Complete Nucleotide Sequence of a Novel Hibiscus-Infecting Cilevirus from Florida and Its Relationship with Closely Associated Cileviruses.

The complete nucleotide sequence of a recently discovered Florida (FL) isolate of hibiscus-infecting cilevirus (HiCV) was determined by Sanger sequencing. The movement and coat protein gene sequences of the HiCV-FL isolate are more divergent than other genes of the previously sequenced HiCV-HI (Hawaii) isolate.

GENOME ANNOUNCEMENT

Hibiscus-infecting cilevirus (HiCV) was first described from Hawaii in 2013 (1). Other than Hibiscus, no other plant genus has been identified as a natural or experimental host for HiCV. Based on sequence identity and phylogenetic relationships, HiCV is most closely related to citrus leprosis virus cytoplasmic type 2 (CiLV-C2) (2, 3). Leprosis-like symptoms in citrus were first reported from Florida in 1911 and disappeared in the mid-1960s (4, 5). This previous outbreak was shown to be due to the nuclear rather than the cytoplasmic type of leprosis (6). HiCV was only recently characterized, and no serological or molecular diagnostic tests are available (1). Although Brevipalpus spp. are considered potential vectors of the genus Cilevirus, transmission of HiCV with Brevipalpus spp. has not yet been demonstrated (1, 7).

Hibiscus rosa-sinensis leaves with typical green ringspot symptoms on senescent leaves were collected in Tampa, FL, USA, in August 2016. Total RNA from HiCV symptomatic and asymptomatic hibiscus leaves was isolated using the RNeasy Plant minikit (Qiagen, Valencia, CA, USA). HiCV-HI (Hawaii) and CiLV-C2 sequences were aligned to design specific and generic primers for reverse transcription (RT)-PCR assay. RT-PCR with CiLV-HI-specific primers produced amplicons of HiCV-HI cilevirus using total RNA as the template. Overlapping RT-PCR amplicons were cloned, and the complete viral genomes were assembled using Sanger sequencing (8). In this study, we report the first complete genomic sequence of an HiCV isolate from Florida.

The complete genome of the HiCV-FL isolate is 13,718 nucleotides (nt) in length. The genome is composed of RNA1 and RNA2 of 8,727 and 4,991 nt, respectively, including the poly(A) tails at the 3′ termini. RNA1 of HiCV-FL has two open reading frames (ORFs). ORF1 of RNA1 encodes a large polyprotein (p285) with methyl-transferase, protease, helicase, and RNA-dependent RNA polymerase domains that share 95 to 98% amino acid (aa) sequence identity with CiLV-C2 (GenBank accession number JX000024) and HiCV-HI (GenBank accession number KC626783), respectively. ORF2 encodes a putative coat protein gene (p29) that is distinct and shares only 92 to 93% aa sequence identity with HiCV-HI and CiLV-C2. RNA2 is organized into five ORFs that encode four hypothetical proteins and one movement protein (MP) in the order of 5′-p15-p9-p61-MP-p24-3′. ORF2 of RNA2 is 243 nt in length, longer than the lengths observed in the HiCV-HI (195 nt) and CiLV-C2 (204 nt) genome sequences. Three other hypothetical proteins of HiCV-FL RNA2, p15, p61, and p24, share 94 to 99% nt and 98 to 100% aa sequence identities with HiCV-HI. Most of the sequence divergence between HiCV-FL RNA2 and HiCV-HI is in the MP region. Interestingly, although HiCV-FL MP shares only 87% nt identity, it has 97% aa sequence identity with CiLV-C2 (GenBank accession number JX000025), followed by 91% nt and 94% aa sequence identities with HiCV-HI (GenBank accession number KC626784). Overall, the ORFs of HiCV-FL RNA1 and RNA2 have 97% and 95% nt sequence identities, respectively, with the HiCV-HI sequences. Further research is needed to determine if HiCV is capable of infecting citrus and producing any symptoms.

Accession number(s).

The genome sequences of the HiCV isolate from Florida have been deposited in GenBank under the accession numbers MG253805 (RNA1) and MG253804 (RNA2).