1. Coronavirus Binding and Entry

David E. Wentworth and Kathryn V. Holmes

Coronaviruses are a diverse family of viruses that bind to host cells primarily through interactions between viral spike glycoproteins and specific host cell surface glycoproteins. Some coronaviruses also bind to sialic acids on glycoproteins and glycolipids via their spike and/or hemagglutinin esterase glycoproteins. The interactions between coronaviruses and host cell receptors are critical determinants of species-specificity, tissue tropism, and virulence. This chapter summarizes how coronaviruses bind to host cells and how the viral envelope fuses with host cell membranes to initiate infection. It will focus on the viral spike and hemagglutinin esterase glycoproteins and their interactions with known host cell receptor proteins, sialyloligosaccharides, and lectins. The functions of the other coronavirus structural proteins, non-structural and accessory proteins, as well as the fascinating replication strategy of coronaviruses will be discussed in later chapters.

2. The Coronavirus Replicase Gene: Special Enzymes for Special Viruses

John Ziebuhr and Eric J. Snijder

Coronaviruses have single-stranded, positive-sense RNA genomes of about 30 kilobases, by far the largest non-segmented RNA virus genomes currently known. The key functions required for coronavirus RNA synthesis are encoded by the viral replicase gene. The gene comprises more than 20,000 nucleotides and encodes two replicase polyproteins, pp1a and pp1ab, that are proteolytically processed by viral proteases. Over the past years, it has become clear that the unique size of the coronavirus genome and the special mechanism that coronaviruses (and several other nidoviruses) have evolved to produce an extensive set of subgenome-length RNAs is linked to the production of a number of nonstructural proteins (nsps) that is unprecedented among RNA viruses. Many of these replicase cleavage products in fact are multidomain proteins themselves, thus further increasing the complexity of protein functions and interactions. Structural studies suggest that several nsps, following their release from larger precursor molecules, form dimers or even multimers. The various pp1a/pp1ab precursors and processing products are thought to assemble into large, membrane-associated complexes that, in a temporally coordinated manner, catalyze the reactions involved in RNA replication and transcription and, most probably, serve yet other functions in the viral life cycle. This article reviews the expression, maturation and the functional and structural properties and peculiarities of coronavirus replicase gene-encoded enzymic and other functions, including protease, polymerase, helicase, ADP-ribose 1"-phosphatase and RNase activities.

3. Genomic Cis-Acting Elements in Coronavirus RNA Replication

Paul S. Masters

In common with the genomes of all other RNA viruses, coronavirus genomes contain cis-acting RNA elements that ensure the specific replication of viral RNA by a virally encoded RNA-dependent RNA polymerase. The embedded cis-acting elements devoted to coronavirus replication constitute a surprisingly small fraction of the total genome, but this is probably a reflection of the fact that coronaviruses have the largest genomes of all RNA viruses. The boundaries of cis-acting elements essential to replication are fairly well-defined, and an increasingly well resolved picture of the RNA secondary structures of these regions is emerging. However, we are only in the early stages of understanding how these cis-acting structures and sequences interact with the viral replicase and host cell components, and much remains to be done before we understand the precise mechanistic roles of such elements in RNA synthesis.

4. Coronavirus RNA Synthesis: Transcription

Luis Enjuanes, Isabel Sola, Sonia Zuñiga, and J. L. Moreno

Coronavirus replication involves both viral and cellular proteins, and takes place in the cytoplasm in a membrane-protected microenvironment. Coronaviruses may control the cell machinery by locating some of their proteins in the host cell nucleus, and their replication and transcription possibly require cross-talk between the 5' and 3' ends. Coronavirus transcription involves a discontinuous RNA synthesis (template switch) during the extension of a negative copy of the subgenomic (sg) mRNAs, a process that is controlled by transcription-regulating sequences (TRSs) preceding each gene. The requirement for base pairing during transcription has been formally demonstrated in arteriviruses and coronaviruses. The template switch requires base pairing between the nascent minus RNA strand and the leader TRS with minimum free energy (DG). Coronavirus N proteins have RNA chaperone activity that may help to overcome the energy barrier threshold associated with template switch. The potential role in transcription of selected proteins will be addressed.

5. Reverse Genetic Analysis of Coronavirus Replication

Volker Thiel

Coronavirus reverse genetics has greatly contributed to our current understanding of coronavirus molecular biology and diseases. Starting in the early 1990's reverse genetic techniques have been applied to the modification of the 3'-third of coronavirus genomes by targeted recombination. In 2000, reverse genetic systems based on full-length cDNAs made the entire coronavirus genome amenable to mutagenesis. Coronavirus reverse genetic techniques have been applied to the analysis of all aspects of coronavirus infections. These include studies on the tropism, pathogenesis, virus entry, virion formation and RNA synthesis of coronaviruses. In addition to the generation of recombinant coronaviruses, reverse genetic approaches have also been used for the development of coronavirus replicon RNAs and novel vectors designed for heterologous gene expression.

