Genetic diversity analysis of lychnis mottle virus and first identification of Angelica sinensis infection.

Gansu Province is a district renowned for the cultivation of Angelica sinensis (Oliv.) Diels, accounting for greater than 90% of China's total annual production. However, virus infection has caused a reduction in A. sinensis yield. Here, we collected suspected virus-infected A. sinensis leaf samples from Gansu Province's A. sinensis cultivation area. For the first time, using small RNA deep sequencing and RT-PCR, lychnis mottle virus (LycMoV) was found to naturally infect A. sinensis. The coat protein (cp) gene of the Gansu A. sinensis LycMoV isolate was obtained through cloning, where its nucleotide and amino acid identity was highest while having the closest affinity to the China Pearl (i.e., Prunus persica) isolate. Recombination analysis indicated that genetic recombination had only a limited influencing effect on the molecular evolution of LycMoV. Moreover, results from genetic diversity analysis indicated that the host, geographic isolation, and genetic drift may be the main factors that contributed to the formation of genetic diversity and differentiation in LycMoV. Furthermore, the LycMoV population trend was expansionary. Selection pressure may also be the main driver for the evolution of the entire LycMoV population, while the driving effect of genetic recombination is limited. This study marks a new LycMoV host (i.e., A. sinensis) for the first time and provides scientific support for the identification, prevention, and control of LycMoV.

Complementary Effects of Virus Population Are Required for Efficient Virus Infection.

It is believed that the virions of a virus infecting a host may share the identical viral genome and characteristics. However, the role of genomic heterogeneity of the virions of a virus in virus infection has not been extensively explored. To address this issue, white spot syndrome virus (WSSV), a DNA virus infecting crustaceans, was characterized in the current study. In WSSV, differences in two nucleotides of the viral genome generated two types of WSSV, forming a virus population that consisted of Type A WSSV (encoding WSSV lncRNA-24) and Type B WSSV (encoding the wsv195 gene) at a ratio of 1:3. The virus populations in all virus-infected cells and tissues of different hosts exhibited a stable 1:3 structure. WSSV lncRNA-24 in Type A WSSV promoted virus infection by binding to shrimp and WSSV miRNAs, while the wsv195 gene in Type B WSSV played an essential role in virus infection. Loss of Type A WSSV or Type B WSSV in the WSSV population led to a 100-fold decrease in viral copy number in shrimp. Simultaneous loss of both types of WSSV prevented virus infection. These results indicated that the virus infection process was completed by two types of WSSV encoding different functional genes, revealing the complementary effects of WSSV population. Therefore, our study highlights the importance of the complementarity of virus population components in virus infection.

Intra- and inter-host evolution of H9N2 influenza A virus in Japanese quail.

Influenza A viruses (IAVs) are constantly evolving. Crucial steps in the infection cycle, such as sialic acid (SA) receptor binding on the host cell surface, can either promote or hamper the emergence of new variants. We previously assessed the relative fitness in Japanese quail of H9N2 variant viruses differing at a single amino acid position, residue 216 in the hemagglutinin (HA) viral surface protein. This site is known to modulate SA recognition. Our prior study generated a valuable set of longitudinal samples from quail transmission groups where the inoculum comprised different mixed populations of HA 216 variant viruses. Here, we leveraged these samples to examine the evolutionary dynamics of viral populations within and between inoculated and naïve contact quails. We found that positive selection dominated HA gene evolution, but fixation of the fittest variant depended on the competition mixture. Analysis of the whole genome revealed further evidence of positive selection acting both within and between hosts. Positive selection drove fixation of variants in non-HA segments within inoculated and contact quails. Importantly, transmission bottlenecks were modulated by the molecular signature at HA 216, revealing viral receptor usage as a determinant of transmitted diversity. Overall, we show that selection strongly shaped the evolutionary dynamics within and between quails. These findings support the notion that selective processes act effectively on IAV populations in poultry hosts, facilitating rapid viral evolution in this ecological niche.

Discovery and Community Dynamics of Novel ssRNA Mycoviruses in the Conifer Pathogen Heterobasidion parviporum.

