LOUPING-ILL AND SEMLIKI FOREST VIRUS INFECTIONS IN THE SHORT-TAILED VTOLE MICROTUS AGRESTIS (L.)

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1 Br. J. exp. Path. (197) 51, 385. LOUPNG-LL AND SEMLK FOREST VRUS NFECTONS N THE SHORT-TALED VTOLE MCROTUS AGRESTS (L.) J. SEAMER AND. ZLOTNK From the Microbiological Research Establishment, Porton Down, Salisbury Received for publication March 14, 197 SUMMARY.-A large proportion of short-tailed voles survived i.c. and footpad inoculations of louping-ill virus and many developed neutralizing antibodies. A short-lived viraemia developed after footpad inoculation, but the levels of virus were low and it seems unlikely that the vole could act as a principal reservoir host to the virus in infected areas. n initial experiments in which a low passage strain of Semliki Forest virus was inoculated i.c. into voles virus multiplication occurred in the brain and moderately severe cerebral lesions developed but neuronal degeneration and necrosis were not seen. Mortality was low and the infection appeared to be self limiting. When a high passage strain of virus was inoculated i.c. into voles high titres of virus were found in the brain. Lesions of neuronal degeneration and necrosis developed and all the inoculated voles died. n later experiments with the low passage strain of virus a higher mortality occurred and the death rate varied from experiment to experiment. The mortality varied independently of virus dosage and the age of the voles. n voles which died high titres of virus were found in the brain and neuronal necrosis was present. n these voles the infection resembled that which followed the inoculation of the high passage strain. By plotting mortality on a monthly basis periods of increased and decreased mortality became evident. Although these periods corresponded roughly with the seasons, the evidence for a seasonal variation was not conclusive. N an earlier report (Seamer, Fitzgeorge and Smith, 1967a) the resistance of wild-trapped Jlicrotus agrestis to the lethal effects of infection with Langat and Semliki Forest viruses was described. Most species of laboratory animal are susceptible to infection with Semliki Forest virus (SFV), so that the resistance of voles to a strain of this virus which multiplied to high titres in their brains seemed worthy of further investigation. Furthermore the resistance of voles to the lethal effects of Langat virus in the previous study suggested thay they might also be resistant to the related louping-ill (L) virus, despite an early report to the contrary (Finidlay and Elton, 1933). nfection of short-tailed voles with L virus is of epidemiological interest because the ecology of the virus in infected districts of Great Britain is not fully understood. Microtus agrestis could act as a reservoir host since it frequently shares the same habitat as sheep, and vole sera from an infected area have been shown to contain haemagglutinin inhibiting antibodies to L virus (Smith, unpublished). MATERALS AND METHODS Voles.-To facilitate the investigation a small breeding colony of Microtus agrestis was established at Allington Farm, Porton Down, with wild-trapped voles obtained from the

