key: cord-0004997-wf0o3whi authors: Sibalin, M.; Gerstl, F.; Pichler, L.; Bürki, F. title: Herpesvirus strigis, a new avian herpesvirus: II. Biochemical and biophysical properties date: 1974 journal: Arch Gesamte Virusforsch DOI: 10.1007/bf01251017 sha: 09e0527d570e76dad36105c6897a92e84a385cbb doc_id: 4997 cord_uid: wf0o3whi A virus (HSIS) originating from dead owls was successfully cultivated in chicken embryo fibroblasts. Its replication was inhibited by IUDR. Tissue cultured virus proved sensitive to ether, chloroform, 0.5 per cent trypsin, and to pH levels of 4.0 or lower. Infectivity was rapidly destroyed at 56° C. Negatively stained naked virions of 100 nm average diameter were seen, and enveloped virions with 160–250 nm size. The capsid was built up of hollow cyclindrical capsomeres, arranged in equilateral triangles, carrying 5 capsomeres along each edge. Cubical symmetry and icosahedron structure yielded a total number of 162 capsomeres. All these biochemical and biophysical data lead to classification of HSIS virus into the genus herpesvirus. Biological properties described in a foregoing paper sustained such grouping, and indicated that the agent was a new avian herpesvirus for which the nameherpesvirus strigis was proposed. A disease syndrom occurring in naturally infected owls and designated as hepato-splenitis in/ectiosa strigum (9) could be experimentally reproduced by a viral agent. In a foregoing paper (7) we classified this virus on behalf of its biological properties as herpesvirus strigis, a new avian herpesvirus. This paper describes its physicochemical properties. I-Ierpesvirus strigis (HSIS), strain x/42/63, originally isolated from an Eagle Owl on CAM (9) , was grown in chicken embryo fibroblasts and used in its 4th to 17th tissue culture passages. Infectious ]aryngotracheitis virus (ILT), strain N5256, was used in its 8th to 10th CAM passages. Infectious bronchitis virus (IB), strain Beaudette, was used after an unknown number of egg passages in its 4th to 9th chicken fibroblast tissue culture passages. Infectious bovine rhinotraeheitis virus (IBR) strain LA, was used in its 13th passage on primary calf kidney monolayers. Bovine enterovirus (ECBO), strain 51/60 (6) , was propagated likewise and tested in its 17th passage. Bovine rhinovirus, strain Sd-1 (4), was cultured in a roller drum at 33 ~ C on primary calf kidney monolayers and was used in its 7th passage. Preparation and handling of tissue cultures and embryonated eggs has been described in the first paper (7) . For all experiments reported in this paper serum-free tissue culture harvests were used. Taking into account the poor viability of herpesviruses at freezing temperatures in the absence of protective colloids, such harvests were stored at +4 ~ C for short periods of time (19) . All titers recorded are expressed as TCD~0 or IDs0/0.1 ml, computed according to REED and MVENCI~ (16). The inhibiting effect of IUDR on virus replication was measured in culture flasks (growth area 50 cm 2) by incorporating 25 ~g/ml of 5-iodo-2-deoxyuridine (Sigma) into maintenance medium. Control flasks received normal maintenance medium. After a preincubation at 37 o C for 3 hours, viruses were added in a multiplicity of approximately 1. One ml samples were withdrawn from each flask after 24, 48, 72 and 96 hours p. i. and immediately titrated for virus content. Ether-resistance was determined according to ANDREWES and 1LIORSTX~,'r (1), chloroform-resistance according to BSGEL and MAYR (5) . Sensitivity to trypsin was tested according to DI~TE~ (13), using crystallized, ]yophilized sterile trypsin and 3 • crystallized, lyophilized soybean trypsin inhibitor (Worthington Biochem. Corp. Freehold, N.Y., U.S.A.). Sensitivity towards different low pH levels was determined as described by Bi3i~I and PICH]~ER (8) . Temperature sensitivity was determined by keeping small aliquots of a given virus batch at --20 ~ C for 48 hours, at +4 ~ C for 48 hours, in a waterbath at 37 ~ C for 24 hours, or