Dengue is the most common mosquito-borne viral disease in humans. The spread of both mosquito vectors and viruses has led to the resurgence of epidemic dengue fever (a self-limited flu-like syndrome) and the emergence of dengue hemorrhagic fever (severe dengue with bleeding abnormalities) in urban centers of the tropics. There are no animal or laboratory models of dengue disease; indirect evidence suggests that dengue viruses differ in virulence, including their pathogenicities for humans and epidemic potential. We developed two assay systems (using human dendritic cells and Aedes aegypti mosquitoes) for measuring differences in virus replication that correlate with the potential to cause hemorrhagic dengue and increased virus transmission. Infection and growth experiments showed that dengue serotype 2 viruses causing dengue hemorrhagic fever epidemics (Southeast Asian genotype) can outcompete viruses that cause dengue fever only (American genotype). This fact implies that Southeast Asian genotype viruses will continue to displace other viruses, causing more hemorrhagic dengue epidemics.

The dengue virus type 2 structures probably involved in human virulence were previously defined by sequencing the complete genome of both American and Southeast (SE) Asian genotype templates in patient serum (K. C. Leitmeyer et al., J. Virol. 73:4738-4747, 1999). We have now evaluated the effects of introducing a mutation in the envelope glycoprotein (E) gene and/or replacement of 5-and 3-nontranslated regions on dengue virus replication in human primary cell cultures. A series of chimeric infectious clones were generated containing different combinations of American and SE Asian genotype sequences. Some of the chimeric viruses had altered plaque morphology in mammalian cells; however, they replicated at similar rates in mosquito cells as measured by quantitative reverse transcription-PCR and plaque assay. Although susceptibility to virus infection varied from donor to donor in experiments using human macrophage and dendritic cells, we were able to measure consistent differences in viral RNA output per infected cell. Using this measurement, we demonstrated that the chimeric virus containing the E mutation had a lower virus output compared to the parental infectious clone. A larger reduction in virus output was observed for the triple mutant and the wild-type, American genotype virus from which chimeric inserts were derived. It appears that the three changes function synergistically, although the E mutation alone gives a lower output compared to the 5-and 3-terminal mutations. The data suggest that these changes may be responsible for decreased dengue virus replication in human target cells and for virulence characteristics during infection.

The coronavirus nucleocapsid (N) protein is a major structural component of virions that associates with the genomic RNA to form a helical nucleocapsid. N appears to be a multifunctional protein since data also suggest that the protein may be involved in viral RNA replication and translation. All of these functions presumably involve interactions between N and viral RNAs. As a step toward understanding how N interacts with viral RNAs, we mapped high-efficiency N-binding sites within BCV- and MHV-defective genomes. Both in vivo and in vitro assays were used to study binding of BCV and MHV N proteins to viral and nonviral RNAs. N-viral RNA complexes were detected in bovine coronavirus (BCV)-infected cells and in cells transiently expressing the N protein. Filter binding was used to map N-binding sites within Drep, a BCV-defective genome that is replicated and packaged in the presence of helper virus. One high-efficiency N-binding site was identified between nucleotides 1441 and 1875 at the 3' end of the N ORF within Drep. For comparative purposes N-binding sites were also mapped for the mouse hepatitis coronavirus (MHV)-defective interfering (DI) RNA MIDI-C. Binding efficiencies similar to those for Drep were measured for RNA transcripts of a region encompassing the MHV packaging signal (nts 3949-4524), as well as a region at the 3' end of the MHV N ORF (nts 4837-5197) within MIDI-C. Binding to the full-length MIDI-C transcript (approximately 5500 nts) and to an approximately 1-kb transcript from the gene 1a region (nts 935-1986) of MIDI-C that excluded the packaging signal were both significantly higher than that measured for the smaller transcripts. This is the first identification of N-binding sequences for BCV. It is also the first report to demonstrate that N interacts in vitro with sequences other than the packaging signal and leader within the MHV genome. The data clearly demonstrate that N binds coronavirus RNAs more efficiently than nonviral RNAs. The results have implications with regard to the multifunctional role of N.

