Open Reading Frame 1a Research Articles

Stem-Pitting Citrus tristeza virus Predominantly Transmitted by the Brown Citrus Aphid from Mixed Infections Containing Non-Stem-Pitting and Stem-Pitting Isolates.

Citrus tristeza virus (CTV) is a phloem-limited Closterovirus that produces a variety of symptoms in various Citrus spp. One of these symptoms is stem pitting (SP). SP does not occur in all Citrus spp. but when it does it may cause low tree vigor, decline, and an economic reduction in fruit size and yield. Historically, the first appearance of CTV-SP in a citrus area often occurs after the introduction of the most efficient CTV vector, the brown citrus aphid (BCA), Toxoptera citricida. Hypotheses for this association range from the introduction of these strains in new planting materials to the increased ability of BCA to transmit SP strains from existing CTV sources. It is known that CTV often exists as a complex of isolates or subisolates. Single and multiple BCA transmissions have been used to separate different genotypes or strains of CTV from mixed CTV infected plants. This study was initiated to determine what the BCA transmits when an exotic severe SP CTV isolate B12 from Brazil or B408 from Dominican Republic are mixed with a non-SP (NSP) isolate, FS627 from Florida. Biological and molecular data was generated from grafted mixtures of these isolates and their aphid-transmitted subisolates. Single-strand conformation polymorphism patterns of the 5' terminal region of open reading frame (ORF) 1a, the overlapping region of ORF1b and ORF2, and the major coat protein gene region of NSP and SP CTV-grafted plants remained unchanged but the patterns of doubly inoculated plants varied. The haplotype diversity within SP isolates B12, B408, and mixtures of NSP and SP isolates (FS627/B12 and FS627/B408) and aphid-transmitted subisolates from doubly inoculated plants was determined by analysis of the haplotype nucleotide sequences. Aphid transmission experiments, symptoms, and molecular analyses showed that SP-CTV was more frequently transmitted with or without NSP-CTV from mixed infections.

Interference of ribosomal frameshifting by antisense peptide nucleic acids suppresses SARS coronavirus replication

The programmed −1 ribosomal frameshifting (−1 PRF) utilized by eukaryotic RNA viruses plays a crucial role for the controlled, limited synthesis of viral RNA replicase polyproteins required for genome replication. The viral RNA replicase polyproteins of severe acute respiratory syndrome coronavirus (SARS-CoV) are encoded by the two overlapping open reading frames 1a and 1b, which are connected by a −1 PRF signal. We evaluated the antiviral effects of antisense peptide nucleic acids (PNAs) targeting a highly conserved RNA sequence on the – PRF signal. The ribosomal frameshifting was inhibited by the PNA, which bound sequence-specifically a pseudoknot structure in the −1 PRF signal, in cell lines as assessed using a dual luciferase-based reporter plasmid containing the −1 PRF signal. Treatment of cells, which were transfected with a SARS-CoV-replicon expressing firefly luciferase, with the PNA fused to a cell-penetrating peptide (CPP) resulted in suppression of the replication of the SARS-CoV replicon, with a 50% inhibitory concentration of 4.4 μM. There was no induction of type I interferon responses by PNA treatment, suggesting that the effect of PNA is not due to innate immune responses. Our results demonstrate that −1 PRF, critical for SARS-CoV viral replication, can be inhibited by CPP-PNA, providing an effective antisense strategy for blocking −1 PRF signals.

