Table of Contents

Porcine circovirus type 2 (PCV2) is closely linked to postweaning multisystemic wasting syndrome (PMWS) and other PCV-associated diseases (PCVADs), which influence the global pig industry. MicroRNAs (miRNAs) are evolutionarily conserved classes of endogenous small non-coding RNA that regulate almost every cellular process. According to our previous transcription study, PCV2 infection causes up-regulation of genes related to inflammation. To reveal the function of miRNAs in PCV2 infection and PCV2-encoded miRNAs, next generation sequencing and data analysis was performed to explore miRNA expression in PCV2-infected cells and non-infected cells. Data analysis found some small RNAs matched the PCV2 genome but PCV2 does not express miRNAs in an in vitro infection (PK-15 cells). More than 297 known and 427 novel miRNAs were identified, of which 44 miRNAs were differently expressed (DE). The pathways of inflammation mediated by chemokine and cytokine signaling pathway (P00031), were more perturbed in PCV2-infected cells than in mock controls. DE miRNAs and DE mRNA interaction network clearly revealed that PCV2 regulates the cellular inflammatory response through dysregulating the cellular miRNA-mRNA network. MiRNA overexpression and down-expression results demonstrated that miRNA dysregulation could affect PCV2 infection-induced cellular inflammatory responses. Our study revealed that host miRNA can be dysregulated by PCV2 infection and play an important role in PCV2-modulated inflammation. Full article

Infectious bronchitis virus (IBV) infection in chickens can lead to an economically important disease, namely, infectious bronchitis (IB). New IBV variants are continuously emerging, which complicates vaccination-based IB control. In this study, five IBVs were isolated from clinical samples submitted to a diagnostic laboratory in Ontario, Canada, and subjected to detailed molecular characterization. Analysis of the spike (S)1 gene showed that these five IBVs were highly related to the Delmarva (DMV/1639) strain (~97.0% nucleotide sequence similarity) that was firstly isolated from an IB outbreak in the Delmarva peninsula, United States of America (USA), in 2011. However, the complete genomic sequence analysis showed a 93.5–93.7% similarity with the Connecticut (Conn) vaccine strain, suggesting that Conn-like viruses contributed to the evolution of the five Canadian IBV/DMV isolates. A SimPlot analysis of the complete genomic sequence showed evidence of recombination for at least three different IBV strains, including a Conn vaccine-like strain, a 4/91 vaccine-like strain, and one strain that is yet-unidentified. The unidentified strain may have contributed the genomic regions of the S, 3, and membrane (M) genes of the five Canadian IBV/DMV isolates. The study outcomes add to the existing knowledge about involvement of recombination in IBV evolution. Full article

The family Parvoviridae includes an ample and most diverse collection of viruses. Exploring the biological diversity and the inherent complexity in these apparently simple viruses has been a continuous commitment for the scientific community since their first discovery more than fifty years ago. The Special Issue of ‘Viruses’ dedicated to the ‘New Insights into Parvovirus Research’ aimed at presenting a ‘state of the art’ in many aspects of research in the field, at collecting the newest contributions on unresolved issues, and at presenting new approaches exploiting systemic (-omic) methodologies. Full article

Traditional pathogenesis studies of alphaviruses involves monitoring survival, viremia, and pathogen dissemination via serial necropsies; however, molecular imaging shifts this paradigm and provides a dynamic assessment of pathogen infection. Positron emission tomography (PET) with PET tracers targeted to study neuroinflammation (N,N-diethyl-2-[4-phenyl]-5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-acetamide, [¹⁸F]DPA-714), apoptosis (caspase-3 substrate, [¹⁸F]CP-18), hypoxia (fluormisonidazole, [¹⁸F]FMISO), blood–brain barrier (BBB) integrity ([¹⁸F]albumin), and metabolism (fluorodeoxyglucose, [¹⁸F]FDG) was performed on C3H/HeN mice infected intranasally with 7000 plaque-forming units (PFU) of Venezuelan equine encephalitis virus (VEEV) TC-83. The main findings are as follows: (1) whole-brain [¹⁸F]DPA-714 and [¹⁸F]CP-18 uptake increased three-fold demonstrating, neuroinflammation and apoptosis, respectively; (2) [¹⁸F]albumin uptake increased by 25% across the brain demonstrating an altered BBB; (3) [¹⁸F]FMISO uptake increased by 50% across the whole brain indicating hypoxic regions; (4) whole-brain [¹⁸F]FDG uptake was unaffected; (5) [¹⁸F]DPA-714 uptake in (a) cortex, thalamus, striatum, hypothalamus, and hippocampus increased through day seven and decreased by day 10 post exposure, (b) olfactory bulb increased at day three, peaked day seven, and decreased day 10, and (c) brain stem and cerebellum increased through day 10. In conclusion, intranasal exposure of C3H/HeN mice to VEEV TC-83 results in both time-dependent and regional increases in brain inflammation, apoptosis, and hypoxia, as well as modest decreases in BBB integrity; however, it has no effect on brain glucose metabolism. Full article