A region of the bovine coronavirus (BCV) genome that functions as a packaging signal has been cloned. The 291-nucleotide clone shares 72% homology with the region of mouse hepatitis coronavirus (MHV) gene 1b that contains the packaging signal. RNA transcripts were packaged into both BCV and MHV virions when the cloned region was appended to a noncoronavirus RNA. This is the first identification of a BCV packaging signal. The data demonstrate that the BCV genome contains a sequence that is conserved at both the sequence and functional levels, thus broadening our insight into coronavirus packaging.The coronavirus genome is a single-stranded, positive-sense, 27-to 31-kb RNA molecule, the largest among all RNA viruses. The genome is encapsidated by multiple copies of the nucleocapsid (N) protein and is packaged as a helical nucleocapsid in the mature enveloped virion (for reviews, see references 14 and 25). During infection, coronaviruses synthesize six or seven subgenomic mRNAs which share 5Ј and 3Ј ends that are identical to the genomic RNA (for a review, see reference 29).How coronaviruses recognize the viral RNA(s) to ensure specific encapsidation and packaging is only beginning to be understood. Thus far, a packaging signal has been identified only for mouse hepatitis coronavirus (MHV). By using MHV defective interfering (DI) RNAs, a 69-nucleotide (nt) packaging signal that maps approximately 20 kb from the 5Ј end of the genome within the gene 1b open reading frame was identified ( Fig. 1) (8,16,28). Inclusion of the packaging signal in a non-MHV RNA is sufficient to allow the RNA to be packaged into MHV virions (30). Mutagenesis of the predicted bulged stem-loop structure of the MHV packaging signal disrupts the ability of the sequence to function as a packaging signal (8). It was also shown that an MHV strain A59 subgenomic DI RNA is packaged into virions when it contains the DI packaging signal, but the subgenomic RNA was reported to be packaged less efficiently than its DI genomic RNA (4).Sequences that are required for RNAs to be packaged by bovine coronavirus (BCV) have not been determined. BCV belongs to the same antigenic subgroup as MHV, and data based on the 3Ј one-third of the genome that has been cloned and sequenced indicate that the two viruses are closely related (1, 2, 13, 15). However, multiple pieces of data suggest that BCV is different from MHV with regard to RNA packaging. The first is that BCV packages subgenomic RNAs in addition to genomic RNA, whereas MHV packages very little, if any, of its subgenomics. BCV N and M mRNAs were shown to be more abundant in virions, on a molar basis, than was genome (11). The second difference is that the BCV defective RNA Drep (Fig. 1), which is replicated and packaged by BCV, does not contain any gene 1b sequence (6).To gain further insight into the requirements for coronavirus packaging, we asked whether the BCV genome contains sequence information equivalent to the MHV packaging signal. Reverse transcription followed by PCR (RT-PCR) was carried out by ...

The coronavirus nucleocapsid protein (N) is involved in encapsidation and packaging of viral RNA. In this study we investigated the ability of the bovine coronavirus (BCV) N protein to interact with RNA. Histidine-tagged BCV N (his-N) protein was expressed in bacteria. A filter binding assay was established to quantitatively measure the binding efficiency of purified his-N to different RNAs. The results indicate that bacterially expressed N bound both BCV and mouse hepatitis coronavirus (MHV) RNAs. Binding to in vitro generated BCV and MHV RNA transcripts was significantly higher than binding to a non-coronavirus RNA. Similar binding efficiencies were measured for a BCV defective genome, pDrep, and a transcript that contained the MHV packaging signal. Interestingly, the entire MHV Dr, pMIDI-C, was bound at a higher efficiency than the packaging signal alone. This is one of the first reports to show that N interacts with the MHV packaging signal.