Archives of Virology 47, 249--255 (1975) © by Springer-Verlag 1975
Thermostability of Newcastle Disease Virus Strains of Different Virulence By B. LOMNIOZI Veterinary Medical Research Inst.itute of the Hungarian Academy of Sciences, Budapest, HungaI~" With 1 Fig~are Received October lI, 1974
Summary The thermal inactivation rate constant for infectivity of a total of 24 Newcastle disease virus (NDV) strains was determined at 50 ° and 56 ° C. The greater part of the examined NDV strains were found, irrespective of virulence properties, to be thermostable, since the loss of infectivity titre did not exceed 2 logarithmic orders after exposure at 50 ° C for 60 minutes. Thermostable (I +) and thermolabile (I-) strains with respect to infectivity were uniformly encountered among the 12 avirulent (lentogenic) and 10 fully virulent (velogenic) strains studied. Strains with both heat stable (Ha +) and heat labile (Ha-) haemagglutinin were found in the lentogenic group, whereas haemagglutinins of all examined velogenic strains were heat stable. On the basis of I / H a character, i.e. combination of thermoscnsitivity of infectivity and haemagglutinin, all examined strains could be classified into one of three categories: I - H a - (8 lentogenic strains), I + H a + (4 lentogenic and 7 vclogenic strains) and I - H a + (2 mesogenic and 3 velogenic strains), The possible fourth combination, I + I t a -, was not encountered among the strains studied. Determination of the I / H a character m a y be helpful in clarifying the origin of a lentogenic NDV strain.
1. Introduction FARI~AS (1930, cit. in reference 15), who was the first to investigate the thermostability of various strains of Newcastle disease virus (NDV), found t h a t most of them lost infectivity on exposure to 50°--55° C for 30 minutes. HA?CSON et al. (1949) and HANSON and BRA~DLEY (1955) concluded from detailed thermostability studies on haemagglutinins from N D V strains of different virulence t h a t at 56 ° C haemagglutinins of lentogenie (avirulent) strains proved to be heat labile whereas those of mesogenic and velogenic strains maintain their activity for at least 30 minutes (ANon., 1971; HANSON et al., 1967). Recently, however, McFERRAN
250
B. Lo~iCZl
:
a n d N~LSO~ (1971) have reported the isolation of a lentogenic s t r a i n whose haema g g l u t i n i n showed a relatively high thermostability. Since no comparative investigations have as yet been carried out on the thermos t a b i l i t y of infectivity of N D V strains of different virulence and, i n addition, a simple, practicable plaque assay has recently been i n t r o d u c e d for i n f e c t i v i t y t i t r a t i o n of lentogenlc strains ( L o ~ I C Z I , 1974), it seemed worthwhile to examine the possible correlations between virulence a n d t h e r m o s t a b i l i t y on the one hand, a n d i n f e c t i v i t y a n d h a e m a g g l u t i n l n t h e r m o s t a b i l i t y on the other hand.
2. Materials and Methods 2.1. Virus Strains Part of the NDV sLrains were the same as used in earlier studies (LoMNICZI, 1973), part were isolated from outbreaks in the field, such as strains NDV-55 and NDV-368 by J. Tanyi (Veterinary Institute, Debreeen), NDV-56, as well as A, B, D, E, K by G. t{rivng~k (Central Veterinary Institute, Budapest,) and the strain Ira from goslings (B. Lo~1~iezi, unpublished data). 2.2..Propagation o/ Virus Virus stocks were prepared by inoculation of embryonated hen's eggs preincubated for 9 - - i 0 days, as described earlier (LosINICZI, 1973). 2.3. Heat Inactivation 0.5 ml amounts of virus containing allantoie fluid were sealed airtightly in vials, and kept at predetermined temperatures for different times, thereafter heat treatment was stopped b y transferring the vials into an ice cold water bath. For the classical thermostability test (50 ° C, 30 minutes), the virus samples were diluted tenfold in bidistilled water and the results were evaluated according to KETLE~ et al. (1962). The decrease of infectivity was characterized by a rate constant (K, expressed in terms of minut~e-i, according to DIGIOIA et al., 1970). t~egression lines were plotted from 4 - - 5 points for each strain. ]for certain strains, which showed a distinetive twocomponent inactivation, the K values of the primary (fast) and secondary (slow) inactivation were determined separately. The rate constants were calculated from the formula K ~= (2.3 log V/V0)t, where V0 is the initial activity and V that of the sample heat-treated for time t. 2.4. Titration o/In]ectivity The infectivity of all samples was determined by plaque-titration in chick embryo lung cell cultures (Lo~NlCZI, 1974). 2.5. Haemagglutinin Titration The haemagglutinin titrcs were determined by Tak~tsy's micro-method (TAK.~TSY, 1955), using 0.5 per cent chicken erythroeyte suspension and t)BS as diluent. The highest dilution still capable to haemagglutinate 50 per cent of the erythroeytes was regarded as the titrat~ion endpoint.