Astroviruses cause disease in a variety of species. Yet, little is known about the epidemiology of a majority of astroviruses including porcine astrovirus type 3 (PoAstV3), which is a putative cause of polioencephalomyelitis in swine. Accordingly, a cross-sectional study was conducted on sow farms with or without reported PoAstV3-associated neurologic disease in growing pigs weaned from those farms. Additionally, a conveniently selected subset of piglets from one farm was selected for gross and histologic evaluation. The distribution of PoAstV3 in the enteric system was evaluated through in situ hybridization. PoAstV3, as detected by RT-qPCR on fecal samples, was frequently detected across sows and piglets (66–90%) on all farms (65–85%). PoAstV3 was detected subsequently at a similar detection frequency (77% vs 85%) on one farm after three months. Viral shedding, as determined by the cycle quantification value, suggests that piglets shed higher quantities of virus than adult swine. No link between gastrointestinal disease and PoAstV3 was found. However, PoAstV3 was detected by in situ in myenteric plexus neurons of piglets elucidating a possible route of spread of the virus from the gastrointestinal tract to the central nervous system. These data suggest PoAstV3 has endemic potential, is shed in the feces at greater quantities by suckling piglets when compared to sows, and infection is widespread on farms in which it is detected. Full article

Hepatitis E virus (HEV) causes predominantly acute and self-limiting hepatitis. However, in HEV-infected pregnant women, the case fatality rate because of fulminant hepatitis can be up to 30%. HEV infection is zoonotic for some genotypes. The HEV genome contains three open reading frames: ORF1 encodes the non-structural polyprotein involved in viral RNA replication; ORF2 encodes the capsid protein; ORF3 encodes a small multifunctional protein. Interferons (IFNs) play a significant role in the early stage of the host antiviral response. In this study, we discovered that the capsid protein antagonizes IFN induction. Mechanistically, the capsid protein blocked the phosphorylation of IFN regulatory factor 3 (IRF3) via interaction with the multiprotein complex consisting of mitochondrial antiviral-signaling protein (MAVS), TANK-binding kinase 1 (TBK1), and IRF3. The N-terminal domain of the capsid protein was found to be responsible for the inhibition of IRF3 activation. Further study showed that the arginine-rich-motif in the N-terminal domain is indispensable for the inhibition as mutations of any of the arginine residues abolished the blockage of IRF3 phosphorylation. These results provide further insight into HEV interference with the host innate immunity. Full article

HBV reactivation (HBVr) can occur due to the ability of HBV to remain latent in the liver as covalently closed circular DNA and by the capacity of HBV to alter the immune system of the infected individuals. HBVr can occur in patients undergoing hematopoietic stem cell transplantation (HSCT) with a clinical spectrum that ranges from asymptomatic infection to fulminant hepatic failure. The risk of HBVr is determined by a complex interplay between host immunity, virus factors, and immunosuppression related to HSCT. All individuals who undergo HSCT should be screened for HBV. HSCT patients positive for HBsAg and also those HBcAb-positive/HBsAg-negative are at high risk of HBV reactivation (HBVr) due to profound and prolonged immunosuppression. Antiviral prophylaxis prevents HBVr, decreases HBVr-related morbidity and mortality in patients with chronic or previous HBV. The optimal duration of antiviral prophylaxis remains to be elucidated. The vaccination of HBV-naïve recipients and their donors against HBV prior to HSCT has an important role in the prevention of acquired HBV infection. This narrative review provides a comprehensive update on the current concepts, risk factors, molecular mechanisms, prevention, and management of HBVr in HSCT. Full article