Heterobasidion species are highly destructive basidiomycetous conifer pathogens of the Boreal forest region. Earlier studies have revealed dsRNA virus infections of families Curvulaviridae and Partitiviridae in Heterobasidion strains, and small RNA deep sequencing has also identified infections of Mitoviridae members in these fungi. In this study, the virome of Heterobasidion parviporum was examined for the first time by RNA-Seq using total RNA depleted of rRNA. This method successfully revealed new viruses representing two established (+)ssRNA virus families not found earlier in Heterobasidion: Narnaviridae and Botourmiaviridae. In addition, we identified the presence of a recently described virus group tentatively named "ambiviruses" in H. parviporum. The H. parviporum isolates included in the study originated from experimental forest sites located within 0.7 km range from each other, and a population analysis including 43 isolates was conducted at one of the experimental plots to establish the prevalence of the newly identified viruses in clonally spreading H. parviporum individuals. Our results indicate that viral infections are considerably more diverse and common among Heterobasidion isolates than known earlier and include ssRNA viruses with high prevalence and interspecies variation.

Influence of Ribavirin on Mumps Virus Population Diversity.

Frequent mumps outbreaks in vaccinated populations and the occurrence of neurological complications (e.g., aseptic meningitis or encephalitis) in patients with mumps indicate the need for the development of more efficient vaccines as well as specific antiviral therapies. RNA viruses are genetically highly heterogeneous populations that exist on the edge of an error threshold, such that additional increases in mutational burden can lead to extinction of the virus population. Deliberate modulation of their natural mutation rate is being exploited as an antiviral strategy and a possibility for rational vaccine design. The aim of this study was to examine the ability of ribavirin, a broad-spectrum antiviral agent, to introduce mutations in the mumps virus (MuV) genome and to investigate if resistance develops during long-term in vitro exposure to ribavirin. An increase in MuV population heterogeneity in the presence of ribavirin has been observed after one passage in cell culture, as well as a bias toward C-to-U and G-to-A transitions, which have previously been defined as ribavirin-related. At higher ribavirin concentration, MuV loses its infectivity during serial passaging and does not recover. At low ribavirin concentration, serial passaging leads to a more significant increase in population diversity and a stronger bias towards ribavirin-related transitions, independently of viral strain or cell culture. In these conditions, the virus retains its initial growth capacity, without development of resistance at a whole-virus population level.

Ecological Approach to Understanding Superinfection Inhibition in Bacteriophage.

In microbial communities, viruses compete with each other for host cells to infect. As a consequence of competition for hosts, viruses evolve inhibitory mechanisms to suppress their competitors. One such mechanism is superinfection exclusion, in which a preexisting viral infection prevents a secondary infection. The bacteriophage ΦX174 exhibits a potential superinfection inhibition mechanism (in which secondary infections are either blocked or resisted) known as the reduction effect. In this auto-inhibitory phenomenon, a plasmid containing a fragment of the ΦX174 genome confers resistance to infection among cells that were once permissive to ΦX174. Taking advantage of this plasmid system, we examine the inhibitory properties of the ΦX174 reduction effect on a range of wild ΦX174-like phages. We then assess how closely the reduction effect in the plasmid system mimics natural superinfection inhibition by carrying out phage-phage competitions in continuous culture, and we evaluate whether the overall competitive advantage can be predicted by phage fitness or by a combination of fitness and reduction effect inhibition. Our results show that viral fitness often correctly predicts the winner. However, a phage's reduction sequence also provides an advantage to the phage in some cases, modulating phage-phage competition and allowing for persistence where competitive exclusion was expected. These findings provide strong evidence for more complex dynamics than were previously thought, in which the reduction effect may inhibit fast-growing viruses, thereby helping to facilitate coexistence.

Walking Together: Cross-Protection, Genome Conservation, and the Replication Machinery of Citrus tristeza virus.

