First identification and molecular characterization of a novel cherry robigovirus Abstract A large contig with sequence similarities to members of the genus Robigovirus was identified by high-throughput sequencing analysis from a symptomless cherry accession. The complete genome sequence of this new virus is 8,384 nucleotides in length, excluding the 3’ poly(A) tail. Its genome organization is very similar to those of four known robigoviruses, encoding a putative replicase, three ‘triple gene block’ proteins, a coat protein, and an unknown protein, 2a. Unlike the four cherry robigoviruses, the new virus does not contain a putative ORF5a. The full-length genome of the virus, which is provisionally named “cherry robigovirus 5” (CRV-5), is 52-57% identical to genome sequences of other robigoviruses. Phylogenetic analysis showed that CRV-5 and other robigoviruses group in a cluster, supporting its assignment to a new species in the genus Robigovirus.

Apple chlorotic fruit spot viroid: a putative new pathogenic viroid on apple characterized by next-generation sequencing Abstract Viroid-like symptoms were observed in 2016 on apple fruits of the cultivar “Ilzer Rose” in southern Burgenland, Austria. Preliminary molecular biological investigations indicated that the symptoms were caused by a new unknown viroid. Therefore, new primers were designed, and the whole genome sequence of the viroid (354 nt) was determined by next-generation amplicon sequencing using the Illumina MiSeq® platform (San Diego, California, USA). The viroid secondary structure has a rod-like conformation and contains conserved regions (the TCR, CCR upper strand, and CCR lower strand) that are characteristic of members of the genus Apscaviroid. Based on our results and the demarcation criteria for viroids, the tentatively named “apple chlorotic fruit spot viroid” should be considered a putative new member of the genus Apscaviroid.

Complete genome sequence of a novel partitivirus from the entomogenous fungus Beauveria bassiana in China Abstract In this study, we report a novel double-stranded RNA (dsRNA) virus, Beauveria bassiana partitivirus 3 (BbPV-3), derived from the entomogenous fungus Beauveria bassiana isolate RCEF5853 from China. The genome of BbPV-3, whose sequence was determined by metagenomic sequencing, RT-PCR, and RACE cloning, comprises two dsRNA genome segments that are 1,856 and 1,719 bp long. The first segment contains a single ORF (ORF-1) encoding a 584-amino-acid-long protein (66.05 kDa) with a conserved RNA-dependent RNA polymerase (RdRp) motif. The second segment also has a single ORF (ORF-2) encoding a 500-amino-acid-long coat protein (CP) (55.9 kDa). The CP and RdRp sequences showed highest identity of 43.4% and 60.2%, respectively, to those of Colletotrichum eremochloae partitivirus 1. Phylogenetic analysis of the RdRp domain of the polyprotein revealed that BbPV-3 grouped together with the members of the genus Epsilonpartitivirus. Hence, we proposed that Beauveria bassiana partitivirus 3 is a novel member of the proposed genus Epsilonpartitivirus.

Highly divergent cattle hepacivirus N in Southern Brazil Abstract The genus Hepacivirus includes 14 species (Hepacivirus A–N). In this study, we determined a partial genome sequence of a highly divergent bovine hepacivirus (hepacivirus N, HNV) isolate from cattle in Southern Brazil. Previously described HNV isolates have shared 80–99.7% nucleotide sequence identity in the NS3 coding region. However, the sequence determined in this study had 72.6% to 73.8% nucleotide sequence identity to known HNV NS3 sequences. This high divergence could be seen in a phylogenetic tree, suggesting that it represents a new genotype of HNV. These data expand our knowledge concerning the genetic variability and evolution of hepaciviruses.

Complete genome sequence of the novel phage vB_EcoS_PHB17, which infects Shiga-toxin-producing Escherichia coli Abstract The complete genome of the novel phage vB_EcoS_PHB17, which infects Shiga-toxin-producing Escherichia coli, was sequenced, revealing a linear double-stranded DNA genome of 48,939 bp with 46% GC content and protruding 150-bp 5ʹ cohesive termini. The genome contained 85 open reading frames, 28 of which were annotated with known functions. No tRNA-encoding genes were detected. Phylogenetic analysis suggested that phage PHB17 is a novel phage of family Siphoviridae.

Molecular characterization of a novel potyvirus infecting noni Abstract The complete genomic sequence of a novel potyvirus from a noni plant in China (Morinda citrifolia) with foliar mosaic and chlorotic symptoms was determined. The genomic RNA consists of 9645 nucleotides (nt) excluding the poly(A) tail, containing the typical open reading frame (ORF) of potyviruses and encoding a large putative polyprotein of 3077 amino acids (aa). Pairwise comparisons showed that the virus shares 48.8%–58.5% sequence identity at the genome sequence level, and 38.5%–53.4% identity at the polyprotein sequence level with other members of the genus Potyvirus. Phylogenetic analysis indicated that the virus is most closely related to jasmine virus T and plum pox virus in the genus Potyvirus. These results suggest that this virus should be considered a distinct member of the genus Potyvirus, and it was tentatively named “noni mosaic virus” (NoMV).

The complete genome sequence of the lumpy skin disease virus vaccine Herbivac LS reveals a mutation in the superoxide dismutase gene homolog Abstract The lumpy skin disease virus (LSDV) vaccine, Herbivac LS, batch 008, was sequenced and found to differ from the Neethling vaccine strain in the locus encoding a superoxide dismutase (SOD) homolog. The presence of a SOD homolog, be it full-length (as in Herbivac LS) or truncated (as in Neethling) may affect vaccine immunogenicity.

