Viruses — Open Access Journal of Virology

Passive administration of broadly neutralizing antibodies (bNAbs) capable of recognizing a broad range of viral strains to non-human primates has led to protection from infection with chimeric SIV/HIV virus (SHIV). This data suggests that generating protective antibody responses could be an effective strategy for an HIV vaccine. However, classic vaccine approaches have failed so far to induce such protective antibodies in HIV vaccine trials. HIV-specific bNAbs identified in natural infection show high levels of somatic hypermutations, demonstrating that they underwent extensive affinity maturation. It is likely that to gain ability to recognize diverse viral strains, vaccine-induced humoral responses will also require complex, iterative maturation. T follicular helper cells (Tfh) are a specialized CD4+ T cell subset that provides help to B cells in the germinal center for the generation of high-affinity and long-lasting humoral responses. It is therefore probable that the quality and quantity of Tfh responses upon vaccination will impact development of bNAbs. Here, we review studies that advanced our understanding of Tfh differentiation, function and regulation. We discuss correlates of Tfh responses and bNAb development in natural HIV infection. Finally, we highlight recent strategies to optimize Tfh responses upon vaccination and their impact on prophylactic HIV vaccine research. Full article

Since their discovery over a decade ago, the class of prokaryotic immune systems known as CRISPR–Cas have afforded a suite of genetic tools that have revolutionized research in model organisms spanning all domains of life. CRISPR-mediated tools have also emerged for the natural targets of CRISPR–Cas immunity, the viruses that specifically infect bacteria, or phages. Despite their status as the most abundant biological entities on the planet, the majority of phage genes have unassigned functions. This reality underscores the need for robust genetic tools to study them. Recent reports have demonstrated that CRISPR–Cas systems, specifically the three major types (I, II, and III), can be harnessed to genetically engineer phages that infect diverse hosts. Here, the mechanisms of each of these systems, specific strategies used, and phage editing efficacies will be reviewed. Due to the relatively wide distribution of CRISPR–Cas systems across bacteria and archaea, it is anticipated that these immune systems will provide generally applicable tools that will advance the mechanistic understanding of prokaryotic viruses and accelerate the development of novel technologies based on these ubiquitous organisms. Full article

The mouse mammary tumor virus (MMTV) Pr77^Gag polypeptide is an essential retroviral structural protein without which infectious viral particles cannot be formed. This process requires specific recognition and packaging of dimerized genomic RNA (gRNA) by Gag during virus assembly. Most of the previous work on retroviral assembly has used either the nucleocapsid portion of Gag, or other truncated Gag derivatives—not the natural substrate for virus assembly. In order to understand the molecular mechanism of MMTV gRNA packaging process, we expressed and purified full-length recombinant Pr77^Gag-His₆-tag fusion protein from soluble fractions of bacterial cultures. We show that the purified Pr77^Gag-His₆-tag protein retained the ability to assemble virus-like particles (VLPs) in vitro with morphologically similar immature intracellular particles. The recombinant proteins (with and without His₆-tag) could both be expressed in prokaryotic and eukaryotic cells and had the ability to form VLPs in vivo. Most importantly, the recombinant Pr77^Gag-His₆-tag fusion proteins capable of making VLPs in eukaryotic cells were competent for packaging sub-genomic MMTV RNAs. The successful expression and purification of a biologically active, full-length MMTV Pr77^Gag should lay down the foundation towards performing RNA–protein interaction(s), especially for structure-function studies and towards understanding molecular intricacies during MMTV gRNA packaging and assembly processes. Full article

Improved vaccine-mediated protection against HIV-1 requires a thorough understanding of the mode of HIV-1 transmission and how various immune responses control transmission. Cell-associated HIV-1 is infectious and contributes to HIV-1 transmission in humans. Non-human primate models of cell-associated SIV infection demonstrate that cell-associated SIV is more infectious than cell-free SIV. In a recently described chimeric simian–human immunodeficiency virus (SHIV) macaque model, it was demonstrated that an occult infection with cell-associated SHIV can be established that evades passive protection with a broadly neutralizing antibody (bnAb). Indeed, considerable in vitro data shows that bnAbs have less efficacy against cell-associated HIV-1 than cell-free HIV-1. Optimizing the protective capacity of immune responses such as bnAbs against cell-associated infections may be needed to maximize their protective efficacy. Full article