H5Nx viruses have continuously emerged in the world, causing poultry industry losses and posing a potential public health risk. Here, we studied the phylogeny, pathogenicity, transmission, and immune response of four H5N6 avian influenza viruses in chickens and mice, which were isolated from waterfowl between 2013 and 2014. Their HA genes belong to Clade 2.3.4.4, circulated in China since 2008. Their NA genes fall into N6-like/Eurasian sublineage. Their internal genes originated from different H5N1 viruses. The results suggested that the four H5N6 viruses were reassortants of the H5N1 and H6N6 viruses. They cause lethal infection with high transmission capability in chickens. They also cause mild to severe pathogenicity in mice and can spread to the brain through the blood–brain barrier. During the infection, the viruses result in the up-regulation of PRRs and cytokine in brains and lungs of chickens and mice. Our results suggested that the high viral loads of several organs may result in disease severity in chickens and mice; there were varying levels of cytokines induced by the H5N6 viruses with different pathogenicity in chickens and mice. Full article

There were five outbreaks of H7N9 influenza virus in humans in China since it emerged in 2013, infecting >1000 people. The H7N9 low pathogenic influenza virus was inserted into four amino acids in the HA protein cleavage site to mutate into the H7N9 highly pathogenic virus. This emerging virus caused 15 outbreaks in chickens from the end of 2016 to date. Two H7N9 avian influenza virus (AIV) strains, A/chicken/Guangdong/A46/2013 (LPAIV) and A/chicken/Guangdong/Q29/2017 (HPAIV), were selected to compare the pathogenicity and transmissibility between H7N9 LPAIVs and HPAIVs in chickens. We inoculated 3- to 4-week-old specific-pathogen-free (SPF) chickens with 6 log₁₀EID₅₀/0.1 mL viruses via the ocular-nasal route and co-housed four chickens in each group. The inoculated chicken mortality rate in the A46 and Q29 groups was 1/5 and 5/5, respectively. Q29 virus replication was more efficient compared to the A46 virus in inoculated chickens. Infected chickens initiated viral shedding to naïve contact chickens through respiratory and digestive routes. Both viruses transmitted between chickens by naïve contact, but the Q29 virus had a higher pathogenicity in contact chickens than the A46 virus. Compared with early H7N9 LPAIVs, the pathogenicity and transmissibility of the emerging H7N9 HPAIV was stronger in chickens, indicating that H7N9 influenza virus may continue to threaten human and poultry health. Full article

A novel avian influenza virus (A/wild duck/Korea/K102/2018) (H2N9) was isolated from wild birds in South Korea in 2018, and phylogenetic and molecular analyses were conducted on complete gene sequences obtained by next-generation sequencing. Phylogenetic analysis indicated that the hemagglutinin (HA) and neuraminidase (NA) genes of the A/wild duck/Korea/K102/2018 (H2N9) virus belonged to the Eurasian countries, whereas other internal genes (polymerase basic protein 1 (PB1), PB2, nucleoprotein (NP), polymerase acidic protein (PA), matrix protein (M), and non-structural protein (NS)) belonged to the East Asian countries. A monobasic amino acid (PQIEPR/GLF) at the HA cleavage site, E627 in the PB2 gene, and no deletion of the stalk region in the NA gene indicated that the A/wild duck/Korea/K102/2018 (H2N9) isolate was a typical low pathogenicity avian influenza (LPAI). Nucleotide sequence similarity analysis of HA revealed that the highest homology (98.34%) is to that of A/duck/Mongolia/482/2015 (H2N3), and amino acid sequence of NA was closely related to that of A/duck/Bangladesh/8987/2010 (H10N9) (96.45%). In contrast, internal genes showed homology higher than 98% compared to those of other isolates derived from duck and wild birds of China or Japan in 2016–2018. The newly isolated A/wild duck/Korea/K102/2018 (H2N9) strain is the first reported avian influenza virus in Korea, and may have evolved from multiple genotypes in wild birds and ducks in Mongolia, China, and Japan. Full article

