scholarly journals Establishment of a Cell Culture Model of Persistent Flaviviral Infection: Usutu Virus Shows Sustained Replication during Passages and Resistance to Extinction by Antiviral Nucleosides

Viruses ◽  
2019 ◽  
Vol 11 (6) ◽  
pp. 560 ◽  
Author(s):  
Raquel Navarro Sempere ◽  
Armando Arias
Keyword(s):  
Cell Culture ◽  
Persistent Infection ◽  
Vero Cells ◽  
Viral Disease ◽  
Viral Rna ◽  
Prolonged Treatment ◽  
Structural Protein ◽  
Usutu Virus ◽  
Antiviral Nucleosides ◽  
Rna Levels

Chronic viral disease constitutes a major global health problem, with several hundred million people affected and an associated elevated number of deaths. An increasing number of disorders caused by human flaviviruses are related to their capacity to establish a persistent infection. Here we show that Usutu virus (USUV), an emerging zoonotic flavivirus linked to sporadic neurologic disease in humans, can establish a persistent infection in cell culture. Two independent lineages of Vero cells surviving USUV lytic infection were cultured over 82 days (41 cell transfers) without any apparent cytopathology crisis associated. We found elevated titers in the supernatant of these cells, with modest fluctuations during passages but no overall tendency towards increased or decreased infectivity. In addition to full-length genomes, viral RNA isolated from these cells at passage 40 revealed the presence of defective genomes, containing different deletions at the 5’ end. These truncated transcripts were all predicted to encode shorter polyprotein products lacking membrane and envelope structural proteins, and most of non-structural protein 1. Treatment with different broad-range antiviral nucleosides revealed that USUV is sensitive to these compounds in the context of a persistent infection, in agreement with previous observations during lytic infections. The exposure of infected cells to prolonged treatment (10 days) with favipiravir and/or ribavirin resulted in the complete clearance of infectivity in the cellular supernatants (decrease of ~5 log10 in virus titers and RNA levels), although modest changes in intracellular viral RNA levels were recorded (<2 log10 decrease). Drug withdrawal after treatment day 10 resulted in a relapse in virus titers. These results encourage the use of persistently-infected cultures as a surrogate system in the identification of improved antivirals against flaviviral chronic disease.

scholarly journals Characterization of Defective Viral RNA Produced during Persistent Infection of Vero Cells with Murray Valley Encephalitis Virus

1998 ◽  
Vol 72 (3) ◽  
pp. 2474-2482 ◽  
Author(s):  
Morag U. Lancaster ◽  
Stuart I. Hodgetts ◽  
John S. Mackenzie ◽  
Nadezda Urosevic
Keyword(s):  
Cell Culture ◽  
Persistent Infection ◽  
Rna Synthesis ◽  
Vero Cells ◽  
Viral Rna ◽  
Parental Virus ◽  
Murray Valley Encephalitis Virus ◽  
Viral Rnas ◽  
Persistently Infected ◽  
First Time

ABSTRACT Defective interfering viral particles are readily produced in cell culture after a high multiplicity of infection with many animal RNA viruses. Due to defects that they carry in their genomes, their life cycle needs to be complemented by the helper functions provided by a parental virus which makes them both dependent on and competitive with the parental virus. In many instances, this may cause the abrogation of a lytic cycle of the parental virus, leading to a persistent infection. In this paper, we describe for the first time the presence of truncated or defective interfering viral RNAs produced in Vero cells persistently infected with the flavivirus Murray Valley encephalitis virus. While these RNAs have not been detected in acutely infected Vero cells, their appearance coincided with the establishment of persistent infection. We also show for the first time that the defective viral RNAs replicate well in both cell culture and cell-free virus replication systems, indicating that they may interfere with the replication of parental virus at the level of viral RNA synthesis. Significantly, structural analyses of these RNA species including nucleotide sequencing have revealed that they carry similar nucleotide deletions encompassing the genes coding for the prM and E proteins and various gene segments coding for the N terminus of the NS1 protein. These deletions are in frame, allowing the synthesis of truncated NS1 proteins to occur in persistently infected cells. This may have further implications for the interference with the parental virus at the level of viral RNA synthesis in addition to a major one at the level of virion assembly and release.

scholarly journals The result of propagation of sheep and goat pox viral vaccine strains into vero cells

2019 ◽  
Vol 26 (01) ◽  
pp. 3-9
Author(s):  
Batmagnai E ◽  
Ariunbold G ◽  
Erdenechimeg D ◽  
Enkhmandakh Yo ◽  
Munkhgerel B ◽  
...  
Keyword(s):  
Cell Culture ◽  
Dna Sequences ◽  
Cytopathic Effect ◽  
Vero Cells ◽  
Viral Disease ◽  
Live Vaccine ◽  
Morbidity Rate ◽  
Produce Cell ◽  
Mortality And Morbidity ◽  
Viral Vaccine