3. Results 3.1. Thermostability o / N D V Strains at 50 ° C The greater p a r t of the e x a m i n e d N D V strains proved to be t h e r m o r e s i s t a n t (Table 1), None of the strains lost more t h a n 2 log orders of i n f e c t i v i t y after 30 m i n u t e s a n d only strains F a n d Texas lost as m u c h after 60 minutes.
Thcrmostability of NDV Strains of Different Virulence
251
As for all strains, except Texas, a single-component i n a c t i v a t i o n curve was obtained, these could be characterized b y a single rate constant. The p r i m a r y ( K = 0 . 1 0 2 ) a n d secondary ( K = 0 . 0 1 3 ) i n a c t i v a t i o n curves for strains Texas intersected a t a b o u t 120 minutes. Table 1. Thermostability o / N D V Strains at 50 ° C Time of 2 log decrease (minutes) Strain of NDV
Rate constant Infectivity Haemagglutination (minute -1)
Per cent survival S
Lentogenic NDV-55 NDV-18 F NDV-368 NDV-56 Ira LaSota B1 Me Sz Queensland V4 Ulster NDV-6
70 70 80 80 90 100 120 150 160 150 140 190 180
50 30 40 50 40 60 30 30 100 120 > 240 > 240 > 240
0.089 0.089 0.062 0.062 0.057 0.057 0.034 0.032 0.029 0.031 0.032 0.026 0.025
2 _ b 1 -10 -10 10 i0 -25 50 10
Mesogenic L i
120 1l0
t00 > 240
0.035 0.038
5 12
Velogenic Texas GB I-Ierts 33 A
60 105 --
>240 > 240 --
0.102-~ 0.013 0.042 --
1 2 3
Of infectivity after 60 minutes heating. b Not done. Comparison of the times required for a c t i v i t y decrease to 1 per cent revealed two different correlations b e t w e e n infectivity a n d the t h e r m o s t a b i l i t y of haemagglutinin. Most lentogenic strains showed a greater t h e r m o s t a b i l i t y of infectivity over h a e m a g g l u t i n i n , whereas 3 lentogenic strains (Queensland V 4, Ulster, NDV-6) 1 mesogenic s t r a i n (H) a n d 2 velogenic strains (Texas GB, Herts) b e h a v e d reversely. The b e h a v i o u r of strains Sz a n d L was i n t e r m e d i a t e between the two patterns. 3.2. Thermostability o~ N D V Strains at 56 ° C The time required for a titre decrease by two (and even three) logarithmic orders was w i t h i n 10 m i n u t e s ( K > 0 . 5 0 ) for five of the e x a m i n e d n i n e lentogenic strains (Table 2). The mesogenic strains a n d strains Texas a n d Herts 33 of the t e n velogenic strains b e h a v e d similarly. The r e m a i n i n g four lentogenic a n d eight velogenie strains h a d a rate c o n s t a n t of < 0 . 5 0 a n d required a t least 10 m i n u t e s or more for a n i n f e c t i v i t y decrease b y two logarithmic orders. All velogenic strains, the mesogenic s t r a i n t I a n d the lentogenic strains Ulster, Q u e e n s l a n d V 4 a n d NDV-6 h a d a t h e r m o s t a b l e h a e m a g g l u t i n i n (survival > 3 0 minutes). W i t h strains
252
B. Lo~:N:ez::
Sz a n d L t h e h a e m a g g l u t i n i n t h e r m o s t a b i l i t y was i n t e r m e d i a t e , w h e r e a s t h e r e s t of t h e t e n t o g e n i c s t r a i n s possessed a t h e r m o l a b i l e h a e m a g g l u t i n i n ( s u r v i v a l 30 >30 >30 >30
1.56-b 0.60 0.86~-0.33 0.48 0.46 0.39 0.36 0.28 [email protected]
I/Ha character ~
Lentogenic LaSota F B1, N D V - 5 5 , -368 Ira
NDV-18 Me Sz Ulster Queensland V 4 NDV-6
I-Ha-
I+H~
±
i+Ha +
Mesogenic I-I L
I-Ha+
I-Ha ±
Velogenic Texas G B H e r t s 33 Vad Gbr D, K B, L E A
I-Ha +
I+Ha+
Criteria in Table 3. b N o t done. Table 3. Criteria/or the Thermostability Character o / N D V Strains T i m e of 2 log decrease in a c t i v i t y (minutes) Activity
50 ° C
56 ° C
Infectivity, I stable I+ labile I-
> 100 < 100
> 10 < 10
Haemagglutinin, Ha stable I-Ia ÷ labile I-Ia-
> 240 _ 30
,:, \z \?
