key: cord-0004999-oa0id89u
authors: Otsuki, K.; Yamamoto, H.; Tsubokura, M.
title: Studies on avian infectious bronchitis virus (IBV): I. Resistance of IBV to chemical and physical treatments
date: 1979
journal: Arch Virol
DOI: 10.1007/bf01318094
sha: 1aa49c5a2eded6e56a51ef7c675fa5c345e03e64
doc_id: 4999
cord_uid: oa0id89u

The resistance of avian infectious bronchitis virus (IBV) to several chemical and physical treatments was studied. Ten strains, including four Japanese strains, were used. 1. All strains were sensitive to heating at 56° C for 15 minutes; although two of them, KH and Massachusetts-41, were resistant to heating at 45° C for 90 minutes. 2. All strains were resistant to pH 3.0 and most of the strains were sensitive to pH 11.0. 3. All strains were completely inactivated by chloroform and sodium deoxycholate and all except Beaudette-42 and Connaught were relatively stable to ether. 4. All strains rapidly lost their infectivities upon ultraviolet irradiation. 5. Trypsin did not affect the infectivity of any strain. 6. From these results, the ten strains were classified into three groups based on their stabilities to exposure to heating at 45° C for 90 minutes and to ether.

Avian infectious bronchitis virus (IBV) is the most intensively studied coronavirus. The sensitivities of some IBV strains, for example Beaudette, Massachusetts, and Connecticut, to chemical and physical treatments have been thouroughly examined (4, 6, 13, 14, 17, 21, 22, 23) , but the results have differed from one report to another. Contradictory results have been particularly reported on the resistance of the strains to exposure to 56 ° C (6, 9, 13, 17, 18, 22) , to pH 3.0 (3, 4, 7, 9, 12, 18, 20) , to pH 12.0 (18, 20) , to trypsin (4, 5, 9, 17, 21, 22) , and to ether (9, 12, 14, 18, 20) . Many other strains have not been examined.

Since the antigenic structure of IBV has not been clarified, the identification of IBV strains is mainly derived from their morphological, chemical and physical characteristics. Thus, it is very important to examine the properties of IBV. In the present investigation, we examined ten typical strains of IBV, including four Japanese strains, for their resistance to various chemical and physicM agents. K. OTSUKI, H. YAMA~OTO, and 5{. TSU~OKUR~:

The t e n strains of I B V e x a m i n e d were: Beaudette-42 (Be-42), Massachusetts-41 (IB-41), Connecticut A-5968 (A-5968), Co~maught, I-Iolte, Iowa-609, Kt~, N e r i m a , Ishida, mad Shiga. The last four strains were isolated in J a p a n . All strains were kindly supplied b y the National I n s t i t u t e of A n i m a l Health, Tokyo (Director Dr. S. Shibalbu). The strains h a d b e e n passaged over t5 times in chick e m b r y o a n d from 4 (Ishida strain) to 122 ( K H strain) times in cultured chicken M d n e y (CK) eelIs before t h e y were analysed in this laboratory. All t h e strains were sho~m, in this laboratory, to produce a e y t o p a t h i c effect (CPE) in p r i m a r y C K cells, aa~d t h e y were -all cloned two or three t i m e s in CK cells prior to examination.

P r i m a r y kidney cells from 2-to 3-week-old chickens were cultured as r e p o r t e d previously (15) .

A ten-fold dilution of each t r e a t e d virus p r e p a r a t i o n was m a d e in H a n k s ' balanced salt solution a n d a 0.1-mI aliquot was inoculated into each of four or five tubes of CK cells. The virus was a d s o r b e d on to t h e cell sheet for 90 m i n u t e s at 37 ° C a n d t h e n a 0.6-ml portior~ of m a i n t e n a n c e m e d i u m was a d d e d to each tube. The TCID~0 t i t e r was calculated as described b y REED and MUENCH (19) .

The h e a t stability of I B V was e x a m i n e d using the m e t h o d of HOFSTAD (13) . Tubes containing a 20-fold dilution of v i r u s d n f e e t e d culture m e d i u m in sterile distilled w a t e r were placed in a w a t e r -b a t h at 56 °, 45 °, or 40 ° C. At 5-minute intervals, tubes were r e m o v e d a n d i m m e d i a t e l y p u t in an ice-bath. The a m o u n t of virus surviving was t h e n t i t r a t e d .

Virus culture m e d i u m was a d j u s t e d to pIK 3.0 or p H 11.0 b y a d d i n g 0.1 x ItCI or 0.1 N NaOt~[, a n d was t h e n dispensed into small tubes at 4 ° C. After 180 m i n u t e s t h e pI-I of the tubes was a d j u s t e d to 7.2 with 0.1 N N a O H or 0.1 N I-ICI, and the virus viability was assayed. Control virus was k e p t at pI-I 7.2.

The sensitivity of I B V to lipid solvents was d e t e r m i n e d as described previously for e t h e r (1), chloroform (I0) a n d sodium deoxyeholate (16) .

Trypsinization of I B V was p e r f o r m e d as follows. One milliliter of virus culture m e d i u m , 13 ml of sterile distilled w a t e r and 2 ml of 2 per cent. t r y p s i n (DIFCO, 1 : 250) were p u t into each test tube. One milliliter of virus culture m e d i u m a n d 15 ml of sterile distilled w a t e r were p u t into each control tube. Test and control tubes were divided into two groups; one group was i n c u b a t e d at r o o m t e m p e r a t u r e and the other at 37 ° C. After 6 hours 4 ml of a 1 per cent t r y p s i n inhibitor (MILES) solution was a d d e d to the t e s t tubes a n d 4 ml of sterile distilled w a t e r to the control tubes. The tubes were t i t r a t e d for virus viability. The sensitivity of I B V to t r y p s i n was judged b y c o m p a r i n g t h e virus titers of the t e s t al~d eongrol tubes.

