RNA Genome Of Positive Polarity Research Articles

The Picornaviridae Family: Knowledge Gaps, Animal Models, Countermeasures, and Prototype Pathogens.

Picornaviruses are nonenveloped particles with a single-stranded RNA genome of positive polarity. This virus family includes poliovirus, hepatitis A virus, rhinoviruses, and Coxsackieviruses. Picornaviruses are common human pathogens, and infection can result in a spectrum of serious illnesses, including acute flaccid myelitis, severe respiratory complications, and hand-foot-mouth disease. Despite research on poliovirus establishing many fundamental principles of RNA virus biology and the first transgenic animal model of disease for infection by a human virus, picornaviruses are understudied. Existing knowledge gaps include, identification of molecules required for virus entry, understanding cellular and humoral immune responses elicited during virus infection, and establishment of immune-competent animal models of virus pathogenesis. Such knowledge is necessary for development of pan-picornavirus countermeasures. Defining enterovirus A71 and D68, human rhinovirus C, and echoviruses 29 as prototype pathogens of this virus family may provide insight into picornavirus biology needed to establish public health strategies necessary for pandemic preparedness.

Therapeutic Applications for Oncolytic Self-Replicating RNA Viruses.

Self-replicating RNA viruses have become attractive delivery vehicles for therapeutic applications. They are easy to handle, can be rapidly produced in large quantities, and can be delivered as recombinant viral particles, naked or nanoparticle-encapsulated RNA, or plasmid DNA-based vectors. The self-replication of RNA in infected host cells provides the means for generating much higher transgene expression levels and the possibility to apply substantially reduced amounts of RNA to achieve similar expression levels or immune responses compared to conventional synthetic mRNA. Alphaviruses and flaviviruses, possessing a single-stranded RNA genome of positive polarity, as well as measles viruses and rhabdoviruses with a negative-stranded RNA genome, have frequently been utilized for therapeutic applications. Both naturally and engineered oncolytic self-replicating RNA viruses providing specific replication in tumor cells have been evaluated for cancer therapy. Therapeutic efficacy has been demonstrated in animal models. Furthermore, the safe application of oncolytic viruses has been confirmed in clinical trials. Multiple myeloma patients treated with an oncolytic measles virus (MV-NIS) resulted in increased T-cell responses against the measles virus and several tumor-associated antigen responses and complete remission in one patient. Furthermore, MV-CEA administration to patients with ovarian cancer resulted in a stable disease and more than doubled the median overall survival.

Calicivirus Non-structural Proteins: Potential Functions in Replication and Host Cell Manipulation.

The Caliciviridae are a family of viruses with a single-stranded, non-segmented RNA genome of positive polarity. The ongoing discovery of caliciviruses has increased the number of genera in this family to 11 (Norovirus, Nebovirus, Sapovirus, Lagovirus, Vesivirus, Nacovirus, Bavovirus, Recovirus, Salovirus, Minovirus, and Valovirus). Caliciviruses infect a wide range of hosts that include fishes, amphibians, reptiles, birds, and marine and land mammals. All caliciviruses have a genome that encodes a major and a minor capsid protein, a genome-linked viral protein, and several non-structural proteins. Of these non-structural proteins, only the helicase, protease, and RNA-dependent RNA polymerase share clear sequence and structural similarities with proteins from other virus families. In addition, all caliciviruses express two or three non-structural proteins for which functions have not been clearly defined. The sequence diversity of these non-structural proteins and a multitude of processing strategies suggest that at least some have evolved independently, possibly to counteract innate and adaptive immune responses in a host-specific manner. Studying these proteins is often difficult as many caliciviruses cannot be grown in cell culture. Nevertheless, the study of recombinant proteins has revealed many of their properties, such as intracellular localization, capacity to oligomerize, and ability to interact with viral and/or cellular proteins; the release of non-structural proteins from transfected cells has also been investigated. Here, we will summarize these findings and discuss recent in silico studies that identified previously overlooked putative functional domains and structural features, including transmembrane domains that suggest the presence of viroporins.