6. Coronavirus Genome Packaging

Krishna Narayanan and Shinji Makino

The assembly of infectious coronavirus particles requires the selection of viral genomic RNA from a cellular pool that contains an abundant excess of non-viral and viral RNAs. Among the seven to ten specific viral mRNAs synthesized in virus-infected cells, only the full length genomic RNA is packaged efficiently into coronavirus particles. Studies have revealed cis-acting elements and trans-acting viral factors involved in coronavirus genome encapsidation and packaging. Understanding the molecular mechanisms of genome selection and packaging is critical for developing antiviral strategies and viral expression vectors based on the coronavirus genome. In this review, we have compiled these collective findings to provide some insights into the initial stages of infectious coronavirus particle assembly.

7. Molecular Evolution of Group 2 Coronaviruses

Leen Vijgen, Els Keyaerts, and Marc Van Ranst

Coronaviruses are well-equipped to adapt rapidly to changing ecological niches based on two major forces that drive their viral evolution: mutation and recombination. Based on the available nucleotide and amino acid sequence information, the evolutionary relationships among coronaviruses can be inferred by using molecular phylogenetic approaches. The calculation of an evolutionary rate provides an indication of the rate at which mutations become fixed in the coronavirus population. The divergence of ancestral strains of existing coronaviruses with a different host-specificity can be dated back in history, thereby estimating the time in which potential interspecies transmission events occurred. A similar cross-species jumping led to the emergence of the SARS coronavirus, that has been proposed to be an early split-off from group 2 coronaviruses. Within group 2 a subdivision into murine hepatitis virus-related and bovine coronavirus-related coronaviruses has been suggested. The remarkably high genetic similarity among the bovine coronavirus-related coronaviruses indicates a relatively recent common evolutionary history. In this chapter, we discuss the molecular evolution of these closely related group 2 coronaviruses.

8. Avian Coronavirus Diseases and Infectious Bronchitis Vaccine Development

Paul Britton and Dave Cavanagh

Infectious bronchitis virus (IBV), a Group 3 coronavirus, is responsible for economic losses and welfare problems with chickens (the domestic fowl) globally. In addition to all respiratory epithelial surfaces it replicates throughout the alimentary tract, kidneys and gonads. Despite live and inactivated vaccines, it continues to be a major problem due to extensive antigenic variation; there is poor cross-protection. Susceptibility to the outcome of IBV infection is genetically determined.

Coronaviruses that are genetically and antigenically similar to IBV cause enteric disease in turkeys, and respiratory and kidney disease in pheasants. IBV-like viruses have recently been reported in several other gallinaceous (fowl-like, order Galliformes) birds, - peafowl, partridge, guinea fowl - and in a duck (teal); some of these isolates may be IBVs that have crossed into other species. Group 3 coronaviruses with additional open reading frames have been detected in greylag goose, pigeon and mallard duck. A group 2 coronavirus has been reportedly isolated from a Manx shearwater (Puffinus puffinus), and a coronavirus of an undetermined Group has been isolated from a parrot.

Inactivated vaccines are ineffective unless preceded by live attenuated vaccines to prime the protective immune response. The S1 spike protein subunit is necessary and sufficient to induce protective immunity. Differences in as few as 5% of the amino acids in S1 can decrease cross-protection. Genetic manipulation of the IBV genome is underway for the rational attenuation of IBV, and for the development of IB vaccines that can be applied in ovo.

9. Feline Coronaviruses: A Tale of Two-Faced Types

Bert Jan Haijema, Peter J.M. Rottier, and Raoul J. de Groot

Feline coronaviruses (FCoVs) are common pathogens of cats. Two serotypes (I and II) have been recognized and, to add further spice, each serotype occurs in two markedly different pathotypes. The most common pathotype, enteric FCoV (eFCoV) causes a mild, often unapparent enteritis, the other one, feline infectious peritonitis virus (FIPV), a devastating, highly lethal systemic infection. During the last ten years, our knowledge of the pathogenesis of feline infectious peritonitis (FIP) and of the relationships between the different FCoV sero- and pathotypes has increased considerably. In this Chapter, we will focus on (i) the pathogenesis and natural history of FCoV infections, (ii) the molecular biology and molecular genetics of FCoVs, and (iii) the development of new and effective vaccines.

10. Control of Neurotropic MHV by Multifactorial Mechanisms

Cornelia C. Bergmann and Stephen A. Stohlman

Infection of the central nervous system (CNS) by mouse hepatitis virus (MHV) can result in fatal encephalitis. Rapid viral growth especially in neurons leads to death prior to effective immunity. Alternatively, MHV can induce an acute encephalomyelitis that resolves into a persistent infection under immune pressure. During both infections there is a coordinated innate immune response which, although unable to control virus, facilitates adaptive immunity during non-fatal infections. During non-lethal infection, virus is controlled by CD8+ T cells which use both perforin mediated cytolysis and IFN-g, depending upon the specific type of cell infected. In contrast to innate and anti-viral cellular mechanisms evident during acute infection, persistence is maintained via virus neutralizing antibody. CD8+ T cells loose anti-viral function as virus is controlled prior to complete virus elimination. Even memory T cells are insufficient to control persisting virus in the absence of antibody. Retention of virus specific antibody secreting within the CNS, despite their delayed appearance relative to T cells, implies local antibody production in providing protection throughout persistence.

