Аннотация
Вирус Западного Нила (ВЗН) — один из самых распространенных арбовирусов в мире, имеющий тенденцию к расширению аре- ала. Инфицирование человека в основном происходит через укусы кровососущих насекомых (трансмиссивный путь передачи).
Следствием заражения могут быть бессимптомная инфекция, развитие лихорадочной (гриппоподобной) формы заболева- ния, в редких случаях поражение центральной нервной системы с развитием менингита, энцефалита, либо острого вялого паралича. Для подтверждения диагноза лихорадки Западного Нила (ЛЗН) применяют в основном иммунологические и молеку- лярно-генетические методы. Используемые серологические исследования могут давать ложноположительные результаты вследствие перекрестных реакций антител с другими ортофлавивирусами. С помощью генодиагностических подходов РНК возбудителя можно выявить в острую фазу заболевания, в период серонегативного окна. Данные методы незаменимы при проведении эпидемиологического мониторинга за численностью зараженных переносчиков возбудителя инфекции, необходи- мого для прогноза заболеваемости ЛЗН. Цель: анализ особенностей лабораторной диагностики ЛЗН молекулярно-генетиче- скими методами, определение тенденций в разработке способов обнаружения РНК ВЗН, сравнение предложенных подходов с учетом их преимуществ и недостатков.
Материал и методы. Поиск источников литературы осуществлён в базах данных Scopus, Web of Science, MedLine, CyberLeninka, РИНЦ.
Результаты и обсуждение. В обзоре обозначены как общие проблемы диагностики ЛЗН (низкая выявляемость случаев ин- фицирования), так и сложности, возникающие при применении молекулярно-генетических подходов: транзиторная виремия, низкая вирусная нагрузка, высокая вариабельность генома ВЗН. Рассмотрены различные варианты полимеразной цепной реакции с обратной транскрипцией (ОТ-ПЦР), разработанные для диагностики ЛЗН, отличающиеся по способу детекции, типу и числу выявляемых мишеней. Отдельный раздел посвящен методам выявления РНК ВЗН, основанным на изотермиче- ской амплификации, в сравнении с ОТ-ПЦР. Охарактеризованы технологии ДНК-микрочипов и секвенирования нуклеотидной последовательности вируса, определены возможные области их применения.
Annotation
West Nile virus (WNV) is one of the most widespread arboviruses in the world, with a tendency to increase its endemic area. Human infection mainly occurs through the bites of blood-sucking insects (vector-borne transmission). Infection may result in asymptomatic and febrile (flu-like) forms of the disease, and, in rare cases, the central nervous system damage with the development of meningitis, encephalitis, or acute flaccid paralysis. Immunologic and molecular genetic approaches method are used to confirm the diagnosis of West Nile fever (WNF). The most commonly used serologic tests may give false-positive results due to cross-reactions of antibodies with other orthoflaviviruses. With the help of genetic approaches, RNA of the pathogen can be detected in the acute phase of the disease, during the seronegative window. In addition, these methods are indispensable for epidemiologic monitoring of the number of infected vectors, which is necessary for predicting the incidence of WNF.
The aim of the review is to analyze the peculiarities of laboratory diagnostics of WNF using molecular genetic methods, to identify trends in the development of West Nile virus RNA detection methods, and to compare the proposed approaches, taking into account their advantages and disadvantages.
Material and methods. Literature sources were searched in Scopus, Web of Science, MedLine, CyberLeninka, and RSCI databases.
Results and discussion. The review outlines both general problems of WNF diagnostics (low detection rate of infection cases) and difficulties arising in the application of molecular genetic approaches: transient viraemia, low viral load, high variability of the WNV genome. Different variants of reverse transcription polymerase chain reaction (RT-PCR) developed for WNF diagnostics, differing in detection method, type and number of targets to be detected, are considered. A separate section is devoted to the methods of detection of WNV RNA based on isothermal amplification in comparison to RT-PCR. The paper also characterizes DNA microarray and virus genome sequencing techniques and identifies their possible applications.