2 386 J. SEAMER AND. ZLOTNK Bureau of Animal Population, Oxford. With the exception of some wild-trapped voles which were obtained from the Bureau, all the voles used in this investigation came from the 1-4th generations bred in the colony. The voles were used at the ages of either 4-5 weeks, subsequently referred to as " young ", or 9-13 weeks old (usually 1-12 weeks) subsequently called" adult ". Mice.-Porton mice weighing 14 g. were used. The infectivity of each inoculum given to voles was established by inoculating it into a group of mice. Viruses.-Two strains of L virus were used. The first was obtained from the Moredun nstitute, Scotland; it was in its 6th mouse brain passage after an unknown number of passages in sheep. This strain is referred to as the " Moredun strain ". A second strain of L virus was isolated from a sheep in Ayrshire by Dr. C. E. G. Smith. This virus was in its first mouse brain passage and is referred to as the " Ayrshire strain ". L virus was assayed in mice by the method used for Langat virus (Seamer and Randles, 1967). Titres are expressed as LD5 per g. of tissue. Two strains of SFV were used. The first, subsequently called the "low passage strain" was that used in the earlier experiments (Seamer et al., 1967a). This virus was obtained from the American Type Culture Collection and was in the 13th passage in mouse brain since the original isolation. The 2nd strain of SFV, subsequently called the "high passage strain " is believed to have had a large but unknown number of passages since the original isolation. SFV was usually assayed on monolayers of chick embryo fibroblasts incubated at 35 as previously described (Seamer, Randles and Fitzgeorge, 1967b). Titres are expressed as plaque forming units (pfu) per g. of tissue. Procedure.-Groups of voles were inoculated i.c. or into the left hind footpad under ether anaesthesia. They were observed for mortality for 15 days (SFV) or 28 days (L virus). Blood samples were taken from the orbital plexus or from the heart. Heparin was used as an anticoagulant. Tissues for virological examination were stored at 7 unless otherwise stated. n some experiments half the brain and cervical cord were fixed in formol saline for histopathological examination: sections were stained by haematoxylin and eosin. Neutralization tests were carried out by the method of Smith and Westgarth (1957). Louping-ill virus RESULTS Moredun strain in wild-trapped voles.-a group of 32 wild-trapped voles was obtained. Sera taken from these voles were examined for neutralizing antibodies against L virus. Fifteen of the voles were then inoculated i.c. with 13-7 LD5 L virus and the remaining 17 received a similar dose into the footpad. Five weeks later sera obtained from the survivors of the footpad inoculation were again examined for neutralizing antibodies. No neutralizing antibodies were found in the pre-inoculation sera. All the voles inoculated i.c. and into the footpad survived for 28 days without signs of illness. Neutralizing antibodies were found in 11 of the 17 sera taken from voles 5 weeks after footpad inoculation. Moredun strain in laboratory-bred voles.-the results above strongly suggested that wild-trapped voles were resistant to L virus, so laboratory-bred voles were next examined. A group of 29 young voles was inoculated with 13-7 LD5L virus into the footpad, 42 received a similar dose, i.c. and 33 were inoculated i.c. with 16 LD5. As usual groups of mice were inoculated similarly. The sera of voles which survived footpad inoculations were examined for neutralizing antibodies. Three voles inoculated into the footpad died, as did one inoculated i.c. with 13. LD5 and 5 inoculated i.c. with 16 LD5. Thus only 9 of a total of 14 voles died. Four of these deaths occurred 3-4 days after inoculation: it is not certain that these were due to L virus since the remaining 5 voles died after 6-8 days and mice inoculated with similar doses rarely died before the 6th day.

3 LOUPNG-LL, SFV AND VOLES 387 Neutralizing antibodies against L virus were found in 22 of the 31 sera which were taken from voles 4-5 weeks after footpad inoculation. Ayshire strain in young voles.-n order to test a second strain of virus 19 young voles were inoculated into the footpad with 16 LD5 Ayshire strain L virus and 18 voles received a similar inoculum i.c. All the mice inoculated i.c. with this dose of virus died 6-7 days later and those inoculated into the footpad died after 8-1 days. Only 2 voles died: 1 death occurred 14 days after inoculation into the footpad and the other 25 days after i.c. inoculation. Viraemia Since the voles survived inoculations of 2 strains of L virus it was of epidemiological interest to see if they became viraemic. n 3 experiments 3 young voles were inoculated into the footpad with 13 7 LD5 Moredun strain L virus. Heparinized blood samples were taken from each vole on 2 occasions during the week after inoculation. n the first experiment '2 ml. inocula of the individual samples were immediately inoculated i.c. into groups of 6 mice under 24 hr old. Portions of the samples were stored at -7 and groups of five 14 g. mice were later inoculated i.c. with 3 ml. n the 2nd and 3rd experiments the samples were examined immediately by i.c. inoculation of five 14 g. mice. t was evident from the first experiment that a number of voles had L virus in their blood but only in 3 instanpes did the whole litter of suckling mice die. When the blood samples were re-examined in 14 g. mice " positive " samples still frequently failed to kill all the group into which they were inoculated: however the older mice appeared to be only slightly less sensitive than suckling mice and the earlier results were substantially confirmed. The numbers of voles with viraemia in the 3 experiments are given in Table : samples which caused the TABLE.-Viraemia in Young Voles after Footpad noculation with 37 LD5 Moredun Strain of Louping-ill Virus Days after inoculation r Positive* Trace* Negative * For definition see text. death of 3 or more mice out of 5 are termed positive, while those which killed only 1 or 2 mice are termed trace. t is apparent that L virus was present in 34 of 47 blood samples taken from voles within 5 days of inoculation. Of 1 voles examined on the 1st and 4th days after inoculation, 7 had L virus in their blood on each occasion, as did 3 of 7 voles examined on the 2nd and 5th days. Semliki Forest virus Low passage strain in adult voles.-nitially, at a time when the animals were in short supply, it was decided to investigate further the results obtained previously (Seamer et al., 1967a) on the growth of the low passage strain of SFV in the organs of voles after i.c. inoculation. Over a period of some weeks 36 adult voles were

