Imūnā atbilde pret HCV vīrusa ARFP proteīnu un HCV genomu core/ARFP reģiona sekvenču analīze
Date
2017
Authors
Akmeņkalns, Gatis
Journal Title
Journal ISSN
Volume Title
Publisher
Latvijas Universitāte
Abstract
C hepatīta vīruss (HCV) ir viens no galveniem smagu aknu slimību izraisītājiem, HCV hroniskā infekcija bieži beidzas ar aknu cirrozi un hepatocelulāro karcinomu. Saskaņā ar Pasaules Veselības Organizācijas datiem aptuveni 71 miljons cilvēku cieš no hroniskas HCV infekcijas. Katru gadu no HCV izraisītām slimībām mirst aptuveni 399000 cilvēku. HCV ir apvalku saturošs vīruss ar vienpaviediena RNS genomu un pieder flavivīrusu (flaviviridae) dzimtai. Tas izplatās, galvenokārt, ar asins starpniecību. HCV vīrusam piemīt augsta ģenētiskā mainība, kas traucē pretvīrusa vakcīnas izstrādi. HCV genomā ir iekodēts poliproteīns, kas pec translācijas tiek procesēts par vīrusa strukturāliem un nestrukturāliem proteīniem. HCV genomā N-galā atrodas alternatīvais nolasīšanas rāmjs, kas pārklajas ar core proteīnu kodēšjošo reģionu. Tās nolasīšanas rāmīs nesauūr ATG kodonu, tāpēc ARFP (alternatīvās nolasīšanas rāmja proteīns) tiek sintezēts ar ribosomas nobīdes mehānismu. Lai gan ir daudz pētīta, precīza ARFP bioloģiskā nozīme HCV dzīves ciklā nav zināma. Darba mērķis ir novērtēt ARFP proteīna imunogentitāti laboratorijas dzivnieku DNS imunizācijas eksperimentā un veikt HCV genomu core/ARFP reģiona sekvenču analīzi ar nolūku novērtēt core/ARFP sekvenču variantu ietekmi uz ARFP proteīna sintēzi. Darba rezultatā tika konstatēts, kā HCV ARFP ir imunogēns DNS imunizācijas eksperimentos, taču gadījumos, kad ARFP tika sintezēts no alternativā nolasišanas rāmja imunoatbilde pret to netika konstatēta. Visos darbā izmantotajos pacientu serumos atklātiem HCV vīrusiem tika noteikts 1b genotips. Visām iegūtajām HCV sekvencēm tika atrasts ARFP sintēzi izraisošs motīvs. Lielākoties noteiktais HCV ARF garums bija 143 kodoni, izņemot vienu, kura ARF garums ir 161 kodons. HCV ARFP sekvencēs tika novērota bieža aminoskābju nomaiņa. Anti-ARFP antivielu klatbūtne analizētos paraugus tika konstatēta pārāk reti, lai veiktu korelācijas analīzi ar augstu būtīskumu.
Hepatitis C virus (HCV) is one of the serious liver disease cause, HCV chronic infection often ends with liver cirrhosis and hepatocellular carcinoma. According to the World Health Organization, approximately 71 million people suffer from chronic HCV infection. Every year from HCV-related diseases dies approximately 399,000 people. HCV is an enveloped virus with sigle strain RNA genome belonging to the flavivirus (Flaviviridae) family. It is spread mainly by blood. HCV virus has a high genetic variability that complicates the development of anti-viral vaccine. HCV genome encodes polyprotein that after translation get processed in the viral structural and nonstructural proteins. HCV genome N-terminal part contains also alternative reading frame that overlaps with the core protein encoding region. That reading frame do not contain ATG codone, so ARFP (alternative reading frame protein) get synthesized using ribosome frameshift. Although there are various studies, the exact biological significance of ARFP for HCV life cycle is unknown. The aim of the study is to assess ARFP protein immunogenicity in laboratory animal DNA immunization experiment and to perform the HCV genome core/ARFP region sequence analysis in order to clarify the effect of core/ARFP sequence variants on the ARFP protein synthesis. It was foud that HCV ARFP is immunogenic in DNA immunization experiment, however, in cases when ARFP were synthesized from an alternative reading frame immune responce against it was not detected. All the HCV viruses discovered in the chronic HCV patients sera used in study belong to genotype 1b. In all acquired HCV sequences was found the motive initiating the synthesis of ARFP. Most of the sequnced HCV ARF are 143 codons long, except the one 161 codon long. In HCV ARFP sequences frequent amino acid replacement were observed. Anti-ARFP presence of antibodies in the analyzed samples were found to be too rare to make a correlation analysis with high signifficance.
Hepatitis C virus (HCV) is one of the serious liver disease cause, HCV chronic infection often ends with liver cirrhosis and hepatocellular carcinoma. According to the World Health Organization, approximately 71 million people suffer from chronic HCV infection. Every year from HCV-related diseases dies approximately 399,000 people. HCV is an enveloped virus with sigle strain RNA genome belonging to the flavivirus (Flaviviridae) family. It is spread mainly by blood. HCV virus has a high genetic variability that complicates the development of anti-viral vaccine. HCV genome encodes polyprotein that after translation get processed in the viral structural and nonstructural proteins. HCV genome N-terminal part contains also alternative reading frame that overlaps with the core protein encoding region. That reading frame do not contain ATG codone, so ARFP (alternative reading frame protein) get synthesized using ribosome frameshift. Although there are various studies, the exact biological significance of ARFP for HCV life cycle is unknown. The aim of the study is to assess ARFP protein immunogenicity in laboratory animal DNA immunization experiment and to perform the HCV genome core/ARFP region sequence analysis in order to clarify the effect of core/ARFP sequence variants on the ARFP protein synthesis. It was foud that HCV ARFP is immunogenic in DNA immunization experiment, however, in cases when ARFP were synthesized from an alternative reading frame immune responce against it was not detected. All the HCV viruses discovered in the chronic HCV patients sera used in study belong to genotype 1b. In all acquired HCV sequences was found the motive initiating the synthesis of ARFP. Most of the sequnced HCV ARF are 143 codons long, except the one 161 codon long. In HCV ARFP sequences frequent amino acid replacement were observed. Anti-ARFP presence of antibodies in the analyzed samples were found to be too rare to make a correlation analysis with high signifficance.
Description
Keywords
Bioloģija , ARFP , PCR , HCV , Genotips