Arenavirus Serological Assay Development Biology Essay

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The Arenaviruses are a diverse family of enveloped single-stranded RNA Viruses, several of which have emerged as causative agents of severe viral hemorrhagic fevers which represent a significant threat to global public health The aim of this project was to develop ELISA serological assays for Arenaviruses potential diagnostics use, following the successful development of antigen-capture ELISAs for Crimean Congo Haemorrhagic Fever. The benefits of such assays include reducing the burden on high-containment diagnostic facilities by replacing current immunofluoresence and molecular methods with a rapid, simple diagnostic assay. Assays for Lassa were developed as a proof of principle before extending the developmental strategy to some of the other Arenaviruses, including Junin, Machupoand Lujo viruses.

Lassa Haemorrhagic Fever Virus-like Particles (VLPs) were generated in a mammalian expression system (co-transfecting?) and purified for use in an ELISA diagnostic assay. (CCHF paper) Recombinant Lassa Nucleoprotein expression in a baculoviral system was attempted with the intention of using the antigen to work-up ELISA assays detecting the presence of antibody to NP in patient sera as the basis for lateral flow assay development. Lassa VLPs were determined to be unsuitable for the assya, so development of assays using lysate from GP and Z-expressing mammalian cells as an alternative was begun. Recombinant baclovirus was expressed in insect cells, but it was found that NP was not expressed due a cloning artefact.

the question(s) you investigated (or purpose), (from Introduction)

state the purpose very clearly in the first or second sentence.

the experimental design and methods used, (from Methods)

clearly express the basic design of the study.

Name or briefly describe the basic methodology used without going into excessive detail-be sure to indicate the key techniques used.

the major findings including key quantitative results, or trends (from Results)

report those results which answer the questions you were asking

identify trends, relative change or differences, etc.

a brief summary of your interpetations and conclusions. (from Discussion)

clearly state the implications of the answers your results gave you.

Introduction (650/1000)



The Arenaviridae are a group of highly pathogenic emerging viruses with worldwide distribution. At least 29 Arenavirus species are currently recognized, of which at least eight cause hemorrhagic fevers in humans (see table 1), and are associated with high morbidity and mortality, representing a significant threat to global public health. The viruses are divided into two serogroups, which differ genetically and by geographical distribution. They are generally associated with rodent-transmitted disease in humans (table 1). Some Arenaviruses, such as Chapare, have caused only outbreaks, while others such as Lassa and Junin are endemic in their respective geographical locations. [proposal][Nunberg][Jay]

Table 1: The Arenaviruses that cause hemorrhagic disease in humans.


Virus Name

Geographial Location

Host Species

Old World Arenaviruses


West Africa

Mastomys nateliensis


Uncertain -outbreaks have only occurred in Lusaka, Zambia and Johannesburg, South Africa

Host unknown


North America, South America, and Europe. Other continents uncertain.

Mus musculus

New World Arenaviruses



Calomys musculunus



Calomys callosus



Host unknown



Host unknown



Zygodontomys brevicauda

Arenaviruses are spherical structures of 90-130µm and are composed of a Z protein matrix surrounded by a lipid envelope of cellular origin(Fields Virology 1635), with glycoprotein spikes composed of trimers of the GP1 & GP2 chains embedded within. (Figure) The Nucleocapsid is helical and composed of 2 proteins - the nucleocapsid ('N') protein, structurally associated with the RNA, and an RNA polymerase ('L'). The genome is linear, bisegmented single-stranded RNA. The large segment (5.7kb) - encodes the L and Z protein, while the small segment (2.8kb) - encodes the G protein and N proteins.

1.2 Lassa Haemorrhagic Fever Virus (LFV)

Figure 1: Geographical Distribution of Lassa Fever Virus in West Africa (From Richmond)

LFV is known to be endemic in Guinea, Liberia, Sierra Leone and parts of Nigeria (WHO) and sporadic outbreaks have also occurred in the Central African Republic, the Democratic Republic of the Congo, Mali, and Senegal. (banister) (CDC)

The virus is named after a northern Nigeria in which the first cases were isolated in 1969, during a nosocomial outbreak at a local hospital. At least 14 laboratory confirmed imported cases of Lassa virus have been reported in the UK since 1970.