Characterization of Equine Humoral Antibody Response to the Nonstructural Proteins of Equine Arteritis Virus

Equine arteritis virus (EAV) replicase consists of two polyproteins (pp1a and pp1ab) that are encoded by open reading frames (ORFs) 1a and 1b of the viral genome. These two replicase polyproteins are posttranslationally processed by three ORF 1a-encoded proteinases to yield at least 13 nonstructural proteins (nsp1 to nsp12, including nsp7α and 7β). These nsps are expressed in EAV-infected cells, but the equine immune response they induce has not been studied. Therefore, the primary purpose of this study was to evaluate the humoral immune response of horses to each of the nsps following EAV infection. Individual nsp coding regions were cloned and expressed in both mammalian and bacterial expression systems. Each recombinant protein was used in an immunoprecipitation assay with equine serum samples from horses (n = 3) that were experimentally infected with three different EAV strains (VB, KY77, and KY84), from stallions (n = 4) that were persistently infected with EAV, and from horses (n = 4) that were vaccinated with the modified live-virus (MLV) vaccine strain. Subsequently, protein-antibody complexes were subjected to Western immunoblotting analysis with individual nsp-specific rabbit antisera, mouse anti-His antibody, or anti-FLAG tag antibody. Nsp2, nsp4, nsp5, and nsp12 were immunoprecipitated by most of the sera from experimentally or persistently infected horses, while sera from vaccinated horses did not react with nsp5 and reacted weakly with nsp4. However, serum samples from vaccinated horses were able to immunoprecipitate nsp2 and nsp12 proteins consistently. Information from this study will assist ongoing efforts to develop improved methods for the serologic diagnosis of EAV infection in horses.

Complete sequence and genetic characterization of Raspberry latent virus, a novel member of the family Reoviridae

A new virus isolated from red raspberry plants and detected in the main production areas in northern Washington State, USA and British Columbia, Canada was fully sequenced and found to be a novel member of the family Reoviridae. The virus was designated as Raspberry latent virus (RpLV) based on the fact that it is symptomless when present in single infections in several Rubus virus indicators and commercial raspberry cultivars. RpLV genome is 26,128 nucleotides (nt) divided into 10 dsRNA segments. The length of the genomic segments (S) was similar to those of other reoviruses ranging from 3948 nt (S1) to 1141 nt (S10). All of the segments, except S8, have the conserved terminal sequences 5′-AGUU----GAAUAC-3′. A point mutation at each terminus of S8 resulted in the sequences 5′-AGUA----GAUUAC-3′. Inverted repeats adjacent to each conserved terminus as well as stem loops and extended pan handles were identified by analyses of secondary structures of the non-coding sequences. All segments, except S3 and S10, contained a single open reading frame (ORF) on the positive sense RNAs. Two out-of-frame overlapping ORFs were identified in segments S3 (ORF S3a and S3b) and S10 (ORF S10a and S10b). Amino acid (aa) alignments of the putative proteins encoded by the main ORF in each segment revealed a high identity to several proteins encoded by reoviruses from different genera including Oryzavirus, Cypovirus, and Dinovernavirus. Alignments of the polymerase, the most conserved protein among reoviruses, revealed a 36% aa identity between RpLV and Rice ragged stunt virus (RRSV), the type member of the genus Oryzavirus, indicating that these two viruses are closely related. Phylogenetic analyses showed that RpLV clusters with members of the genera Oryzavirus, Cypovirus, Dinovernavirus and Fijivirus. These genera belong to the subfamily Spinareovirinae which includes reoviruses with spiked core particles (‘turreted’ reoviruses). In addition, two nucleotide binding motifs, regarded as ‘signature’ sequences among turreted reoviruses, were also found in RpLV P8, suggesting that RpLV is a novel dicot-infecting reovirus in the subfamily Spinareovirinae.

Evidence of a Recombinant Wild-Type Human Astrovirus Strain from a Kenyan Child with Gastroenteritis

A human astrovirus (HAstV) strain from Kenya was characterized by nucleotide sequence analysis. Sequences from open reading frame 1a (ORF1a) clustered with genotype 6/7, those from ORF1b clustered with genotype 3, and those from ORF2 clustered with genotype 2. A recombination point in the ORF1b-ORF2 junction was identified, with a second possible recombination point within the ORF1a region.