Key words: West Nile virus (WNV); West Nile fever (WNF); molecular genetic methods; review
Список литературы
Л И Т Е РАТ У РА ( п п . 2 , 3 , 5 — 1 6 , 1 8 , 1 9 , 2 1 , 2 3 — 3 0 , 3 2 — 4 9 с м . R E F E R E N C E S )
1 . Городин В .Н ., Нежурин А .В ., Жукова Л .И . Современные аспекты лихорадки Западного Нила . Инфекционные болезни. 2023; 21(1): 140-7 . DOI: 10 .20953/1729-9225-2023-1-140-147
2 . Путинцева Е .В ., Удовиченко С .К ., Никитин Д .Н ., Бородай Н .В ., Колоскова А .Ю ., Антонов А .С . и др . Лихорадка Западного Нила в Российской Федерации в 2024 г ., прогноз на 2025 г . Проблемы осо- бо опасных инфекций . 2025; (1): 84-95 . DOI: 10 .21055/0370-1069- 2025-1-84-95
17 . Красовская Т .Ю ., Шарова И .Н ., Щербакова С .А ., Яшечкин Ю .И ., Хуторецкая Н .В ., Ларичев В .Ф . и др . Разработка и внедрение тест- системы для лабораторной диагностики лихорадки Западного
Нила методом ПЦР . Здоровье населения и среда обитания. 2007;
6(171): 42-5 . DOI: 10 .21055/0370-1069-2011-3(109)-13-17
20 . Платонов А .Е ., Карань Л .С ., Шопенская Т .А . и др . Генотипирова- ние штаммов вируса лихорадки Западного Нила, циркулирующих на юге России, как метод эпидемиологического расследования:
принципы и результаты . Журнал микробиологии, эпидемиологии и
иммунобиологии. 2011; 2: 29-37 22 . Батурин А .А ., Ткаченко Г .А ., Леденева М .Л ., Лемасова Л .В ., Бон- дарева О .С ., Кайсаров и др . Молекулярно-генетический анализ вариантов вируса Западного Нила, циркулировавших на террито- рии европейской части России в 2010-2019 гг. Журнал микробио-
логии, эпидемиологии и иммунобиологии. 2021; 98(3): 308-18 . DOI:
10 .36233/0372-9311-85
31 . Прохватилова Е .В ., Ткаченко Г .А ., Батурин А .А ., Белицкая Л .И ., Топорков А .В . Оценка диагностической эффективности набора реагентов для in vitro диагностики лихорадки Западного Нила ме- тодом полимеразной цепной реакции с обратной транскрипцией и гибридизационно-флуоресцентной детекцией . БИОпрепараты.
Профилактика, диагностика, лечение. 2023; 23(1): 90-101 . DOI:
10 .30895/2221-996X-2023-23-1-90-101
R E F E R E N C E S
1 . Gorodin V .N ., Nezhurin A .V ., Zhukova L .I . Current aspects of West Nile fever . Infectious Diseases. 2023; 21(1): 140-7 . DOI: 10 .20953/1729-9225-2023-1-140-147 . (in Russian) 2 . Singh P ., Khatib M .N ., Ballal S . Kaur M ., Nathiya D ., Sharma S . et al . West Nile Virus in a Changing Climate: epidemiology, pathology, advances in diagnosis and treatment, vaccine designing and control strategies, emerging public health challenges — a
comprehensive review . Emerg. Microbes Infect. 2024; 2437244 . DOI: 10 .1080/22221751 .2024 .2437244
3 . Habarugira G ., Suen W .W ., Hobson-Peters J . Hall R .A ., Bielefeldt- Ohmann H . West Nile virus: an update on pathobiology, epidemiology, diagnostics, control and «one health» implications . Pathogens . 2020; 9(7): 589 . DOI: 10 .3390/pathogens9070589 4 . Putintseva E .V ., Udovichenko S .K ., Nikitin D .N ., Boroday N .V ., Koloskova A .Yu ., Antonov A .S . et al . West Nile Fever in the Russian Federation in 2024, Forecast for 2025 . Problemy osobo opasnykh infektsiy. 2025; (1): 84-95 . DOI: 10 .21055/0370-1069-2025-1-84-95 (in Russian) 5 . Ronca S .E ., Ruff J .C ., Murray K .O . A 20-year historical review of West Nile virus since its initial emergence in North America: Has West Nile virus become a neglected tropical disease? Vasconcelos PFC, ed- itor . PLOS Neglected Tropical Diseases. 2021; 15(5): e0009190 . DOI: 10 .1371/journal .pntd .0009190 6 . Rios M ., Daniel S ., Chancey C ., Hewlett I .K ., Stramer S .L . West Nile virus adheres to human red blood cells in whole blood . Clin. Infect.