L.
o
\
,
50
",,5"., -,
+--
~E
I....
I
!
48
50
52
"tp
54
56
Temperetu re,°C Fig. I. Stability of the haemagglutinin of lentogenic strains of ~NDV temperature
at different
4. Discussion The thermoresistance and the virulence of NDV strains are not related to one another. Strains of higher or lower heat sensitivity occurred in both lentogenic and velogenic groups. I t is, nevertheless, remarkable that while the haemagglutinin of all examined vetogenic strains survived 30 minutes at 56 ° C, the lentogenic strains can be divided, according to their haemaggtutinin resistance, into at least two groups: i) thermolabile and ii) thermostable strains. Formerly, thermolabile strains whose haemagglutinin became inactivated within 5 minutes at 56 ° C were chiefly found in the lentogenie group (HA~'so~ and BRA~DL~Y, 1955; Hx~SO~+ et al., 1967; A~+o~., 1971). The infectivity of these strains decreased by at least three logarithmic orders within less than 10 minutes and the thermostability of their haemagglutinin was usually less than one hour at, 50 ° C, thus showing a thermostability character of I - H a - . On the other hand, the haemagglutinin of the
254
B. LoM~iczi:
thermostable lentogenic strains survives longer than 30 minutes at 56 ° C, more than 4 hours at 50°C and these strains require at least 20 minutes for a 2 log decrease of infectivity titre at 56 ° C, thus, their thermostability character is I + H a +. The existence of thermostable lentogenic strains was not detected until the recent description of the Ulster strain b y I~IcF~I~I~.A~and NELSON (197i). In the present study, strain Queensland V4 isolated by SI~Mo~s (1967) in Australia and strain NDV-6 isolated by DI~N and BA~t~I~GER (1967) from slaughterhouse earcases in Hungary, were additionally identified as heat stable lentogenic strains. Strain Sz is intermediate in respect of heat sta,bility (B. L o ~ I C Z I , unpublished data), but can at all events be easily differentiated from thermolabile strains. FOSTEI~ and TI~IOMPSo~ (t957) who studied the survival of four NDV strains of different. virulence at various temperatures found them to have an equal thermostabi]ity. I t does not seem improbable that the thermolabile lentogenic strains, which occur in nature and can not infrequently be isolated from poultry flocks healthy or showing respiratory symptoms (Me, Ita, NDV-55, NDV-56, NDV-368), are in fact the descendants of one of the strains used as live vaccines (L~Sota, B1) being also thermolabile. Nevertheless, new markers (HANso~-, et al. 1967) would be required to substantiate this assumption. The origin of the thermostable lentogenie strains is unclear, and two hypothetical explanations can be considered; one being that they arose by attenuation of heat-stable mesogenic or velogenic strains, the other that the lentogenie strains m a y have acquired thermostable haemagglutinin and/or other structural proteins responsible for high heat resistance through completion of the original genetic pool. Comparison of the times required for activity decrease by two logarithmic orders showed that in most strains, the haemagglntinin is considerably more resistant to exposure at 56 ° C than the infectivity. This is in accordance with the fhldings of others (DIGIOI)~ et al., 1970 ; PIERC~ and I-IAYwOOD,1973). Lentogenie strains, however, showed either an almost equal thermoresistanee of both activities or a greater resistance of infectivity at 56 ° C and a consequently lower resistance of haemagglutinin at 50 ° C. Yet, not a single strain was found to have the character I+Ha -. In a recent paper, however, published by ESTOLA (1974), an exceptionally high thermostable velogenic NDV strain has been described. Though exact thermal inactivation curves for infectivity are not presented, the Finnish strain seems to fit into the I + H a - category. Paramyxoviruses were generally found to be thermolabile in the classical thermostability tests (50 ° C, 30 minutes, I~[cc~:, 1964), and in some virus groups thermostability even appeared to be related to the acid-fast property (KETLEI~ et al., 1962). In the present study not a single NDV strain lost two logaiithmic ordeis of infectivity titre within 30 minutes, whilst the titre of six thermostable NDV strains felt off b y more than 4 logarithmic orders within one hour at p H 3.0 (B. Los~ICZI, unpublished data), thus, the stability to heat or low p H values should be considered independent qualities of NDV strains.
