Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

Summary

The first step in the arenavirus life cycle is attachment of viral particles to host cells. We report a quantitative (q)RT-PCR-based assay for ultrasensitive detection and quantitation of arenavirus attachment events.

Abstract

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.

Introduction

Arenaviruses are a family of enveloped, single-stranded RNA viruses that are maintained primarily in rodents in nature^1-3. While these viruses typically establish an asymptomatic, persistent infection in rodent reservoirs^1,2, they can cause severe disease in humans³. Important pathogenic species include the New World Junin virus (JUNV)⁴ and the Old World Lassa virus⁵, which are the etiologic agents of Argentine hemorrhagic fever in South America and Lassa fever in Africa, respectively. Lymphocytic choriomeningitis virus (LCMV), the prototypic virus of the family⁶, has a worldwide distribution and is res....

Protocol

Note: It is critical that the described protocol be carried out using strict pre-PCR technique (see ²⁸ for an excellent overview on how to set up a pre-PCR laboratory and how to conduct experiments using good pre-PCR technique). It is imperative that all reagents and equipment are free of amplified DNA containing the qPCR target sequence and that appropriate controls are in place to detect such contamination. An overview of the protocol is shown in Figure 1.

1. Prepare Media and Seed Cells in 48-well Plates

1. Prepare 500 ml of complete Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum, 1% penic....

Results

To demonstrate the sensitivity and dynamic range of the qPCR assay, known quantities of a plasmid encoding the NP gene of JUNV C#1 (range 5-5 x 10⁷ copies) were subjected to qPCR as described in Section 4 of the protocol. The assay is sensitive and can reliably detect as few as 5 copies of the target nucleic acid (Figure 2). Further, the dynamic range of the assay is large and, as shown in Figure 2, can cover at least 7 logs of template. While .......

Discussion

The protocol we describe is straightforward and robust provided the following critical considerations are observed. First, strict pre-PCR technique must be employed (see ²⁸ for an excellent review). It is imperative that all reagents and equipment are free of amplified DNA containing the qPCR target sequence and that appropriate controls are in place to detect such contamination. Second, for the virus-cell attachment portion of the protocol, the virus, cells, and media must be kept at 4 °C to prevent.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Life Technologies	11965-118
Penicillin-Streptomycin	Life Technologies	15140-163	100x
HEPES Buffer Solution	Life Technologies	15630-130	100x
PBS pH 7.4	Life Technologies	10010-023	1x
Vero E6 cells	American Type Culture Collection	CRL-1586
Costar 48-well plate	Corning	3548
RNeasy mini kit	Qiagen	74106	do not mix buffers RLT or  RW1 with bleach
QIAshredder homogenizer	Qiagen	79654
Taqman Universal PCR Master Mix	Life Technologies	4326614
Multiscribe Reverse Transcriptase (50U/µl)	Life Technologies	4311235
dNTP Mixture (10 mM; 2.5 mM each dNTP)	Life Technologies	N808-0260
GeneAmp 10X PCR Buffer II and 25 mM MgCl2 solution	Life Technologies	N808-0010
RNase Inhibitor (5000U/100 µl)	Life Technologies	N808-0119
RT primer 5’-AAGGGTTTAAAAAT GGTAGCAGAC-3’	Integrated DNA Technologies	250 nmol DNA oligo	standard desalting
Forward qPCR primer  5’-CATGGAGGTCAAACAACTTCCT-3’	Life Technologies	4304971	standard desalting
Reverse qPCR primer 5’-GCCTCCAGACATGGTTGTGA-3’	Life Technologies	4304971	standard desalting
TaqMan Probe 5’-6FAM-ATGTCATCGGATCCTT-MGBNFQ-3’	Life Technologies	4316033	HPLC purified
MicroAmp Fast Optical 96-well reaction plate (0.1 mL)	Life Technologies	4346906
StepOnePlus Real-Time PCR System	Life Technologies	4376598	or other qPCR instrument