Dis. 2007; 45: 181-6 . DOI: 10 .1086/518850 7 . Busch M .P ., Kleinman S .H ., Tobler L .H ., Kamel H .T ., Norris P .J ., Walsh I . et al . Virus and antibody dynamics in acute west nile virus infection . J. Infect. Dis. 2008; 198(7): 984-93 . DOI: 10 .1086/591467 8 . Kasule S ., Fernholz E ., Grant L ., Kole A ., Grys T .E ., Kaleta E . et al . Whole-Blood PCR Preferred for Timely Diagnosis of Neuroin- vasive West Nile Virus Infections: Lessons From the 2021 Arizona Outbreak . Open Forum Infect. Dis. 2024; 11(5): 188 . DOI: 10 .1093/ ofid/ofae188 9 . Lustig Y ., Sofer D ., Bucris E .D ., Mendelson E . Surveillance and Di- agnosis of West Nile Virus in the Face of Flavivirus Cross-Reactivi- ty . Front. Microbiol. 2018; 9: 2421 . DOI: 10 .3389/fmicb .2018 .02421 10 . Gdoura M ., Fares W ., Bougatef S ., Inoubli A ., Touzi H ., Hogga N . et al . The value of West Nile virus RNA detection by real-time RT-PCR in urine samples from patients with neuroinvasive forms . Arch Micro- biol. 2022; 204(5): 238 . DOI: 10 .1007/s00203-022-02829-6 11 . Cvjetković I .H ., Radovanov J ., Kovačević G ., Turkulov V ., Patić A Diagnostic value of urine qRT-PCR for the diagnosis of West Nile virus neuroinvasive disease . Diagn. Microbiol. Infect. Dis. 2023; 107(1): 115920 . DOI: 10 .1016/j .diagmicrobio .2023 .115920 12 . Lanciotti R .S ., Ebel G .D ., Deubel V ., Kerst AJ, Murri S, Meyer R . et al Complete genome sequences and phylogenetic analysis of West Nile virus strains isolated from the United States, Europe, and the Middle East . Virology . 2002; 298(1): 96-105 . DOI: 10 .1006/viro .2002 .1449 13 . Pachler K ., Lebl K ., Berer D ., Rudolf I ., Hubalek Z ., Nowotny N Putative new West Nile virus lineage in Uranotaenia unguiculata mos- quitoes, Austria, 2013 . Emerg. Infect. Dis. 2014; 12: 2119-22 . DOI: 10 .3201/eid2012 .140921
14 . Antonov A .S ., Shpak I .M ., Ustinov D .V ., Izhberdeeva M .P ., Guseva A .N ., Galkina A .Y . et al . Phylogenetic analysis and molecular genetic characteristics of West Nile virus lineage 2 isolates circulating in the Russian Federation . Virus Genes. 2024; 60(4): 370-6 . DOI: 10 .1007/ s11262-024-02079-2 15 . Shah-Hosseini N ., Chinikar S ., Ataei B ., Fooks A .R ., Groschup M .H . Phylogenetic analysis of West Nile virus genome, Iran . Emerg.
Infect. Dis. 2014; 20(8): 1419-21 . DOI: https://doi .org/10 .1099/ vir .0 .046888-0 16 . McMullen A .R ., Albayrak H ., May F .J ., Davis C .T ., Beasley D .W .C ., Barrett A .D .T . Molecular evolution of lineage 2 West Nile virus . J.
