De novo genome assembly of the partial homozygous dihaploid potato identified PVY resistance gene (Rychc) derived from Solanum chacoense.

The isolation of disease resistance genes introduced from wild or related cultivated species is essential for understanding their mechanisms, spectrum and risk of breakdown. To identify target genes not included in reference genomes, genomic sequences with the target locus must be reconstructed. However, de novo assembly approaches of the entire genome, such as those used for constructing reference genomes, are complicated in higher plants. Moreover, in the autotetraploid potato, the heterozygous regions and repetitive structures located around disease resistance gene clusters fragment the genomes into short contigs, making it challenging to identify resistance genes. In this study, we report that a de novo assembly approach of a target gene-specific homozygous dihaploid developed through haploid induction was suitable for gene isolation in potatoes using the potato virus Y resistance gene Rychc as a model. The assembled contig containing Rychc-linked markers was 3.3 Mb in length and could be joined with gene location information from the fine mapping analysis. Rychc was successfully identified in a repeated island located on the distal end of the long arm of chromosome 9 as a Toll/interleukin-1 receptor-nucleotide-binding site-leucine rich repeat (TIR-NBS-LRR) type resistance gene. This approach will be practical for other gene isolation projects in potatoes.

First report of barley virus G infecting wheat (Triticum aestivum) in Japan.

Barley virus G (BVG) is a single stranded, positive sense RNA virus and tentative species of the genus Polerovirus in the family Solemoviridae. BVG was first identified in barley (Hordeum vulgare) in Korea with symptoms that were similar to those of barley yellow dwarf disease (Zhao et al. 2016). It has also been identified from proso millet (Park et al. 2017), barley (Erickson and Falk, 2021; Nancarrow et al. 2019; Svanella-Dumas et al. 2022), maize (Gavrili et al. 2021), wheat (Nancarrow et al. 2019), and oats (Nancarrow et al. 2019) in various countries. In the spring of 2019, wheat (Triticum aestivum) plants that exhibited symptoms of yellow leaves, necrosis, and stunting were observed in a few fields of the Chugoku region (western main island) in Japan. The four soil-borne viruses, wheat yellow mosaic virus (WYMV), Chinese yellow mosaic virus (CWMV), Japanese soil-borne wheat mosaic virus (JSBWMV), and soil-borne wheat mosaic virus (SBWMV), that often occur in winter wheat in Japan, were not detected by DAS-ELISA for WYMV, CWMV, and JSBWMV (Netsu et al. 2011) and ELISA Reagent Set for SBWMV (Agdia, IN, USA). To identify the pathogen, total RNA was isolated from the leaves and petioles using the PureLink RNA Mini kit (Thermo Fisher Scientific, MA, USA), and RT-PCR was performed using the PrimeScript One Step RT-PCR Kit Ver.2 (Dye plus) (Takara Bio Inc, Shiga, Japan). Based on the symptoms, luteoviruses and poleroviruses, which are transmitted by aphids, were suspected and RT-PCR was performed using known primers (Malmstrom and Shu, 2004; Mustafayev et al. 2013). This resulted in an amplicon of approximately 300 bp being obtained from RT-PCR using the Luteo2F/YanR-new primers (Mustafayev et al. 2013). The amplicon was directly sequenced by Sanger sequencing and a nucleotide BLAST search of the database revealed that the sequence was highly similar (99% identity, query coverage 95%) to the genome of BVG. In samples of a single field, four out of six plants that were showing necrosis and stunting were positive by RT-PCR using the primers BVG-CP-F (5´- GCGGGAAACATTTGTATTTTCG-3´)/BVG-CP-R (5´- GATTTTGGGTTAGAACATCCATCG-3´). In addition, five out of six plants with some yellowing of the leaves in the same field were also positive. Other luteoviruses and poleroviruses were not detected by RT-PCR using known primers. The full-length genome sequence of the Chugoku isolate was amplified using the primers BVG-F (5´-ACAAAAGGGACCCAGAGGG-3´)/BVG-R (5´-TACCAAGGATACTAGAGAGAGA-3´), which were designed from the 5´ and 3´ end sequences of the known BVG sequence. The resultant amplicon was directly sequenced by Sanger sequencing and the sequence was deposited into the DNA Data Bank of Japan (Chugoku isolate, LC649801). The sequence comprised of 5,620 bp and the genomic structures were consistent with that of BVG. The sequence showed more than 97% nucleotide identity with the BVG Gimji (KT962089), Uiseong (LC259081), NL1 (MF960779), and California (LC259081) isolates by pairwise comparisons. This is the first report of BVG in wheat in Japan to the best of our knowledge. The correlation between BVG and the observed symptoms and the impact of BVG on wheat production in Japan requires further investigation. Reference Erickson, A. C. and Falk, B. 2021. Plant Dis. doi:10.1094/PDIS-03-21-0478-PDN Gavrili, V. et al. 2021. J. Plant Pathol. 103:1331. doi:10.1007/s42161-021-00903-4 Malmstrom, C. M. and Shu, R. 2004. J. Virol. Methods. 120:69. doi:10.1016/j.jviromet.2004.04.005 Mustafayev, E. S. et al. 2013. Plant Dis. 97:849. doi:10.1094/PDIS-07-12-0656-PDN Nancarrow, N. et al. 2019. Plant Dis. 103:1799. doi: 10.1094/PDIS-01-19-0166-PDN Netsu, O. et al. 2011. Plant Dis. 58:13. doi.org/10.11337/ktpps.2011.13 Park, C.Y. et al. 2017. Plant Dis. 101:393. doi: 10.1094/PDIS-07-16-0952-PDN Svanella-Dumas, L. et al. 2022. Plant Dis. doi: 10.1094/PDIS-06-22-1294-PDN Zhao, F. et al. 2016. Arch. Virol. 161: 2047. doi:10.1007/s00705-016-2881-0.