Flavivirus: From Structure to Therapeutics Development.

Flaviviruses are still a hidden threat to global human safety, as we are reminded by recent reports of dengue virus infections in Singapore and African-lineage-like Zika virus infections in Brazil. Therapeutic drugs or vaccines for flavivirus infections are in urgent need but are not well developed. The Flaviviridae family comprises a large group of enveloped viruses with a single-strand RNA genome of positive polarity. The genome of flavivirus encodes ten proteins, and each of them plays a different and important role in viral infection. In this review, we briefly summarized the major information of flavivirus and further introduced some strategies for the design and development of vaccines and anti-flavivirus compound drugs based on the structure of the viral proteins. There is no doubt that in the past few years, studies of antiviral drugs have achieved solid progress based on better understanding of the flavivirus biology. However, currently, there are no fully effective antiviral drugs or vaccines for most flaviviruses. We hope that this review may provide useful information for future development of anti-flavivirus drugs and vaccines.

Rewiring cellular networks by members of the Flaviviridae family.

Members of the Flaviviridae virus family comprise a large group of enveloped viruses with a single-strand RNA genome of positive polarity. Several genera belong to this family, including the Hepacivirus genus, of which hepatitis C virus (HCV) is the prototype member, and the Flavivirus genus, which contains both dengue virus and Zika virus. Viruses of these genera differ in many respects, such as the mode of transmission or the course of infection, which is either predominantly persistent in the case of HCV or acutely self-limiting in the case of flaviviruses. Although the fundamental replication strategy of Flaviviridae members is similar, during the past few years, important differences have been discovered, including the way in which these viruses exploit cellular resources to facilitate viral propagation. These differences might be responsible, at least in part, for the various biological properties of these viruses, thus offering the possibility to learn from comparisons. In this Review, we discuss the current understanding of how Flaviviridae viruses manipulate and usurp cellular pathways in infected cells. Specifically, we focus on comparing strategies employed by flaviviruses with those employed by hepaciviruses, and we discuss the importance of these interactions in the context of viral replication and antiviral therapies.

The Regulation of Translation in Alphavirus-Infected Cells

Sindbis virus (SINV) contains an RNA genome of positive polarity with two open reading frames (ORFs). The first ORF is translated from the genomic RNA (gRNA), rendering the viral non-structural proteins, whereas the second ORF is translated from a subgenomic mRNA (sgRNA), which directs the synthesis of viral structural proteins. SINV infection strongly inhibits host cell translation through a variety of different mechanisms, including the phosphorylation of the eukaryotic initiation factor eIF2α and the redistribution of cellular proteins from the nucleus to the cytoplasm. A number of motifs have been identified in SINV sgRNA, including a hairpin downstream of the AUG initiation codon, which is involved in the translatability of the viral sgRNA when eIF2 is inactivated. Moreover, a 3′-UTR motif containing three stem-loop structures is involved in the enhancement of translation in insect cells, but not in mammalian cells. Accordingly, SINV sgRNA has evolved several structures to efficiently compete for the cellular translational machinery. Mechanistically, sgRNA translation involves scanning of the 5′-UTR following a non-canonical mode and without the requirement for several initiation factors. Indeed, sgRNA-directed polypeptide synthesis occurs even after eIF4G cleavage or inactivation of eIF4A by selective inhibitors. Remarkably, eIF2α phosphorylation does not hamper sgRNA translation during the late phase of SINV infection. SINV sgRNA thus constitutes a unique model of a capped viral mRNA that is efficiently translated in the absence of several canonical initiation factors. The present review will mainly focus in the non-canonical mechanism of translation of SINV sgRNA.

