Wheat streak mosaic virus (WSMV; genus Tritimovirus; family Potyviridae) is an economically important wheat virus that is transmitted by the wheat curl mite (WCM; Aceria tosichella Keifer) in a persistent manner. Virus–vector coevolution may potentially influence vector gene expression to prolong viral association and thus increase virus transmission efficiency and spread. To understand the transcriptomic responses of WCM to WSMV, RNA sequencing was performed to assemble and analyse transcriptomes of WSMV viruliferous and aviruliferous mites. Among 7291 de novo-assembled unigenes, 1020 were differentially expressed between viruliferous and aviruliferous WCMs using edgeR at a false discovery rate ≤0.05. Differentially expressed unigenes were enriched for 108 gene ontology terms, with the majority of the unigenes showing downregulation in viruliferous mites in comparison to only a few unigenes that were upregulated. Protein family and metabolic pathway enrichment analyses revealed that most downregulated unigenes encoded enzymes and proteins linked to stress response, immunity and development. Mechanistically, these predicted changes in mite physiology induced by viral association could be suggestive of pathways needed for promoting virus–vector interactions. Overall, our data suggest that transcriptional changes in viruliferous mites facilitate prolonged viral association and alter WCM development to expedite population expansion, both of which could enhance viral transmission.
We previously showed that single amino acid substitutions at seven positions in haemagglutinin determined major antigenic change of influenza H3N2 virus. Here, the impact of two such substitutions was tested in 11 representative H3 haemagglutinins to investigate context-dependence effects. The antigenic effect of substitutions introduced at haemagglutinin position 145 was fully independent of the amino acid context of the representative haemagglutinins. Antigenic change caused by substitutions introduced at haemagglutinin position 155 was variable and context-dependent. Our results suggest that epistatic interactions with contextual amino acids in the haemagglutinin can moderate the magnitude of antigenic change.
Sequences for Lloviu virus (LLOV), a putative novel filovirus, were first identified in Miniopterus schreibersii bats in Spain following a massive bat die-off in 2002, and also recently found in bats in Hungary. However, until now it is unclear if these sequences correspond to a fully functional, infectious virus, and whether it will show a pathogenic phenotype like African filoviruses, such as ebola- and marburgviruses, or be apathogenic for humans, like the Asian filovirus Reston virus. Since no infectious virus has been recovered, the only opportunity to study infectious LLOV is to use a reverse genetics-based full-length clone system to de novo generate LLOV. As a first step in this process, and to investigate whether the identified sequences indeed correspond to functional viral proteins, we have developed life cycle modelling systems for LLOV, which allow us to study genome replication and transcription as well as entry of this virus. We show that all LLOV proteins fulfill their canonical role in the virus life cycle as expected based on the well-studied related filovirus Ebola virus. Further, we have analysed the intergenus-compatibility of proteins that have to act in concert to facilitate the virus life cycle. We show that some but not all proteins from LLOV and Ebola virus are compatible with each other, emphasizing the close relationship of these viruses, and informing future studies of filovirus biology with respect to the generation of genus-chimeric proteins in order to probe virus protein–protein interactions on a functional level.
Influenza A virus mutates rapidly, allowing it to escape natural and vaccine-induced immunity. Neuraminidase (NA) is a surface protein capable of cleaving the glycosidic linkages of neuraminic acids to release newly formed virions from infected cells. Genetic variants within a viral population can influence the emergence of pandemic viruses as well as drug susceptibility and vaccine effectiveness. In the present study, 55 clinical specimens from patients infected with the 2009 pandemic influenza A/H1N1 virus, abbreviated as A(H1N1)pdm09, during the 2015–2016 outbreak season in Taiwan were collected. Whole genomes were obtained through next-generation sequencing. Based on the published sequences from A(H1N1)pdm09 strains worldwide, a mixed population of two distinct variants at NA position 151 was revealed. We initially reasoned that such a mixed population may have emerged during cell culture. However, additional investigations confirmed that these mixed variants were detectable in the specimens of patients. To further investigate the role of the two NA-151 variants in a dynamic population, a reverse genetics system was employed to generate recombinant A(H1N1)pdm09 viruses. It was observed that the mixture of the two distinct variants was characterized by a higher replication rate compared to the recombinant viruses harbouring a single variant. Moreover, an NA inhibition assay revealed that a high frequency of the minor NA-151 variant in A(H1N1)pdm09 was associated with a reduced susceptibility to NA inhibitors. We conclude that two distinct NA-151 variants can be identified in patient specimens and that such variants may increase viral replication and NA activity.
Persistent propagative plant viruses are usually transmitted between a vector insect and a host plant. To adapt to the two different organisms, viruses may show distinct genomic replication or gene expression patterns. To verify this hypothesis, we applied an aboslute real-time quantitative PCR method to measure and compare the replication levels of four genomic RNA segments and the expression levels of seven genes of rice stripe virus (RSV) according to the infection time in the small brown planthopper and rice plant, respectively. In the vector insect, RNA3 began replicating later than the other segments, and RNA2 remained nearly constant during the infection process. RNA1 was the dominant segment, and a difference of over 300-fold appeared among the four segments. In rice plants, the size of the four segments increased with infection time, but decreased to a low level in the late infection period. The ratios of the four segments varied by no more than 15-fold. In planthoppers, three expression patterns were observed for the seven viral genes during viral infection, while in rice plants, the expression patterns of the seven viral genes were similar. These results reflect distinct genomic replication and gene expression patterns in a persistent propagative plant virus in adapting to vector insects and host plants.
