Development of an infectious clone and replicon system of norovirus GII.4.

Human norovirus (HuNoV) is one of the main causes of acute gastroenteritis worldwide and is responsible for at least 20% of all cases. The detailed molecular mechanism of this norovirus remains unknown due to the lack of a suitable in vitro culturing system. An infectious clone of HuNoV would be a useful tool for elucidating the processes of viral infection and the mechanisms of replication. We developed an infectious cDNA clone of HuNoV using the rapid technique of Gibson Assembly. The complete genome of the HuNoV GII.4 Sydney subtype was cloned into a previously modified pcDNA3.1-based plasmid vector downstream from a cytomegaloviral promoter. We monitored the viral infection in vitro by inserting the reporter gene of the green fluorescent protein (GFP) between the NTPase and p22 genes, also by Gibson Assembly, to construct a HuNoV-GFP replicon. Human Caco-2 cells were transfected with the full-length genomic clone and the replicon containing GFP. The gene encoding the VP1/VP2 capsid protein was expressed, which was indirect evidence of the synthesis of subgenomic RNAs and thus the negative strand of the genome. We successfully constructed the infectious clone and its replicon containing GFP for the HuNoV GII.4 Sydney subtype, a valuable tool that will help the study of noroviral infection and replication.

The First Report of Tomato chlorotic spot virus (TCSV) Infecting Long Beans and Chili Peppers in the Dominican Republic.

The Dominican Republic has a significant area of the country cultivated with vegetables. In July 2013, in the provinces of Moca and La Vega, horticultural crops showed typical tospovirus symptoms (>30% incidence), including bronzing, chlorosis, necrosis, and ring spots on leaves and fruits. Samples were collected from potatoes (Solanum tuberosum), long beans (Vignaun guiculata), chili peppers (Capsicum frutescens), sweet peppers (C. annuum), and tomatoes (S. lycopersicum). Serological tests were clearly positive for infection by Tomato spotted wilt virus (TSWV) and/or related tospoviruses when tested with AgDia immunostrips. The viral RNA extracted from five plants per host was pooled to construct a cDNA library that was sequenced using an Illumina HiSeq 2000 platform. The paired-end reads were assembled using CLC Genomic Workbench version 6.0.3. The assembled contigs were submitted to BLASTx against a viral genome database. The results confirmed the presence of Tomato chlorotic spot virus (TCSV) and TSWV. Then, PCR tests were performed with primers pairs TSWV-LF 5' CTGTTGTCTATTGAGGATTGTG 3' AND TSWV-LR 5' CAGAGAGCTTGTTAATGCAGGAC 3' to amplify part of the TSWV L RNA, the pairs TCSV-SF 5' AACTGGGAAAGCAGAAAACC 3' and TCSV-SR 5' CCTTACTCCGAACATTGCA 3', and GRSV-SF 5' CTGTCAGGAAAATCTTGACCTG 3' and GRSV-SR 5' CTTGACTCCAAACATCTCGT 3' to detect part of the TCSV and Groundnut ringspot virus (GRSV) S segments. In the long bean and chili pepper samples from La Vega, only TCSV was detected (40% of the all samples) based on amplification of the expected size fragment with the S RNA specific primer pair. All the other samples were positive for TSWV and no GRSV was detected. The complete N gene of TCSV and TSWV were amplified using the primer pairs TCSV-NR2 5' CACACTGAACTGAACTATAACACAC 3' and TCSV-NF 5' ACCTTGAATCATATCTCTCG 3' and primers N-TSWV_FW 5' TACGGATCCGATGTCTAAGGTTAAGCTCAC 3' and N-TSWV_RV 5' TTATCTCGAGTCAAGCAAGTTCTGCGAG 3'. The TCSV N protein sequences (KJ399303 and KJ399304) were 99% identical with the TCSV found in processing tomatoes in the Dominican Republic (1) and the United States (2). The TSWV N protein sequences (KJ399313, KJ399314 and KJ399315) shared 96 to 98% identity with the TSWV N sequences available. Dot blot hybridization tests (1) using DIG-labeled specific TCSV N gene probe confirmed TCSV infection in PCR-positive long bean and chili pepper samples, whereas no hybridization signal was detected for TSWV-infected tomatoes, potatoes, sweet peppers, or healthy samples. In addition, no reassortants were detected based on amplification of the expected size RNA fragments (3). These other amplicons (KJ399301, KJ399299, KJ399302, and KJ399300) showed 98% identity with the L and M segments of TCSV. Thrips collected from symptomatic plants were identified mainly as Frankliniella schultzei, consistent with the main thrips species transmitting TCSV. In the last two years, TCSV was reported in North and Central America and in the Caribbean Basin (1,2,4). These findings have an important epidemiological impact since TCSV represents a new threat to other horticultural crops affected by this tospovirus. References: (1) O. Batuman et al. Plant Dis. 98:286, 2014. (2) A. Londono et al. Trop. Plant Pathol. 37:333, 2012. (3) C. G. Webster et al. Virology 413:216, 2011. (4) C. G. Webster et al. Plant Health Progress. Online publication. doi:10.1094/PHP-2013-0812-01-BR, 2013.

Characterization of a member of a new Potyvirus species infecting arracacha in Brazil.

An arracacha (Arracacia xanthorrhiza) plant collected in Brazil was found to be infected by a new virus. This viral isolate (named C17) systemically infected Nicotiana benthamiana and Apium graveolens. A polyclonal antibody was raised, and analysis of our arracacha germplasm collection showed a high infection rate of C17-like viruses (93% infection). Sequencing of the ca. 1.7 kb 3'-terminal genomic region revealed a typical potyvirus genome organization. It shared less than 70% nucleotide identity with any other potyvirus sequence, which thus indicated that it is possibly a member of a new Potyvirus species tentatively named Arracacha mottle virus (AMoV).