LFV causes an acute and sometimes severe hemorrhagic illness with approximately 5,000 fatalities per year. LFV is distributed across Western Africa (see figure 1). 300,000 - 500,000 cases of Lassa fever are estimated to occur each year with approximately 5,000 deaths, while mortality rates of up to 50% have been recorded during epidemics. (Branco)

Infection results from contact with faeces and urine of the reservoir host Mastomys natalensis, commonly known as the "multimammate rat," and also through consumption of rodent meat. Secondary and Nosocomial transmission of LASV between humans occurs through direct contact with infected blood or bodily secretions. There is no reliable treatment for Arenaviral infection. Ribavirin has been used with success in Lassa fever patients, but is most effective when used early, which highlights the importance of rapid diagnosis to prevent death and further transmission.

1.3 Diagnostics and Treatment

There is currently no diagnostic gold standard for Lassa or any of the other Arenaviruses. Serology-based assays, such as Enzyme-linked immunosorbent assays (ELISAs) are often used to detect IgM or IgG and have many advantages (including...), but there are currently no commercial assays available for Lassa virus. Immunofluorescence assays are also used, but have many drawbacks including long assay time and the use of the need to isolate the virus through cell culture, so it is constrained to specialist maximum containment laboratories which are not always available. Molecular assays such as RT-PCR do not require cell culture and results are available more quickly, but they are beyond the resources and capabilities of labs in many developing countries. Therefore there is a pressing need to develop new serology- based assays to allow rapid detection and response to cases of Arenavirus infection imported to the UK as well as those in the field. (cfsph) (WHO) (Branco)

assay and allowing standardisation between laboratories, resulting in more efficient diagnosis and subsequent care of infected patients.

There is currently no licensed vaccine or reliable treatment for Arenavirus infections, although the antiviral drug ribavirin can shorten the course of certain hemorrhagic fevers and help prevent complications and relapses. The drug is only effective, however, if administered early in the course of infection. This, together with the severity of Arenavirus infections and their potential use as bio-weapons, highlights the need for rapid diagnostic tests that can be easily deployed in remote endemic areas with few laboratory resources, where molecular diagnostic techniques may not be available. [CDC] [Branco] (WHO)(Barber)

1.4 Virus-Like Particles (VLP)

VLPs are morphologically similar to virions but contain no genetic material and are therefore non-infectious, representing an alternative to the use of authentic viruses or virus antigens for diagnostic assay development. VLPs have been produced fro varied RNA viruses, including Filoviridae and Hepatitis B Virus. They can be produced by co-expressing GP and Z matrix protein, after which VLPs self-assemble and Z protein drives budding from the cell membrane. VLPs are released into the cell culture supernatant and can be purified like authentic virions. They recognised by the immune system and can elicit antibody responses, making them a useful tool in studies of virus structure, vaccine development and as an immunogen for generation of monoclonal antibodies for neutralisation studies.

1.5 Introduction to the Project

The Aim of the project was to develop ELISA assays to viruses of the Arenaviridae family for potential diagnostics use to allow rapid detection and response to cases of Arenavirus infection (Proposal), by utilizing recombinant technologies to reduce the burden on high-containment labs by using a known immunogenic component of the virus, rather than live virus or authentic antigen. The end-product was intended to be a lateral-flow device that can be distributed to hospitals in endemic countries, as well as diagnostic and reference labs in non-endemic countries that may require detection of disease in travellers, allowing a rapid diagnosis of on-going or previous infection with only a small drop of blood and minimal laboratory equipment. Additionally, all reagents can be harmonized allowing the standardization of tests across international boundaries and between diagnostic facilities.