A New Member of Cardiovirus: Unknown Pathogenicity to Humans

Members of the family Picornaviridae are nonenveloped viruses with a single-stranded RNA genome of positive polarity. ‘Fields Virology’ reads that the family contains many important human and animal pathogens and is composed of nine genera; aphthovirus, cardiovirus, enterovirus, erbovirus, hepatovirus, kobugvirus, parechovirus, rhinovirus, and teschovirus in the fifth edition [1]. The genus Cardiovirus contains Encephalomyocarditis virus (EMCV) and Theiler’s murine encephalomyelitis virus (TMEV) and has long been believed to infect mainly rodents. However, in 2007, Saffold virus (SAFV) is identified from the stool sample with fever of unknown origin as a novel human cardiovirus [2]. Subsequently, SAFV was isolated from nasal and stool specimens from infants presenting with respiratory or gastrointestinal symptoms. In addition, the virus was isolated from the cerebrospinal fluid specimen of patient with aseptic meningitis. Since some seroepidemiological studies demonstrated that the seroprevalence to SAFV reaches > 90% in older than 24 months children and adults in several countries, SAFV is thought to be spread world-wide. Although several epidemiological studies have been reported up to now, they have failed to provide a clear answer of the relationship between SAFV and human diseases [3]. Animal experiments have been carried out in order to study the pathogenicity of SAFV, although SAFV does not infect rodents naturally. The experiments of different two groups suggest that SAFV is neurotropic in mice. Of special note is the presence of inflammation in the spinal cord white matter, because the closely related mouse cardiovirus, TMEV, causes a demyelinating central nervous system disease that resembles multiple sclerosis [3]. Therefore, the recombination between SAFV and TMEV, which could alter the host range, will be a serious problem to humans. However, Himeda et al. reported in the other issue of J Plant Pathol Microbiol that the recombination of capsid proteins between SAFV and TMEV did not occur [4]. Another important issue is that the major viral load following SAFV intraperitoneal inoculation is the pancreas. Several viruses in the family Picornaviridae, particularly Coxsackie B viruses have long been implicated in the etiology of type I diabetes. In addition, EMCV is known to induce pancreatitis and type I diabetes in rodents [3]. Recently, several European groups started the study to investigate the relationship between SAFV and type I diabetes. The answer to this issue awaits the data from them. From these observations, the pathogenicity of SAFV still remains unclear, although the potential pathogenicity of SAFV to humans is thought to be varied (respiratory, gastrointestinal and neurological diseases and type I diabetes etc.). In order to clarify the pathogenicity of SAFV, the further epidemiological studies including the data of healthy persons as a control group is required. In addition, the researches of viral factors involved in the pathogenicity of SAFV using a reverse genetic technique [5] are needed. Furthermore, the identification of the receptor(s) for SAFV infection is also important in order to establish the transgenic mice as a novel animal model to study the pathogenicity of SAFV [3].

Performance of a Foot-and-Mouth Disease virus Reverse Transcription-Polymerase Chain Reaction with Amplification Controls between Three Real-Time Instruments

The foot-and-mouth disease virus (FMDV) is a member of the picornavirus family, possessing an 8-kb single-stranded RNA genome of positive polarity. It is highly contagious among several livestock species and can lead to severe economic consequences, as evidenced by the UK outbreak in 2001. The usage of real-time polymerase chain reaction has facilitated rapid detection of FMDV. Several real-time PCR instruments are available with various capabilities, such as portability and high sample volume analysis. Primers and a dual-labeled TaqMan probe were optimized to detect a single, highly conserved 88-bp segment of the FMDV 3D (RNA polymerase) gene. To increase the confidence of the RT-PCR result, a positive amplification control was synthesized to detect potential false-positive results due to contamination if a wild-type virus is used as positive control. In addition, a preventative measure against false-negative results was developed in which endogenous beta actin mRNA is coamplified by RT-PCR. Assay performance was compared on the LightCycler1.2 (Roche), the SmartCyclerII (Cepheid), and the SDS 7900HT (ABI). These assays successfully identified the FMDV genome and beta actin mRNA from several sources of infected nasal and oral swabs, as well as probang samples.