"Cross-protection", a nearly 100 years-old virological term, is suggested to be changed to "close protection". Evidence for the need of such change has accumulated over the past six decades from the laboratory experiments and field tests conducted by plant pathologists and plant virologists working with different plant viruses, and, in particular, from research on Citrus tristeza virus (CTV). A direct confirmation of such close protection came with the finding that "pre-immunization" of citrus plants with the variants of the T36 strain of CTV but not with variants of other virus strains was providing protection against a fluorescent protein-tagged T36-based recombinant virus variant. Under natural conditions close protection is functional and is closely associated both with the conservation of the CTV genome sequence and prevention of superinfection by closely similar isolates. It is suggested that the mechanism is primarily directed to prevent the danger of virus population collapse that could be expected to result through quasispecies divergence of large RNA genomes of the CTV variants continuously replicating within long-living and highly voluminous fruit trees. This review article provides an overview of the CTV cross-protection research, along with a discussion of the phenomenon in the context of the CTV biology and genetics.

Virus population structure in the ectomycorrhizal fungi Lactarius rufus and L. tabidus at two forest sites in Southern Finland.

Lactarius fungi belong to the Russulaceae family and have an important ecological role as ectomycorrhizal symbionts of coniferous and deciduous trees. Two Lactarius species, L. tabidus and L. rufus have been shown to harbor bisegmented dsRNA viruses belonging to an unclassified virus group including the mutualistic Curvularia thermal tolerance virus (CThTV). In this study, we characterized the first complete genome sequences of these viruses designated as Lactarius tabidus RNA virus 1 (LtRV1) and Lactarius rufus RNA virus 1 (LrRV1), both of which included two genome segments of 2241 and 2049 bp. We also analyzed spatial distribution and sequence diversity of the viruses in sixty host strains at two forest sites, and showed that the viruses are species-specific at sites where both host species co-occur. We also found that single virus isolates inhabited several different conspecific host strains, and were involved in persistent infections during up to eight years.

Infectious Bronchitis Virus Population Structure Defines Immune Response and Protection.

A commercial Arkansas (Ark) Delmarva Poultry Industry (DPI)-type vaccine and a more homogeneous population of that vaccine obtained previously through adaptation to chicken embryo kidney (CEK) cells (CEK-ArkDPI) were used as a model to further understand the impact of population genetic structure on generation of immune responses and protection. In a first experiment, vaccinated chickens were challenged with an IBV Ark99-type virulent strain (AL/4614/98). Despite extensive sequence similarity between the vaccines, the more heterogeneous commercial ArkDPI was more efficient at reducing viral loads in challenged chickens, while respiratory signs and tracheal lesions were reduced similarly by either vaccine. A distinct subpopulation of the Ark challenge virus showing asparagine at S1 position 56 was consistently negatively selected by immune pressure originating from vaccination with either vaccine. Antibody levels and antibody avidity to Ark-type S1 protein were greater in CEK-ArkDPI-vaccinated chickens compared to chickens vaccinated with the more diverse commercial ArkDPI vaccine. Synchronous replication of a homogeneous virus population likely elicits clonal expansion and affinity maturation of a greater number of responding B cells compared to a diverse virus population continuously changing its proportion of phenotypes during replication. The results of a second experiment showed that during initial vaccine virus replication (24 and 48 hr postvaccination), the virus population showing increased diversity (commercial ArkDPI) achieved higher concentrations of IBV RNA in the trachea compared to the more homogenous virus. mRNA expression of genes associated with innate immune responses in the trachea 48 hr postvaccination generally showed greater upregulation in chickens vaccinated with the heterogeneous commercial ArkDPI vaccine compared to the CEK-adapted virus. The greater upregulation of these genes is likely associated with higher virus replication achieved by the heterogeneous commercial vaccine. Thus, while the adaptive antibody response was favored by the more homogenous structure of the CEK-ArkDPI vaccine population (higher antibody levels and antibody avidity), the innate immune response was favored by the more diverse viral population of the commercial ArkDPI. We confirmed previous results that distinct subpopulations in wild Ark challenge virus become selected by immune pressure originating from vaccination, and we concluded that the population structure of IBV vaccines impacts innate immune response, antibody avidity, and protection.