Background: Sheep and goat pox viral disease, which affects negatively to our country’s economy by prevalence and infection, has high mortality and morbidity rate. Although our country manufactures sheep and goat pox viral vaccine using lamb’s testicle tissue in the Biocombinat (Bio-factory), in winter, there is high number of diseased animals, lamb testicle is scarce, therefore there is a need to produce cell-culture based sheep and goat pox vaccine. Materials and methods: Russian VNIIZJ strain type 2 sheep pox vaccine and Chinese goat pox live vaccine (serial number 010030) antigens were used after 20 times of dilution and propogated into Vero and BHK-21 cell culture. By PCR the result was examined and sequenced by ABI3130xl sequencer machine and sequences were compared by MEGA7 program Results: 3 days after infection sheep pox and 4 days after infection goat pox were shown CPE (cytopathic effect or cytopathogenic effect) respectively, 6 days after the infection both of them has been shown full CPE. From these infected materials we isolated viral DNA and run PCR assay using Biocombinat’s strain as a positive control. PCR products were all equal, 289 bp long. When we compare these DNA sequences of sheep and goat pox viruses, they were 92% identical to complete genome of Indian sheep and goat pox virus. Conclusion: We can produce cell-culture based live vaccine and diagnostic tests for sheep and goat pox viral disease by re-propagating these strains into Vero. Хонь, ямааны цэцгийн вирусын вакцины омгийг vero эсэд дасгасан дүн Хураангуй: Хонь, ямааны цэцгийн вируст өвчин халдвар, тархалт өндөртэй бөгөөд өвчлөл, хорогдлынтүвшин өндөр байгаа нь манай улсын эдийн засагт сөргөөр нөлөөлж байна. Манай улс Биокомбинат (Био-үйлдвэр) үйлдвэрт хонины цэцэг өвчний эсрэг вакциныг хурганы төмсөгний анхдагч эдэд өсгөвөрлөх замаар амьд вакцин, ямааны цэцэг өвчний эсрэг вакциныг ямаанд халдвар хийж ам, хамрын орчинд үүссэн шархны эдийг химийн бодисоор идэвхгүйжүүлэх замаар тус тус үйлдвэрлэж байна. Ялангуяа хонины цэцэг өвчний вакциныг зөвхөн мал төллөх хаврын цагт үйлдвэрлэх боломжтой байдаг нь өвлийн цагт гарсан өвчнийг хянах аргагүйд хүрч байна. Иймээс цаг хугацаанаас хамааралгүй вакцин үйлдвэрлэхэд дамжмал эсийн технологи хэрэглэх зайлшгүй шаардлагатай байна. Оросын VNIIZJ омогашигласан 2 төрлийн хонины цэцгийн амьд вакцин, Хятадын ямааны цэцгийн амьд вакцин (серийн дугаар 010030) шингэлж, Vero эсийн өсгөвөрт халдаасан. Стандарт ПГУ-аар үр дүнг шалгаж, ABI3130xl sequencer машинаар үүсгэгчийн нуклеотидын дарааллыг тодорхойлсон ба MEGA7 програмаар дарааллуудыг харьцуулж удам зүйн мод байгуулсан. Эсэд халдвар хийснээс 3 хоногийн дараа хонины цэцэг, 4 хоногийн дараа ямааны цэцэг CPE (cytopathic effect) буюу эс эмгэгшүүлэх нөлөө тус тус үзүүлсэн бөгөөд 6 хоногийн дараа хоёулаа бүрэн CPE үзүүлсэн байна. Эдгээр вирус агуулсан эсийн тэжээлт орчноос бид вирусын ДНХ ялгаж Биокомбинат-ын хонины цэцгийн Перего омгийг эерэг хяналт болгон ашиглаж ПГУ-ын шинжилгээг явуулсан. ПГУ-ын бүтээгдэхүүн бүгд адилхан, 289 хос суурийн урттай байсан. Хонь, ямааны цэцгийн вирусын ДНХ-ийн нуклеотидын дарааллыг харьцуулж үзэхэд хониныцэцгийн вирусын бүтэн геномтой 92%, ямааны цэцгийн вирусын бүтэн геномтой 90% адилханбайв. Бид эдгээр өвчний үүсгэгчийг Vero эсэд халдвар хийх нөхцлийг тогтворжуулсан ньдамжмал эсийн өсгөвөрт суурилсан амьд вакцины түүхий эдийг ихээр бэлтгэх боломжтойболлоо. Түлхүүр үг: эсийн өсгөвөр, эсийн эмгэгшил, днх дараалал, амьд вакцин

scholarly journals Production of Newcastle Disease Virus by Vero Cells Grown on Cytodex 1 Microcarriers in a 2-Litre Stirred Tank Bioreactor