Engineering the genome of Grapevine virus A into a vector for expression of proteins in herbaceous plants

Grapevine virus A (GVA), a species of the genus Vitivirus, consists of a ∼7.4 kb single-stranded RNA genome of positive polarity, organized into five open reading frames (ORFs). In addition to grape varieties, GVA infects Nicotiana benthamiana plants and protoplasts. We engineered the genome of GVA as a vector that includes duplication of homologous sequences that contain the promoter of the movement protein (MP) sgRNA, supplemented by enzymatic restriction sites to be used as a convenient tool for transient expression of foreign genes from an individual sgRNA. The resulting vector was able to infect and to move in N. benthamiana plants in a manner similar to the wild-type GVA, but it was not stable and the inserted sequence was lost from the genome. Replacing the duplicated promoter with a GVA-MP promoter derived from a distantly related isolate of GVA improved the stability of the inserted sequence. The resulting vector was successfully used to express the reporter gene beta-glucuronidase (GUS) and the coat protein gene of Citrus tristeza virus in inoculated N. benthamiana plants. Development of a useful GVA vector is expected to find a use as a biotechnological tool for improvement of grapevines and it may enable vine breeders to bypass obstacles involved in genetic manipulation of perennial and fruiting plants.

Analysis of hepatitis C virus/classical swine fever virus chimeric 5'NTRs: sequences within the hepatitis C virus IRES are required for viral RNA replication.

Hepatitis C virus (HCV) is classified in the genus Hepacivirus of the family Flaviviridae, whose members have a single-stranded RNA genome of positive polarity, which encodes a single polyprotein. Within this family, HCV is closely related to viruses of the genus Pestivirus, which includes classical swine fever virus (CSFV). Translation of the hepaci- and pestiviral polyprotein is initiated by internal entry of ribosomes, promoted by the 5'NTR. The secondary and tertiary RNA structures of the HCV and pestivirus 5'NTRs are well conserved, despite the fact that their sequences differ significantly from one another. By analogy with other positive-stranded RNA viruses, the 5'NTR of HCV is likely to contain cis-acting determinants for replication as well as the determinants for translation. Studies on both signals could be complicated, as these signals might overlap. In this study, this problem was addressed by constructing chimeric HCV/CSFV 5'NTRs. A two-step analysis of these 5'NTRs was performed: (a) in a translation assay, which provided the possibility to study translation independently of the possible effects on replication; and (b) in a replication assay, in which were studied only the chimeric 5'NTRs for which IRES-dependent translation was demonstrated. An overlap was observed between HCV RNA elements involved in these processes. Exchange of domain II had a minor effect on the translation efficiency of the chimeric 5'NTRs, while replication of subgenomic replicons with these chimeric 5'NTRs was abolished. Exchange of domain III subdomains severely decreased translation activity, while replication was maintained.

Two classes of subgenomic RNA of grapevine virus A produced by internal controller elements

Grapevine virus A (GVA), a species of the recently established genus Vitivirus, consists of an ∼7.3-kb single-stranded RNA genome of positive polarity, organized into five open reading frames (ORFs). The virus, which is closely associated with the grapevine rugose wood disease complex, has been poorly investigated genetically. We explored the production of viral RNAs in a GVA-infected Nicotiana benthamiana herbaceous host and characterized one nested set of three 5′-terminal sgRNAs of 5.1, 5.5, and 6.0 kb, and another, of three 3′-terminal sgRNAs of 2.2, 1.8, and 1.0 kb that could serve for expression of ORFs 2–3, respectively. Neither 3′- nor 5′-terminal sgRNAs, which would correspond to ORF5, was detected, suggesting that expression of this ORF occurs via a bi- or polycistronic mRNA. The 5′-terminal sgRNAs were abundant in dsRNA-enriched extracts. Cloning and sequence analysis of the 3′ end of 5.5-kb 5′-terminal sgRNA and the 5′ end of the 1.8-kb 3′-terminal sgRNA suggested that a mechanism other than specific cleavage was involved in production of these sgRNAs. Apparently, the production of the 5′- and 3′-terminal sgRNAs was controlled by sequences upstream of the 5′-terminus of each of ORFs 2–4. Detection of both plus and minus strands of the 5′- and 3′-terminal sgRNAs, though in different levels of accumulation, suggested that each of these cis-acting elements is involved in production of four RNAs: a 3′-terminal plus-strand sgRNA which could act as an mRNA, the corresponding 3′-terminal minus-strand RNA, a 5′-terminal plus-strand sgRNA, and the corresponding 5′-terminal minus-strand RNA.