A 10 ml portion of virus culture m e d i u m in a petri dish (6 cm in diameter) was irradiated for 20 seconds w i t h a IJV lamp (15 ~3 '~, 0.5 A) at a distance of 80-cm. Virus viability was assayed after irradiation.

All strains of IBV were rapidly inactivated on exposure to 56 ° C (Fig. 1) . Virus viability was reduced in most strMns b y 4 log10 or more in 15 minutes. Figure 2 shows the resistance to exposure to 45 ° C for the I B V strains. The tilers of the I B -4 i a n d K H strains were reduced by less t h a n 1 log10 in 90 minutes. However, greatly decreased infeetivities were observed after 30 minutes and no infectious virus were observed ~fter 90 minutes, with the other eight I B V strains. 

The results are shown in Table 1 . All the strains were relatively resistant to p H 3.0 treatment. However, most strains were more inactivated on exposure to p H 11.0. Here the decrease in the infectivity titer of the A-5968 strain was 1.5 log10 in 120 minutes, while the other strains shows decreases in their infectivity titers of 3.3 to 4.2 log10 in 120 minutes. Thus, only the A-5968 strain was regarded as stable at p H 11.0.

The results are summarized in Table 2 . No viable virus from a n y strain was detected after exposure to chloroform or sodium deoxycholate. On the other hand, all the strains except Be-42 and Connaught retained some activity after exposure to ether, although their titer~ decreased by 2.0 to 3.8 log10. The strains showing some activity after exposure to ether were regarded as ether-stable.

Virus titer~ reduction at Virus strain pI-I 3.0 pI-I 11.0 Table 3) K. OTSUKI, I-I. YAMAMOTO, and M. TSUBOKURA:

The results of the different treatments are summarized in Table 4 . All strains were sensitive to heating at 56 ° C for 15 minutes, pH 11.0 (although the A-5968 strain was partially resistant), and to treatment with chloroform, sodium deoxychelate and UV irradiation. All strains were resistant to pH 3.0, and to trypsin treatment. On the other hand, resistance to heating at 45 ° C for 90 minutes, and to ether treatment varied with each strain. The ten strains tested can be classified into three gronps on the basis of their sensitivities to the last two treatments ( Table 5 ). Group I includes the Be-42 and Connaught strains, which are sensitive to both treatments; group II includes six strains, which are relatively resistant to ether but sensitive to heating at 45 ° C for 90 minutes; and group III includes the IB41 and KH strains, which are resistant to both treatments. 

Although IBV is the most extensively studied eoronavirus, the sensitivity of IBV strains to chemical and physical treatments has not been clarified. Thus, in the present investigation, ten IBV strains, including four Japanese strains, were examined for their sensitivities to various treatments.

CU~IXG~{AM et al. (5, 17) have shown that trypsin inactivates several United States strains of IBV. However, the results obtained in the present investigation and those obtained by others (4, 9, 21, 22) show that most IBV strains are trypsinresistant.

All the strains tested were sensitive to chloroform and sodium deoxyeholate as shown by other investigators (8, 11, 17, 21) and this indicates that lipid is an essential component of IBV. Coronaviruses are defined as being ether sensitive (9, 12, 14, 20) although Q~TIROZ and H~tNsos- (18) reported that IBV is relatively resistant to ether. In the present investigation, Be-42 and Connaught strains completely lost their infectivity on exposure to ether, whereas the other eight strains were relatively stable to ether treatment.

ESTOLA (9) regarded IBV as acid-labile, while others reported that IBV is acid-stable (4, 7, t2, 18, 20) or variable (3) . Some difficulty has arisen in determinating when IBV strains are acid-stable and this may have caused these inconsistent results. In fact, some reports have regarded IBV as acid-stable when only a small portion of virus particles survived exposure to pH 2 or 3 (12, 18, 20) . In the present investigation, since the infectivity titers of all the strains were reduced by less than 2 ]ogl0 on treatment at pH 3.0 for 6 hours at 4 ° C, we consider that these IBV strains are pH 3.0-resistant. 0 n l y one thermostable I B V strain has been reported (13) and most I B V have been shown to be sensitive to heating (6, 9, 17, 18, 22) . I n this s t u d y all strains were sensitive to heating at 56 ° C and only two of the strains were resistant to heating at 45 ° C for 90 minutes. F r o m these results, we consider t h a t thermostability depends on the intrinsic characteristics of the I B V strain rather t h a n the n u m b e r of passages (13) . The n u m b e r of passages for the strains were different, a n d there was no relationship between resistance to heat a n d the n u m b e r of passage.

No difference was observed between the strains with respect to resistance to 56 ° C, p H 3.0, p i t 11.0 (except the A-5968 strain), chloroform, sodium deoxycholate, UV irradiation or trypsin.

I B V strains tested were classified into three groups, I, I I and I I I , on the basis of their sensitivities to ether a n d to heating at 45 ° C for 90 minutes. Since I B V serotyping has not been established, a satisfactory classification method is necessary. The t e n t a t i v e grouping of the I B V strains described in this paper m a y be useful as a basis for the subdivision of IBV strains.

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Authors' address: Dr. IK. O~suKI, Department of Veterinary Microbiolegy, Faculty of Agriculture, Tottori University, Tottori City, Japan.iR, eceived June 5, 1978