One-step real-time multiplex reverse transcription-polymerase chain reaction assay with melt curve analysis for detection of potato leafroll virus, potato virus S, potato virus X, and potato virus Y.

BACKGROUND: Certification of seed potato as free of viruses is essential for stable potato production. Among more than 30 virus species infecting potato, potato leafroll virus (PLRV), potato virus S (PVS), potato virus X (PVX), and potato virus Y (PVY) predominate worldwide and should be the targets of a high-throughput detection protocol for seed potato quarantine. RESULTS: We developed an assay based on one-step real-time multiplex reverse transcription-polymerase chain reaction (mRT-PCR) with melt curve analysis for the four viruses and one internal control, potato elongation factor 1 alpha gene (EF1α). Virus-specific primers were derived from conserved regions among randomly selected representatives considering viral genomic diversity. Our assay simultaneously detected representative Japanese isolates of PLRV, O lineage of PVS, PVX, and NTN strain of PVY. The variability of melting temperature (Tm) values for each virus was confirmed using Japanese isolates, and virus species could be identified by the values of 87.6 for PLRV, 85.9 for PVX, 82.2 (Ordinary lineage) to 83.1 (Andean lineage) for PVS, and 79.4 (NA-N strain) to 80.5 (O strain and NTN strain) for PVY on average. The reliability of calculation was validated by comparing the calculated Tm values and measured Tm values and the values had a strong linear correlation (correlation of determination: R2 = 0.9875). Based on the calculated Tm values, representative non-Japanese isolates could also be identified by our assay. For removing false positives, two criteria were set for the evaluation of result; successful amplification was considered as 30.0 ≥ threshold cycle value, and the virus-specific peak higher than the EF1α-specific peak was considered as positive. According to these criteria, our assay could detect PLRV and PVS from 100-fold dilution of potato leaf homogenate and PVX and PVY from 1000-fold in a model assay. CONCLUSION: This new high-throughput detection protocol using one-step real-time mRT-PCR was sensitive enough to detect viruses in a 100-fold dilution of singly-virus contaminated homogenate in a model assay. This protocol can detect the four viruses in one assay and yield faster results for a vast number of samples, and greatly save the labor for seed potato quarantine and field surveys.

Complete nucleotide sequence of soybean leaf rugose mosaic virus, an atypical member of the genus Bymovirus.