Porcine reproductive and respiratory syndrome virus (PRRSV) is a huge threat to the modern pig industry, and current vaccine prevention strategies could not provide full protection against it. Therefore, exploring new anti-PRRSV strategies is urgently needed. Ginsenoside Rg1, derived from ginseng and notoginseng, is shown to exert anti-inflammatory, neuronal apoptosis-suppressing and anti-oxidant effects. Here we demonstrate Rg1-inhibited PRRSV infection both in Marc-145 cells and porcine alveolar macrophages (PAMs) in a dose-dependent manner. Rg1 treatment affected multiple steps of the PRRSV lifecycle, including virus attachment, replication and release at concentrations of 10 or 50 µM. Meanwhile, Rg1 exhibited broad inhibitory activities against Type 2 PRRSV, including highly pathogenic PRRSV (HP-PRRSV) XH-GD and JXA1, NADC-30-like strain HNLY and classical strain VR2332. Mechanistically, Rg1 reduced mRNA levels of the pro-inflammatory cytokines, including IL-1β, IL-8, IL-6 and TNF-α, and decreased NF-κB signaling activation triggered by PRRSV infection. Furthermore, 4-week old piglets intramuscularly treated with Rg1 after being challenged with the HP-PRRSV JXA1 strain display moderate lung injury, decreased viral load in serum and tissues, and an improved survival rate. Collectively, our study provides research basis and supportive clinical data for using Ginsenoside Rg1 in PRRSV therapies in swine. Full article

The prion-like spreading and accumulation of specific protein aggregates appear to be central to the pathogenesis of many human diseases, including Alzheimer’s and Parkinson’s. Accumulating evidence indicates that inoculation of tissue extracts from diseased individuals into suitable experimental animals can in many cases induce the aggregation of the disease-associated protein, as well as related pathological lesions. These findings, together with the history of the prion field, have raised the questions about whether such disease-associated protein aggregates are transmissible between humans by casual or iatrogenic routes, and, if so, do they propagate enough in the new host to cause disease? These practical considerations are important because real, and perhaps even only imagined, risks of human-to-human transmission of diseases such as Alzheimer’s and Parkinson’s may force costly changes in clinical practice that, in turn, are likely to have unintended consequences. The prion field has taught us that a single protein, PrP, can aggregate into forms that can propagate exponentially in vitro, but range from being innocuous to deadly when injected into experimental animals in ways that depend strongly on factors such as conformational subtleties, routes of inoculation, and host responses. In assessing the hazards posed by various disease-associated, self-propagating protein aggregates, it is imperative to consider both their actual transmissibilities and the pathological consequences of their propagation, if any, in recipient hosts. Full article

Viruses are a highly abundant, dynamic, and diverse component of planktonic communities that have key roles in marine ecosystems. We aimed to reveal the diversity and dynamics of marine large dsDNA viruses infecting algae in the Northern Skagerrak, South Norway through the year by metabarcoding, targeting the major capsid protein (MCP) and its correlation to protist diversity and dynamics. Metabarcoding results demonstrated a high diversity of algal viruses compared to previous metabarcoding surveys in Norwegian coastal waters. We obtained 313 putative algal virus operational taxonomic units (vOTUs), all classified by phylogenetic analyses to either the Phycodnaviridae or Mimiviridae families, most of them in clades without any cultured or environmental reference sequences. The viral community showed a clear temporal variation, with some vOTUs persisting for several months. The results indicate co-occurrences between abundant viruses and potential hosts during long periods. This study gives new insights into the virus-algal host dynamics and provides a baseline for future studies of algal virus diversity and temporal dynamics. Full article

Thermobifida fusca is of biotechnological interest due to its ability to produce an array of plant cell wall hydrolytic enzymes. Nonetheless, only one T. fusca bacteriophage with genome information has been reported to date. This study was aimed at discovering more relevant bacteriophages to expand the existing knowledge of phage diversity for this host species. With this end in view, a thermostable T. fusca bacteriophage P318, which belongs to the Siphoviridae family, was isolated and characterized. P318 has a double-stranded DNA genome of 48,045 base pairs with 3′-extended COS ends, on which 52 putative ORFs are organized into clusters responsible for the order of genome replication, virion morphogenesis, and the regulation of the lytic/lysogenic cycle. In comparison with T. fusca and the previously discovered bacteriophage P1312, P318 has a much lower G+C content in its genome except at the region encompassing ORF42, which produced a protein with unknown function. P1312 and P318 share very few similarities in their genomes except for the regions encompassing ORF42 of P318 and ORF51 of P1312 that are homologous. Thus, acquisition of ORF42 by lateral gene transfer might be an important step in the evolution of P318. Full article