Aeknowledfments The skillful technical assistance of Miss Ildik5 Sornod is appreciated.
Thermostability of N D V Strains of Different Virulence
255
Referenees 1. A~ox., in: Methods for Examining Poultry Biologics and for Identifying and Quantifying Avian Pathogens. pp. 66--93. National Academy of Sciences, ~¥a.shington, D.C., 1971. 2. DIGIOlA, G . A . , J. J. LICCIAI~D:ELLO, J. T. R. NICKERSON, and S. A. GOLD]3LITH: Thermal inactivation of Newcastle disease virus. Appl. Microbiol. 19, 451--454 (1970). 3. Dm~N, Cs., and K. BAMBERGE~g: Comparison of Newcastle disease virus strains isolated from broiler chickens (in Hungarian). Magy. Ao. Lap. 22, 160--t62 (1967). 4. ESTOLA, T.: Isolation of a Finnish Newcastle disease virus with an exceptionally high thermostability. Avian Dis. 18, 274--277 (t974). 5. FAnlNAS, E. C. : Avian pest, a disease of birds hitherto unknown in the Philippine Islands. Ptfilipp. J. Agr. 1, 311--366 (1930). 6. FOSTER, N . M . , and C. H. T~oMPsoN: The comparative thermolability of four strains of Newcastle disease virus of widely varying virulence. Vet. Med. 52, 119 to t21 (1957). 7. HANSON, R. P., and C. A. BRAN])LY: Identification of vaccine strains of Newcastle disease virus. Science 122, 156~ 157 (1955). 8. HANSO~~, R. P., E. UPTON, C. A. Bt~ANt)LEY, and N. S. ~VINsLow: Heat. stability of hemagglutinin of various strains of Newcastle disease virus. Proc. Soe. exp. Biol. (N.Y.) 70, 283--287 (1949). 9. t-IANsON, R . P . , J. SPALATIN, J. ES:['I~INAN, and G. SOl,LOEb: Identification of lentogenic strains of Newcastle disease virus. Avian Dis. 11, 49--53 (1967). 10. HvCK, R. A. : The classification of viruses. Vet. Bull. (Weybridge) 34, 239--253 (1964). 11. KETLF,R, A., V. V. HA~PA~IAN, and M. R. HILLE~AN: Characterization and classification of ECHO28 rhinovirus-coryzavirus agents. Proe. Soc. exp. Biol. (N.Y.) 110, 821--831 (1962). 12. Los~NlCZI, B. : Studies on interferon production and interferon sensitivity of different strains of the Newcastle disease virus. J. gen. Virot. 21, 305--313 (1973). 13. Lo~NleZi, B. : Plaque assay for avirulent (lentogenic) strains of Newcastle disease virus, Appl. Mierobiol. 27, 1162--t 163 (1974). 14. MCFE~RAN, J . B . , and 1~. NELSON: Some properties of an avirulent Newcastle disease virus. Arch. gas. Virusforsch. 34, 64--74 (1971). t5. PICKEN, J. C., J]~.: Thermostability of Newcastle disease virus. I n : Newcastle Disease Virus: An Evolving Pathogen (R. P. HAnsoN, ed.), pp. 167--188. The University of Wisconsin Press, Madison and Milwaukee, 1964. 16. PIERCE, J. S., and A. 2¢I. ttAYWOOD : Thermal inactivation of Newcastle disease virus. I. Coupled inactivation rates of hemagglutinating and neuraminidase activities. J. Virol. 11, 168--176 (1973). 17. SI~Mo~s, G. C. : The isolation of Newcastle disease virus in Queensland. Aust. vet. J. 43, 29--30 (1967). 18. TA~:X~sw, G¥. : The use of spiral loops in serological and virological micro-methods. Acta microbiol. Acad. Sci. hung. 3, 191--202 (1955). Author's address: Dr. B. Los~iNiczI, Pf. 18, It-t581 Budapest, Hungary.