The host range of previously reported bymoviruses is restricted to plants belonging to the family Poaceae. Soybean leaf rugose mosaic virus (SbLRMV) from non-Poaceae plants is related to bymoviruses based on a partial genome sequence. However, unlike bymoviruses, this virus infects plants of at least four dicotyledonous families, including Fabaceae, and causes disease in soybean. Complete nucleotide sequences of two variants of SbLRMV were determined, and its taxonomic position was clarified. RNA1 is 7109 nucleotides (nt) long with one large open reading frame (ORF), possibly encoding a polyprotein of 257 kDa. This polyprotein is likely processed into eight mature proteins. The entire RNA1 ORF shares 52%-55% nucleotide sequence identity and 27%-43% amino acid sequence identity, and the coat protein shares 49%-54% nucleotide sequence identity and 30%-34% amino acid sequence identity to other bymoviruses. The similarity to other viruses in the family Potyviridae is generally lower. RNA2 is 3413 or 3415 nt long and putatively encodes a polyprotein of 108 kDa. This protein is probably cleaved into two mature proteins. The sequences of these two RNAs are very similar to those of bymoviruses. Phylogenetic analysis of members of the family Potyviridae showed that RNA1 and RNA2 of SbLRMV formed a basal clade with known bymoviruses. Inoculation tests using leaf samples suggested that SbLRMV RNA1 can systemically infect and cause disease in soybean without the presence of RNA2. In conclusion, SbLRMV is an atypical member of the genus Bymovirus that infects soybean (Fabaceae) and other dicots rather than gramineous hosts.

First report of soft rot of cabbage caused by Pectobacterium wasabiae in Japan.

Cabbage (Brassica oleracea var. capitata) is one of the important vegetables in Japan. In the summer of 2019, some cabbages with soft rot were found in commercial fields in Hokkaido, the northern island in Japan. All diseased plants showed grey to brown discoloration and expanding water-soaked lesions on leaves. We obtained two independent strains (NACAB191 and NACAB192) from diseased leaves. DNA from these strains yielded an expected single size amplicon with the primer set of PhF/PhR for P. wasabiae (De Boer et al. 2012) by PCR, but did not yield the expected amplicon with the primer set of BR1f/L1r for P. carotovorum subsp. brasiliense (Duarte et al. 2004) and Eca1f/Eca2r for P. atrosepticum (De Boer et al., 1995) by PCR. These two strains grew at 37°C, and their ability to utilize raffinose and lactose. These bacterial strains were gram-negative and rod-shaped. The bacterium was positive for O-nitrophenyl-beta-D-galactopyranoside, N-acetylglucosaminyl transferase, gelatin liquefaction, and acid production from D-galactose, lactose, melibiose, raffinose, citrate, and trehalose. The bacterium was negative for indole production and acid production from maltose, α-methyl-D-glucoside, sorbitol, D-arabitol, inositol, inulin, and melezitose. All strains exhibited pectolytic activity on potato slices. The sequence analysis of 16S rDNA (LC597897 and LC597898) showed more than 98% identities to P. wasabiae strain (e.g. HAFL01 in Switzerland) by BLAST analysis. In addition, Multi-locus sequence analysis (Ma et al. 2007) was performed by MEGA10 (Kumer et al. 2018) using concatenated DNA sequences of seven housekeeping genes (aconitate hydratase(acnA, LC597923 and LC597924), glyceraldehyde-3-phosphate dehydrogenase A(gapA, LC597970 and LC597971), isocitrate dehydrogenase (icdA, LC597996 and LC597997), malate dehydrogenase(mdh, LC598022 and LC598023), mannitol-1-phosphate dehydrogenase (mtlD, LC598048 and LC598049), glucose-6-phosphate isomerase (pgi, LC598074 and LC598075) and gamma-glutamyl phospate reductase (proA, LC598079 and LC598080)), and all clustered NACAB191 and NACAB192 into a clade containing other confirmed strains of P. wasabiae. As a result, these two strains shared high identity with each other (>98%, E-Values showed 0). The clade containing these two strains was consistently placed in a larger clade with the other P. wasabiae and 100% bootstrap support for its separation from other Pectobacterium species available in GenBank when the consensus tree constructed using Maximum Likelihood method. Pathogenicity of these strains against cabbage (cv. 'Rakuen') was confirmed by the field experiments with five weeks growth plants sprayed with bacterial suspension (1×107cfu/ml). Thirty cabbages per strain were used in this study, 12 plants treated the suspension of NACAB191 and 16 plants treated the suspension of NACAB192 which died with the same soft rot symptoms about four weeks after inoculation. Whereas water-inoculated plants remained symptomless. Strains re-isolated from the artificially diseased stems were confirmed as P. wasabiae using the methods as biochemical characterization and multiple genetic analyses. Based on the disease symptoms, the cultural, molecular, and pathological features of the strains, we conclude that the soft rot symptoms of cabbage in Hokkaido in 2019 were caused by P. wasabiae. To our knowledge, this is the first report of P. wasabiae as the soft rot disease agent of cabbage in Japan.