4 388 J. SEAMER AND,, ZLOTNK inoculated with 13 6 pfu virus. At intervals after inoculation 21 voles, all of which appeared healthy, were killed for virological and histopathological examination. The remaining 15 voles were observed for mortality. One of the 15 voles died 5 days after inoculation: the brain of this vole was found to have a titre of 18 pfu per g. The virus contents of the voles' tissues are shown in Table. With one exception, titres of pfu per g. were found in all the brains examined within 4 days of inoculation, indicating that considerable virus multiplication had occurred. On the 5th day the brain titres decreased and no virus was detected on the 7th day. Virus was present in other tissues examined on the first 2 days after inoculation, but the titres were lower than those found in the brain. TABLE l.-tissue Virus Levels (log 1 pfu per g.) of Adult Voles noculated i.c. with 13.6 pfu Low Passage Semliki Forest Virus Days after inoculation Vole, - - Tissue number /11 Blood Spleen *4 2 5 Liver Brown fat Brain *1 Histopathological changes were evident in the brain on the 1st day after inoculation: there was swelling of capillaries and a cellular exudate in the meninges, mainly around the gyrus subcallosus and spreading towards the pyriform cortex. On the 2nd day the lesions were more widespread. There were small microglial infiltrations in the paraterminal body, pyriform cortex, corpus striatum and thalamus. Additionally one brain contained small foci of polymorphonuclear cell infiltrations. The first perivascular cuffings consisting of single layers of cdlls, were seen 3 days after inoculation. Cuffing occurred mainly in the area parolfactoria, gyrus hippocampus and hippocampus. On the 4th day similar lesions were also seen in the thalamus, corpus striatum, mesencephalon and temporal cortex. At this time a moderate meningeal exudate was present along the ventral aspect of the brain, extending forwards to the olfactory cortex and lobes. Seven days after inoculation the lesions either began to regress or they assumed a subacute EXPLANATON OF PLATES FG. 1.-Subacute reaction in low passage Semliki Forest virus infection. Vole brainhippocampus. x 35. FiG. 2. Degenerative changes in high passage Semliki Forest virus infection. Vole brainhippocampus, x 24.

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6 LOUPNG-LL, SFV AND VOLES 389 character, with diffuse microglial infiltrations in affected areas. These showed a moderate degree of nuclear transformation into elongated, hypertrophied forms. Most of the round cells in the perivascular cuffs were replaced by microglial elements. At no time did any of the brains show any evidence of neuronal damage (Fig. 1). High passage strain SFV in adult voles.-the high passage strain of SFV was known to be more virulent for mice than the low passage strain, and it was therefore of interest to see if voles were resistant to it. Eighteen adult voles which were inoculated i.c. with l3- pfu high passage virus were observed for mortality, while the tissues of others were harvested for examination on the 1st and 2nd days after inoculation. n addition 7 young voles and 7 wild-trapped voles were inoculated i.c. with 14i5 pfu virus. These inoculations of the high passage strain of SFV were uniformly fatal to voles, which all died 2-4 days later. The levels of virus found in the tissues harvested from adult voles after inoculation with 135 pfu virus are shown in Table. On the 1st day titres around 15 pfu per g. were found in the blood and tissues while titres in the brain exceeded 18 pfu per g. On the 2nd day the titres in the blood and tissues were lower, but the levels in the brain were still very high. n addition the brains of 8 moribund or recently dead voles harvested on the 2nd day after inoculation all had titres in excess of 19 pfu per g. TABLE.-Tissue Virus Levels (log 1 pfu per g.) of Adult Voles noculated i.c. with 13 pfu High Passage Semnliki Forest Virus Days after inoculation A Vole, 5 Tissue number 1 2 Blood Spleen * Liver * Brown fat Brain On the 1st day after inoculation lesions in the brain resembled those found in voles one day after inoculation with the low passage strain of SFV. However on the 2nd day lesions of meningoencephalitis with moderate or severe neuronal degeneration and necrosis were seen. n each vole cellular infiltrations of lymphocytes, microglia and typical polymorphs were seen. The cellular infiltrations were either focal or diffuse and were present in most cortical and subcortical centres ofthe brain and spinal cord. Perivascular cuffings were seldom if ever composed of more than one layer of cells, and as a rule they were surrounded by a loose zone of microglial cells. Degenerative changes were scattered throughout the brain but the most conspicuous lesions were seen in the hippocampus where spongy