Thermostability of Newcastle Disease Virus Strains of Different Virulence By B. LOMNIOZI Veterinary Medical Research Inst.itute of the Hungarian Academy of Sciences, Budapest, HungaI~" With 1 Fig~are Received October lI, 1974
Summary The thermal inactivation rate constant for infectivity of a total of 24 Newcastle disease virus (NDV) strains was determined at 50 ° and 56 ° C. The greater part of the examined NDV strains were found, irrespective of virulence properties, to be thermostable, since the loss of infectivity titre did not exceed 2 logarithmic orders after exposure at 50 ° C for 60 minutes. Thermostable (I +) and thermolabile (I-) strains with respect to infectivity were uniformly encountered among the 12 avirulent (lentogenic) and 10 fully virulent (velogenic) strains studied. Strains with both heat stable (Ha +) and heat labile (Ha-) haemagglutinin were found in the lentogenic group, whereas haemagglutinins of all examined velogenic strains were heat stable. On the basis of I / H a character, i.e. combination of thermoscnsitivity of infectivity and haemagglutinin, all examined strains could be classified into one of three categories: I - H a - (8 lentogenic strains), I + H a + (4 lentogenic and 7 vclogenic strains) and I - H a + (2 mesogenic and 3 velogenic strains), The possible fourth combination, I + I t a -, was not encountered among the strains studied. Determination of the I / H a character m a y be helpful in clarifying the origin of a lentogenic NDV strain.
1. Introduction FARI~AS (1930, cit. in reference 15), who was the first to investigate the thermostability of various strains of Newcastle disease virus (NDV), found t h a t most of them lost infectivity on exposure to 50°--55° C for 30 minutes. HA?CSON et al. (1949) and HANSON and BRA~DLEY (1955) concluded from detailed thermostability studies on haemagglutinins from N D V strains of different virulence t h a t at 56 ° C haemagglutinins of lentogenie (avirulent) strains proved to be heat labile whereas those of mesogenic and velogenic strains maintain their activity for at least 30 minutes (ANon., 1971; HANSON et al., 1967). Recently, however, McFERRAN
250
B. Lo~iCZl
:
a n d N~LSO~ (1971) have reported the isolation of a lentogenic s t r a i n whose haema g g l u t i n i n showed a relatively high thermostability. Since no comparative investigations have as yet been carried out on the thermos t a b i l i t y of infectivity of N D V strains of different virulence and, i n addition, a simple, practicable plaque assay has recently been i n t r o d u c e d for i n f e c t i v i t y t i t r a t i o n of lentogenlc strains ( L o ~ I C Z I , 1974), it seemed worthwhile to examine the possible correlations between virulence a n d t h e r m o s t a b i l i t y on the one hand, a n d i n f e c t i v i t y a n d h a e m a g g l u t i n l n t h e r m o s t a b i l i t y on the other hand.
2. Materials and Methods 2.1. Virus Strains Part of the NDV sLrains were the same as used in earlier studies (LoMNICZI, 1973), part were isolated from outbreaks in the field, such as strains NDV-55 and NDV-368 by J. Tanyi (Veterinary Institute, Debreeen), NDV-56, as well as A, B, D, E, K by G. t{rivng~k (Central Veterinary Institute, Budapest,) and the strain Ira from goslings (B. Lo~1~iezi, unpublished data). 2.2..Propagation o/ Virus Virus stocks were prepared by inoculation of embryonated hen's eggs preincubated for 9 - - i 0 days, as described earlier (LosINICZI, 1973). 2.3. Heat Inactivation 0.5 ml amounts of virus containing allantoie fluid were sealed airtightly in vials, and kept at predetermined temperatures for different times, thereafter heat treatment was stopped b y transferring the vials into an ice cold water bath. For the classical thermostability test (50 ° C, 30 minutes), the virus samples were diluted tenfold in bidistilled water and the results were evaluated according to KETLE~ et al. (1962). The decrease of infectivity was characterized by a rate constant (K, expressed in terms of minut~e-i, according to DIGIOIA et al., 1970). t~egression lines were plotted from 4 - - 5 points for each strain. ]for certain strains, which showed a distinetive twocomponent inactivation, the K values of the primary (fast) and secondary (slow) inactivation were determined separately. The rate constants were calculated from the formula K ~= (2.3 log V/V0)t, where V0 is the initial activity and V that of the sample heat-treated for time t. 2.4. Titration o/In]ectivity The infectivity of all samples was determined by plaque-titration in chick embryo lung cell cultures (Lo~NlCZI, 1974). 2.5. Haemagglutinin Titration The haemagglutinin titrcs were determined by Tak~tsy's micro-method (TAK.~TSY, 1955), using 0.5 per cent chicken erythroeyte suspension and t)BS as diluent. The highest dilution still capable to haemagglutinate 50 per cent of the erythroeytes was regarded as the titrat~ion endpoint.