Campylobacter jejuni is a frequent foodborne pathogen of humans. As C. jejuni infections commonly arise from contaminated poultry, phage treatments have been proposed to reduce the C. jejuni load on farms to prevent human infections. While a prior report documented the transcriptome of C. jejuni phages during the carrier state life cycle, transcriptomic analysis of a lytic C. jejuni phage infection has not been reported. We used RNA-sequencing to profile the infection of C. jejuni NCTC 11168 by the lytic T4-like myovirus NCTC 12673. Interestingly, we found that the most highly upregulated host genes upon infection make up an uncharacterized operon (cj0423–cj0425), which includes genes with similarity to T4 superinfection exclusion and antitoxin genes. Other significantly upregulated genes include those involved in oxidative stress defense and the Campylobactermultidrug efflux pump (CmeABC). We found that phage infectivity is altered by mutagenesis of the oxidative stress defense genes catalase (katA), alkyl-hydroxyperoxidase (ahpC), and superoxide dismutase (sodB), and by mutagenesis of the efflux pump genes cmeA and cmeB. This suggests a role for these gene products in phage infection. Together, our results shed light on the phage-host dynamics of an important foodborne pathogen during lytic infection by a T4-like phage. Full article

Despite the abundance, ubiquity and impact of environmental viruses, their inherent genomic plasticity and extreme diversity pose significant challenges for the examination of bacteriophages on Earth. Viral metagenomic studies have offered insight into broader aspects of phage ecology and repeatedly uncover genes to which we are currently unable to assign function. A combined effort of phage isolation and metagenomic survey of Chicago’s nearshore waters of Lake Michigan revealed the presence of Pbunaviruses, relatives of the Pseudomonas phage PB1. This prompted our expansive investigation of PB1-like phages. Genomic signatures of PB1-like phages and Pbunaviruses were identified, permitting the unambiguous distinction between the presence/absence of these phages in soils, freshwater and wastewater samples, as well as publicly available viral metagenomic datasets. This bioinformatic analysis led to the de novo assembly of nine novel PB1-like phage genomes from a metagenomic survey of samples collected from Lake Michigan. While this study finds that Pbunaviruses are abundant in various environments of Northern Illinois, genomic variation also exists to a considerable extent within individual communities. Full article

The productive infection of influenza A virus (IAV) depends on host factors. However, the involvement of long non-coding RNAs (lncRNAs) in IAV infection remains largely uninvestigated. In this work, we have discovered a human lncRNA, named lncRNA-PAAN (PA-associated noncoding RNA) that enhances IAV replication. The level of lncRNA-PAAN increases upon infection of IAV, but not other viruses, nor interferon treatment, suggesting specific up-regulation of lncRNA-PAAN expression by IAV. Silencing lncRNA-PAAN significantly decreases IAV replication through impairing the activity of viral RNA-dependent RNA polymerase (RdRp). This function of lncRNA-PAAN is a result of its association with viral PA protein, a key component of IAV RNA polymerase complex. Consequently, depletion of lncRNA-PAAN prevents the formation of functional RdRp. Together, these results suggest that lncRNA-PAAN promotes the assembly of viral RNA polymerase, thus warranting efficient viral RNA synthesis. Elucidating the functions of lncRNAs in IAV infection is expected to advance our understanding of IAV pathogenesis and open new avenues to the development of novel anti-IAV therapeutics. Full article

