation (i.e.), harvested on
Day 6, and was later given 16 passages intracerebrally in baby hamsters. The virus was then designated QM3H 16. For experimental infection of mice a 10% suspension of QM3H16 hamster brain in borate-buffered saline having a pH of 8-0 and containing antibiotics and 0 75% bovine albumin was used. This produced a cytopathic effect (CPE) in LLC-MK2 tissue culture cells (TC) and had a titre of 10 9- 3median tissue culture infective doses (TCID50) per ml. The mice were 6 week old female Porton specific-pathogen-free animals. They were lightly anaesthetized with ether and each was given 0 03 ml of v,irus suspension intranasally. An experiment with 100 mice was carried out to study the mortality after this dose of virus, and the time after infection at which deaths occurred was recorded. For sequential pathological examination and for virus isolation studies a further 1000 mice were infected. Ten mice were killed at each of the following stages: daily from Days 1-9 after infection, at Day 11, 12 and 14, and 5 mice at 3, 6 and 8 weeks. For histopathology .5 inice at each stage were used. The trachea and lungs were removed en bloc and these and the brain, liver, spleen and kidneys were fixed in 10% formol saline. The nose was skinned and separated from the cranium at the junction of the nasal and frontal bones and was also fixed in formol saline. The nasal structures were subsequently decalcified in modified Gooding and Stewart's fluid and transverse blocks from them were embedded in paraffin wax by standard methods. Paraffin sections
PATHOLOGY OF EXPERIMENTAL QUARANFIL VIRUS INFECTION
of all tissues were stained with haematoxylin and eosin (H. and E.) and selected tissues and sections were also stained by Cajal's method for astrocytes, by Gordon and Sweet's method for reticulin fibres, and by the Van Gieson technique. For virus isolation, 5 mice were taken at the above stages up to Day 14, and from each mouse the brain, lungs and spleen were dissected out, and 20% homogenates were prepared in tissue culture media by grinding in sterile sand followed by centrifugation. Of the supernatant, 0-1 ml was inoculated into LLC-MK2 tissue culture cells initially sown at 0 5 X 105 viable cells per ml. The cells were maintained in Parker's 199 medium, supplemented with 2% foetal calf serum, 0 025% NaHCO3 and 1% crystamycin. Identification of Quaranfil virus was by neutralization test in TC using sheep anti-Quaranfil serum prepared in the laboratory. For estimation of virus titre the respective tissues homogenates were inoculated i.c. into 3 week old mice at dilutions in the range 10-1-10- 9. As control animals 75 anaesthetized mice were inoculated intranasally with 0 03 ml of a 10% suspension of normal hamster brain in PBS containing antibiotic. Groups of 5 mice were killed at intervals from Day 1 to 1 month after inoculation and from each mouse the trachea, lungs and brain were taken for histopathology. The lungs, brain and spleen from the remaining 25 mice were taken periodically as controls for virological isolation.
RESULTS
Clinical signs.-These are described for the mortality experiment, which was typical of the series as a whole. Clinical signs were first noticed on Day 3, when 8 mice were hunched and disinclined to move and had laboured respiratory movements. By Day 4 more mice were affected in this way, and in addition the eyelids of several were closed and encrusted with exudate. The earliest sign of neurological disturbance was on Day 6, when a small number of mice trailed one or both hind legs behind the body. Two mice died at this stage. On Day 7 20 mice died and a further 20 had paralysis of one or both hind limbs. These legs
had little motor power but responded to a foot-pad pinch with a brisk reflex twitch. Most mice were now weak and either disinclined or unable to move. Convulsions lasting for several minutes could be provoked by touching the body or by a sudden loud noise. A total of 55 animals died on Days 8 and 9. All mice surviving to Day 9 had clinical neurological signs. The hind limb reflexes were absent at this stage and one or both hind limbs were in flaccid paralysis. The 3 animals which survived beyond Day 12 gradually recovered and were able to eat, but 1 month later they had not regained the use of affected limbs.
Mortality The number of deaths and the stages at which they occurred are summarized in Table I. The overall mortality was 97/100 (97%). Virus isolation The CPE had 3 recognizable phases: 1. Changes appeared in discrete and localized areas throughout the monolayer. Affected cells were reduced in size and exhibited varying degrees of pleomorphism and degeneration. 2. The characteristic epithelial type of morphology of the cells was lost. Some cells were refractile and rounded in outline while others appeared stellate. 3. Complete destruction of the monolayer. The period between stages 1 and 3 varied from 6-24 hours. It was noted that the CPE was most marked when the media had a pH value of between 5 and 6 at Stage 1. The time required for primary isolation of Quaranfil virus from experimental tissues was found to vary between 8-14 days. Subsequent passage reduced this interval to 6-12 days after the first passage, and to 2-6 days after the fourth passage. The results of virus isolation from the brain
TABLE I Days after infection Number of mice died
153
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 0 0 0 0 0 2 20 26 29 9 6 5 0 0 0
154
A. BASKERVILLE AND G. LLOYD
TABLE II 1 Days after infection Number of virus fLung 4/5 isolations 1 Brain 0/5
2
3
4
5/5 3/5
4/5 4/5
4/5 4/5
and lungs at different stages are presented in Table II. Since the spleen samples were consistently negative for virus this organ is not included in the Table. The titre of virus in the brain and lungs during the course of the infection is shown in Fig. 1. The maximum titre reached in the lungs was 105 mouse lethal doses (MLD50) on Day 1 and this fell progressively to Day 9. Virus was first detected in the brain in low concentration (