Identification of specific variations within the HE, S1, and ORF4 genes of bovine coronaviruses associated with enteric and respiratory diseases in dairy cattle.

The bovine Coronavirus (BCV) is the causative agent of enteric diseases including neonatal calf diarrhea (NCD) (Dea et al., 1980; Mebus et al., 1973), winter dysentery (WD) (Benfield and Saif, 1990; Dea et al., 1995), and chronic shedding in adult cattle (AD) (Tsunemitsu et Saif, 1995). BCV can also infect the upper respiratory tract of growing calves causing pneumonia (Chouljenko et al., 1998; Reynolds et al., 1985). The respiratory BCV (RBCV) is now recognized as an important agent associated to shipping fever (Storz et al., 1996). The virion possesses a single stranded, non segmented RNA genome of positive polarity, and is made of 5 structural proteins: the nucleocapsid phosphoprotein (N: 52 kDa), the matrix glycoprotein (M: 25 kDa), the peplomer glycoprotein (S: 200 kDa), the small membrane protein (E: 9.5 kDa) and the hemagglutinin esterase glycoprotein (HE: 140 kDa) (Spaan et al., 1988). Both S and HE are able to agglutinate red blood cells and elicit production of neutralizing antibodies (Dea et Tijssen, 1989; Deregt and Babiuk, 1987; Vautherot et al, 1992). The S protein has an important role to play in tropism being responsible for binding to the host cell (Spaan et al., 1988). The regions situated between the S and M genes (ORF4 and ORF5) may also be implicated in the tropism of coronaviruses (Mounir et al., 1992).

Translating ribosomes inhibit poliovirus negative-strand RNA synthesis.

Poliovirus has a single-stranded RNA genome of positive polarity that serves two essential functions at the start of the viral replication cycle in infected cells. First, it is translated to synthesize viral proteins and, second, it is copied by the viral polymerase to synthesize negative-strand RNA. We investigated these two reactions by using HeLa S10 in vitro translation-RNA replication reactions. Preinitiation RNA replication complexes were isolated from these reactions and then used to measure the sequential synthesis of negative- and positive-strand RNAs in the presence of different protein synthesis inhibitors. Puromycin was found to stimulate RNA replication overall. In contrast, RNA replication was inhibited by diphtheria toxin, cycloheximide, anisomycin, and ricin A chain. Dose-response experiments showed that precisely the same concentration of a specific drug was required to inhibit protein synthesis and to either stimulate or inhibit RNA replication. This suggested that the ability of these drugs to affect RNA replication was linked to their ability to alter the normal clearance of translating ribosomes from the input viral RNA. Consistent with this idea was the finding that the protein synthesis inhibitors had no measurable effect on positive-strand synthesis in normal RNA replication complexes. In marked contrast, negative-strand synthesis was stimulated by puromycin and was inhibited by cycloheximide. Puromycin causes polypeptide chain termination and induces the dissociation of polyribosomes from mRNA. Cycloheximide and other inhibitors of polypeptide chain elongation "freeze" ribosomes on mRNA and prevent the normal clearance of ribosomes from viral RNA templates. Therefore, it appears that the poliovirus polymerase was not able to dislodge translating ribosomes from viral RNA templates and mediate the switch from translation to negative-strand synthesis. Instead, the initiation of negative-strand synthesis appears to be coordinately regulated with the natural clearance of translating ribosomes to avoid the dilemma of ribosome-polymerase collisions.

Enteroviral RNA replication in the myocardium of patients with left ventricular dysfunction and clinically suspected myocarditis.