2010 ◽  
Vol 2010 ◽  
pp. 1-7 ◽  
Author(s):  
Mohd Azmir Arifin ◽  
Maizirwan Mel ◽  
Mohamed Ismail Abdul Karim ◽  
Aini Ideris
Keyword(s):  
Cell Culture ◽  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Newcastle Disease ◽  
High Speed ◽  
Virus Titer ◽  
Vero Cells ◽  
Stirred Tank ◽  
Stirred Tank Bioreactor ◽  
Maximum Cell

The aim of this study is to prepare a model for the production of Newcastle disease virus (NDV) lentogenic F strain using cell culture in bioreactor for live attenuated vaccine preparation. In this study, firstly we investigated the growth of Vero cells in several culture media. The maximum cell number was yielded by culture of Vero cells in Dulbecco's Modified Eagle Medium (DMEM) which was1.93×106 cells/ml. Secondly Vero cells were grown in two-litre stirred tank bioreactor by using several commercial microcarriers. We achieved the maximum cell concentration about7.95×105 cells/ml when using Cytodex 1. Later we produced Newcastle Disease virus in stirred tank bioreactor based on the design developed using Taguchi L4 method. Results reveal that higher multiplicity of infection (MOI) and size of cell inoculums can yield higher virus titer. Finally, virus samples were purified using high-speed centrifugation based on3∗∗(3-1) Fractional Factorial Design. Statistical analysis showed that the maximum virus titer can be achieved at virus sample concentration of 58.45% (v/v), centrifugation speed of 13729 rpm, and centrifugation time of 4 hours. As a conclusion, high yield of virus titer could be achieved through optimization of cell culture in bioreactor and separation by high-speed centrifugation.

Mycobacterium abscessus Infection in an Owl Monkey (Aotus trivirgatus)

1970 ◽  
Vol 7 (5) ◽  
pp. 448-454 ◽  
Author(s):  
Alfred G. Karlson ◽  
Herman R. Seibold ◽  
Robert H. Wolf
Keyword(s):  
Cell Culture ◽  
Vero Cell ◽  
Intraperitoneal Injection ◽  
Herpes Virus ◽  
Vero Cells ◽  
Mycobacterium Abscessus ◽  
Vero Cell Culture ◽  
Owl Monkey ◽  
Herpès Virus

Mycobacterium abscessus was isolated from the lungs of an owl monkey which died 27 days after intraperitoneal injection of herpes virus-infected Vero cells. The lungs and liver had multiple microscopic granulomas with acid-fast microorganisms. The mycobacteria also were isolated from a Vero-cell culture inoculated with a suspension of lung and liver. The same microorganism was eventually isolated from Vero cells of the same source as that used to propagate the herpes virus for the original attempt to infect the monkey.

scholarly journals Dengue Viral RNA Levels in Peripheral Blood Mononuclear Cells Are Associated with Disease Severity and Preexisting Dengue Immune Status

PLoS ONE ◽  
2012 ◽  
Vol 7 (12) ◽  
pp. e51335 ◽  
Author(s):  
Anon Srikiatkhachorn ◽  
Sineewanlaya Wichit ◽  
Robert V. Gibbons ◽  
Sharone Green ◽  
Daniel H. Libraty ◽  
...  
Keyword(s):  
Disease Severity ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Immune Status ◽  
Viral Rna ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Rna Levels

scholarly journals An Alanine-to-Valine Substitution in the Residue 175 of Zika Virus NS2A Protein Affects Viral RNA Synthesis and Attenuates the Virus In Vivo

Viruses ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 547 ◽  
Author(s):  
Silvia Márquez-Jurado ◽  
Aitor Nogales ◽  
Ginés Ávila-Pérez ◽  
Francisco Iborra ◽  
Luis Martínez-Sobrido ◽  
...  
Keyword(s):  
Cdna Clone ◽  
Zika Virus ◽  
Rna Synthesis ◽  
Infectious Clone ◽  
Vero Cells ◽  
Viral Rna ◽  
Single Amino Acid ◽  
Reverse Genetic System ◽  
Infectious Clones