Molecular Mapping of the RNA Cap 2′-O-Methyltransferase Activation Interface between Severe Acute Respiratory Syndrome Coronavirus nsp10 and nsp16*

Several protein-protein interactions within the SARS-CoV proteome have been identified, one of them being between non-structural proteins nsp10 and nsp16. In this work, we have mapped key residues on the nsp10 surface involved in this interaction. Alanine-scanning mutagenesis, bioinformatics, and molecular modeling were used to identify several “hot spots,” such as Val42, Met44, Ala71, Lys93, Gly94, and Tyr96, forming a continuous protein-protein surface of about 830 Å2, bearing very conserved amino acids among coronaviruses. Because nsp16 carries RNA cap 2′-O-methyltransferase (2′O-MTase) activity only in the presence of its interacting partner nsp10 (Bouvet, M., Debarnot, C., Imbert, I., Selisko, B., Snijder, E. J., Canard, B., and Decroly, E. (2010) PLoS Pathog. 6, e1000863), functional consequences of mutations on this surface were evaluated biochemically. Most changes that disrupted the nsp10-nsp16 interaction without structural perturbations were shown to abrogate stimulation of nsp16 RNA cap 2′O-MTase activity. More strikingly, the Y96A mutation abrogates stimulation of nsp16 2′O-MTase activity, whereas Y96F overstimulates it. Thus, the nsp10-nsp16 interface may represent an attractive target for antivirals against human and animal pathogenic coronaviruses.

Molecular characterization of partial-open reading frames 1a and 2 of the human astroviruses in South Korea

Human astroviruses (HAstVs) are among the major causes of gastroenteritis in South Korea. In this study, the partial regions of the open reading frame (ORF) 1a and ORF2 genes of HAstVs from gastroenteritis patients in nine hospitals were sequenced, and the molecular characterization of the viruses was revealed. 89 partial nucleotide sequences of ORF1a and 88 partial nucleotide sequences of ORF2 were amplified from 120 stool specimens. Phylogenetic analysis showed that most of the nucleotide sequences of ORF1a and ORF2 were grouped with HAstV type 1 but had evolutionary genetic distance compared with the reference sequences, such as the HAstV-1 prototype, Dresden strain, and Oxford strain. According to the phylogenetic analysis, some nucleotide sequences including SE0506041, SE0506043, and SE0506058, showed the discrepancy of the genotypes, but there was no proof of recombination among the HAstV types. In conclusion, this study showed that the dominant HAstV isolated from the Seoul metropolitan area in 2004-2005 was HAstV type 1, and that Korean HAstV-1 had the genetic distance in evolution compared with the reference sequences of HAstVs. Lots of nucleotide sequences of the ORF1a and ORF2 genes of HAstV will be useful for studying for the control and prevention of HAstV gastroenteritis in South Korea.

A New Cistron in the Murine Hepatitis Virus Replicase Gene

We report an RNA-negative, temperature-sensitive (ts) mutant of Murine hepatitis virus, Bristol ts31 (MHV-Brts31), that defines a new complementation group within the MHV replicase gene locus. MHV-Brts31 has near-normal levels of RNA synthesis at the permissive temperature of 33 degrees C but is unable to synthesize viral RNA when the infection is initiated and maintained at the nonpermissive temperature of 39.5 degrees C. Sequence analysis of MHV-Brts31 RNA indicated that a single G-to-A transition at codon 1307 in open reading frame 1a, which results in a replacement of methionine-475 with isoleucine in nonstructural protein 3 (nsp3), was responsible for the ts phenotype. This conclusion was confirmed using a vaccinia virus-based reverse genetics system to produce a recombinant virus, Bristol tsc31 (MHV-Brtsc31), which has the same RNA-negative ts phenotype and complementation profile as those of MHV-Brts31. The analysis of protein synthesis in virus-infected cells showed that, at the nonpermissive temperature, MHV-Brtsc31 was not able to proteolytically process either p150, the precursor polypeptide of the replicase nonstructural proteins nsp4 to nsp10, or the replicase polyprotein pp1ab to produce nsp12. The processing of replicase polyprotein pp1a in the region of nsp1 to nsp3 was not affected. Transmission electron microscopy showed that, compared to revertant virus, the number of double-membrane vesicles in MHV-Brts31-infected cells is reduced at the nonpermissive temperature. These results identify a new cistron in the MHV replicase gene locus and show that nsp3 has an essential role in the assembly of a functional MHV replication-transcription complex.