The Asian yellow-legged hornet Vespa velutina nigrithorax, a major predator of honeybees, is spreading in Europe in part due to a lack of efficient control methods. In this study, as a first step to identify biological control agents, we characterized viral RNA sequences present in asymptomatic or symptomatic hornets. Among 19 detected viruses, the honey bee virus Deformed wing virus-B was predominant in all the samples, particularly in muscles from the symptomatic hornet, suggesting a putative cause of the deformed wing symptom. Interestingly, two new viruses closely related to Acyrthosiphon pisum virus and Himetobi P virus and viruses typically associated with honey bees, Acute bee paralysis virus and Black queen cell virus, were detected in the brain and muscles, and may correspond to the circulation and possible replication forms of these viruses in the hornet. Aphid lethal paralysis virus, Bee Macula-like virus, and Moku virus, which are known to infect honey bees, were also identified in the gut virus metagenome of hornets. Therefore, our study underlined the urgent need to study the host range of these newly discovered viruses in hornets to determine whether they represent a new threat for honey bees or a hope for the biocontrol of V. velutina. Full article

The prevalence and variation of the H9N2 avian influenza virus (AIV) pose a threat to public health. A total of eight viruses isolated from farmed poultry in South China during 2017–2018 were selected as representative strains for further systematic study. Phylogenetic analyses indicated that these prevalent viruses belong to the Y280-like lineage and that the internal genes are highly similar to those of recently circulating human H7N9 viruses. The receptor-binding assay showed that most of the H9N2 isolates preferentially bound to the human-like receptor, increasing the risk of them crossing the species barrier and causing human infection. Our in vitro, multi-step growth curve results indicate these viruses can effectively replicate in mammalian cells. Infection in mice showed that three viruses effectively replicated in the lung of mice. Infection in swine revealed that the viruses readily replicated in the upper respiratory tract of pig and effectively induced viral shedding. Our findings suggested that the H9N2 AIVs circulating in poultry recently acquired an enhanced ability to transmit from avian to mammalians, including humans. Based on our findings, we propose that it is essential to strengthen the efforts to surveil and test the pathogenicity of H9N2 AIVs. Full article

RNA viruses are highly successful pathogens and are the causative agents for many important diseases. To fully understand the replication of these viruses it is necessary to address the roles of both positive-strand RNA ((+)RNA) and negative-strand RNA ((−)RNA), and their interplay with viral and host proteins. Here we used branched DNA (bDNA) fluorescence in situ hybridization (FISH) to stain both the abundant (+)RNA and the far less abundant (−)RNA in both hepatitis C virus (HCV)- and Zika virus-infected cells, and combined these analyses with visualization of viral proteins through confocal imaging. We were able to phenotypically examine HCV-infected cells in the presence of uninfected cells and revealed the effect of direct-acting antivirals on HCV (+)RNA, (−)RNA, and protein, within hours of commencing treatment. Herein, we demonstrate that bDNA FISH is a powerful tool for the study of RNA viruses that can provide insights into drug efficacy and mechanism of action. Full article

Background: Rhinovirus C is an important pathogen of asthmatic and non-asthmatic children hospitalised with episodic wheeze. Previous studies on other respiratory viruses have shown that several host cytokines correlate with duration of hospitalisation, but this has yet to be investigated in children with RV-C infection. We determined the nasal cytokine profiles of these children and investigated their relationship with RV-C load and clinical outcome. Flocked nasal swabs were collected from children aged 24–72 months presenting to the Emergency Department at Princess Margaret Hospital with a clinical diagnosis of acute wheeze and an acute upper respiratory tract viral infection. RV-C load was determined by quantitative RT-PCR and cytokine profiles were characterised by a commercial human cytokine 34-plex panel. RV-C was the most commonly detected virus in pre-school-aged children hospitalised with an episodic wheeze. RV-C load did not significantly differ between asthmatic and non-asthmatic patients. Both groups showed a Th2-based cytokine profile. However, Th17 response cytokines IL-17 and IL-1β were only elevated in RV-C-infected children with pre-existing asthma. Neither RV-C load nor any specific cytokines were associated illness severity in this study. Medically attended RV-C-induced wheeze is characterised by a Th2 inflammatory pattern, independent of viral load. Any therapeutic interventions should be aimed at modulating the host response following infection. Full article

