FLAVIVIRUSES 1103 Background and Classification 1103 Structure and Physical Properties of the Virion 1104 Binding and Entry 1105 Genome Structure 1106 Translation and Proteolytic Processing 1107 Features of the Structural Proteins 1108 Features of the Nonstructural Proteins 1109 RNA Replication 1112 Membrane Reorganization and the Compartmentalization of Flavivirus Replication 1112 Assembly and Release of Particles from Flavivirus-infected Cells 1112 Host Resistance to Flavivirus Infection 1113

Flaviviridae :T he Viruses and Their Replication

The nucleotide sequence of the RNA genome of the human hepatitis C virus (HCV) has been determined from overlapping cDNA clones. The sequence (9379 nucleotides) has a single large open reading frame that could encode a viral polyprotein precursor of 3011 amino acids. While there as little overall amino acid and nucleotide sequence homology with other viruses, the 5' HCV nucleotide sequence upstream of this large open reading frame has substantial similarity to the 5' termini of pestiviral genomes. The polyprotein also has significant sequence similarity to helicases encoded by animal pestiviruses, plant potyviruses, and human flaviviruses, and it contains sequence motifs widely conserved among viral replicases and trypsin-like proteases. A basic, presumed nucleocapsid domain is located at the N terminus upstream of a region containing numerous potential N-linked glycosylation sites. These HCV domains are located in the same relative position as observed in the pestiviruses and flaviviruses and the hydrophobic profiles of all three viral polyproteins are similar. These combined data indicate that HCV is an unusual virus that is most related to the pestiviruses. Significant genome diversity is apparent within the putative 5' structural gene region of different HCV isolates, suggesting the presence of closely related but distinct viral genotypes.

https://www.pnas.org/content/pnas/88/6/2451.full.pdf

Genetic organization and diversity of the hepatitis C virus.

The possible use of pig organs and tissues as xenografts in humans is actively being considered in biomedical research. We therefore examined whether pig endogenous retrovirus (PERV) genomes can be infectiously transmitted to human cells in culture. Two pig kidney cell lines spontaneously produce C-type retrovirus particles. Cell-free retrovirus produced by the PK-15 kidney cell line (PERV-PK) infected pig, mink and human kidney 293 cell lines and co-cultivation of X-irradiated PK-15 cells with human cells resulted in a broader range of human cell infection, including human diploid fibroblasts and B- and T-cell lines. Kidney, heart and spleen tissue obtained from domestic pigs contained multiple copies of integrated PERV genomes and expressed viral RNA. Upon passage in human cells PERV-PK could rescue a Moloney retroviral vector and acquired resistance to lysis by human complement.

Infection of human cells by an endogenous retrovirus of pigs

This chapter discusses the manipulation of clones of coronavirus and of complementary DNAs (cDNAs) of defective-interfering (DI) RNAs to study coronavirus RNA replication, transcription, recombination, processing and transport of proteins, virion assembly, identification of cell receptors for coronaviruses, and processing of the polymerase. The nature of the coronavirus genome is nonsegmented, single-stranded, and positive-sense RNA. Its size ranges from 27 to 32 kb, which is significantly larger when compared with other RNA viruses. The gene encoding the large surface glycoprotein is up to 4.4 kb, encoding an imposing trimeric, highly glycosylated protein. This soars some 20 nm above the virion envelope, giving the virus the appearance-with a little imagination-of a crown or coronet. Coronavirus research has contributed to the understanding of many aspects of molecular biology in general, such as the mechanism of RNA synthesis, translational control, and protein transport and processing. It remains a treasure capable of generating unexpected insights.

The molecular biology of coronaviruses

We have developed a novel DNA expression system, based on the Semliki Forest virus (SFV) replicon, which combines a wide choice of animal cell hosts, high efficiency and ease of use. DNA of interest is cloned into SFV plasmid vectors that serve as templates for in vitro synthesis of recombinant RNA. The RNA is transfected with virtually 100% efficiency into animal tissue culture cells by means of electroporation. Within the cell, the recombinant RNA drives its own replication and capping and leads to massive production of the heterologous protein while competing out the host protein synthesis. The expression system also includes an in vivo packaging procedure whereby recombinant RNA is packaged into infectious virus particles using cotransfection with packaging-deficient helper RNA molecules. The resulting high titer recombinant virus stock can be used to infect a wide range of animal cells with subsequent high expression of the heterologous gene product, but without expression of any structural proteins of the helper. The infected cells produce protein for up to 75 hours post infection after which the heterologous product can constitute as much as 25% of the total cell protein. The general utility of the system is demonstrated through the expression of human transferrin receptor, mouse dihydrofolate reductase, chick lysozyme and Escherichia coli beta-galactosidase.

A new generation of animal cell expression vectors based on the Semliki Forest virus replicon.