First Report of Cucumber mosaic virus Infecting Pinewood Coneflower (Rudbeckia bicolor) in Korea

Department of Plant Medicine, Chungbuk National University, Cheongju 361-763, Korea(Received on December 21, 2009; Accepted on February 15, 2010)A virus isolate causing symptoms of yellow mosaic, fernleaves, malformation and plant necrosis on Rudbeckiabicolor was prevalent around Pyeongchang area inKorea. The causal virus was identified as Cucumbermosaic virus (CMV) using characteristics from bio-logical, serological and molecular analyses and namedas CMV-Rb. CMV-Rb caused mosaic on Nicotianabenthamiana, N. tabacum, Capsicum annuum, and Lyco-persicon esculentum. However, typical local lesions didnot develop on inoculated Pisum sativum, Cucurbitamoschata, Datura stramonium and Tetragonia expansaplants. Full-length genome sequences of CMV-RbRNAs 1, 2 and 3 were obtained using 12 primer pairs byRT-PCR analysis. The genome of CMV-Rb RNA seg-ments 1, 2, and 3 consists of 3363nt, 3049nt, and 2214ntin length, respectively. In order to ascertain their taxo-nomic identity, nucleotide and the deduced amino acidsequence analyses RNAs 1, 2 and 3 of CMV-Rb isolateswere conducted with previously reported sequences ofCMV strains and/or isolates. CMV-Rb RNAs showedabout 90 to 99% sequence identity to those of subgroupI strains suggesting that CMV-Rb is more closely relat-ed to CMV isolates belong to subgroup I. To our know-ledge, this is the first report of CMV on Rudbeckiabicolor in Korea.Keywords : CMV-Rb, phylogenetic analyses, Rudbeckiabicolor, subgroup ICucumber mosaic virus (CMV), the type member of thegenus Cucumovirus, infects more than 1,000 species ofplants and induces the typical mosaic symptoms in manyhost plants. Some of the intermediate symptoms, however,include blight, fernleaf, ringspot, and stunt. In Korea, atleast 109 natural host plant species has been reported, i.e.,20 in upland crops, 20 in vegetable crops, 28 in floral crops,acacia in wood, 40 in wild plants (List of plant disease inKorea, 2004, The Korean Society of Plant Pathology).Recently, CMV infections were also reported on Hydrangeamacrophylla (Lee et al., 2001), water chickweed (Choi etal., 2004), Isodon inflexus Kudo, Jeffersonia dubia Benth,Phryma leptostachya var. asiatica Hara (Lee et al., 2008).However, no natural outbreaks by CMV have been reportedon Rudbeckia bicolor, but Bidens mottle virus (BiMoV)was only identified (Brunt et al., 1996; Choi, 2001; Jin,2003; Lee, 1981; Lee et al., 2003; Logan et al., 1984).The genome of CMV consists of three genomic RNAs(RNA 1, 2 and 3; Roossinck, 2001). In infected host plants,it also produces two subgenomic RNAs (4 and 4A; Palukaitiset al., 1992). Based upon biological, serological, and, mole-cular biological characteristics of CMV isolates, they wereclassified into two subgroups, i.e., subgroup I and II (Owenand Palukaitis, 1988). The nucleotide sequence identitybetween subgroup I and II isolates range from 69 to 77%,while showing above 90% nucleotide sequence identitywithin each subgroup isolates. More detailed nucleotidesequence analyses, especially with the coat protein (CP)and 5' nontranslated region (NTR) of RNA 3 further sug-gested subdivision of subgroup I into subgroups IA and IB(Roossinck et al., 1999). This subgrouping was more clear-ly supported from the analysis of nucleotide sequences of1a and 3a open reading frames (ORFs) than those of the 2aand CP ORFs (Roossinck, 2002). In this paper, we reportthe biological and serological characteristics as well ascomplete nucleotide sequence analyses of CMV-Rb isolate.We believe this is the first report of natural CMV infectionon Rudbeckia bicolor in Korea.Pinewood coneflower (R. bicolor), family Asteraceae, iswidely grown as an ornamental crop and a wildflower. In2005, we could easily observe yellow mosaic, sometimes