7 3.9 J. SEAMER AND. ZLOTNK degeneration was present in addition to degeneration and necrosis of ganglion cells (Fig. 2). Further experiments with low passage SF V. The results of a preliminary experiment suggested that young voles were more susceptible to the low passage strain of virus than adults. n further experiments into the susceptibility of voles to this strain of SFV young voles and larger doses (15.1 pfu) of virus were used. These experiments were controlled by inoculating adult voles with the same dose of virus used earlier (samples of the low passage strain containing 13 6 pfu virus which had been prepared at the start of the investigation and stored at -7 were used for several months). The results of a series of these experiments showed that there was a considerable variation in mortality between groups of voles inoculated with the low passage strain of SFV. Most of the voles which died did so from 3-6 days after inoculation. The day of death did not appear to depend upon the dose of virus inoculated or upon the age of the voles. Mortality also varied independently of the virus dose and the age of the voles as is shown by the figures presented in Table V. Thus of 4 young voles inoculated with 13.6 pfu virus in July 1967, 2 died; in February and March 1968 the death rates were 3/13 and 4/12 respectively, while in the latter month mortality among adult voles which were similarly inoculated was 7/13. noculation of young and adult voles with 15.1 pfu virus in August and September resulted in a high mortality, but mortality was low in a similar experiment in November. TABLE V. = Variation in Mortality Among Voles noculated with Low passage Semliki Forest Virus During Different Months of the Year Month of Age of Dose of inioculation voles virus AMortalitv -July 1967 Young 13-6 pfu 2/4 Feb Young 136 pfu 3/13 March Young 13.6 pfu 4/12 March Adult 1O3- pfu 7/13 Aug Adult pfu 6/13 Sept 1968 Young 15.1 pfu 8/12 Nov Adult 13-6 pfu 1/12 Nov Adult 15l pfu. 2/11 Nov Young 151 p)fu 2/1 n Fig. 3 the deaths which occurred in all experiments with the low passage strain of SFV are plotted against the month of inoculation. Two peaks of mortality are evident, one in the spring and summer months of 1968 and a second, smaller peak in May and June Although the numbers were small, it is evident that mortality was low in the autumn of 1967 when the first experiments were carried out. Mortality was also low in the winter months of and when the experiments ceased in September As the experiments continued during 1968 and 1969 brains from moribund or recently dead adult voles which had been inoculated with 13.6 pfu virus were harvested for examination. n Fig. 4 the titres found in the brains of these voles are compared with the brain titres given in Table. t is apparent that in voles which died in the later experiments considerably more virus multiplication occurred in the brain over a longer period than was found in the experiments recorded earlier.

