Department of Bacteriology, Nagoya University School of Medicine, Nagoya, Aichi 466-8550, Japan1
Author for correspondence: Nobuo Kato. Present address: Aichi Medical University, Nagakute, Aichi 480-1195, Japan. Fax +81 561 63 4940. e-mail 2015{at}gk.amu.aichi-med-u.ac.jp
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Human adenoviruses primarily cause respiratory, gastrointestinal and ocular infections in humans and are divided into six subgroups (A to F) based on several biochemical and antigenic characteristics (Horwitz, 1996 ; Shenk, 1996
). Respiratory tract adenovirus infection exhibits various clinical forms, including pharyngitis, exudative tonsillitis, pharyngoconjunctival fever and pneumonia (Horwitz, 1996
). In most of these clinical forms, fever is no doubt one of the dominant clinical symptoms. There have been a number of earlier reports dealing with adenovirus pyrogenicity and these have reported that adenoviruses failed to induce fever in rabbits (King, 1962
; Siegert, 1967
; Grossgebauer, 1967
). In this communication, I have re-examined this phenomenon and show that adenoviruses can exhibit pyrogenicity in rabbits.
Human adenovirus types 3, 5 and 8 were used because they belong to different groups (B, C and D, respectively) and produce distinct disease syndromes in typical cases (Horwitz, 1996 ). Types 5 and 8 were originally isolated from patients and type 3 was the prototype strain (Trim). Adenoviruses were propagated and assayed in HeLa cell cultures. HeLa cells were grown in Eagles minimal essential medium (MEM) supplemented with 10% heat-inactivated foetal bovine serum. Eagles MEM without serum was used for maintenance of cells after virus inoculation. For large-scale virus production, 10 cultures of HeLa cells in 18 oz. bottles were inoculated with virus, harvested after 5 days of infection, suspended in 30 ml PBS, subjected to five cycles of freezing and thawing and homogenized by the fluorocarbon technique (Valentine & Pereira, 1965
). The extract of infected HeLa cells was centrifuged at 40000 g for 1 h to sediment virus particles and purified virus was obtained by two cycles of CsCl gradient ultracentrifugation (Norrby, 1969
). The titres of purified preparations were determined by a plaque assay and found usually to contain 13x109 p.f.u./ml. For a control, an extract of uninfected HeLa cells was prepared in the same way and was confirmed to exhibit no detectable pyrogenicity. Parainfluenza virus type 1 (HVJ or Sendai virus) Nagoya 1-60 strain (Kato et al., 1961
) used as paramyxovirus pyrogen was propagated in the allantoic cavity of embryonated eggs, purified by differential centrifugation (Kato & Hara, 1961
) and assayed by haemagglutinin (HA) titrations (Kato et al., 1965
). For preparation of antisera to adenoviruses, rabbits were inoculated intramuscularly with 5 ml extract of infected HeLa cells mixed with Freunds complete adjuvant. An intravenous booster injection of 2 ml of the extract was given 4 weeks later and the rabbits were exsanguinated after another week. Antiserum to HVJ was produced in rabbits by three intravenous injections (34 days apart) of 3 ml UV-inactivated purified vaccine containing 10240 HA units/ml (Kato, 1967
). Bacterial lipopolysaccharide (LPS)was extracted from Salmonella enteritidis NUB1 strain by the phenolwater method (Westphal & Jann, 1965
) and further purified (Kato et al., 1976
).
For the pyrogen test, rabbits weighing 23 kg were used. Rectal temperatures were taken by a thermistor thermometer inserted 5 cm into the rectum. Temperature measurements were made at 30 min intervals for 1 h before injection of test material and for 6 h after injection. Material to be tested was injected into the marginal auricular vein of rabbits and elevation in temperature above a baseline temperature was determined. The mean response of three animals was plotted against time. Fever was considered significant if the rectal temperature increased by 0·4 °C above the mean pre-inoculation temperature. The fever index was assessed by determining the area under each temperature curve up to 6 h with a planimeter and was expressed as °Ch (Coates et al., 1986 ). Glassware used in the assay of pyrogen was sterilized by heating in an oven at 160 °C for 2 h. For injections, sterile pyrogen-free disposable syringes and needles were used. Water used for solutions and buffers was first deionized and then made pyrogen-free by distillation in a glass still.