Detection of Newly Described Astrovirus MLB1 in Stool Samples from Children

To the Editor: We read with interest the article by Finkbeiner et al. describing an epidemiologic survey of newly described astrovirus MLB1 (AstV-MLB1) conducted in the United States in 2008 (1). This study was an extension of recently published reports of characterization of AstV-MLB1 from a fecal sample obtained in Australia in 1999 (2,3). These studies provide evidence of a divergent group of astroviruses and their etiologic association with human disease. However, the occurrence of a MLB1-like AstV in humans has already been documented. Walter identified a novel AstV in an 8-month-old child with diarrhea in Mexico in 1991 (4). In that study, Walter screened fecal samples for AstVs by using a variety of techniques. Sequencing of selected PCR products identified a unique virus that had typical AstV morphologic appearance but was nonreactive with human AstV-specific monoclonal or polyclonal antibodies. Phylogenetic analysis of fragments of open reading frame 1a (ORF1a) and ORF2 genome regions of this virus strain (M3363) showed that it was only distantly related to other mammalian AstVs, including human AstVs (4). This sequence divergence from canonical human AstVs suggested that M3363 might have been transmitted from an animal reservoir (4,5). When we reanalyzed ORF1a of M3363, we found that this strain was actually an MLB1-like AstV with >98% amino acid similarity to prototype and strains from the United States (Figure) that dated back to 1991. The fact that such closely related viruses were found in a scattered temporal and spatial pattern in children may indicate that MLB1-like AstVs represent a true human enteric virus, which was probably overlooked in the absence of adequate diagnostic reagents and protocols. Figure Neighbor-joining tree based on partial sequences of open reading frame 1a protein of human and animal astroviruses. Amino acid sequences of the Mexican M3363 strain (arrow) were obtained from Walter (4); other sequences were obtained from GenBank. Bootstrap ...

Detection of Newly Described Astrovirus MLB1 in Stool Samples from Children

To the Editor: We read with interest the article by Finkbeiner et al. describing an epidemiologic survey of newly described astrovirus MLB1 (AstV-MLB1) conducted in the United States in 2008 (1). This study was an extension of recently published reports of characterization of AstV-MLB1 from a fecal sample obtained in Australia in 1999 (2,3). These studies provide evidence of a divergent group of astroviruses and their etiologic association with human disease. However, the occurrence of a MLB1-like AstV in humans has already been documented. Walter identified a novel AstV in an 8-month-old child with diarrhea in Mexico in 1991 (4). In that study, Walter screened fecal samples for AstVs by using a variety of techniques. Sequencing of selected PCR products identified a unique virus that had typical AstV morphologic appearance but was nonreactive with human AstV-specific monoclonal or polyclonal antibodies. Phylogenetic analysis of fragments of open reading frame 1a (ORF1a) and ORF2 genome regions of this virus strain (M3363) showed that it was only distantly related to other mammalian AstVs, including human AstVs (4). This sequence divergence from canonical human AstVs suggested that M3363 might have been transmitted from an animal reservoir (4,5). When we reanalyzed ORF1a of M3363, we found that this strain was actually an MLB1-like AstV with >98% amino acid similarity to prototype and strains from the United States (Figure) that dated back to 1991. The fact that such closely related viruses were found in a scattered temporal and spatial pattern in children may indicate that MLB1-like AstVs represent a true human enteric virus, which was probably overlooked in the absence of adequate diagnostic reagents and protocols. Figure Neighbor-joining tree based on partial sequences of open reading frame 1a protein of human and animal astroviruses. Amino acid sequences of the Mexican M3363 strain (arrow) were obtained from Walter (4); other sequences were obtained from GenBank. Bootstrap ...

