Current Laboratory Techniques in Rabies Diagnosis, Research and Prevention, Volume 1

Laboratory Techniques in Rabies Diagnosis, Research and Prevention provides a basic understanding of the current trends in rabies. It establishes a new facility for rabies surveillance, vaccine and antibody manufacturing. It offers clarity about the choice of laboratory methods for diagnosis and virus typing, of systems for producing monoclonal and polyclonal antibodies and of methods for testing potency of vaccines and antibodies.

The book covers advancements in the classical methods described as well as recent methods and approaches pertaining to rabies diagnosis and research.

• Supplies techniques pertaining to rabies diagnosis and research
• Provides an update on the conventional and modern vaccines for rabies prevention
• Offers updates on the full length antibodies and antibody fragments for post exposure prophylaxis of rabies
• Presents technique descriptions that can be used to be compared to industry protocols to identify and establish potential new techniques

• Language: English
• Publisher: Academic Press
• Release date: Jul 30, 2014
• ISBN: 9780128004654

Book preview

Brazil

Part One

Introduction

Outline

Chapter One Basic Facts about Lyssaviruses

Chapter One

Basic Facts about Lyssaviruses

Ivan V. Kuzmin,    Global Alliance for Rabies Control, Manhattan, KS, USA

Rabies is caused by negative-sense single-stranded RNA (ssRNA) viruses in the genus Lyssavirus, family Rhabdoviridae. The bullet-shaped lyssavirus virion consists of two units. The external unit represents a lipid membrane with protruding spikes of glycoprotein. The internal nucleocapsid core consists of a ribonucleoprotein complex comprising the genomic RNA bound to the nucleoprotein, viral polymerase, and phosphoprotein. The matrix protein condenses the nucleocapsid and interacts with the external unit. The genome includes five major genes common to all rhabdoviruses. Lyssaviruses are distributed globally, except in Antarctica and several insular locations. Bats and carnivores are their major hosts. At present, the genus includes 14 viral species, with an additional putative member, Lleida bat lyssavirus. Phylogenetically, lyssaviruses are segregated into two phylogroups. Serologic cross-reactivity usually exists within, but not between, phylogroups. This raises concerns on the protective ability of rabies biologics against the variety of lyssaviruses, and on the capability of diagnostic tests.

Keywords

Distribution; Genome; Lyssavirus; Pathobiology; Phylogeny; Rabies; Structure; Taxonomy

Chapter Contents

1.1 Introduction 3

1.2 Virion and Genome Organization 4

1.3 Phylogeny and Serologic Cross-Reactivity of Lyssaviruses 6

1.4 Host Range 12

1.5 Pathobiology 15

References 17

1.1 Introduction

Rabies is one of the most feared infectious diseases known to humankind since ancient times.¹ The name of the disease may originate from the Sanskrit word rabhas (violence). Rabies (an acute progressive encephalomyelitis) is caused by negative-sense single-stranded RNA (ssRNA) viruses from the genus Lyssavirus, family Rhabdoviridae.² It is neither scientifically nor clinically correct to say that lyssaviruses other than the rabies virus (RABV) cause a rabies-like disease, as the disease is the same. Despite the significant progress in rabies prevention and control achieved during recent decades, the disease still causes more than 55,000 human deaths every year.³ Nearly 99% of these cases are associated with dog-transmitted rabies in the developing countries of Asia and Africa. Nevertheless, even in many developed countries where canine rabies has been eliminated, the disease is still present in wildlife, which poses public health concerns and a possibility of re-introduction of dog rabies.⁴ As with other diseases, efficient strategies for rabies control, prevention, prophylaxis, and treatment depend on the availability of effective diagnostic tests, laboratory-based surveillance, and safe and potent biologics. This introductory chapter is dedicated to a basic description of causative agents of rabies, including their taxonomy, morphology, serologic cross-reactivity, and generic pathobiological and ecological patterns that are significant for the development of reliable diagnostic tests, and for the creation of efficient protective biologics.

1.2 Virion and Genome Organization

Lyssaviruses have relatively large bullet-shaped virions, 130–250 nm in length and 60–100 nm in diameter (Figure 1.1). The lipid bilayer envelope is acquired from host cell membranes during budding. The envelope is studded with glycoprotein (G) spikes organized in trimers. The internal nucleocapsid core consists of a ribonucleoprotein (RNP) complex comprising the genomic RNA tightly bound to the nucleoprotein (N), together with an RNA-dependent RNA polymerase (L) and phosphoprotein (P). The nucleocapsid is active for transcription and replication: the N-RNA template is processed by the L protein, which contains most enzymatic activities, and its cofactor the P protein. The nucleocapsid has a helical symmetry, is uncoiled and filamentous, about 700×20 nm in size. In the virion, the matrix protein (M) condenses the nucleocapsid, interacts with the N-RNA complex, and associates with the host-derived lipid bilayer containing the transmembrane G protein.⁵

Figure 1.1 Schematic illustration of a lyssavirus virion.