for 15, 30, and 60 minutes respectively at 56 ~ C. All samples were then kept at +4~ up to the time of titration for residual virus content. Such determinations were done concomitantly in a single batch of cell cultures. Chicken embryo fibroblast flask cultures showing a CPE of 70 per cent (7) were decanted and the cell sheets collected with a rubber policeman in a small quantity of MILLo~m buffer (15) . The cells were pelleted at 2500g for 5 minutes, washed twice with the same buffer and examined immediately. After decanting the buffer, 4--6 drops of a 5 per cent ammoniumacetate solution containing 0.01 per cent of a 16 per cent bovine albumin solution were thouroughly mixed with sedimented cells (23) . Two drops of this suspension were then mixed with 4 drops of a 2 per cent sodium PTA solution of pH 6.5 to 7.0. This mixture was transferred to Formvar grids previously fortified by a carbon layer. Photographs were made on Gevaert Scientia 23D50 plates in a Siemens Elmiskop I using 80KV and double condenser illumination. As evident from Figure 8 , the titer of HSIS virus in normal culture medium increased steadily over an observation period of 96 hours. In the presence of IUDR, however, no t/SIS could be detected, except a low titer of probably residual input virus in the sample taken at 24 hours p. i. This behaviour indicated that tile essential nucleic acid of HSIS is DNA. On the other hand, IUDI~ failed to have any inhibiting effect on the replication of the RNA containing IB virus, a known coronavirus (2) ; the decrease in titer of IB virus observed after 72 hours p.i. might be due to thermal inactivation. According to Table 1 , both herpesviruses tested, i.e. HSIS and IBI~, got completely inactivated by ether as well as chloroform indicating that their virions were sensitive to lipid solvents. The ECBO virus, however, remained unaffected by both lipid solvents as an enterovirus should according to definition (2). Purified trypsin in 0.5 per cent concentration had a strongly inactivating effect on HSIS virus, as evident from Table 2 . Likewise, IBt~ virus, a bovine herpesvirus determined earlier as sensitive to trypsin treatment (8) and retested for control purposes alongside to HSIS virus, was confirmed as being sensitive to trypsin. ECBO virus, known from previous experiments as resistant to trypsin (8) , resisted to trypsin treatment as expected. As evident from Table 4 , HSIS virus for short periods of time was stable at --20 ~ +4 ~ or 37 ~ C. It proved, however, very sensitive to heating at 56 ~ C. IBR tested alongside displayed a similar temperature sensitivity fulfilling thereby another criterion of the herpesvirus group (2, 19) . ECBO virus, on the other hand, partially withstood heating to 56~ even during prolonged incubation, this in accordance with previous reports (MOLL and FI~LAYSO~, cit. 2). Using negative staining technique, about 60 per cent naked and 40 per cent enveloped HSIS virus particles were found. Their average size, based on a great number of determinations, measured 100 nm when naked, and 160--200 nm (occasionally up to 250 nm) when enveloped. Some capsids as well as most of their PTA-penetrated cores showed a hexagonal outline (Figs. 1, 4) . The inner diameter of cores measured 75 nm on the average. Within some virions core structures could be recognized. In some instances such structures contained a central ring approximating 35 nm in diameter and penetrated by PTA. (Fig. 1, upper left corner; Fig. 4 .) The capsomeres had the shape of hollow cylinders (Figs. 6 and 7) measuring 10 nm in length and 10 nm in width, with a central hole approximating 2.5 nm (Fig. 6 ). Capsomeres were arranged in equilateral triangles with 5 capsomeres along one edge (Fig. 7, arrows) . As expected, size and form of envelopes varied greatly (Figs. 3, 4 and 5) . Quite frequently, spike-like projections were visible, forming fringes at the outer contour of the envelope (Fig. 4) . Herpesvirus strigis was shown to possess a number of biochemical