The Open Reading Frame 3a Protein of Severe Acute Respiratory Syndrome-Associated Coronavirus Promotes Membrane Rearrangement and Cell Death

The genome of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) contains eight open reading frames (ORFs) that encode novel proteins. These accessory proteins are dispensable for in vitro and in vivo replication and thus may be important for other aspects of virus-host interactions. We investigated the functions of the largest of the accessory proteins, the ORF 3a protein, using a 3a-deficient strain of SARS-CoV. Cell death of Vero cells after infection with SARS-CoV was reduced upon deletion of ORF 3a. Electron microscopy of infected cells revealed a role for ORF 3a in SARS-CoV induced vesicle formation, a prominent feature of cells from SARS patients. In addition, we report that ORF 3a is both necessary and sufficient for SARS-CoV-induced Golgi fragmentation and that the 3a protein accumulates and localizes to vesicles containing markers for late endosomes. Finally, overexpression of ADP-ribosylation factor 1 (Arf1), a small GTPase essential for the maintenance of the Golgi apparatus, restored Golgi morphology during infection. These results establish an important role for ORF 3a in SARS-CoV-induced cell death, Golgi fragmentation, and the accumulation of intracellular vesicles.

Population Dynamics of a Florida Citrus tristeza virus Isolate and Aphid-Transmitted Subisolates: Identification of Three Genotypic Groups and Recombinants After Aphid Transmission

Tristeza is an important citrus disease affecting the viability and productivity of citrus worldwide. The causal agent, Citrus tristeza virus (CTV), usually occurs as a mixture of genotypes in nature, with one of the genotypes often dominating the population. CTV has a monopartite, positive-sense RNA genome of approximately 19.3 kb and exhibits over 30% diversity in the 5' half and less than 10% in the 3' half among different genotypes. A Florida CTV isolate, FS627, was selected for this study. Isolate FS627 was analyzed by reverse-transcription polymerase chain reaction (RT-PCR) using primers to three regions: 788-bp region in the 5' (697 to 1,484 nucleotides), open reading frame (ORF)1a, 696 or 718 bp from the overlapping region of the RdRp (ORF1b) and p33 (ORF2) gene, and a 672-bp major coat protein gene (ORF7) in the 3' end of the CTV genome. The presence of T36, T30, and VT genotypes in isolate FS627 was confirmed utilizing the genotype specific overlapping region of RdRp primer pairs for RT-PCR amplification followed by cloning and sequence analysis. Analysis of single-strand conformational polymorphisms and sequences of RT-PCR-amplified products of the above regions were used to determine the presence of genotypes in both the parent and aphid-transmitted (AT) subisolates. Although the parent isolate had T36 as the major genotype, T30 was the major genotype in most of the AT subisolates. Some intermediate genotypes were detected that differed from the parental or AT subisolates. These intermediate genotypes were considered to be recombinants of the T30 and T36 genotypes and also were observed in the second level of AT subisolates generated from the of first-level AT subisolates of CTV-FS627. This work provides advance information on the population dynamics in CTV mixtures and the generation of virus recombinants after aphid transmission.