Human enteroviruses and human parechoviruses are associated with a broad range of diseases and even severe and fatal conditions. For human cosaviruses, the etiological role is yet unknown. Little is known about the circulation of non-polio enteroviruses, human parechoviruses, and human cosaviruses in Nigeria. A total of 113 stool samples were collected from healthy individuals in Osun State between February 2016 and May 2017. RT-PCR assays targeting the 5′ non-coding region (5′ -NCR) were used to screen for human enteroviruses, human parechoviruses, and human cosaviruses. For human enteroviruses, species-specific RT-PCR assays targeting the VP1 regions were used for molecular typing. Inoculation was carried out on RD-A, CaCo-2, HEp-2C, and L20B cell lines to compare molecular and virological assays. Ten samples tested positive for enterovirus RNA with 11 strains detected, including CV-A13 (n = 3), E-18 (n = 2), CV-A20 (n = 1), CV-A24 (n = 1), EV-C99 (n = 1), and EV-C116 (n = 2). Three samples tested positive for human parechovirus RNA, and full genome sequencing on two samples allowed assignment to a new Parechovirus A type (HPeV-19). Thirty-three samples tested positive for cosavirus with assignment to species Cosavirus D and Cosavirus A based on the 5′-NCR region. Screening of stool samples collected from healthy individuals in Nigeria in 2016 and 2017 revealed a high diversity of circulating human enteroviruses, human parechoviruses, and human cosaviruses. Molecular assays for genotyping showed substantial benefits compared with those of cell-culture assays. Full article

Typical viral propagation involves sequential viral entry, uncoating, replication, gene transcription and protein synthesis, and virion assembly and release. Some viral proteins must be transported into host nucleus to facilitate viral propagation, which is essential for the production of mature virions. During the transport process, nuclear localization signals (NLSs) play an important role in guiding target proteins into nucleus through the nuclear pore. To date, some classical nuclear localization signals (cNLSs) and non-classical NLSs (ncNLSs) have been identified in a number of viral proteins. These proteins are involved in viral replication, expression regulation of viral genes and virion assembly. Moreover, other proteins are transported into nucleus with unknown mechanisms. This review highlights our current knowledge about the nuclear trafficking of cellular proteins associated with viral propagation. Full article

Analyses of pospiviroids in commercial seed lots of tomato and capsicum, determined by testing of 12,000 to 40,000 seeds per lot, have enabled the development of empirically-derived distribution curves for the observed prevalences of viroids in those commodities. Those distribution curves can be considered in conjunction with statistically-based estimates of detection that would be achieved using other sample sizes. Statistical calculations using binomial distributions show that sample sizes of 3000 and 9400 seeds allow detection of viroid prevalences as low as 0.1% and 0.032%, respectively, with 95% confidence. Applying those calculations to observed viroid prevalences in contaminated tomato seed lots, it is estimated that the use of sample sizes of 3000 and 9400 seeds would detect 15% and 42%, respectively, of the contaminated seed lots identified using the larger sample sizes of approximately 20,000 seeds reported in this study. It is concluded that the higher costs associated with testing of larger sample sizes represent a worthwhile investment in agricultural biosecurity. Full article

Mosquitoes harbor an extensive diversity of ‘insect-specific’ RNA viruses in addition to those important to human and animal health. However, because most studies of the mosquito virome have been conducted at lower latitudes, little is known about the diversity and evolutionary history of RNA viruses sampled from mosquitoes in northerly regions. Here, we compared the RNA virome of two common northern mosquito species, Culex pipiens and Culex torrentium, collected in south-central Sweden. Following bulk RNA-sequencing (meta-transcriptomics) of 12 libraries, comprising 120 specimens of Cx. pipiens and 150 specimens of Cx. torrentium, we identified 40 viruses (representing 14 virus families) of which 28 were novel based on phylogenetic analysis of the RNA-dependent RNA polymerase (RdRp) protein. Hence, we documented similar levels of virome diversity as in mosquitoes sampled from the more biodiverse lower latitudes. Many viruses were also related to those sampled on other continents, indicative of a widespread global movement and/or long host–virus co-evolution. Although the two mosquito species investigated have overlapping geographical distributions and share many viruses, several viruses were only found at a specific location at this scale of sampling, such that local habitat and geography may play an important role in shaping viral diversity in Culex mosquitoes. Full article