First report of soft rot of onion caused by Pectobacterium wasabiae in Japan.

Onion (Allium cepa L.) is one of the important vegetables in Japan. In the summer of 2019, onions with soft rot were found in commercial fields in Hokkaido, the northern island in Japan. Diseased onion showed chlorosis, maceration of leaves, and rotted bulbs. We sampled some diseased onions and isolated three independent isolations (NAONI191, NAONI192 and NAONI193) from infected bulbs on LB medium. These strains were identified as Pectobacterium wasabiae based on their inability to grow at 37°C, and their ability to utilize raffinose and lactose. These bacterial strains were gram-negative, rod-shaped, N-acetylglucosaminyl transferase, gelatin liquefaction. The bacterium was positive for O-nitrophenyl-beta-D-galactopyranoside, N-acetylglucosaminyl transferase, gelatin liquefaction, and acid production from D-galactose, lactose, melibiose, raffinose, citrate, and trehalose. The bacterium was negative for indole production and acid production from maltose, α-methyl-D-glucoside, sorbitol, D-arabitol, inositol, inulin, and melezitose. All the strains exhibited pectolytic activity on potato slices. DNA from these strains yielded a single size amplicon with the primer set of PhF/PhR for P. wasabiae (De Boer et al. 2012) by PCR. However, DNA from these strains did not yield the expected amplicon with the primer set of BR1f/L1r for P. carotovorum subsp. brasiliense (Duarte et al. 2004) and Eca1f/Eca2r for P. atrosepticum (De Boer et al., 1995) by PCR. The sequence analysis of 16S rDNA (LC597917- LC597919) showed more than 98% identities to P. wasabiae strains (e.g. HAFL01 in Switzerland) by BLAST analysis. In addition, Multi-locus sequence analysis (Ma et al. 2007) was performed by MEGA6.06 using concatenated DNA sequences of seven housekeeping genes (aconitate hydratase(acnA, LC597925- LC597927), glyceraldehyde-3-phosphate dehydrogenase A(gapA, LC597972-LC597974), isocitrate dehydrogenase (icdA, LC597998- LC597998LC598000), malate dehydrogenase(mdh, LC598024- LC598026), mannitol-1-phosphate dehydrogenase (mtlD, LC598050- LC598052), glucose-6-phosphate isomerase (pgi, LC598076- LC598078) and gamma-glutamyl phospate reductase (proA, LC598099- LC598101)), and all clustered into a clade containing other confirmed strains of P. wasabiae. As a result, these three strains shared high identity with each other (>98%, E-Values showed 0). The clade containing these three strains was consistently placed in a larger clade with the other P. wasabiae and 100% bootstrap support for its separation from other Pectobacterium species available in GenBank when the consensus tree constructed using Maximum Likelihood method. Pathogenicity of these strains against onion (cv. 'Hayate') was confirmed by the field experiments with 5 weeks growth plants sprayed with bacterial suspension (1×107cfu/ml) resulting in soft rot on the plants about four weeks after inoculation, whereas water-inoculated plants remained symptomless. Strains re-isolated from the artificially diseased stems were confirmed as P. wasabiae using the methods as biochemical characterization and multiple genetic analyses. Based on the disease symptoms, the cultural, molecular, and pathological features of the strains, we conclude that the soft rot symptoms of onion in Hokkaido in 2019 were caused by P. wasabiae. To our knowledge, this is the first report of P. wasabiae as the soft rot disease agent of onion in Japan.

Cylindrical Inclusion Protein of Wheat Yellow Mosaic Virus Is Involved in Differential Infection of Wheat Cultivars.