Previous studies dealing with the detection of enteroviral RNA in human endomyocardial biopsies have not differentiated between latent persistence of the enteroviral genome and active viral replication. Enteroviruses that are considered important factors for the development of myocarditis have a single-strand RNA genome of positive polarity that is transcribed by a virus-encoded RNA polymerase into a minus-strand mRNA during active viral replication. The synthesis of multiple copies of minus-strand enteroviral RNA therefore occurs only at sites of active viral replication but not in tissues with mere persistence of the viral genome. We investigated enteroviral RNA replication versus enteroviral RNA persistence in endomyocardial biopsies of 45 patients with left ventricular dysfunction and clinically suspected myocarditis. Using reverse-transcriptase polymerase chain reaction in conjunction with Southern blot hybridization, we established a highly sensitive assay to specifically detect plus-strand versus minus-strand enteroviral RNA in the biopsies. Plus-strand enteroviral RNA was detected in endomyocardial biopsies of 18 (40%) of 45 patients, whereas minus-strand RNA as an indication of active enteroviral RNA replication was detected in only 10 (56%) of these 18 plus-strand-positive patients. Enteroviral RNA was not found in biopsies of the control group (n=26). These data demonstrate that a significant fraction of patients with left ventricular dysfunction and clinically suspected myocarditis had active enteroviral RNA replication in their myocardium (22%). Differentiation between patients with active viral replication and latent viral persistence should be particularly important in future studies evaluating different therapeutic strategies. In addition, molecular genetic detection of enteroviral genome and differentiation between replicating versus persistent viruses is possible in a single endomyocardial biopsy.

Human MxA protein confers resistance to Semliki Forest virus and inhibits the amplification of a Semliki Forest virus-based replicon in the absence of viral structural proteins.

Mx proteins form a small family of interferon (IFN)-induced GTPases with potent antiviral activity against various negative-strand RNA viruses. To examine the antiviral spectrum of human MxA in homologous cells, we stably transfected HEp-2 cells with a plasmid directing the expression of MxA cDNA. HEp-2 cells are permissive for many viruses and are unable to express endogenous MxA in response to IFN. Experimental infection with various RNA and DNA viruses revealed that MxA-expressing HEp-2 cells were protected not only against influenza virus and vesicular stomatitis virus (VSV) but also against Semliki Forest virus (SFV), a togavirus with a single-stranded RNA genome of positive polarity. In MxA-transfected cells, viral yields were reduced up to 1,700-fold, and the degree of inhibition correlated well with the expression level of MxA. Furthermore, expression of MxA prevented the accumulation of 49S RNA and 26S RNA, indicating that SFV was inhibited early in its replication cycle. Very similar results were obtained with MxA-transfected cells of the human monocytic cell line U937. The results demonstrate that the antiviral spectrum of MxA is not restricted to negative-strand RNA viruses but also includes SFV, which contains an RNA genome of positive polarity. To test whether MxA protein exerts its inhibitory activity against SFV in the absence of viral structural proteins, we took advantage of a recombinant vector based on the SFV replicon. The vector contains only the coding sequence for the viral nonstructural proteins and the bacterial LacZ gene, which was cloned in place of the viral structural genes. Upon transfection of vector-derived recombinant RNA, expression of the beta-galactosidase reporter gene was strongly reduced in the presence of MxA. This finding indicates that viral components other than the structural proteins are the target of MxA action.

Potyviral HC-Pro: a multifunctional protein.

Introduction. The genus Potyvirus, family Potyviridae, is the largest genus of plant viruses with 180 members or possible members (Brunt, 1992). Potyviruses are flexuous filamentous particles which contain a single-stranded RNA genome of positive polarity possessing a covalently linked 5′-terminal viral protein (VPg) and a 3′-terminal poly(A) tail (reviewed in Riechmann et al., 1992). They are transmitted from plant to plant by aphids in a non-persistent manner, and this process is dependent on the presence of two virus-encoded proteins (reviewed in Pirone, 1991). One of these, the helper component-proteinase (HC-Pro) has attracted renewed attention during the last few years due to its multifunctionality and to it being implicated in different steps of the potyvirus life cycle. The properties, as well as the established and postulated functions of this protein, are reviewed.