Purified preparations of adenovirus types 3, 5 and 8 were adjusted so as to contain 109 p.f.u./ml virus. One ml aliquots of undiluted suspension and suspensions diluted 1:4, 1:16, 1:64, 1:256 and 1:1024 were injected intravenously into rabbits and febrile responses were assessed. The results with type 3 adenovirus are shown in Fig. 1(a). Injection of undiluted preparations resulted in a sharp elevation in body temperature, which started without a significant lag period and reached a peak 1 h post-injection (p.i.). Thereafter, the fever tended to decline rapidly, but it elevated again slightly later than 2·5 h and produced a second peak, which was much lower than the first peak, 3·5 h p.i. Fever returned to levels <0·4 °C by 6 h p.i. With decreasing doses of purified virus preparations, the peak level of temperature became lower and the length of time to reach the peak temperature was elongated depending upon the dose of virus injected. The highest dilution (the lowest virus dose) retaining significant pyrogenic activity (>0·4 °C) was 1:64 (1·56x107 p.f.u.). Fever patterns and the lowest pyrogenic virus dose with injections of adenovirus types 5 and 8 were very similar to those resulting from injection of type 3, except that the second peak was not detectable after injection of type 5. After UV irradiation capable of inactivating the infectivity completely (Kato, 1967
), the pyrogenicity of adenovirus types 3, 5 and 8 was retained. Heating at 56 °C for 30 min efficiently inactivated both the infectivity and the pyrogenicity of all three types of adenovirus.
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Cross-tolerance between adenovirus pyrogens and paramyxovirus pyrogen (HVJ) or LPS was studied (Fig. 2). Adenovirus type 8 was used as adenovirus pyrogen. No cross-tolerance was found between adenovirus type 8 and HVJ or LPS. The febrile reaction following intravenous injection of HVJ had a longer lag period of about 1 h, after which monophasic fever ensued, reaching a peak level 34·5 h p.i. LPS induced a febrile response without a significant lag period and reached a peak 1·52 h p.i. These fever patterns resulting from HVJ and LPS did not differ significantly regardless of whether each pyrogen was administered first or it was administered on the second day after adenovirus type 8 was administered on the first day. Also, the fever pattern caused by adenovirus type 8 was not changed by a prior injection of HVJ or LPS. Experimental results very similar to those with type 8 described here were obtained with adenovirus types 3 and 5.
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Adenovirus types 3, 5 and 8 are antigenically distinct from each other. The pyrogenicity of adenovirus types 3, 5 and 8 was never neutralized by homologous antiserum. Pyrogenic cross-tolerance could be established between every combination of these types. These findings suggest that the virion component(s) responsible for pyrogenicity does not correspond to any type-specific antigen and seems to be common to different adenovirus types, at least types 3, 5 and 8. The pyrogenicity of adenoviruses was not inactivated by UV irradiation, in contrast to infectivity, but both pyrogenicity and infectivity were inactivated by heating at 56 °C for 30 min. The simplest conclusion derived from the heat lability of adenovirus pyrogenicity is that the pyrogenic virion component(s) is easily inactivated by heating at 56 °C for 30 min. However, it was found that, when adenovirus type 5 was heated at 56 °C, the icosahedral capsid of the virion is ruptured at the twelve vertices (Russell et al., 1967 ). If the behaviour of adenovirus types 3 and 8 on heating is similar to that of type 5, the finding that adenoviruses lose pyrogenic activity when heated at 56 °C may also support the hypothesis that the complete structure of the virion is needed for its action as a pyrogen. Influenza virus pyrogenicity was shown to depend upon the integrity of virions, but haemagglutinin and/or neuraminidase appear to be essential and lipid may be involved (Pickering et al., 1992
; Alluwaimi et al., 1994
).
In contrast to the high dose (109 p.f.u.) of adenovirus required to exhibit a definite febrile response, a much lower dose (107 p.f.u.), that is one that is non-pyrogenic in itself, became highly pyrogenic in the presence of homologous type-specific antibodies at an optimal virus:antibody ratio. The pathogenesis of the febrile response in humans infected with adenovirus may involve two different kinds of mechanism; one that depends upon the pyrogenic activity of virions and the other that depends upon the pyrogenic activity of the virusantibody complex. From the results of the present study, however, it is probable that the latter mechanism is the main contributor to fever in adenovirus infection under actual physiological conditions.
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References |
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Received 27 June 2000;
accepted 7 August 2000.
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