Gen. Virol. 2013; 94(Pt 2): 318-25 . DOI: 10 .1099/vir .0 .046888-0 17 . Krasovskaya T .Y ., Sharova I .N ., Shcherbakova S .A . et al . Develop- ment and implementation of a test system for laboratory diagnosis
of West Nile fever by PCR . Zdorov`e naseleniya I sreda obitaniya
2007; 6(171): 42-5 . DOI: 10 .21055/0370-1069-2011-3 (109)-13-17
(in Russian) 18 . Johnson N ., Wakeley P .R ., Mansfield K .L ., McCracken F ., Haxton B ., Phipps L .P . et al . Assessment of a novel real-time pan-flavivirus RT-polymerase chain reaction . Vector Borne Zoonotic Dis. 2010; 10: 665-71 . DOI: 1089/vbz .2009 .0210 19 . Lanciotti R .S ., Kerst A .J ., Nasci R .S ., Godsey M .S ., Mitchell C .J ., Savage H .M . et al . Rapid detection of West Nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a TaqMan Reverse Transcriptase-PCR assay . Journal of Clinical Microbiology. 2000; 38(11): 4066-71 . DOI: 10 .1128/jcm .38 .11 .4066- 4071 .2000
20 . Platonov A .E ., Karan’ L .S ., Shopenskaia T .A . et al . Genotyping of West Nile fever virus strains circulating in southern Russia as an epidemio- logical investigation method: principles and results . Zhurnal mikrobi-
ologii, epidemiologii I immunobiologii. 2011; 2: 29-37 . (in Russian)
21 . Lee D .H ., Mathew J ., Pfahler W ., Ma D ., Valinsky J ., Prince A .M . et al . Individual donor nucleic acid amplification testing for detection of West Nile virus . J. Clin. Microbiol. 2005; 43(10): 5111-6 . DOI: 10 .1128/JCM .43 .10 .5111-5116 .2005
22 . Baturin A .A ., Tkachenko G .A ., Ledeneva M .L ., Lemasova L .V ., Bondareva O .S ., Kaysarov I .D . et al . Molecular genetic analysis of West Nile virus variants circulating in European Russia between 2010
and 2019 . Zhurnal mikrobiologii, epidemiologii i immunobiologii.
2021; 98(3): 308-18 . DOI: 10 .36233/0372-9311-85 . (in Russian) 23 . De Filette M ., Ulbert S ., Diamond M .S ., Sanders N .N . Recent progress in West Nile virus diagnosis and vaccination . Vet. Res. 2012; 43(16) DOI: 10 .1186/1297-9716-43-16
24 . Warang A ., Zhang M ., Zhang S ., Shen Z . A panel of real-time PCR assays for the detection of Bourbon virus, Heartland virus, West Nile virus, and Trypanosoma cruzi in major disease-transmitting vectors . J. Vet. Diagn. Invest. 2021; 33(6): 1115-22 . DOI: 10 .1177/10406387211039549
25 . Xu Z ., Peng Y ., Yang M ., Li X ., Wang J ., Zou R . Simultaneous detection of Zika, chikungunya, dengue, yellow fever, West Nile, and Japanese encephalitis viruses by a two-tube multiplex real-time RT-PCR assay . J. Med. Virol. 2022; 94(6): 2528-36 . DOI: 10 .1002/ jmv .27658 26 . Mishra N ., Ng J ., Rakeman J .L ., Perry M .J ., Centurioni D .A ., Dean A .B ., Price A . et al . One-step pentaplex real-time polymerase chain reaction assay for detection of zika, dengue, chikungunya, West nile viruses and a human housekeeping gene. J. Clin. Virol. 2019; 120: 44- 50 . DOI: 10 .1016/j .jcv .2019 .08 .011 27 . Boga J .A ., Alvarez-Arguelles M .E ., Rojo-Alba S ., Rodríguez M ., de Oña M ., Melón S . Simultaneous detection of Dengue virus, Chikungunya virus, Zika virus, Yellow fever virus and West Nile virus . J. Virol. Methods. 2019; 268: 53-5 . DOI: 10 .1016/j jviromet .2019 .03 .014 28 . Nikiforova M .A ., Kuznetsova N .A ., Shchetinin A .M ., Butenko A .M ., Kozlova A .A ., Larichev V .P . Arboviruses in the Astrakhan region of Russia for 2018 season: The development of multiplex PCR assays and analysis of mosquitoes, ticks, and human blood sera . Infect.
