Sequence analysis of the thymidine kinase genes of acyclovir-resistant varicella zoster virus isolates

128 In V i t r o and In V i v o C h a r a c t e r i z a t i o n of H e r p e s S i m p l e x V i r u s I s o l a t e s R e c o v e r e d from HIV+ Patients. E. L. Hill, G.A. Hunter, and M. N. Ellis. B u r r o u g h s W e l l c o m e Co., Res. Tri. Park, N.C., USA. In p a t i e n t s w i t h n o r m a l i m m u n i t y A C V r e s i s t a n c e has not b e e n a s i g n i f i c a n t c l i n i c a l problem. However, in t h e i m m u n o c o m p r o m i s e d p a t i e n t A C V - r e s i s t a n t H S V can c a u s e l o c a l l y progressive d i s e a s e . C o n c e r n t h a t i s o l a t e s shed by H I V + p a t i e n t s m i g h t p o s s e s s novel m e c h a n i s m s of r e s i s t a n c e or b e more virulent than previously reported resistant isolates led us to e v a l u a t e t h e s e i s o l a t e s in v i t r o and in vivo. In v i t r o a n a l y s i s of t h e c l i n i c a l i s o l a t e s w a s by t h e d y e u p t a k e assay, differential p l a q u e a u t o r a d i o g r a p h y , and e n z y m e k i n e t i c s . Of t h e i00 i s o l a t e s r e c o v e r e d from 51 patients, 77 w e r e A C V r e s i s t a n t w i t h ED50 v a l u e s of >3 ug/ml. Of t h e s e 77 r e s i s t a n t viruses, 75 h a d a t h y m i d i n e k i n a s e - d e f i c i e n t (TKd) p h e n o t y p e and 2 h a d a T K - a l t e r e d (TKa) p h e n o t y p e . All A C V - s e n s i t i v e i s o l a t e s p o s s e s s e d a w i l d type TK. T h e c u t a n e o u s v i r u l e n c e of s e l e c t e d i s o l a t e s was d e t e r m i n e d in h a i r l e s s (HRS/J) and a t h y m i c mice, w h i l e n e u r o v i r u l e n c e w a s d e t e r m i n e d in B A L B / C mice. O u r r e s u l t s i n d i c a t e t h a t H S V i s o l a t e s r e c o v e r e d from H I V + p a t i e n t s are s i m i l a r in d r u g s u s c e p t i b i l i t y and animal p a t h o g e n i c i t y to H S V i s o l a t e s r e c o v e r e d from normal p a t i e n t s and n o n - A I D S i m m u n o c o m p r o m i s e d p a t i e n t s . At the p r e s e n t time, it is d i f f i c u l t to d e t e r m i n e t h e i n c i d e n c e of r e s i s t a n c e in t h i s p o p u l a t i o n b e c a u s e only i s o l a t e s from t r e a t m e n t f a i l u r e s are sent to us for analysis.

129 Sequence Analysis of the Thymidine Kinase Genes of Acyclovir-Resistant Varicella Zoster Virus Isolates. C. Talarico, W. Phelps, K. Biron. Burroughs Wellcome Co., Research Triangle Park, NC, USA AIDS patients often experience recurrent infections with varicella zoster virus (VZV) which require repeated or prolonged treatment with acyclovir. We have documented in vitro resistance of isolates recovered from four patients after treatment failure (Jacobson, et al. Annals of Internal Med. 112:187-191, 1990.). Sequence analysis of PCR-amplified DNA encoding the thymidine kinase (TK) of plaque-purified virus from these clinical isolates showed sequence changes. Viral strains isolated from two patients had nucleotide deletions in the TK DNA. These deletions introduced a premature termination codon which is expected to result in the production of a truncated protein. Immunoprecipitation of extracts from cells infected with these virus strains showed no detectable TK. Viral strains from the other two patients had nucleotide substitutions located in the proposed nucleoside-binding site of the TK protein which may be expected to change the substrate specificity of the TK. Immunoprecipitation of extracts from cells infected with these virus strains suggested that a full length TK protein was synthesized. Susceptibililities of these virds strains to TK dependent, candidate anti-VZV agents in the 5substituted uracil arabinoside class were determined. 112