Wheat yellow mosaic virus (WYMV) belongs to the genus Bymovirus in the family Potyviridae and has a bipartite genome (RNA1 and RNA2). WYMV in Japan is classified into three pathotypes (I to III) based on its pathogenicity to wheat cultivars. Among these three, pathotypes I and II are discriminated by their pathogenicity to the wheat cultivar Fukuho; pathotype I infects Fukuho but pathotype II does not. In the present study, the genomic regions that are involved in such pathogenicity were examined using infectious viral cDNA clones of pathotypes I and II. Reassortant experiments between viral RNA1 and RNA2 revealed the presence of a viral factor related to pathogenicity in RNA1. A chimeric pathotype II virus harboring a cylindrical inclusion (CI) cistron from pathotype I facilitated systemic infection of Fukuho, indicating that CI protein is involved in pathogenicity. Furthermore, analysis of chimeric and site-directed mutants revealed that three amino acids at the N-terminal region of CI protein were involved in pathogenicity to Fukuho. On the other hand, at the single-cell level, pathotype II replicated in protoplasts of Fukuho similar to that of pathotype I virus. These data suggest that differential pathogenicity between pathotypes I and II was considered to depend on the ability of cell-to-cell or long-distance viral movement, in which CI protein is involved. To the best of our knowledge, this is the first report to show the involvement of the bymoviral CI protein in pathogenicity.

Species Composition of Alate Aphids (Hemiptera: Aphididae) Harboring Potato Virus Y and the Harbored Virus Strains in Hokkaido, Northern Japan.

Many studies have evaluated transmission abilities of laboratory-reared aphids for potato virus Y (PVY), but few have focused on PVY-harboring species of field-collected aphids and the strains of PVY harbored by aphids. In the present study, we collected alate aphids in yellow pan traps in potato fields with Japanese commercial cultivars in Hokkaido, northern Japan in single 24-h periods during the tuber bulking stage and examined whether individual whole aphids harbored PVY by nested RT-PCR. PVY-positive individuals were identified to species using the gene sequence for cytochrome c oxidase subunit I and, when needed, morphological data and distribution records. In addition, individual strains of PVY harbored were determined using partial sequences of coat protein. Among 1,857 aphids trapped, 195 aphids had PVY and comprised 19 species; 17 species were identified to species-group taxa. Most of the aphid species detected as PVY positive colonize weeds that are common around potato fields in Hokkaido. Five species-group taxa had not been reported previously as a vector aphid of PVY and might be new PVY-vector species. PVYNTN was most frequently detected from PVY-positive aphids as found recently in PVY-infected potatoes in commercial fields in Hokkaido. Two or three PVY strains were rarely detected from a single aphid, and no obvious difference was found in the proportion of the harbored PVY strains among positive aphid species. The first documentation of the species composition of PVY-harboring aphids and the strains of PVY harbored in East Asia should aid understanding of the epidemiology of PVY in Japan.

Biological and genetic diversity of Wheat yellow mosaic virus (Genus Bymovirus).

The biological and genetic diversity of Wheat yellow mosaic virus (WYMV) isolates in Japan was characterized. On the basis of wheat cultivar reactions, 14 WYMV isolates from various places were classified into pathotypes I, II, or III. These were distributed in central, northern, and southern areas of Japan, respectively. WYMV isolates comprised three genotypes (A, A' and B) based on amino acid differences in RNA1 and two genotypes (a and b) based on amino acid differences in RNA2. A correlation was found between the WYMV RNA1-based genotype and pathotype, suggesting that factors associated with pathogenicity map to RNA1. Genotype Aa and A'a were distributed mainly in the central to southern areas of Japan, and genotype Bb was found in northern areas of Japan, as shown by reverse-transcription polymerase chain reaction restriction fragment length polymorphism analysis. Chinese isolates YA and YZ were closely related to genotypes Bb and Aa, respectively. Wheat was introduced from China to Japan in the 4th and 5th centuries, and the two genotypes of WYMV might also have been introduced with the crop from China and later adapted to local wheat cultivars in Japan.

The protruding domain of the coat protein of Melon necrotic spot virus is involved in compatibility with and transmission by the fungal vector Olpidium bornovanus.

The Chi and W strains of Melon necrotic spot virus (MNSV) are efficiently transmitted by isolates Y1 and NW1, respectively, of the fungal vector Olpidium bornovanus. Analysis of chimeric viruses constructed by switching the coat protein (CP) gene between the two strains unveiled the involvement of the CP in the attachment of MNSV to zoospores of a compatible isolate of O. bornovanus and in the fungal transmission of the virus. Furthermore, analysis of the chimeric virus based on the Chi strain with the protruding domain of the CP from strain W suggested the involvement of the domain in compatibility with zoospore. Comparison of the three-dimensional structures between the CP of the two MNSV strains showed that many of the differences in these amino acid residues are present on the surface of the virus particles, suggesting that these affects the recognition of fungal vectors by the virus.