Identification and genomic characterization of grapevine Kizil Sapak virus, a novel grapevine-infecting member of the family Betaflexiviridae Abstract A novel virus with a (+) single-stranded RNA genome was detected by high-throughput sequencing (HTS) in a sample of grapevine (Vitis vinifera) cv. Kizil Sapak (sample/isolate 127) that originated from Turkmenistan. The complete genome of the virus, tentatively named “grapevine Kizil Sapak virus” (GKSV), is 7,604 nucleotides in length, excluding the poly(A) tail. The genome organization of GKSV, encoded genes, and sequence domains are typical for members of the family Betaflexiviridae, specifically those belonging to the subfamily Trivirinae. Phylogenetic analysis placed GKSV within the subfamily Trivirinae, in the same clade as fig latent virus 1 (FLV-1) but distinct from the clades formed by members of other genera. A comparative analysis of GKSV-127 with the HTS-derived sequences obtained from two additional isolates showed that they are genetic variants of the same virus species. Based on current ICTV species and genus demarcation criteria, and the results of the sequence and phylogenetic analyses, we propose that GKSV and FLV-1 represent a new genus within the subfamily Trivirinae.

Circulation of multiple subtypes (A, G and CRFs 02_AG) of human immunodeficiency virus type 1 (HIV-1) in selected districts of Punjab province, Pakistan Abstract Owing to consistent genetic mutation and recombination, various escape mutants and/or drug-resistant mutants of human immunodeficiency virus (HIV-1) are now emerging worldwide. Therefore, an understanding of the genetic characteristics of prevailing strains, particularly with regard to drug-resistance-associated substitutions, is essential for devising and implementing treatments and disease control interventions in endemic settings such as Pakistan. We processed a total of 130 plasma samples originating from HIV-treatment centers in selected districts of Punjab province, Pakistan. The samples were first screened using an HIV-1 Ag/Ab Combo test followed by amplification of the pol gene (1084 bp) from samples that were positive either for the antigen or for both the antigen and antibodies simultaneously. Screening revealed that a total of 45 samples were positive (34.62%; 95% CI: 26.99-43.13) for either antigen or both antigen and antibodies (n = 18, 40%; 95% CI: 27.02-54.55) or for antibodies alone (n = 27, 60%; 95% CI: 45.45-72.98). A largest number of positive samples was from the district of Lahore (n = 19/43, 44.18%; 95% CI: 30.44-58.9) followed by Faisalabad (n= 12/36, 33.33%; 95% CI: 20.21-49.66), Gujranwala (n = 05/23, 21.7%; 95% CI: 9.66-41.9) and Sargodha (n = 09/28, 32.1%; 95% CI: 17.93-50.66). The probability of occurrence of HIV infection was significantly associated with individuals having a history of injecting drug use (68.08%; OR = 11.15; 95% CI: 53.84-79.61, p = 0.0001). Phylogenetic analysis based on the pol gene showed that the sequences from this study clustered into three distinct clades representing recombinant form 02_AG (n = 14, 77.0%; 95% CI: 54.79-91.00), and subtypes A (n = 2, 11.1%; 95% CI: 3.1-32.8) and G (n = 2, 11.1%; 95% CI: 3.1-32.8). Although we screened 18 samples for drug-resistance-associated mutations, except for an accessory mutation (M46K) in the protease (PR) region in one subject, we found a lack of drug-resistance-associated substitutions in the PR region. On the other hand, we found two subjects (2/18) carrying a resistance-associated mutation (V106I) conferring a low level of resistance against non-nucleoside reverse transcriptase inhibitors. The present study shows that multiple subtypes of HIV-1 are present in the affected population. Continuous disease surveillance coupled with evaluation of drug resistance at higher resolution should be done in future studies.

Intranasal immunization with coxsackievirus A16 virus-like particles confers protection against lethal infection in neonatal mice Abstract Coxsackievirus A16 (CV-A16) is one of the main causative agents of hand, foot and mouth disease (HFMD) in young children and has become prevalent in the Asia-Pacific region in recent years. However, no approved vaccines or drugs are available for CV-A16 infection. CV-A16 virus-like particles (VLPs) are a potential vaccine candidate; however, whether the intranasal route of immunization is suitable for inducing immune responses against CV-A16 infection has not been clarified. In this study, the comprehensive immunogenicity and protective efficacy of the CV-A16 VLP vaccine were evaluated by multiple methods in a mouse model. In mice, a high neutralizing antibody (NTAb) titre could be elicited by intranasal immunization with CV-A16 VLPs, which produced NTAb levels similar to those induced by intranasal immunization with inactivated CV-A16. Passive immunity with NTAbs provided very good protection, as the survival rate of the immunized neonatal mice was 100% after challenges with CV-A16 at a dose of 1000 LD50. Passive protective effects were transferred to the neonates via the mother, thus protecting all the pups against challenges with the homologous or heterologous strains of CV-A16 at a dose of 1000 LD50. In addition, intranasal immunization with CV-A16 VLPs also induced the production of mucosal secretory IgA (s-IgA) antibodies, which may inhibit CV-A16 virus invasion. This study provides valuable supplemental information to facilitate our understanding of the specific protective efficacy of CV-A16 VLPs and has significance for development of the candidate vaccine into a safe and effective vaccine.