and biophysical properties compatible with its classification into the genus herpesvirus. Its replication was suppressed by IUDI~, indicating its essential nucleic acid is DNA. It proved sensitive to ether, chloroform and trypsin. It was labile towards low pH and heat. All these features are common properties of established herpesviruses. Most of these properties were also shared by a German isolate of I-ISIS virus (18) . The observation of naked as well as enveloped virions in our negatively stained preparations could be expected. BURTSCttER and Se~U~MAem~R (10) in ultrathin sections earlier demonstrated that herpesvirus strigis is present in the naked form in the nuclei, acquires an envelope upon passage through the nuclear membrane [common feature of herpesvirus replication (21) ], and occurs only in enveloped form in the cytoplasm. The relatively high percentage of naked virions encountered in our PTA stained preparations might be explained by the early harvest of infected cells and the slow liberation (7) of herpesvirus strigis from infected cells. With an average diameter of 100 nm for naked and of 160 to 250 nm for enveloped HSIS virions our values are in good agreement with those determined (17), varicella virus (3), infectious laryngotracheitis virus (12, 20) . In ultrathin sections (10) virions of herpe~viru8 strigis measured in diameter some 20 per cent less than in negatively stained preparations. The same observation has been made with infectious laryngotracheitis virus (20) , another avian herpesvirus. In Figure 7 a herpesvirus strigis virion clearly displays a triangular facet with 5 capsomeres along each edge to the viewer. This facet is one of 20 in a virion with icosahedral symmetry (11) . Using the formula of I-Iot~N]~ and WlnnY (14) x=10(n--1)2+2, and putting n:5, we arrive at a total number of 162 capsomeres, which unequivocally classifies I-ISIS virus as an herpesvirus (14) . Other authors had observed similar core structures with some of their herpesviruses (12, 20) , and also fringes on the viral envelope (3, 12, 20) , of the size recorded here for herpesvirus strigis. Thus structurally HSIS virus is a typical representative of the genus herpesvirus. Based on its biological properties described in our first paper (7), which allowed to differentiate it from other herpesviruses, we proposed tISIS as a new avian virus species to be named herpesvirus strigis. The susceptibility of viruses to ethyl ether London : Bailli~re, Tindall and Cassell Morphology of varicella (chicken pox) virus H~I: Ein Rhinovirus des Rindes Untersuchungen fiber die Chloroformresistenz der Enteroviren des/Zindes und des Schweines Studien fiber bovine Enteroviren. 1. Mitteilung. Zbl. Vet SIBALI~: Herpesvirus strigis, a new avian species of the genus herpesvirus. I. Biological properties Further biochemical testing of feline picornaviruses Die virusbedingte t:Iepatosplenitis infectiosa strigum. 1. Mitteilung: Morphologische Untersuehungen Morphologisehe Untersuchungen zur Virus-~tiologie der Hepatosplenitis infectiosa s~rigum Physical principles in the construction of regular viruses The fine structure of infectious laryngotracheitis virus Relationship between bovine virus diarrhea virus and hog cholera virus WILI)~r: Symmetry in virus architecture Advantages of a phosphate buffer for 0sea solutions in fixation A simple method of estimating fifty percent endpoints The morphology of noninfeetive pseudorabies virus produced by cells treated with 5-fluorouracil In vitro Untersuchungen fiber die Eigenschaften des Virus der Hepatitis et Splenitis Infectiosa Strigum Uber die Haltbarkeit yon drei Herpes simplex Virusst/~mmen bei tiefen Temperaturen The structure of infectious laryngotracheitis virus Classification and nomenclature of viruses The morphology of herpesvirus RKI: On the capsid structure of small feline and bovine RNA viruses Authors' address: Dr. IVi. SIBALIN, Institute of Virology, Veterinary University, Linke Bahngasse 11, A-I030 Wien, Austria.