Tick-borne flaviviruses have a global distribution and cause significant human disease, including encephalitis and hemorrhagic fever, and often result in neurologic sequelae. There are two distinct properties that determine the neuropathogenesis of a virus. The ability to invade the central nervous system (CNS) is referred to as the neuroinvasiveness of the agent, while the ability to infect and damage cells within the CNS is referred to as its neurovirulence. Examination of laboratory variants, cDNA clones, natural isolates with varying pathogenicity, and virally encoded immune evasion strategies have contributed extensively to our understanding of these properties. Here we will review the major viral determinants of virulence that contribute to pathogenesis and influence both neuroinvasiveness and neurovirulence properties of tick-borne flaviviruses, focusing particularly on the envelope protein (E), nonstructural protein 5 (NS5), and the 3′ untranslated region (UTR). Full article

Recombination among porcine reproductive and respiratory syndrome viruses (PRRSVs) is thought to contribute to the emergence of new PRRSV variants. In this study, two newly emerged PRRSV strains, designated SCcd16 and SCya17, are isolated from lung tissues of piglets in Southwestern China. Genome comparative analysis reveals that SCcd16/SCya17 exhibit 93.1%/93.2%, 86.9%/87.0%, 85.3%/85.7%, and 83.6%/82.0% nucleotide similarity to PRRSVs JXA1, VR-2332, QYYZ and NADC30, respectively. They only exhibit 44.8%/45.1% sequence identity with LV (PRRSV-1), indicating that both emergent strains belong to the PRRSV-2 genotype. Genomic sequence alignment shows that SCcd16 and SCya17 have the same discontinuous 30-amino acid (aa) deletion in Nsp2 of the highly pathogenic Chinese PRRSV strain JXA1, when compared to strain VR-2332. Notably, SCya17 shows a unique 5-nt deletion in its 3’-UTR. Phylogenetic analysis shows that both of the isolates are classified in the QYYZ-like lineage based on ORF5 genotyping, whereas they appear to constitute an inter-lineage between JXA1-like and QYYZ-like lineages based on their genomic sequences. Furthermore, recombination analyses reveal that the two newly emerged PRRSV isolates share the same novel recombination pattern. They have both likely originated from multiple recombination events between lineage 8 (JXA1-like), lineage 1 (NADC30-like), and lineage 3 (QYYZ-like) strains that have circulated in China recently. The genomic data from SCcd16 and SCya17 indicate that there is on going evolution of PRRSV field strains through genetic recombination, leading to outbreaks in the pig populations in Southwestern China. Full article

Phage therapy is based on a simple concept: the use of a virus (bacteriophage) that is capable of killing specific pathogenic bacteria to treat bacterial infections. Since the pioneering work of Félix d’Herelle, bacteriophages (phages) isolated in vitro have been shown to be of therapeutic value. Over decades of study, a large number of rather complex mechanisms that are used by phages to hijack bacterial resources and to produce their progeny have been deciphered. While these mechanisms have been identified and have been studied under optimal conditions in vitro, much less is known about the requirements for successful viral infections in relevant natural conditions. This is particularly true in the context of phage therapy. Here, we highlight the parameters affecting phage replication in both in vitro and in vivo environments, focusing, in particular, on the mammalian digestive tract. We propose avenues for increasing the knowledge-guided implementation of phages as therapeutic tools. Full article

Dengue is a worldwide problem characterized by a multifactorial pathogenesis. Considering the viral components, it is known that high viremia or high levels of the secreted nonstructural protein 1 (NS1) may be associated with a more severe disease. We aimed to characterize the NS1 antigenemia and viremia in dengue fatal and non-fatal cases, as potential markers of progression to a fatal outcome. NS1 antigenemia and viremia were determined in Brazilian dengue fatal cases (n = 40) and non-fatal cases (n = 40), representative of the four dengue virus (DENV) serotypes. Overall, the fatal cases presented higher NS1 levels and viremia. Moreover, the fatal cases from secondary infections showed significantly higher NS1 levels than the non-fatal ones. Here, irrespective of the disease outcome, DENV-1 cases presented higher NS1 levels than the other serotypes. However, DENV-2 and DENV-4 fatal cases had higher NS1 antigenemia than the non-fatal cases with the same serotype. The viremia in the fatal cases was higher than in the non-fatal ones, with DENV-3 and DENV-4 presenting higher viral loads. Viral components, such as NS1 and viral RNA, may be factors influencing the disease outcome. However, the host immune status, comorbidities, and access to adequate medical support cannot be ruled out as interfering in the disease outcome. Full article