Hepatitis B virus has been classified into 10 genotypes and 48 subgenotypes worldwide. We found previously, through polymerase chain reaction (PCR) amplification of a sample collected in 2011, that an HBsAg carrier was infected with two genotypes (B and D) of HBV. We carried out cloning, sequencing and phylogenetic analysis of the complete genomes and, for confirmation, analysed a sample collected from the same individual in 2018. Fifteen complete sequences were obtained from each sample. The carrier was infected in 2011 by genotypes B and D and by various recombinants, but only genotype D was present in 2018. The major and minor parents of the recombinants are genotypes B and D, respectively, although the recombination breakpoints vary among them. All 23 genotype D isolates form a cluster, branching out from other subgenotype D sequences and supported by a 100 % bootstrap value. Based on complete genome sequences, almost all of the estimated intragroup nucleotide divergence values between our isolates and HBV subgenotypes D1–D10 exceed 4 %. Compared to the other subgenotypes (D1–D10), 35 unique amino acids were present in our isolates. Our data provide evidence for a novel subgenotype, provisionally designated HBV subgenotype D11.
Foot-and-mouth disease virus (FMDV) displays various epitopes on the capsid outer surface. In addition to the five neutralizing antigenic sites, there is evidence of the existence of other, yet unidentified, epitopes that are believed to play a role in antibody-mediated protection. Previous attempts to identify these epitopes revealed two additional substitutions at positions VP2-74 and -191 (5M2/5 virus) to be of antigenic significance. However, complete resistance to neutralization was not obtained in the neutralization assay, indicating the existence of other, undisclosed epitopes. Results from this study provides evidence of at least two new neutralizing epitopes involving residues VP3-116 and -195 around the threefold axis that have significant impact on the antigenic nature of the virus. These findings extend our knowledge of the surface features of the FMDV capsid known to elicit neutralizing antibodies, and should help with rational vaccine design.
The yak (Bosgrunniens) is a unique domestic bovine species that plays an indispensable role for herdsmen in the Qinghai–Tibet Plateau. Here, 336 diarrhoeic samples were collected from yaks on 29 farms in the Qinghai–Tibet Plateau from 2015 to 2017. Approximately 69.05 % (232/336) of the diarrhoeic samples were assessed as bovine coronavirus (BCoV)-positive by RT-PCR assay, and most of the detected strains showed a unique evolution based on 40 spike (S), nucleocapsid (N) and haemagglutinin-esterase (HE) gene fragments. Notably, the 12 complete S genes detected shared 1 identical amino acid mutation (E121V) in the S1 subunit compared with the other 150 complete S genes in the GenBank database. Furthermore, a BCoV strain (designated YAK/HY24/CH/2017) was isolated from one diarrhoeic sample (virus titre : 108.17TCID50 ml−1), and a phylogenetic analysis based on complete genome sequences revealed that strain YAK/HY24/CH/2017 has the closest genetic relationship with the BCoV prototype strain Mebus. Interestingly, 2 significant characteristics were observed in the genome of strain YAK/HY24/CH/2017 : (1) the strain had 26 unique amino acid variations in the S gene compared with the other 150 BCoV S genes in the GenBank database and (2) a recombination event was identified between the esterase and lectin domains of the HE gene. In conclusion, this study revealed the high prevalence of BCoV in yaks in the Qinghai–Tibet Plateau. To the best of our knowledge, this is the first description of the molecular prevalence of BCoV in yaks and of a BCoV genome with an HE gene recombination.
Parvovirus B19 (B19V) possesses a linear single-stranded DNA genome of either positive or negative polarity. Due to intramolecular sequence homologies, either strand may theoretically be folded in several alternative ways. Viral DNA, when extracted from virions by several procedures, presents as linear single-stranded and/or linear double-stranded molecules, except when one particular commercial kit is used. This protocol yields DNA with an aberrant electrophoretic mobility in addition to linear double-stranded molecules, but never any single-stranded molecules. This peculiar kind of DNA was found in all plasma or serum samples tested and so we decided to analyse its secondary structure. In line with our results for one- and two-dimensional electrophoresis, mobility shift assays, DNA preparation by an in-house extraction method with moderate denaturing conditions, density gradient ultracentrifugation, DNA digestion experiments and competition hybridization assays, we conclude that (i) the unique internal portions of this distinctive single-stranded molecules are folded into tight tangles and (ii) the two terminal redundant regions are associated with each other, yielding non-covalently closed pseudo-circular molecules stabilized by a short (18 nucleotides) intramolecular stem, whereas the extreme 3′- and 5′-ends are folded back on themselves, forming a structure resembling a twin hairpin. The question arises as to whether this fairly unstable structure represents the encapsidated genome structure. The answer to this question remains quite relevant in terms of comprehending the initiation and end of B19V genome replication.