La estructura poblacional del virus de la bronquitis infecciosa define la respuesta inmune y la protección. Se utilizaron una vacuna comercial del tipo Arkansas (Ark) Industria Avícola de Delmarva (Delmarva Poultry Industry: DPI) y una población más homogénea de esa vacuna obtenida previamente a través de su adaptación a las células de riñón embrionario de pollo (CEK) (CEK-ArkDPI) como modelos para comprender mejor el impacto de la estructura genética de la población en la generación de respuestas inmunes y protección. En un primer experimento, los pollos vacunados fueron desafiados con una cepa virulenta de tipo Ark99 del virus de la bronquitis infecciosa (AL/4614/98). A pesar de la extensa similitud en las secuencias entre las vacunas, la cepa comercial Arkansas DPI más heterogénea fue más eficiente en la reducción de las cargas virales en los pollos desafiados, mientras que los signos respiratorios y las lesiones traqueales se redujeron de manera similar con cualquiera de las dos vacunas. Una subpoblación distinta del virus de desafío Arkansas que muestra asparagina en la posición 56 de la proteína S1 fue seleccionada de forma negativa consistentemente por la presión inmunitaria originada por la vacunación con cualquiera de las dos vacunas. Los niveles de anticuerpos y su avidez hacia la proteína S1 tipo Arkansas fueron mayores en los pollos inmunizados con la vacuna CEK-ArkDPI en comparación con los pollos vacunados con la vacuna comercial Arkansas DPI más diversa. La replicación sincrónica de una población de virus homogénea probablemente provoca la expansión clonal y la maduración por afinidad de un mayor número de células B con capacidad de respuesta en comparación con una población de virus diversa que cambia continuamente su proporción de fenotipos durante la replicación. Los resultados de un segundo experimento mostraron que durante la replicación inicial del virus de la vacuna (24 y 48 horas después de la vacunación), la población de virus que mostró una mayor diversidad (Arkansas DPI comercial) alcanzó mayores concentraciones de ARN del virus de bronquitis infecciosa en la tráquea en comparación con el virus más homogéneo. La expresión de ARNm de genes asociados con respuestas inmunes innatas en la tráquea 48 h después de la vacunación generalmente mostró una mayor regulación positiva en pollos vacunados con la vacuna comercial heterogénea Arkansas DPI en comparación con el virus adaptado a células de riñón embrionario de pollo. Es probable que la mayor regulación positiva de estos genes esté asociada con una mayor replicación del virus lograda por la vacuna comercial heterogénea. Por lo tanto, mientras que la respuesta de anticuerpos adaptativos fue favorecida por la estructura más homogénea de la población de vacunas CEK-ArkDPI (mayores niveles de anticuerpos y mayor avidez de anticuerpos), la respuesta inmune innata fue favorecida por la población viral más diversa de la vacuna Arkansas DPI comercial. Se confirman resultados previos de que distintas subpoblaciones en el virus de desafío Arkansas de tipo silvestre se seleccionan por la presión inmune originada por la vacunación y se concluye que la estructura de la población de las vacunas contra el virus de la bronquitis infecciosa afecta la respuesta inmune innata, la avidez de los anticuerpos y la protección.

Harnessed viruses in the age of metagenomics and synthetic biology: an update on infectious clone assembly and biotechnologies of plant viruses.

Recent metagenomic studies have provided an unprecedented wealth of data, which are revolutionizing our understanding of virus diversity. A redrawn landscape highlights viruses as active players in the phytobiome, and surveys have uncovered their positive roles in environmental stress tolerance of plants. Viral infectious clones are key tools for functional characterization of known and newly identified viruses. Knowledge of viruses and their components has been instrumental for the development of modern plant molecular biology and biotechnology. In this review, we provide extensive guidelines built on current synthetic biology advances that streamline infectious clone assembly, thus lessening a major technical constraint of plant virology. The focus is on generation of infectious clones in binary T-DNA vectors, which are delivered efficiently to plants by Agrobacterium. We then summarize recent applications of plant viruses and explore emerging trends in microbiology, bacterial and human virology that, once translated to plant virology, could lead to the development of virus-based gene therapies for ad hoc engineering of plant traits. The systematic characterization of plant virus roles in the phytobiome and next-generation virus-based tools will be indispensable landmarks in the synthetic biology roadmap to better crops.