11. SARS Coronavirus - Pathogenesis and Correlation With Clinical Disease

John M. Nicholls and Joseph S.M. Peiris

Human infection by SARS coronavirus appears to be limited to the respiratory tract where infection of susceptible cells leads to damage to the pneumocytes resulting in a histological picture of diffuse alveolar damage and a clinical picture of adult respiratory distress syndrome. Diarrhoea is also present but there is limited evidence of damage to the intestinal epithelium. The damage to the respiratory tree appears limited to the lower respiratory tract and there is evidence that the immune response plays a part in the outcome of patients with SARS.

12. SARS-Coronavirus and the Antiviral Cytokine Response

Martin Spiegel and Friedemann Weber

Intracellular pathogens such as SARS-Coronavirus (SARS-CoV) have to cope intensively with the innate immune system. Type I interferons (IFN-a/b) represent an important part of this host defense. IFNs are potent, antivirally active cytokines which can be produced by all nucleated cells in response to infection. Moreover, professional IFN producer cells such as dendritic cells are able to secrete immense amounts of IFNs within short time. IFNs trigger the synthesis of antivirally active proteins and shape adaptive immunity. Thus, in order to establish infection, viruses were forced to evolve efficient anti-IFN strategies. There is growing evidence that SARS-CoV is no exception to that rule. Here, we attempt to summarize the current state of knowledge regarding the interaction SARS-CoV with components of the IFN system, its connection to the induction of other cytokines, and the consequences for the pathogenesis of SARS.

13. Grand Challenges in Human Coronavirus Vaccine Development

Barry Rockx and Ralph S. Baric

The emergence and identification of several common and rare human coronaviruses that cause severe lower respiratory tract infection argues for the judicious development of robust coronavirus vaccines and vector platforms.

Currently, limited information is available on the correlates of protection against SARS-CoV and other severe lower respiratory tract human coronavirus infections, a clear priority for future research. Passive immunization has been successful in establishing protection from SARS-CoV in animal models suggesting an important role for neutralizing antibodies. One important property of future vaccine candidates is the ability to confer protection against multiple variant strains of SARS-CoV, especially in senescent populations that are most at risk for severe disease.

Many vaccine candidates are capable of inducing humoral and cellular responses and protection from subsequent homologous challenge has been reported in animal models. The development of infectious clones for coronaviruses has facilitated the identification of attenuating mutations, deletions and recombinations which could ultimately result in live attenuated vaccine candidates. Stable vaccine platforms should be developed that allow for rapid intervention strategies against any future emergence coronaviruses.

Vaccine correlates that enhance disease after challenge should be thoroughly investigated and mechanisms devised to circumvent vaccine-associated complications.

14. SARS and Other 'New' Coronaviruses

Leo L.M. Poon

SARS coronavirus is a novel human pathogen identified in 2003. Its emergence caused severe impacts on human health. This chapter will summarize the commonly used diagnostic methods for SARS. The origin of this novel virus will be discussed. Besides, other novel coronaviruses found in the post-SARS era will also be reviewed.

15. Human Coronavirus NL63, a Long Lost Brother

Krzysztof Pyrc and Lia van der Hoek

Over the last decade a number of novel human respiratory viruses have emerged or have been recognized. A novel group I coronavirus, human coronavirus NL63 (HCoV-NL63) was identified in 2004 in the Netherlands in children and adults with acute respiratory illness. Worldwide analysis of clinical specimen indicated that this virus has widely spread within the human population. It is observed mostly in young children, elderly people and immunocompromised patients with upper and lower acute respiratory tract disease (1-10% of all respiratory infections). This chapter will discuss current knowledge on HCoV-NL63 replication, pathogenesis, disease burden and advances in treatment strategies.

16. Current Status of Antiviral Severe Acute Respiratory Syndrome Coronavirus Research

Els Keyaerts, Leen Vijgen, and Marc Van Ranst

Before the emergence of the severe acute respiratory syndrome associated coronavirus (SARS-CoV), no efforts were put into the search for antivirals against coronaviruses. The rapid transmission and high mortality rate made SARS a global threat for which no efficacious therapy was available and empirical strategies had to be used to treat the patients. New insights into the field of the SARS-CoV genome structure and pathogenesis revealed novel potential anti-coronavirus targets. Several proteins encoded by the SARS-CoV could be considered as targets for therapeutic intervention: the spike protein, the main protease, the NTPase/helicase, the RNA dependent RNA polymerase and different other viral protein-mediated processes. Different animal models have now been established to enable the evaluation of potential anti-SARS-CoV drugs in vivo. The development of effective drugs against SARS-CoV may also provide new strategies for the prevention or treatment of other coronavirus diseases in animals or humans. In this chapter, we review and discuss experimental and clinical data about possible coronavirus antivirals.