3. Results 3.1. Thermostability o / N D V Strains at 50 ° C The greater p a r t of the e x a m i n e d N D V strains proved to be t h e r m o r e s i s t a n t (Table 1), None of the strains lost more t h a n 2 log orders of i n f e c t i v i t y after 30 m i n u t e s a n d only strains F a n d Texas lost as m u c h after 60 minutes.
Thcrmostability of NDV Strains of Different Virulence
251
As for all strains, except Texas, a single-component i n a c t i v a t i o n curve was obtained, these could be characterized b y a single rate constant. The p r i m a r y ( K = 0 . 1 0 2 ) a n d secondary ( K = 0 . 0 1 3 ) i n a c t i v a t i o n curves for strains Texas intersected a t a b o u t 120 minutes. Table 1. Thermostability o / N D V Strains at 50 ° C Time of 2 log decrease (minutes) Strain of NDV
Rate constant Infectivity Haemagglutination (minute -1)
Per cent survival S
Lentogenic NDV-55 NDV-18 F NDV-368 NDV-56 Ira LaSota B1 Me Sz Queensland V4 Ulster NDV-6
70 70 80 80 90 100 120 150 160 150 140 190 180
50 30 40 50 40 60 30 30 100 120 > 240 > 240 > 240
0.089 0.089 0.062 0.062 0.057 0.057 0.034 0.032 0.029 0.031 0.032 0.026 0.025
2 _ b 1 -10 -10 10 i0 -25 50 10
Mesogenic L i
120 1l0
t00 > 240
0.035 0.038
5 12
Velogenic Texas GB I-Ierts 33 A
60 105 --
>240 > 240 --
0.102-~ 0.013 0.042 --
1 2 3
Of infectivity after 60 minutes heating. b Not done. Comparison of the times required for a c t i v i t y decrease to 1 per cent revealed two different correlations b e t w e e n infectivity a n d the t h e r m o s t a b i l i t y of haemagglutinin. Most lentogenic strains showed a greater t h e r m o s t a b i l i t y of infectivity over h a e m a g g l u t i n i n , whereas 3 lentogenic strains (Queensland V 4, Ulster, NDV-6) 1 mesogenic s t r a i n (H) a n d 2 velogenic strains (Texas GB, Herts) b e h a v e d reversely. The b e h a v i o u r of strains Sz a n d L was i n t e r m e d i a t e between the two patterns. 3.2. Thermostability o~ N D V Strains at 56 ° C The time required for a titre decrease by two (and even three) logarithmic orders was w i t h i n 10 m i n u t e s ( K > 0 . 5 0 ) for five of the e x a m i n e d n i n e lentogenic strains (Table 2). The mesogenic strains a n d strains Texas a n d Herts 33 of the t e n velogenic strains b e h a v e d similarly. The r e m a i n i n g four lentogenic a n d eight velogenie strains h a d a rate c o n s t a n t of < 0 . 5 0 a n d required a t least 10 m i n u t e s or more for a n i n f e c t i v i t y decrease b y two logarithmic orders. All velogenic strains, the mesogenic s t r a i n t I a n d the lentogenic strains Ulster, Q u e e n s l a n d V 4 a n d NDV-6 h a d a t h e r m o s t a b l e h a e m a g g l u t i n i n (survival > 3 0 minutes). W i t h strains
252
B. Lo~:N:ez::
Sz a n d L t h e h a e m a g g l u t i n i n t h e r m o s t a b i l i t y was i n t e r m e d i a t e , w h e r e a s t h e r e s t of t h e t e n t o g e n i c s t r a i n s possessed a t h e r m o l a b i l e h a e m a g g l u t i n i n ( s u r v i v a l 30 >30 >30 >30
1.56-b 0.60 0.86~-0.33 0.48 0.46 0.39 0.36 0.28 [email protected]
I/Ha character ~
Lentogenic LaSota F B1, N D V - 5 5 , -368 Ira
NDV-18 Me Sz Ulster Queensland V 4 NDV-6
I-Ha-
I+H~
±
i+Ha +
Mesogenic I-I L
I-Ha+
I-Ha ±
Velogenic Texas G B H e r t s 33 Vad Gbr D, K B, L E A
I-Ha +
I+Ha+
Criteria in Table 3. b N o t done. Table 3. Criteria/or the Thermostability Character o / N D V Strains T i m e of 2 log decrease in a c t i v i t y (minutes) Activity
50 ° C
56 ° C
Infectivity, I stable I+ labile I-
> 100 < 100
> 10 < 10
Haemagglutinin, Ha stable I-Ia ÷ labile I-Ia-
> 240 _ 30
,:, \z \?