Genet. Evol. 2021; 88: 104711 . DOI: 10 .1016/j .meegid .2021 .104711 29 . Fall G ., Faye M ., Weidmann M ., Kaiser M ., Dupressoir A ., Ndiaye E .H . et al . Real-Time RT-PCR assays for detection and genotyping of West Nile Virus lineages circulating in Africa . Vector Borne Zoonotic Dis. 2016; 16(12): 781-9 . DOI: 10 .1089/vbz .2016 .1967 30 . Vázquez A ., Herrero L ., Negredo A ., Hernández L ., Sánchez-Seco M .P ., Tenorio A . et al . Real time PCR assay for detection of all known
lineages of West Nile virus . J. Virol. Methods. 2016; 236: 266-70 DOI: 10 .1016/j .jviromet .2016 .07 .026 31 . Prokhvatilova E .V ., Tkachenko G .A ., Baturin A .A ., Belickaya L .I ., Toporkov A .V . Evaluation of the diagnostic efficacy of a reagent kit for in vitro diagnosis of West Nile fever using reverse transcription polymerase chain reaction with fluorescent probe-based detection
BIOpreparaty. Profilaktika, diagnostika, lechenie. 2023; 23(1): 90-
101 . DOI: 10 .30895/2221-996X-2023-23-1-90-101 . (in Russian) 32 . Parida M ., Posadas G ., Inoue S ., Hasebe F ., Morita K . Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of West Nile virus . Journal of Clinical Microbiology. 2004; 42(1): 257-63 . DOI: 10 .1128/jcm .42 .1 .257-263 .2004 33 . Kim D ., DeBriere T .J ., Eastmond B .H ., Alomar A .A ., Yaren O ., McCarter J . et al . Rapid detection of West Nile and Dengue viruses from mosquito saliva by loop-mediated isothermal amplification and displaced probes . PLoS One. 2024; 19(2): e0298805 . DOI: 10 .1371/ journal .pone .0298805 34 . Wheeler S .S ., Ball C .S ., Langevin S .A ., Fang Y ., Coffey L .L ., Meagher R .J . Surveillance for Western Equine Encephalitis, St . Louis Encephalitis, and West Nile viruses using reverse transcription loop- mediated isothermal amplification . PLoS One. 2016; 11(1): e0147962 DOI: 10 .1371/journal .pone .0147962 35 . Khedhiri M ., Chaouch M ., Ayouni K ., Chouikha A ., Gdoura M ., Touzi H . et al . Development and evaluation of an easy to use real-time reverse-transcription loop-mediated isothermal amplification assay for clinical diagnosis of West Nile virus . J. Clin. Virol. 2024; 170: 105633 . DOI: 10 .1016/j .jcv .2023 .105633 36 . Tomar P .S ., Patel S ., Dash P .K ., Kumar J .S . Simple and field amenable loop-mediated isothermal amplification-lateral flow dipstick assay for detection of west Nile virus in human clinical samples . J. Appl.
Microbiol. 2022; 133(6): 3512-22 . DOI: 10 .1111/jam .15783 37 . Burkhalter K . L ., O’Keefe M ., Holbert-Watson Z ., Green T ., Savage H .M ., Markowski D .M . Laboratory and field evaluations of a commercially available real-time Loop-Mediated Isothermal Amplification assay for the detection of West Nile Virus in mosquito pools . J. Am. Mosq. Control Assoc. 2021; 37(4): 256-62 . DOI: 10 .2987/21-7033
38 . Lanciotti R .S ., Kerst A .J . Nucleic acid sequence-based amplification assays for rapid detection of West Nile and St . Louis encephalitis
viruses . Journal of clinical microbiology. 2001; 39(12): 4506-13
DOI: 10 .1128/JCM .39 .12 .4506-4513 .2001
39 . Ziermann R ., Sánchez-Guerrero S .A . PROCLEIX® West Nile virus assay based on transcription-mediated amplification . Expert
Review of Molecular Diagnostics . 2008; 8(3): 239-45 . DOI:
10 .1586/14737159 .8 .3 .239
40 . Tomar P .S ., Kumar S ., Patel S ., Kumar J .S . Development and Evaluation of Real-Time Reverse Transcription Recombinase Polymerase Amplification Assay for Rapid and Sensitive Detection of West Nile Virus in Human Clinical Samples . Front Cell Infect.