Cis preferential replication of Lettuce infectious yellows virus (LIYV) RNA 1: The initial step in the asynchronous replication of the LIYV genomic RNAs

A series of Lettuce infectious yellows virus (LIYV) RNA 1 mutants was created to evaluate their ability to replicate in tobacco protoplasts. Mutants ΔEcoRI, ΔE-LINK, and Δ1B, having deletions in open reading frames (ORFs) 1A and 1B, did not replicate when individually inoculated to protoplasts or when co-inoculated with wild-type RNA1 as a helper virus. A fragment of the green fluorescent protein (GFP) gene was inserted into the RNA 1 ORF 2 (P34) in order to provide a unique sequence tag. This mutant, P34-GFP TAG, was capable of independent replication in protoplasts. Mutants derived from P34-GFP TAG having frameshift mutations in the ORF 1A or 1B were unable to replicate in protoplasts alone or in trans when co-inoculated with wild-type RNA1 as a helper virus. Taken together, these data strongly suggest that LIYV RNA 1 replication is cis-preferential.

Murine coronaviruses encoding nsp2 at different genomic loci have altered replication, protein expression, and localization.

Partial or complete deletion of several coronavirus nonstructural proteins (nsps), including open reading frame 1a (ORF1a)-encoded nsp2, results in viable mutant proteins with specific replication defects. It is not known whether expression of nsps from alternate locations in the genome can complement replication defects. In this report, we show that the murine hepatitis virus nsp2 sequence was tolerated in ORF1b with an in-frame insertion between nsp13 and nsp14 and in place of ORF4. Alternate encoding or duplication of the nsp2 gene sequence resulted in differences in nsp2 expression, processing, and localization, was neutral or detrimental to replication, and did not complement an ORF1a Deltansp2 replication defect. The results suggest that wild-type genomic organization and expression of nsps are required for optimal replication.

Reverse transcription loop-mediated isothermal amplification for the detection of highly pathogenic porcine reproductive and respiratory syndrome virus

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay targeting the open reading frames 1a of highly pathogenic porcine reproductive and respiratory syndrome virus genome was developed. The 10 reference strains, 1 clinical isolation strain and 122 positive samples were tested. Positive reactions were confirmed for all strains and specimens by reverse transcription loop-mediated isothermal amplification and nested reverse transcription polymerase chain reaction (RT-PCR). The results showed this detection technique is more reliable and convenient for rapid and sensitive diagnosis of highly pathogenic porcine reproductive and respiratory syndrome virus infection.

Mouse-Passaged Severe Acute Respiratory Syndrome-Associated Coronavirus Leads to Lethal Pulmonary Edema and Diffuse Alveolar Damage in Adult but Not Young Mice

Advanced age is a risk factor of severe acute respiratory syndrome (SARS) in humans. To understand its pathogenesis, we developed an animal model using BALB/c mice and the mouse-passaged Frankfurt 1 isolate of SARS coronavirus (SARS-CoV). We examined the immune responses to SARS-CoV in both young and adult mice. SARS-CoV induced severe respiratory illness in all adult, but not young, mice on day 2 after inoculation with a mortality rate of 30 to 50%. Moribund adult mice showed severe pulmonary edema and diffuse alveolar damage accompanied by virus replication. Adult murine lungs, which had significantly higher interleukin (IL)-4 and lower IL-10 and IL-13 levels before infection than young murine lungs, rapidly produced high levels of proinflammatory chemokines and cytokines known to induce macrophage and neutrophil infiltration and activation (eg, tumor necrosis factor-alpha). On day 2 after inoculation, young murine lungs produced not only proinflammatory cytokines but also IL-2, interferon-gamma, IL-10, and IL-13. Adult mice showed early and acute excessive proinflammatory responses (ie, cytokine storm) in the lungs after SARS-CoV infection, which led to severe pulmonary edema and diffuse alveolar damage. Intravenous injection with anti-tumor necrosis factor-alpha antibody 3 hours after infection had no effect on SARS-CoV infection. However, intraperitoneal interferon-gamma injection protected adult mice from the lethal respiratory illness. The experimental model described here may be useful for elucidating the pathophysiology of SARS and for evaluating therapies to treat SARS-CoV infection.