The recent outbreaks of Zika virus (ZIKV), its association with Guillain–Barré syndrome and fetal abnormalities, and the lack of approved vaccines and antivirals, highlight the importance of developing countermeasures to combat ZIKV disease. In this respect, infectious clones constitute excellent tools to accomplish these goals. However, flavivirus infectious clones are often difficult to work with due to the toxicity of some flavivirus sequences in bacteria. To bypass this problem, several alternative approaches have been applied for the generation of ZIKV clones including, among others, in vitro ligation, insertions of introns and using infectious subgenomic amplicons. Here, we report a simple and novel DNA-launched approach based on the use of a bacterial artificial chromosome (BAC) to generate a cDNA clone of Rio Grande do Norte Natal ZIKV strain. The sequence was identified from the brain tissue of an aborted fetus with microcephaly. The BAC clone was fully stable in bacteria and the infectious virus was efficiently recovered in Vero cells through direct delivery of the cDNA clone. The rescued virus yielded high titers in Vero cells and was pathogenic in a validated mouse model (A129 mice) of ZIKV infection. Furthermore, using this infectious clone we have generated a mutant ZIKV containing a single amino acid substitution (A175V) in the NS2A protein that presented reduced viral RNA synthesis in cell cultures, was highly attenuated in vivo and induced fully protection against a lethal challenge with ZIKV wild-type. This BAC approach provides a stable and reliable reverse genetic system for ZIKV that will help to identify viral determinants of virulence and facilitate the development of vaccine and therapeutic strategies.

scholarly journals Quantitative measurement of infectious virus in SARS-CoV-2 Alpha, Delta and Epsilon variants reveals higher infectivity (viral titer:RNA ratio) in clinical samples containing the Delta and Epsilon variants.

2021 ◽  
Author(s):  
Hannah W Despres ◽  
Margaret G Mills ◽  
David J Shirley ◽  
Madaline M Schmidt ◽  
Meei-Li Huang ◽  
...  
Keyword(s):  
Quantitative Measurement ◽  
Infectious Virus ◽  
Viral Rna ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Genome Copy ◽  
Live Virus ◽  
High Degree ◽  
The Relationship ◽  
Rna Levels

ABSTRACT Background Novel SARS-CoV-2 Variants of Concern (VoC) pose a challenge to controlling the COVID-19 pandemic. Previous studies indicate that clinical samples collected from individuals infected with the Delta variant may contain higher levels of RNA than previous variants, but the relationship between viral RNA and infectious virus for individual variants is unknown. Methods We measured infectious viral titer (using a micro-focus forming assay) as well as total and subgenomic viral RNA levels (using RT-PCR) in a set of 165 clinical samples containing SARS-CoV-2 Alpha, Delta and Epsilon variants that were processed within two days of collection from the patient. Results We observed a high degree of variation in the relationship between viral titers and RNA levels. Despite the variability we observed for individual samples the overall infectivity differed among the three variants. Both Delta and Epsilon had significantly higher infectivity than Alpha, as measured by the number of infectious units per quantity of viral E gene RNA (6 and 4 times as much, p=0.0002 and 0.009 respectively) or subgenomic E RNA (11 and 7 times as much, p<0.0001 and 0.006 respectively). Conclusion In addition to higher viral RNA levels reported for the Delta variant, the infectivity (amount of replication competent virus per viral genome copy) may also be increased compared to Alpha. Measuring the relationship between live virus and viral RNA is an important step in assessing the infectivity of novel SARS-CoV-2 variants. An increase in the infectivity of the Delta variant may further explain increased spread and suggests a need for increased measures to prevent viral transmission.

scholarly journals One-step RNA extraction for RT-qPCR detection of 2019-nCoV

2020 ◽  
Author(s):  
Monica Sentmanat ◽  
Evguenia Kouranova ◽  
Xiaoxia Cui
Keyword(s):  
Real Time ◽  
Rna Extraction ◽  
Virus Transmission ◽  
Extraction Methods ◽  
Viral Rna ◽  
Taqman Probe ◽  
N Gene ◽  
Structural Protein ◽  
Diagnostic Panel ◽  
Primer Sets

ABSTRACTThe global outbreak of coronavirus disease 2019 (COVID-19) has placed an unprecedented burden on healthcare systems as the virus spread from the initial 27 reported cases in the city of Wuhan, China to a global pandemic in under three month[1]. Resources essential to monitoring virus transmission have been challenged with a demand for expanded surveillance. The CDC 2019-nCoV Real-Time Diagnostic Panel uses a real-time reverse transcription polymerase chain reaction (RT-PCR) consisting of two TaqMan probe and primer sets specific for the 2019-nCoV N gene, which codes for the nucleocapsid structural protein that encapsulates viral RNA, for the qualitative detection of 2019-nCoV viral RNA in respiratory samples. To isolate RNA from respiratory samples, the CDC lists RNA extraction kits from four manufacturers. In anticipation of a limited supply chain of RNA extraction kits and the need for test scalability, we sought to identify alternative RNA extraction methods. Here we show that direct lysis of respiratory samples can be used in place of RNA extraction kits to run the CDC 2019-nCoV Real-Time Diagnostic assay with the additional benefits of higher throughput, lower cost, faster turnaround and possibly higher sensitivity and improved safety.

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