Change of specific T cells in an emerging neonatal infectious disease induced by a bacterial superantigen.

A new epidemic, NTED, has recently occurred in Japan. The cause of NTED is a bacterial superantigen, TSST-1. The aim of the present study was to analyze the change in Vbeta2(+) T cells reactive to TSST-1 in NTED in order to establish T-cell-targeted diagnostic criteria for NTED. Blood samples from 75 patients with clinically diagnosed NTED were collected from 13 neonatal intensive care units throughout Japan. We investigated the percentages of Vbeta2(+), Vbeta3(+) and Vbeta12(+) T cells and their CD45RO expressions in the samples using flow cytometry. In 18 of the 75 patients, we conducted multiple examinations of the T cells and monitored serial changes. The Vbeta2(+) T-cell population rapidly changed over three phases of the disease. Whereas the percentage of Vbeta2(+) T cells was widely distributed over the entire control range, CD45RO expression on Vbeta2(+) T cells in CD4(+) in all 75 patients was consistently higher than the control range. Patients cannot necessarily be diagnosed as having NTED based on expansion of Vbeta2(+) T cells alone in the early acute phase. Instead, CD45RO expression on specific Vbeta2(+) cells is a potential diagnostic marker for a rapid diagnosis of NTED. We present three diagnostic categories of NTED. Fifty patients (66.7%) were included in the category 'definitive NTED'. It is important to demonstrate an increase of Vbeta2(+) T cells in the following phase in cases of 'probable NTED' or 'possible NTED'.

Induction of necrosis via mitochondrial targeting of Melon necrotic spot virus replication protein p29 by its second transmembrane domain.

The virulence factor of Melon necrotic spot virus (MNSV), a virus that induces systemic necrotic spot disease on melon plants, was investigated. When the replication protein p29 was expressed in N. benthamiana using a Cucumber mosaic virus vector, necrotic spots appeared on the leaf tissue. Transmission electron microscopy revealed abnormal mitochondrial aggregation in these tissues. Fractionation of tissues expressing p29 and confocal imaging using GFP-tagged p29 revealed that p29 associated with the mitochondrial membrane as an integral membrane protein. Expression analysis of p29 deletion fragments and prediction of hydrophobic transmembrane domains (TMDs) in p29 showed that deletion of the second putative TMD from p29 led to deficiencies in both the mitochondrial localization and virulence of p29. Taken together, these results indicated that MNSV p29 interacts with the mitochondrial membrane and that p29 may be a virulence factor causing the observed necrosis.

Selective excretion of anti-inflammatory cytokine interleukin-10 in a superantigen-inducing neonatal infectious disease.

Neonatal toxic shock syndrome (TSS)-like exanthematous disease (NTED) is an emerging neonatal infectious disease caused by TSS toxin-1 (TSST-1). Although NTED and TSS are caused by the same superantigenic exotoxin, NTED is less severe than TSS. The mechanism of this reduced severity in NTED has not been elucidated. Thirteen patients with NTED were enrolled in the study. We investigated serum cytokine profile using a cytometric bead array system with a cytokine panel. Expression of Vbeta2 and CD45RO in CD4(+) T cells was investigated in mononuclear cells by using flowcytometry. Ten patients with other bacterial infections and eight patients without any infections were also enrolled as control groups. The mean serum level of IL-10 was 1209.9 pg/mL in patients with NTED at the time of admission into the study. The other inhibitory cytokine, IL-4, exhibited a minimum level. The high level of IL-10 rapidly decreased within 3-9 days of the onset of NTED. The cytokine profile of NTED, with its high IL-10 level, was clearly different from that of the other bacterial infections. The increased level of IL-10 seems to be related to the reduced severity of NTED. Th2 shift is not thought to be the cause of this IL-10 excretion.

A new strain of Melon necrotic spot virus that is unable to systemically infect Cucumis melo.