Statistical phylogenetic methods are a powerful tool for inferring the evolutionary history of viruses through time and space. The selection of mathematical models and analysis parameters has a major impact on the outcome, and has been relatively well-described in the literature. The preparation of a sequence dataset is less formalized, but its impact can be even more profound. This article used simulated datasets of enterovirus sequences to evaluate the effect of sample bias on picornavirus phylogenetic studies. Possible approaches to the reduction of large datasets and their potential for introducing additional artefacts were demonstrated. The most consistent results were obtained using “smart sampling”, which reduced sequence subsets from large studies more than those from smaller ones in order to preserve the rare sequences in a dataset. The effect of sequences with technical or annotation errors in the Bayesian framework was also analyzed. Sequences with about 0.5% sequencing errors or incorrect isolation dates altered by just 5 years could be detected by various approaches, but the efficiency of identification depended upon sequence position in a phylogenetic tree. Even a single erroneous sequence could profoundly destabilize the whole analysis by increasing the variance of the inferred evolutionary parameters. Full article

Respiratory Syncytial Virus (RSV) is a very important viral pathogen in children, immunocompromised and cardiopulmonary diseased patients and the elderly. Most of the published research with RSV was performed on RSV Long and RSV A2, isolated in 1956 and 1961, yet recent RSV isolates differ from these prototype strains. Additionally, these viruses have been serially passaged in cell culture, which may result in adaptations that affect virus–host interactions. We have isolated RSV from mucosal secretions of 12 patients in the winters 2016–2017 and 2017–2018, of which eight RSV-A subtypes and four RSV-B subtypes. Passage 3 of the isolates was assessed for viral replication kinetics and infectious virus production in HEp-2, A549 and BEAS-2B cells, thermal stability at 37 °C, 32 °C and 4 °C, syncytia formation, neutralization by palivizumab and mucin mRNA expression in infected A549 cells. We observed that viruses isolated in one RSV season show differences on the tested assays. Furthermore, comparison with RSV A2 and RSV B1 reveals for some RSV isolates differences in viral replication kinetics, thermal stability and fusion capacity. Major differences are, however, not observed and differences between the recent isolates and reference strains is, overall, similar to the observed variation in between the recent isolates. One clinical isolate (BE/ANT-A11/17) replicated very efficiently in all cell lines, and remarkably, even better than RSV A2 in the HEp-2 cell line. Full article

Porcine deltacoronavirus (PDCoV) was first identified in Hong Kong in 2012 from samples taken from pigs in 2009. PDCoV was subsequently identified in the USA in 2014 in pigs with a history of severe diarrhea. The virus has now been detected in pigs in several countries around the world. Following the development of tissue culture adapted strains of PDCoV, it is now possible to address questions regarding virus–host cell interactions for this genera of coronavirus. Here, we presented a detailed study of PDCoV-induced replication organelles. All positive-strand RNA viruses induce the rearrangement of cellular membranes during virus replication to support viral RNA synthesis, forming the replication organelle. Replication organelles for the Alpha-, Beta-, and Gammacoronavirus genera have been characterized. All coronavirus genera induced the formation of double-membrane vesicles (DMVs). In addition, Alpha- and Betacoronaviruses induce the formation of convoluted membranes, while Gammacoronaviruses induce the formation of zippered endoplasmic reticulum (ER) with tethered double-membrane spherules. However, the structures induced by Deltacoronaviruses, particularly the presence of convoluted membranes or double-membrane spherules, are unknown. Initially, the dynamics of PDCoV strain OH-FD22 replication were assessed with the onset of viral RNA synthesis, protein synthesis, and progeny particle release determined. Subsequently, virus-induced membrane rearrangements were identified in infected cells by electron microscopy. As has been observed for all other coronaviruses studied to date, PDCoV replication was found to induce the formation of double-membrane vesicles. Significantly, however, PDCoV replication was also found to induce the formation of regions of zippered endoplasmic reticulum, small associated tethered vesicles, and double-membrane spherules. These structures strongly resemble the replication organelle induced by avian Gammacoronavirus infectious bronchitis virus. Full article