Influenza A virus (IAV) causes seasonal epidemics and occasional but devastating pandemics, which are major public health concerns. Because the effectiveness of seasonal vaccines is highly variable and the currently available drugs are limited in their efficacy because of the emergence of drug resistance, there is an urgent need to develop novel antivirals. In this study, we characterized a recombinant IAV-carrying Gaussia luciferase (Gluc) gene and determined its potential as a tool for evaluating therapeutics. We demonstrated that this recombinant IAV is replication-competent in tissue culture and pathogenic in mice, although it is slightly attenuated compared to the parental virus. Luciferase expression correlated well with virus propagation both in vitro and in vivo, providing a simple measure for viral replication in tissue culture and in mouse lungs. To demonstrate the utility of this virus, ribavirin and oseltamivir phosphate were used to treat the IAV-infected cells and mice, and we observed the dose-dependent inhibition of viral replication by a luciferase assay. Moreover, the decreased luciferase expression in the infected lungs could predict the protective efficacy of antiviral interventions as early as day 2 post virus challenge. In summary, this study provides a new and quantitative approach to evaluate antivirals against IAV. Full article

Applications for bacteriophages as antimicrobial agents are increasing. The industrial use of these bacterial viruses requires the production of large amounts of suitable strictly lytic phages, particularly for food and agricultural applications. This work describes a new approach for phage production. Phages H387 (Siphoviridae) and A511 (Myoviridae) were propagated separately using Listeria ivanovii host cells immobilised in alginate beads. The same batch of alginate beads could be used for four successive and efficient phage productions. This technique enables the production of large volumes of high-titer phage lysates in continuous or semi-continuous (fed-batch) cultures. Full article

Antibiotic resistance evolution in bacteria indicates that one of the challenges faced by phage therapy is that, sooner or later, bacteria will evolve resistance to phages. Evidently, this is the case of every known antimicrobial therapy, but here this is also part of a ubiquitous natural process of co-evolution between phages and bacteria. Fundamental evolutionary studies hold some clues that are crucial to limit the problematic process of bacterial resistance during phage applications. First, I discuss here the importance of defining evolutionary and ecological factors influencing bacterial resistance and phage counter-defense mechanisms. Then, I comment on the interest of determining the co-evolutionary dynamics between phages and bacteria that may allow for selecting the conditions that will increase the probability of therapeutic success. I go on to suggest the varied strategies that may ensure the long-term success of phage therapy, including analysis of internal phage parameters and personalized treatments. In practical terms, these types of approaches will define evolutionary criteria regarding how to develop, and when to apply, therapeutic phage cocktails. Integrating this perspective in antimicrobial treatments, such as phage therapy, is among the necessary steps to expand its use in the near future, and to ensure its durability and success. Full article

Highly periodic and uniform nanostructures, based on a genetically engineered M13 bacteriophage, displayed unique properties at the nanoscale that have the potential for a variety of applications. In this work, we report a multilayer biofilm with self-assembled nanoporous surfaces involving a nanofiber-like genetically engineered 4E-type M13 bacteriophage, which was fabricated using a simple pulling method. The nanoporous surfaces were effectively formed by using the networking-like structural layers of the M13 bacteriophage during self-assembly. Therefore, an external template was not required. The actual M13 bacteriophage-based fabricated multilayered biofilm with porous nanostructures agreed well with experimental and simulation results. Pores formed in the final layer had a diameter of about 150–500 nm and a depth of about 15–30 nm. We outline a filter application for this multilayered biofilm that enables selected ions to be extracted from a sodium chloride solution. Here, we describe a simple, environmentally friendly, and inexpensive fabrication approach with large-scale production potential. The technique and the multi-layered biofilms produced may be applied to sensor, filter, plasmonics, and bio-mimetic fields. Full article