The lyssavirus genome is represented by a non-segmented, linear, negative-sense ssRNA about 12 kb in length. It includes five major genes that are arranged in the conserved linear order 3′-N-P-M-G-L-5′. Each of the individual genes is flanked by transcription initiation and termination/polyadenylation signals that are largely conserved among members of the same species. Transcription units are separated by short untranscribed intergenic regions. The ends of the genomic RNA, termed 3′ leader and 5′ trailer sequences, exhibit terminal complementarity and contain promoter sequences that initiate replication of the genome and antigenome, respectively.⁵

Lyssaviruses have five structural proteins designated L, G, N, P, and M.⁵ The L (220–240 kDa) has multiple domains and performs the functions required for genome transcription and replication, including RNA-dependent RNA polymerase, mRNA 5′ capping enzyme, cap methyltransferase, 3′ poly (A) polymerase, and protein kinase activities. The G (65–90 kDa) is assembled into trimers to form the virion surface spikes. The G protein binds to host cell receptors, induces virus endocytosis, and mediates fusion of viral and endosomal membranes. The G protein induces production of virus-neutralizing antibodies and elicits cell-mediated immune responses. The N (47–62 kDa) is the major component of the nucleocapsid, and actively interacts with RNA, and L and P proteins. The P (20–30 kDa) plays multiple roles during transcription and replication as a non-catalytic cofactor of the viral polymerase. It mediates the physical link and proper positioning of the L protein on the N-RNA template, and acts as a chaperone during synthesis of N by forming N-P complexes that prevent N from self-aggregating and binding to cellular RNA.⁵ In addition, P was shown to have multiple binding sites for different viral or host proteins to allow their assembly into multi-molecular complexes. The P protein forms elongated and flexible dimers, and has a modular organization composed of three structured domains separated by intrinsically disordered regions. The highly acidic N-terminal domain has binding sites for the viral L and N proteins, host cell kinases, and importins. The central domain has an L protein binding site and dimerization domain, and the C-terminal basic domain is involved in N-RNA binding.⁶ The P may also interact with the host cellular transport systems such as the dynein motor complex, nucleo-cytoplasmic transporters, and microtubules⁷–⁹ to facilitate intracellular movement of viral components. Furthermore, P interferes with the innate immune response by inhibiting different steps of the host cell interferon response. The M is an inner component of the virion (20–30 kDa). It appears to be involved in regulation of the genome RNA transcription. The M binds to nucleocapsids and the cytoplasmic domain of the G, thereby facilitating the budding process, and also mediates such pathobiological effects as cell rounding, intracellular membrane redistribution, and apoptosis.⁵

1.3 Phylogeny and Serologic Cross-Reactivity of Lyssaviruses

Until the 1950s, it was believed that RABV was unique. The discoveries of serologically related viruses in Nigeria, Lagos bat virus (LBV) from a Pteropodid bat,¹⁰ and Mokola virus from a shrew¹¹ demonstrated that the structure of this virus group was more complex, and the terms rabies-related viruses (RRVs) and rabies serogroup were introduced.¹¹ Another serologically related virus, Duvenhage virus, was isolated from a man who died of rabies after a bite from an insectivorous bat in 1970 in South Africa;¹² it was recognized as a fourth viral serotype.¹³ As viruses regularly isolated from bats in Europe since the 1950s were related serologically to Duvenhage virus, they were initially included in the Duvenhage serotype.¹⁴,¹⁵

Later, application of monoclonal antibodies (MAbs) refined the classification of the rabies serogroup:¹⁶ European bat lyssaviruses were not only distinguished from the African Duvenhage virus,¹⁷ but separated into two distinct serotypes, temporally termed biotypes.¹⁸ This differentiation was later supported by gene sequencing and phylogenetic analysis.¹⁹ Extensive phylogenetic studies on the diversity of RRVs led to the creation of the novel operational term genotype, which has been broadly used in the scientific literature for over a decade. New genotypes were discovered, and quantitative criteria for their demarcation were proposed.¹⁹–²¹

To accommodate the growing variety of RRVs, the genus Lyssavirus was established under the auspices of the International Committee on the Taxonomy of Viruses (ICTV). The name of the genus originated from Greek mythology: Lyssa (Λυσσα) was a goddess, or spirit of rage, fury, madness, or frenzy.²² The existing genotypes served as a basis for lyssavirus taxonomy, but were refined to satisfy the official ICTV taxonomy rules, which operate with more complex entities such as viral species. The present taxonomy of lyssaviruses includes 14 species. One additional representative, Lleida bat virus, is known by a fragment of genome sequence only²³ and therefore does not have taxonomic status (Table 1.1, Figure 1.2).

Table 1.1

Viruses Currently Included in the Genus Lyssavirus

*NA: Neither a member of Phylogroup I nor II. Other Phylogroup not assigned.

Figure 1.2 Maximum likelihood phylogenetic tree of lyssaviruses based on partial nucleoprotein gene sequences (850 nucleotides). Branch lengths are drawn to scale, and bootstrap values (1,000 replicates) are shown for key