8 LOUPNG-LL, SFV AND VOLES 391 The histology of 14 brains was also examined. Apart from differences in the severity of the lesions, the changes in each appeared to be very similar. The disease process seemed to start around the site of inoculation, giving rise to a large necrotic lesion which penetrated from the temporal cortex into the hippocampus and thalamus. As a rule the lesion consisted of a severe microglial reaction which spread to different parts of the brain, accompanied by varying degrees of neuronal degeneration with areas of necrosis. The perivascular reaction was usually very mild..7 C 6C - 5C -J - 4C w < 3 z w U cr w 2 a. J A S N DJ F M A M J J A S O N D J F M A M J J A S u A u A u L N L N L FG Percentage mortality in all voles inoculated with low passage Semliki Forest virus plotted against the month of inoculation. Figures at each point = number of deaths/ numfber inoculated. DSCUSSON n contrast to the earlier findings of Findlay and Elton (1933), mortality among short-tailed voles inoculated with L virus in the present study was low. Although tissue virus titres were not examined, it seems probable that virus multiplication followed both i.c. and footpad inoculations of L virus. Voles inoculated by the footpad route developed viraemia and many developed neutralizing antibodies to the virus. The results suggest that only a limited amount of virus multiplication occurred; however, in view of the results obtained with SFV in this investigation it is possible that other strains of L virus, or different experimental circumstances might lead to greater multiplication with the consequence of increased fatalities and increased viraemia. The amounts of virus present in the blood of voles with viraemia appeared to 35

9 392 J. SEAMER AND. ZLOTNK be low and the viraemia itself short-lived. Varma and Smith (unpublished) have also found a short-lived viraemia with low levels of L virus in experimentally inoculated voles and xodes ricinub larvae fed upon them did not become infected with virus. t therefore seems unlikely, upon present evidence, that the shorttailed vole could be a principal reservoir host for L virus in nature. The findings with SFV are more complex. The high passage strain was fatal to all the voles into which it was inoculated. These deaths were accompanied by Li. % 5 J w 6. L ou5 x S. X x x A i- w4 E: S. 3 X 2 x X --wd DAYS AFTER NOCULATON FG. 4.-Brain virus titres (log 1 pfu per g.) of adult voles after inoculation with 13.6 pfu low passage Semliki Forest virus. Voles which died or were killed when moribund. x Values from Table for voles which were killed while apparently healthy. very high virus titres in the brain and by severe cerebral lesions with neuronal degeneration and necrosis. On the other hand, in the initial experiments with the low passage strain of virus the low mortality reported earlier (Seamer et al., 1967a) occurred again. n these voles brain virus titres tended to decline to very low levels by the 5th day, and although severe cerebral lesions occurred, neuronal degeneration and necrosis did not occur. n later experiments, however, there was frequently a higher mortality and the numbers of deaths fluctuated independently of virus dosage and the age of the voles. n these later experiments the virus titres in the brain were considerably higher than those found earlier. n addition the cerebral lesions were more severe and neuronal degeneration and necrosis were frequently found. The development of neuronal lesions and death thus

10 LOUPNG-LL, SFV AND VOLES 393 appeared to be related to very high levels of virus multiplication, and in these respects there is a clear resemblance to the events in voles inoculated with the high passage strain of SFV. t is difficult to find a satisfactory explanation for the fluctuations in mortality in voles inoculated with the low passage strain of virus. Experimental conditions were stabilized as far as practicable and many of the virus inocula used were prepared at one time. The breeding history of the voles was examined, but deaths did not appear to have a familial incidence, and since all the voles came from the first 4 generations no great overall change in genotype seems likely. By plotting the mortality of successive experiments against the month of inoculation, periods of increased and decreased mortality became evident. These periods coincided roughly with the seasons, but the coincidence is not sufficiently striking to be convincing. The fluctuations in mortality observed in this investigation may therefore have been due to seasonal influences, but conclusive evidence on this point is lacking. We are grateful to Mr. H. W. Cox, Allington Farm, Porton Down, for management of the breeding colony of voles; to Mr. E. T. W. Bowen who performed the neutralization tests, and to Mrs. P. Stenhouse for skilled technical assistance. REFERENCES FNDLAY, G. M. AND ELTON, C. (1933) J. comp. Path., 46, 126. SEAMER, J., FTZGEORGE, R. AND SMTH, C. E. G. (1967a) Br. J. exp. Path., 48, 463. SEAMER, J. AND RANDLES, W. J.-(1967) Br. J. exp. Path., 48, 43. SEAMER, J., RANDLES, W. J. AND FTZGEORGE, R.-(1967b) Br. J. exp. Path., 48, 395. SMTH, C. E. G. AND WESTGARTH, D. R.-(1957) J. Hyg., Camb., 55, 224.