The papillomavirus (PV) protein E2 is one of only two proteins required for viral DNA replication. E2 is the viral transcriptional regulator/activation protein as well as the initiator of viral DNA replication. E2 is known to interact with various cellular DNA replication proteins, including the PV E1 protein, the cellular ssDNA binding complex (RPA), and topoisomerase I. Recently, we observed that cellular DNA polymerase ε (pol ε) interacts with the PV helicase protein, E1. E1 stimulates its activity with a very high degree of specificity, implicating pol ε in PV DNA replication. In this paper, we evaluated whether E2 also shows a functional interaction with pol ε. We found that E2 stimulates the DNA synthesis activity of pol ε, independently of pol ε’ s processivity factors, RFC, PCNA, and RPA, or E1. This appears to be specific for pol ε, as cellular DNA polymerase δ is unaffected by E1. However, unlike other known stimulatory factors of pol ε, E2 does not affect the processivity of pol ε. The domains of E2 were analyzed individually and in combination for their ability to stimulate pol ε. Both the transactivation and hinge domains were found to be important for this stimulation, while the E2 DNA-binding domain was dispensable. These findings support a role for E2 beyond E1 recruitment in viral DNA replication, demonstrate a novel functional interaction in PV DNA replication, and further implicate cellular pol ε in PV DNA replication. Full article

Enterovirus 71 (EV-A71) is a major etiological agent of hand, foot and mouth disease (HFMD) that mainly affects young children less than five years old. The onset of severe HFMD is due to neurological complications bringing about acute flaccid paralysis and pulmonary oedema. In this review, we address how genetic events such as recombination and spontaneous mutations could change the genomic organization of EV-A71, leading to an impact on viral virulence. An understanding of the recombination mechanism of the poliovirus and non-polio enteroviruses will provide further evidence of the emergence of novel strains responsible for fatal HFMD outbreaks. We aim to see if the virulence of EV-A71 is contributed solely by the presence of fatal strains or is due to the co-operation of quasispecies within a viral population. The phenomenon of quasispecies within the poliovirus is discussed to reflect viral fitness, virulence and its implications for EV-A71. Ultimately, this review gives an insight into the evolution patterns of EV-A71 by looking into its recombination history and how spontaneous mutations would affect its virulence. Full article

Murray Valley Encephalitis virus (MVEV) is a mosquito-borne Flavivirus. Clinical presentation is rare but severe, with a case fatality rate of 15–30%. Here we report a case of MVEV from the cerebrospinal fluid (CSF) of a patient in the Northern Territory in Australia. Initial diagnosis was performed using both MVEV-specific real-time, and Pan-Flavivirus conventional, Polymerase Chain Reaction (PCR), with confirmation by Sanger sequencing. Subsequent isolation, the first from CSF, was conducted in Vero cells and the observed cytopathic effect was confirmed by increasing viral titre in the real-time PCR. Isolation allowed for full genome sequencing using the Scriptseq V2 RNASeq library preparation kit. A consensus genome for VIDRL-MVE was generated and phylogenetic analysis identified it as Genotype 2. This is the first reported isolation, and full genome sequencing of MVEV from CSF. It is also the first time Genotype 2 has been identified in humans. As such, this case has significant implications for public health surveillance, epidemiology, and the understanding of MVEV evolution. Full article

Fruit trees, such as apricot trees, are constantly exposed to the attack of viruses. As they are propagated in a vegetative way, this risk is present not only in the field, where they remain for decades, but also during their propagation. Metagenomic diagnostic methods, based on next generation sequencing (NGS), offer unique possibilities to reveal all the present pathogens in the investigated sample. Using NGS of small RNAs, a special field of these techniques, we tested leaf samples of different varieties of apricot originating from an isolator house or open field stock nursery. As a result, we identified Cherry virus A (CVA) and little cherry virus 1 (LChV-1) for the first time in Hungary. The NGS results were validated by RT-PCR and also by Northern blot in the case of CVA. Cloned and Sanger sequenced viral-specific PCR products enabled us to investigate their phylogenetic relationships. However, since these pathogens have not been described in our country before, their role in symptom development and modification during co-infection with other viruses requires further investigation. Full article