Microbiol. 2021; 10: 619071 . DOI: 10 .3389/fcimb .2020 .619071 41 . Myhrvold C ., Freije C .A ., Gootenberg J .S ., Abudayyeh O .O ., Metsky H .C ., Durbin A .F . et al . Field-deployable viral diagnostics using CRISPR-Cas13 . Science . 2018; 360 (6387): 444-8 . DOI: 10 .1126/ science .aas8836 42 . Nordström H ., Falk K .I ., Lindegren G ., Mouzavi-Jazi M ., Waldén A ., Elgh F . et al . DNA microarray technique for detection and identification of seven flaviviruses pathogenic for man . J. Med. Virol. 2005; 77(4): 528-40 . DOI: 10 .1002/jmv .20489 43 . Berthet N ., Paulous S ., Coffey L .L ., Frenkiel M .P ., Moltini I ., Tran C . et al . Resequencing microarray method for molecular diagnosis of human arboviral diseases . J. Clin. Virol. 2013; 56(3): 238-43 . DOI: 10 .1016/j .jcv .2012 .10 .022 44 . De Giorgi V ., Zhou H ., Alter H .J ., Allison R .D . A microarray-based pathogen chip for simultaneous molecular detection of transfusion- transmitted infectious agents. J. Transl. Med. 2019; 17(1): 156 . DOI: 10 .1186/s12967-019-1905-4 45 . Wollants E ., Smolders D ., Naesens R ., Bruynseels P ., Lagrou K ., Matthijnssens J . et al . Use of next-generation sequencing for diagnosis of West Nile Virus infection in patient returning to Belgium from Hungary . Emerg. Infect. Dis . 2018; 24(12): 2380-2 . DOI: 10 .3201/ eid2412 .180494 46 . Wilson M . R ., Zimmermann L . L ., Crawford E . D ., Sample H .A ., Soni P .R ., Baker A .N . et al . Acute west nile virus meningoencephalitis diagnosed via metagenomic deep sequencing of cerebrospinal fluid in a renal transplant patient . Am. J. Transplant. 2017; 17: 803-8 . DOI: 10 .1111/ajt .14058 47 . Williams S .H ., Cordey S ., Bhuva N ., Laubscher F ., Hartley M .A ., Boillat-Blanco N . et al . Investigation of the Plasma Virome from Cases of Unexplained Febrile Illness in Tanzania from 2013 to 2014: a Comparative Analysis between Unbiased and VirCapSeq-VERT High-Throughput Sequencing Approaches . mSphere . 2018; 3(4): e00311-18 . DOI: 10 .1128/mSphere .00311-18 48 . Tešović B ., Nišavić J ., Banović Đeri B ., Petrović T ., Radalj A .,
Šekler M . et al . Development of multiplex PCR based NGS protocol for whole genome sequencing of West Nile virus lineage 2 directly from biological samples using Oxford Nanopore platform . Diagn Microbiol. Infect. Dis. 2023; 105(2): 115852 . DOI: 10 .1016/j diagmicrobio .2022 .115852 49 . Hyeon J .Y ., Helal Z .H ., Appel A ., Tocco N ., Hunt A ., Lee D .H . et al Whole genome sequencing and phylogenetic analysis of West Nile viruses from animals in New England, United States, 2021 . Front. Vet.
Sci. 2023; 10: 1085554 . DOI: 10 .3389/fvets .2023 .1085554