Publisher Summary The last comprehensive reviews of nonarbotogaviruses included discussions on pestiviruses, rubella virus, lactate dehydrogenase-elevating virus (LDV), equine arteritis virus (EAV), simian hemorrhagic fever virus (SHFV), cell fusion agent, and nonarboflaviviruses. The inclusion of all these viruses in the family Togaviridae was largely based on the similarities in morphological and physical–chemical properties of these viruses, and in the sizes and polarities of their genomes. In the intervening years, considerable new information on the replication strategies of these viruses and the structure and organization of their genomes has become available that has led to the reclassification or suggestions for reclassification of some of them. The replication strategy of EAV resembles that of the coronaviruses, involving a 3'-coterminal nested set of mRNAs. Therefore, EAV has been suggested to be included in a virus superfamily, along with coronaviruses and toroviruses. Recent evidence indicates that LDV not only resembles EAV in morphology, virion and genome size, and number and size of their structural proteins, but also in genome organization and replication via a 3'-coterminal set of mRNAs. SHFV, although not fully characterized, exhibits properties resembling those of LDV and EAV, and the recent evidence suggest that it may possess the same genome organization as these viruses. The three viruses may, therefore, represent a new family of positive-strand RNA viruses and are reviewed together in this chapter. In this chapter, emphasis is on the recent information concerning their molecular properties and pathogenesis in vitro and in vivo and on the host immune responses to infections by these viruses.

Lactate dehydrogenase-elevating virus, equine arteritis virus, and simian hemorrhagic fever virus: a new group of positive-strand RNA viruses.

Although classical swine fever (CSF) has been well known for decades and epidemics still occur, clinical diagnosis continues to cause problems for veterinary practitioners. This is due to the extensive differential diagnosis, further complicated by the emergence of new diseases such as porcine reproductive and respiratory syndrome (PRRS) and porcine dermatitis and nephropathy syndrome (PDNS). In addition, acute, chronic and prenatal courses of CSF have to be distinguished. As a cause of considerable economical losses within the EU, control of CSF requires knowledge of the primary outbreaks and spread of the disease. Genetic typing of CSF virus isolates has proved to be a potent method of supporting epidemiological investigations. Phylogenetic analysis of CSF virus strains and isolates originating from different continents has allowed three genetic groups and several subgroups within these groups to be distinguished. Whereas isolates belonging to group 3 seem to occur solely in Asia, all CSF virus isolates of the 1990s isolated in the EU belonged to one of the subgroups within group 2 (2.1, 2.2, or 2.3) and were clearly distinct from former CSF reference viruses, which belong to group 1. Within the EU, different strategies are followed for the eradication of CSF in domestic pigs and in wild boar. While a strict non-vaccination policy is followed for domestic pigs, eradication of the disease in wild boar is more complex, and oral immunisation together with special hunting strategies have been applied. Recently, marker vaccines with a companion discriminatory test designed to allow differentiation between vaccinated animals and animals having recovered from field virus infection have been developed. Preliminary studies indicated that the discriminatory tests had a reduced sensitivity and specificity. Further improvements are therefore necessary before marker vaccines can be considered for emergency use in EU Member States. Prevention of CSF remains the main objective within the EU.

Clinical signs and epidemiology of classical swine fever: a review of new knowledge.

Introduction to classical swine fever: virus, disease and control policy.

Epidemiology of classical swine fever in Germany in the 1990s.

Introduction. The term ‘pestivirus’ was coined in 1973 to group together two antigenically related enveloped RNA viruses: hog cholera virus (HCV) and bovine viral diarrhoea virus (BVDV; Horzinek, 1973). A third animal pathogen, the border disease virus (BDV) of sheep, was found later to be a close relative of BVDV. Pestiviruses are among the smallest enveloped animal RNA viruses (about 40 nm in diameter) and possess a nucleocapsid of non-helical, probably icosahedral symmetry (Horzinek et al., 1967); they share these traits with the numerous flaviviruses, of which the arthropod-borne yellow fever virus is the prototype. The pestiviruses are not arthropod-borne and currently hold generic status in the family Togaviridae. Previously, flaviviruses also held generic status in this family. However, when details of flavivirus molecular structure, replication strategy and gene sequence became known in the early 1980s, the Togaviridae Study Group recognized the fundamental differences and proposed the creation of the new family Flaviviridae with Flavivirus as the only genus (Westaway et al., 1985).

Volker Moennig

Papers

Lactate dehydrogenase-elevating virus, equine arteritis virus, and simian hemorrhagic fever virus: a new group of positive-strand RNA viruses.

Clinical signs and epidemiology of classical swine fever: a review of new knowledge.

Introduction to classical swine fever: virus, disease and control policy.

Epidemiology of classical swine fever in Germany in the 1990s.

Recent Advances in Pestivirus Research