The project followed the same assay development strategy that recently proved successful for developing an ELISA-based Lateral-Flow device for Crimean-Congo Haemorrhagic Fever (CCHF). Two types of Arenavirus antigen were expressed and used to begin development of serological assays: Nucleoprotein and Virus-like particles. It was envisaged that an assay for Lassa would be developed as a proof of concept following on from the CHHF work, and then the developmental strategy would be applied to other Arenaviruses, focusing on the viruses highlighted here.

1.6 Overview of Assay Development

Expression of Arenaviral Nucleoprotein for antibody-capture ELISA and lateral-flow development:

The objectives of this section of the project were to:

Clone Arenavirus NP into pBACgus baculoviral expression vector.

Express NP by transfecting recombinant baculovirus plasmid in sF9 insect cells.

Purify NP sing current FPLC protocols.

Use pure recombinant NP as antigen for an IgG Ab capture ELISA development, to be used as the basis for lateral flow ('dip stick') assay development detecting the presence of antibody to NP in patient sera - in collaboration with Forsite Diagnostics ltd .

Use the recombinant NP used to generate monoclonal antibody for antigen capture ELISAs and possible neutralization screens.

Generation of Lassa VLP as antigen for antibody-capture ELISA

The objectives of this section of the project were to:

Clone Arenavirus GP and Z protein into pcDNA3.1 mammalian expression vector.

Express Arenavirus Virus-like Particles (VLP) by co-transfecting Human Embryonic Kidney producer cells with recombinant plasmids.

Use the purified VLPs as an antigen for Antibody-capture ELISA development to capture antibody to the viral Glycoprotein (GP) and Matrix (Z) proteins in potential patient sera.

Generate Z-protein:Beta Lactamase fusion protein construct and work up a new in-vitro infectivity assay for LFV neutralisation screening

Raise mAb to GP/Z by immunization of mice, to create antibody reagents for future research/assays.


This project will follow the same cloning, expression, purification and ELISA assay development strategy that recently proved extremely successful for developing an ELISA to detect antibody to Crimean-Congo Haemorrhagic Fever (CCHF). Our CCHF ELISA was generated entirely in-house using these techniques and is now used by several international partners (e.g. Turkey, Kosovo, Kazakhstan, etc) as a diagnostic assay - method paper currently under submission. It is currently being automated for use in SPRU and the recombinant CCHF-NP reagent has been used to generate hybridomas secreting mAb to CCHF - now being used to develop an antigen capture ELISA. This ELISA is also being worked-up to a lateral flow 'point of care' test for use in the field. It is this success that provides a template for the proposed project above.

Value to Microbiology Services - Porton:

This project will provide Microbiology Services - Porton with a unique set of assays to a highly pathogenic group of viruses to which there is a pressing need to develop serological detection tests. It will enhance our ability to diagnose such infections and increase our capability to respond to suspected arenavirus infection - particularly in cases imported to the UK. It will maintain HPA-Porton and the group's reputation as a world leader in the research, detection and assay development of Viral Haemorrhagic fever viruses and fulfil our remit in providing a key diagnostic capability to such diseases.

However, diagnosis of Lassa Fever cases can be difficult due to variable and non-specific symptoms that are similar to those of other diseases, including but not limited to; Yellow Fever, Typhoid Fever, Malaria and other viral Haemorrhagic fevers.

Many of these techniques necessitate the use of maximum containment facilities, which severely restricts the number of laboratories able to carry out these tests. (Branco)

Advantages of ELISA using recombinant antigen:

Are that it is non-infectious - it can used with heat-inactivated sera and antigens

Sensitive - probability that a person having a disease will be correctly identified by the test.

Simple and low-tech, useful in the field - as in the CCHF project, (picture of LFD) ELISAs can be worked up into lateral flow devices, which use only a drop of the patients' blood, much more convenient than molecular assays both in field and in imported countries.

Rapid - current diagnostics: cell culture and IF, which are time-consuming and labour-intensive.