Saint Louis encephalitis virus (SLEV) is a mosquito-borne flavivirus that occurs throughout the Americas, and is considered a public health threat. In Brazil, SLEV has been detected from human cases associated with dengue-like disease, but no neurological symptoms were reported. Furthermore, the epidemiology of SLEV in human populations is still poorly explored in the country. We reported serological and molecular detection of SLEV in a healthy population of equids and humans from rural areas in Southeast Brazil. A plaque reduction neutralization test was applied, and neutralizing antibodies were detected in 11 individuals (4.6%) and 60 horses (21.5%). A qPCR targeting the 5′UTR region and reverse transcription-PCR (RT-PCR) targeting the non-structural protein (NS5) gene were performed and three individuals tested positive in both assays. Subsequent phylogenetic analysis confirmed SLEV circulation and its findings suggest the occurrence of an asymptomatic or subclinical presence in human and animal cases, correlating with the risks for outbreaks and consequently burden of SLEV infections to public health. Preventive strategies should include improved surveillance in regions with a high probability of SLEV occurrence, improvement in diagnostic methods, and evaluation of exposure/risk factors that can favor SLEV emergence. Full article

Influenza A viruses (IAVs) are important animal and human emerging and re-emerging pathogens that are responsible for yearly seasonal epidemics and sporadic pandemics. IAVs cause a wide range of clinical illnesses, from relatively mild infections by seasonal strains, to acute respiratory distress during infections with highly pathogenic avian IAVs (HPAI). For this study, we infected A549 human lung cells with lab prototype A/PR/8/34 (H1N1) (PR8), a seasonal H1N1 (RV733), the 2009 pandemic H1N1 (pdm09), or with two avian strains, an H5N1 HPAI strain or an H7N9 strain that has low pathogenicity in birds but high pathogenicity in humans. We used a newly-developed aptamer-based multiplexed technique (SOMAscan^®) to examine >1300 human lung cell proteins affected by the different IAV strains, and identified more than 500 significantly dysregulated cellular proteins. Our analyses indicated that the avian strains induced more profound changes in the A549 global proteome compared to all tested low-pathogenicity H1N1 strains. The PR8 strain induced a general activation, primarily by upregulating many immune molecules, the seasonal RV733 and pdm09 strains had minimal effect upon assayed molecules, and the avian strains induced significant downregulation, primarily in antimicrobial response, cardiovascular and post-translational modification systems. Full article

Metagenomic studies of mosquitoes have revealed that their virome is far more diverse and includes many more viruses than just the pathogenic arboviruses vectored by mosquitoes. In this study, the virome of 953 female mosquitoes collected in the summer of 2017, representing six mosquito species from two geographic locations in Mid-Eastern Sweden, were characterized. In addition, the near-complete genome of nine RNA viruses were characterized and phylogenetically analysed. These viruses showed association to the viral orders Bunyavirales, Picornavirales, Articulavirales, and Tymovirales, and to the realm Ribovira. Hence, through this study, we expand the knowledge of the virome composition of different mosquito species in Sweden. In addition, by providing viral reference genomes from wider geographic regions and different mosquito species, future in silico recognition and assembly of viral genomes in metagenomic datasets will be facilitated. Full article

The transmission of Macacine alphaherpesvirus 1 (McHV-1) from macaques, the natural host, to humans causes encephalitis. In contrast, human infection with Cercopithecine alphaherpesvirus 2 (CeHV-2), a closely related alphaherpesvirus from African vervet monkeys and baboons, has not been reported and it is believed that CeHV-2 is apathogenic in humans. The reasons for the differential neurovirulence of McHV-1 and CeHV-2 have not been explored on a molecular level, in part due to the absence of systems for the production of recombinant viruses. Here, we report the generation of a fosmid-based system for rescue of recombinant CeHV-2. Moreover, we show that, in this system, recombineering can be used to equip CeHV-2 with reporter genes. The recombinant CeHV-2 viruses replicated with the same efficiency as uncloned, wt virus and allowed the identification of cell lines that are highly susceptible to CeHV-2 infection. Collectively, we report a system that allows rescue and genetic modification of CeHV-2 and likely other alphaherpesviruses. This system should aid future analysis of CeHV-2 biology. Full article