Three genes of Citrus tristeza virus are dispensable for infection and movement throughout some varieties of citrus trees

Citrus tristeza virus (CTV), a member of the Closteroviridae, possesses a 19.3-kb positive-stranded RNA genome that is organized into twelve open reading frames (ORFs). The CTV genome contains two sets of conserved genes, which are characteristic of this virus group, the replication gene block (ORF 1a and 1b) and the quintuple gene block (p6, HSP70 h, p61, CPm, and CP). With the exception of the p6 gene, they are required for replication and virion assembly. CTV contains five additional genes, p33, p18, p13, p20 and p23, in the 3′ half of the genome, some of which (p33, p18 and p13) are not conserved among other members of this virus group, and have been proposed to have evolved for specific interactions with the citrus host. In the present study, the requirements for systemic infection of citrus trees of p33, p6, p18, p13 and p20 were examined. Viral mutants with a deletion in the p6 or the p20 ORF failed to infect citrus plants systemically, suggesting their possible roles in virus translocation/systemic infection. However, we found that deletions within the p33, p18 or p13 ORF individually resulted in no significant loss of ability of the virus to infect, multiply, and spread throughout citrus trees. Furthermore, deletions in the p33, p18 and p13 genes in all possible combinations including deletions in all three genes allowed the virus to systemically invade citrus trees. Green fluorescent protein-tagged CTV variants with deletions in the p33 ORF or the p33, p18 and p13 ORFs demonstrated that the movement and distribution of these deletion mutants were similar to that of the wild-type virus.

Identification of the avian infectious bronchitis coronaviruses with mutations in gene 3

The sequence of a 6.0-kb fragment was compared in the 3′-encoding region of the genome in 27 infectious bronchitis virus (IBV) strains. All these strains have the same S-3-M-5-N gene order, as is the case for other IBVs. However, the sizes of the corresponding open reading frames (ORFs) of some genes varied among the virus strains. Phylogenetic analysis and sequence alignments demonstrated that recombination events had occurred in the origin and evolution of the strains CK/CH/LSD/03I and CK/CH/LLN/98I and the possible recombinant junction sites might be located at the 3c and M genes, respectively. The normal product of ORF 3a is 57 amino acids long, whereas a 43-bp deletion at the 3′-end of the CK/CH/LSD/03I 3a gene was detected, resulting in a frameshift event and C-terminally truncated protein with 47 amino acids. Comparison of the growth ability in embryos and replication and pathogenicity in chickens with IBV carrying the normal 3a gene indicated that this deleted sequence in the 3a gene of CK/CH/LSD/03I was not necessary for viral pathogenesis and replication either in vitro or in vivo. Occurrence of a mutation at the corresponding position of the CK/CH/LLN/98I start codon in the 3a gene led to the absence of ORF 3a in this virus, resulting in a novel genomic organization at the 3′-encoding regions: S-3b, 3c-M-5a, 5b-N. Comparison with other viruses carrying the normal 3a gene revealed that CK/CH/LLN/98I had replication and pathogenicity abilities in vivo similar to those of other IBVs; however, its growth ability in embryos was lower, although the relationship between the lower growth ability and the ORF 3a defect requires further confirmation.

Characterization of a defective RNA derived from RNA 3 of the Y strain of cucumber mosaic virus

A defective (D) RNA 3 naturally generated from the Y strain of cucumber mosaic virus (CMV-Y) was characterized using a biologically active cDNA clone. Sequencing of the clone revealed that the D RNA 3, named D RNA 3Yalpha, was derived from CMV-Y RNA 3 and contained a 10 nt deletion in the 5' untranslated region and a 162 nt deletion within the 3a open reading frame. Co-inoculation of D RNA 3Yalpha with CMV-Y derived from in vitro transcripts facilitated propagation of CMV-Y containing D RNA 3Yalpha in Nicotiana benthamiana or Nicotiana tabacum plants. CMV-Y, however, did not produce deletion mutants upon serial mechanical passages in the plants.