The 5′-Untranslated Region of Picornaviral Genomes

Publisher SummaryPicornaviruses are small naked icosahedral viruses with a single-stranded RNA genome of positive polarity. According to current taxonomy, the family includes four genera: Enterouirus (polioviruses, coxsackieviruses, echoviruses, and other enteroviruses), Rhinovirus, Curdiouirus [encephalomyocarditis virus (EMCV), mengovirus, Theiler's murine encephalomyelitis virus (TMEV)], and Aphthouirus [foot-and-mouth disease viruses (FMDV)]. There are also some, as yet, unclassified picornaviruses [e.g., hepatitis A virus (HAW] that should certainly be assessed as a separate genus. Studies on the molecular biology of picornaviruses might be divided into two periods: those before and after the first sequencing of the poliovirus genome. The 5'-untranslated region (5-UTR) of the viral genome was one of the unexpected problems. This segment proved to be immensely long: about 750 nucleotides or ∼10% of the genome length. There were also other unusual features (e.g., multiple AUG triplets preceding the single open reading frame (ORF) that encodes the viral polyprotein). This chapter shows that the picornaviral 5-UTRs are not only involved in such essential events as the synthesis of viral proteins and RNAs that could be expected to some extent, although some of the underlying mechanisms appeared to be quite a surprise, but also may determine diverse biological phenotypes from the plaque size or thermosensitivity of reproduction to attenuation of neurovirulence. Furthermore, a close inspection of the 5-UTR structure unravels certain hidden facets of the evolution of the picornaviral genome. Finally, the conclusions drawn from the experiments with the picornaviral5-UTRs provide important clues for understanding the functional capabilities of the eukaryotic ribosomes.

Expression of porcine transmissible gastroenteritis virus genes in E. coli as beta-galactosidase chimaeric proteins.

Transmissible gastroenteritis virus (TGEV) causes gastroenteritis in pigs of all ages but has a high mortality in neonatal piglets resulting in severe economical losses in affected pig farms. In piglets, under two weeks of age, the first clinical sign is usually vomiting 18–24h after infection rapidly followed by a diarrhoea, resulting in loss of weight and dehydration; death usually occurs after 2–5 days (Garwes, 1982). The virus is enveloped, contains a single stranded RNA genome of positive polarity and three structural proteins; a 200 000 dalton surface glycoprotein (the spike or peplomer protein), a 30 000 dalton glycoprotein associated with the viral envelope (the integral membrane protein) and a basic 47 000 dalton phosphorylated protein associated with the viral RNA (the nucleoprotein). During replication of the virus in infected cells four subgenomic species of RNA are produced of which three have been shown to produce the three structural proteins (Millson et al. unpublished results). The fourth RNA species does not appear to produce a polypeptide detectable either in infected cells or by in vitro translation of the RNA though it has the capacity to produce a polypeptide of about 33 000 daltons. The nucleoprotein gene has been copied from the smallest subgenomic RNA species, shown by in vitro translation to produce the nucleoprotein (Millson et al. impublished results), into cDNA. The complete DNA sequence of the gene has been determined (Britton et al. unpublished results) and a 1.38kb fragment composed of 245 amino acids, corresponding to 68% of the complete gene product, has been fused to the 3′ end of the E. coli lacz gene. The fused genes produced a β-galactosidase-TGEV nucleoprotein fragment chimaeric protein in E. coli. The chimaeric protein has been purified and used to raise antibodies in mice which immune precipitated only the viral nucleoprotein confirming that the DNA sequence assumed to contain the nucleoprotein gene sequence does so. This method provides a useful source of TGEV antigen for virus diagnostic tests and viral protein for use in a research programme aimed at understanding the mechanisms of antigen processing and immune stimulation. The method is useful for confirming the presence of open reading frames (ORFs) and identifying the true product of genes on cDNA either suspected to contain a particular gene or for those previously identified by DNA sequencing.