We report a new strain of Melon necrotic spot virus (MNSV) that is unable to systemically infect Cucumis melo. A spherical virus (W-isolate), about 30 nm in diameter like a carmovirus, was isolated from watermelons with necrotic symptoms. The W-isolate had little serological similarity to MNSV, and it did not cause any symptoms in six melon cultivars susceptible to MNSV; however, the host range of the W-isolate was limited exclusively to cucurbitaceous plants, and transmission by O. bornovanus was confirmed. Its genomic structure was identical to that of MNSV, and its p89 protein and coat protein (CP) showed 81.6 to 83.2% and 74.1 to 75.1% identity to those of MNSV, respectively. Analysis of protoplast showed that the W-isolate replicated in melons at the single-cell level. Furthermore, chimeric clones carrying the CP of MNSV induced necrotic spots in melons. These results suggested that the absence of symptoms in melons was due to a lack of ability of the W-isolate to move from cell to cell. In view of these findings, we propose that the new isolate should be classified as a novel MNSV watermelon strain.

Pathogenicity of Pepper mild mottle virus Is Controlled by the RNA Silencing Suppression Activity of Its Replication Protein but Not the Viral Accumulation.

ABSTRACT Pepper mild mottle virus (PMMoV) infects pepper plants, causing mosaic symptoms on the upper developing leaves. We investigated the relationship between a virus pathogenicity determinant domain and the appearance of mosaic symptoms. Genetically modified PMMoV mutants were constructed, which had a base substitution in the 130K replication protein gene causing an amino acid change or a truncation of the 3' terminal pseudoknot structure. Only one substitution mutant (at amino acid residue 349) failed to cause symptoms, although its accumulation was relatively high. Conversely, the pseudoknot mutants showed the lower accumulation, but they still caused mosaic symptoms as severe as the wild-type virus. Therefore, the level of virus accumulation in a plant does not necessarily correlate with the development of mosaic symptoms. The activity to suppress posttranscriptional gene silencing (PTGS) was impaired in the asymptomatic mutant. Consequently, pathogenicity causing mosaic symptoms should be controlled by combat between host PTGS and its suppression by the 130K replication protein rather than virus accumulation.

Hemodynamic effect of the ventricular pacing site in fetal lambs with complete atrioventricular block.

Complete atrioventricular (AV) block in hydrops fetalis is associated with high mortality. Fetal ventricular pacing to restore ventricular rate can be an effective procedure, however, no fetal data has shown an appropriate epicardial ventricular pacing site. To evaluate the hemodynamic effect of right and left ventricular pacing in fetal lambs with complete AV block, a fetal complete AV block model was created. Aortic pressure, central venous pressure, and QRS duration were measured, and right and left ventricular output was estimated in seven fetal lambs. The uterus was opened under maternal anesthesia, and under local anesthesia, catheters were inserted into the fetal superior vena cava and ascending aorta through a neck incision. Pacing leads were then sutured onto the fetal right and left ventricular epicardium via a midline thoracotomy. Complete AV block was created by cryoablation of the AV node. Ventricular output was estimated using echocardiography by a transuterine approach. Fetal hemodynamics were observed before AV block creation (control), and after complete AV block creation with the right and left ventricular pacing set at 150/min. The right ventricular output was 320 +/- 66 mL/kg per minute at control, decreased to 243 +/- 65 mL/kg per minute during right ventricular pacing (P < 0.05), and was 254 +/- 61 mL/kg per minute during left ventricular pacing. The left ventricular output was 224 +/- 98 mL/kg per minute at control, 176 +/- 77 mL/kg minute during right ventricular pacing, and 178 +/- 67 mL/kg per minute during left ventricular pacing. Biventricular (combined ventricular) output was 544 +/- 134 mL/kg per minute at control, 419 +/- 114 mL/kg per minute during right ventricular pacing, and 432 +/- 100 mL/kg minute during left ventricular pacing. Systolic aortic pressure was 62.2 +/- 8.7 mmHg at control, 55.2 +/- 9.5 mmHg during right ventricular pacing, and 53.4 +/- 9.1 mmHg during left ventricular pacing. Central venous pressure (CVP) was 2.6 +/- 0.5 mmHg at control, 4.0 +/- 2.7 mmHg during right ventricular pacing, and 4.4 +/- 2.5 mmHg during left ventricular pacing. The QRS duration was 51 +/- 54 ms at control, but lengthened to 87 +/- 19 ms during right ventricular pacing and to 78 +/- 21 ms during left ventricular pacing (P < 0.05). In conclusion, the right ventricular output decreased during right ventricular pacing in fetal lambs with complete AV block, while it was preserved during the left ventricular pacing. Left ventricular pacing might be superior for treating hydropic fetuses with complete AV block.