L.
o
\
,
50
",,5"., -,
+--
~E
I....
I
!
48
50
52
"tp
54
56
Temperetu re,°C Fig. I. Stability of the haemagglutinin of lentogenic strains of ~NDV temperature
at different
4. Discussion The thermoresistance and the virulence of NDV strains are not related to one another. Strains of higher or lower heat sensitivity occurred in both lentogenic and velogenic groups. I t is, nevertheless, remarkable that while the haemagglutinin of all examined vetogenic strains survived 30 minutes at 56 ° C, the lentogenic strains can be divided, according to their haemaggtutinin resistance, into at least two groups: i) thermolabile and ii) thermostable strains. Formerly, thermolabile strains whose haemagglutinin became inactivated within 5 minutes at 56 ° C were chiefly found in the lentogenie group (HA~'so~ and BRA~DL~Y, 1955; Hx~SO~+ et al., 1967; A~+o~., 1971). The infectivity of these strains decreased by at least three logarithmic orders within less than 10 minutes and the thermostability of their haemagglutinin was usually less than one hour at, 50 ° C, thus showing a thermostability character of I - H a - . On the other hand, the haemagglutinin of the
254
B. LoM~iczi:
thermostable lentogenic strains survives longer than 30 minutes at 56 ° C, more than 4 hours at 50°C and these strains require at least 20 minutes for a 2 log decrease of infectivity titre at 56 ° C, thus, their thermostability character is I + H a +. The existence of thermostable lentogenic strains was not detected until the recent description of the Ulster strain b y I~IcF~I~I~.A~and NELSON (197i). In the present study, strain Queensland V4 isolated by SI~Mo~s (1967) in Australia and strain NDV-6 isolated by DI~N and BA~t~I~GER (1967) from slaughterhouse earcases in Hungary, were additionally identified as heat stable lentogenic strains. Strain Sz is intermediate in respect of heat sta,bility (B. L o ~ I C Z I , unpublished data), but can at all events be easily differentiated from thermolabile strains. FOSTEI~ and TI~IOMPSo~ (t957) who studied the survival of four NDV strains of different. virulence at various temperatures found them to have an equal thermostabi]ity. I t does not seem improbable that the thermolabile lentogenic strains, which occur in nature and can not infrequently be isolated from poultry flocks healthy or showing respiratory symptoms (Me, Ita, NDV-55, NDV-56, NDV-368), are in fact the descendants of one of the strains used as live vaccines (L~Sota, B1) being also thermolabile. Nevertheless, new markers (HANso~-, et al. 1967) would be required to substantiate this assumption. The origin of the thermostable lentogenie strains is unclear, and two hypothetical explanations can be considered; one being that they arose by attenuation of heat-stable mesogenic or velogenic strains, the other that the lentogenie strains m a y have acquired thermostable haemagglutinin and/or other structural proteins responsible for high heat resistance through completion of the original genetic pool. Comparison of the times required for activity decrease by two logarithmic orders showed that in most strains, the haemagglntinin is considerably more resistant to exposure at 56 ° C than the infectivity. This is in accordance with the fhldings of others (DIGIOI)~ et al., 1970 ; PIERC~ and I-IAYwOOD,1973). Lentogenie strains, however, showed either an almost equal thermoresistanee of both activities or a greater resistance of infectivity at 56 ° C and a consequently lower resistance of haemagglutinin at 50 ° C. Yet, not a single strain was found to have the character I+Ha -. In a recent paper, however, published by ESTOLA (1974), an exceptionally high thermostable velogenic NDV strain has been described. Though exact thermal inactivation curves for infectivity are not presented, the Finnish strain seems to fit into the I + H a - category. Paramyxoviruses were generally found to be thermolabile in the classical thermostability tests (50 ° C, 30 minutes, I~[cc~:, 1964), and in some virus groups thermostability even appeared to be related to the acid-fast property (KETLEI~ et al., 1962). In the present study not a single NDV strain lost two logaiithmic ordeis of infectivity titre within 30 minutes, whilst the titre of six thermostable NDV strains felt off b y more than 4 logarithmic orders within one hour at p H 3.0 (B. Los~ICZI, unpublished data), thus, the stability to heat or low p H values should be considered independent qualities of NDV strains.
Aeknowledfments The skillful technical assistance of Miss Ildik5 Sornod is appreciated.
Thermostability of N D V Strains of Different Virulence
255
Referenees 1. A~ox., in: Methods for Examining Poultry Biologics and for Identifying and Quantifying Avian Pathogens. pp. 66--93. National Academy of Sciences, ~¥a.shington, D.C., 1971. 2. DIGIOlA, G . A . , J. J. LICCIAI~D:ELLO, J. T. R. NICKERSON, and S. A. GOLD]3LITH: Thermal inactivation of Newcastle disease virus. Appl. Microbiol. 19, 451--454 (1970). 3. Dm~N, Cs., and K. BAMBERGE~g: Comparison of Newcastle disease virus strains isolated from broiler chickens (in Hungarian). Magy. Ao. Lap. 22, 160--t62 (1967). 4. ESTOLA, T.: Isolation of a Finnish Newcastle disease virus with an exceptionally high thermostability. Avian Dis. 18, 274--277 (t974). 5. FAnlNAS, E. C. : Avian pest, a disease of birds hitherto unknown in the Philippine Islands. Ptfilipp. J. Agr. 1, 311--366 (1930). 6. FOSTER, N . M . , and C. H. T~oMPsoN: The comparative thermolability of four strains of Newcastle disease virus of widely varying virulence. Vet. Med. 52, 119 to t21 (1957). 7. HANSON, R. P., and C. A. BRAN])LY: Identification of vaccine strains of Newcastle disease virus. Science 122, 156~ 157 (1955). 8. HANSO~~, R. P., E. UPTON, C. A. Bt~ANt)LEY, and N. S. ~VINsLow: Heat. stability of hemagglutinin of various strains of Newcastle disease virus. Proc. Soe. exp. Biol. (N.Y.) 70, 283--287 (1949). 9. t-IANsON, R . P . , J. SPALATIN, J. ES:['I~INAN, and G. SOl,LOEb: Identification of lentogenic strains of Newcastle disease virus. Avian Dis. 11, 49--53 (1967). 10. HvCK, R. A. : The classification of viruses. Vet. Bull. (Weybridge) 34, 239--253 (1964). 11. KETLF,R, A., V. V. HA~PA~IAN, and M. R. HILLE~AN: Characterization and classification of ECHO28 rhinovirus-coryzavirus agents. Proe. Soc. exp. Biol. (N.Y.) 110, 821--831 (1962). 12. Los~NlCZI, B. : Studies on interferon production and interferon sensitivity of different strains of the Newcastle disease virus. J. gen. Virot. 21, 305--313 (1973). 13. Lo~NleZi, B. : Plaque assay for avirulent (lentogenic) strains of Newcastle disease virus, Appl. Mierobiol. 27, 1162--t 163 (1974). 14. MCFE~RAN, J . B . , and 1~. NELSON: Some properties of an avirulent Newcastle disease virus. Arch. gas. Virusforsch. 34, 64--74 (1971). t5. PICKEN, J. C., J]~.: Thermostability of Newcastle disease virus. I n : Newcastle Disease Virus: An Evolving Pathogen (R. P. HAnsoN, ed.), pp. 167--188. The University of Wisconsin Press, Madison and Milwaukee, 1964. 16. PIERCE, J. S., and A. 2¢I. ttAYWOOD : Thermal inactivation of Newcastle disease virus. I. Coupled inactivation rates of hemagglutinating and neuraminidase activities. J. Virol. 11, 168--176 (1973). 17. SI~Mo~s, G. C. : The isolation of Newcastle disease virus in Queensland. Aust. vet. J. 43, 29--30 (1967). 18. TA~:X~sw, G¥. : The use of spiral loops in serological and virological micro-methods. Acta microbiol. Acad. Sci. hung. 3, 191--202 (1955). Author's address: Dr. B. Los~iNiczI, Pf. 18, It-t581 Budapest, Hungary.
