Viruses — Open Access Journal

The Australasian Virology Society (AVS) aims to promote, support and advocate for the discipline of virology in the Australasian region. The society was incorporated in 2011 after 10 years operating as the Australian Virology Group (AVG) founded in 2001, coinciding with the inaugural biennial scientific meeting. AVS conferences aim to provide a forum for the dissemination of all aspects of virology, foster collaboration, and encourage participation by students and post-doctoral researchers. The tenth Australasian Virology Society (AVS10) scientific meeting was held on 2–5 December 2019 in Queenstown, New Zealand. This report highlights the latest research presented at the meeting, which included cutting-edge virology presented by our international plenary speakers Ana Fernandez-Sesma and Benjamin tenOever, and keynote Richard Kuhn. AVS10 honoured female pioneers in Australian virology, Lorena Brown and Barbara Coulson. We report outcomes from the AVS10 career development session on “Successfully transitioning from post-doc to lab head”, winners of best presentation awards, and the AVS gender equity policy, initiated in 2013. Plans for the 2021 meeting are underway which will celebrate the 20th anniversary of AVS where it all began, in Fraser Island, Queensland, Australia. Full article

YerA41 is a Myoviridae bacteriophage that was originally isolated due its ability to infect Yersinia ruckeri bacteria, the causative agent of enteric redmouth disease of salmonid fish. Several attempts to determine its genomic DNA sequence using traditional and next generation sequencing technologies failed, indicating that the phage genome is modified in such a way that it is an unsuitable template for PCR amplification and for conventional sequencing. To determine the YerA41 genome sequence, we performed RNA-sequencing from phage-infected Y. ruckeri cells at different time points post-infection. The host-genome specific reads were subtracted and de novo assembly was performed on the remaining unaligned reads. This resulted in nine phage-specific scaffolds with a total length of 143 kb that shared only low level and scattered identity to known sequences deposited in DNA databases. Annotation of the sequences revealed 201 predicted genes, most of which found no homologs in the databases. Proteome studies identified altogether 63 phage particle-associated proteins. The RNA-sequencing data were used to characterize the transcriptional control of YerA41 and to investigate its impact on the bacterial gene expression. Overall, our results indicate that RNA-sequencing can be successfully used to obtain the genomic sequence of non-sequencable phages, providing simultaneous information about the phage–host interactions during the process of infection. Full article

Noroviruses are a leading cause of gastroenteritis worldwide. Although infections in healthy individuals are self-resolving, immunocompromised individuals are at risk for chronic disease and severe complications. Chronic norovirus infections in immunocompromised hosts are often characterized by long-term virus shedding, but it is unclear whether this shed virus remains infectious. We investigated the prevalence, genetic heterogeneity, and temporal aspects of norovirus infections in 1140 patients treated during a 6-year period at a pediatric research hospital. Additionally, we identified 20 patients with chronic infections lasting 37 to >418 days. Using a new human norovirus in vitro assay, we confirmed the continuous shedding of infectious virus for the first time. Shedding lasted longer in male patients and those with diarrheal symptoms. Prolonged shedding of infectious norovirus in immunocompromised hosts can potentially increase the likelihood of transmission, highlighting the importance of isolation precautions to prevent nosocomial infections. Full article

Protein-shelled viruses have been thought as “tin cans” that merely carry the genomic cargo from cell to cell. However, through the years, it has become clear that viruses such as rhinoviruses and caliciviruses are active and dynamic structures waiting for the right environmental cues to deliver their genomic payload to the host cell. In the case of human rhinoviruses, the capsid has empty cavities that decrease the energy required to cause conformational changes, resulting in the capsids “breathing”, waiting for the moment when the receptor binds for it to release its genome. Most strikingly, the buried N-termini of VP1 and VP4 are transiently exposed during this process. A more recent example of a “living” protein capsid is mouse norovirus (MNV). This family of viruses have a large protruding (P) domain that is loosely attached to the shell via a single-polypeptide tether. Small molecules found in the gut, such as bile salts, cause the P domains to rotate and collapse onto the shell surface. Concomitantly, bile alters the conformation of the P domain itself from one that binds antibodies to one that recognizes receptors. In this way, MNV appears to use capsid flexibility to present one face to the immune system and a completely different one to attack the host tissue. Therefore, it appears that even protein-shelled viruses have developed an impressive array of tricks to dodge our immune system and efficiently attack the host. Full article

Higher accessibility and decreasing costs of next generation sequencing (NGS), availability of commercial kits, and development of dedicated analysis pipelines, have allowed an increasing number of laboratories to adopt this technology for HIV drug resistance (HIVDR) genotyping. Conventional HIVDR genotyping is traditionally carried out using population-based Sanger sequencing, which has a limited capacity for reliable detection of variants present at intra-host frequencies below a threshold of approximately 20%. NGS has the potential to improve sensitivity and quantitatively identify low-abundance variants, improving efficiency and lowering costs. However, some challenges exist for the standardization and quality assurance of NGS-based HIVDR genotyping. In this paper, we highlight considerations of these challenges as related to laboratory, clinical, and implementation of NGS for HIV drug resistance testing. Several sources of variation and bias occur in each step of the general NGS workflow, i.e., starting material, sample type, PCR amplification, library preparation method, instrument and sequencing chemistry-inherent errors, and data analysis options and limitations. Additionally, adoption of NGS-based HIVDR genotyping, especially for clinical care, poses pressing challenges, especially for resource-poor settings, including infrastructure and equipment requirements and cost, logistic and supply chains, instrument service availability, personnel training, validated laboratory protocols, and standardized analysis outputs. The establishment of external quality assessment programs may help to address some of these challenges and is needed to proceed with NGS-based HIVDR genotyping adoption. Full article

Infections due to arboviruses (arthropod-borne viruses) have dramatically increased worldwide during the last few years. In humans, symptoms associated with acute infection of most arboviruses are often described as “dengue-like syndrome”, including fever, rash, conjunctivitis, arthralgia, and muscular symptoms such as myalgia, myositis, or rhabdomyolysis. In some cases, muscular symptoms may persist over months, especially following flavivirus and alphavirus infections. However, in humans the cellular targets of infection in muscle have been rarely identified. Animal models provide insights to elucidate pathological mechanisms through studying viral tropism, viral-induced inflammation, or potential viral persistence in the muscle compartment. The tropism of arboviruses for muscle cells as well as the viral-induced cytopathic effect and cellular alterations can be confirmed in vitro using cellular models. This review describes the link between muscle alterations and arbovirus infection, and the underlying mechanisms. Full article

African swine fever (ASF) is a devastating disease in pigs, with no vaccines for control. The genetic manipulation of African swine fever virus (ASFV) is often tedious and time consuming. Here, we describe a method to manipulate the virus genome to produce gene deletion viruses in a much-reduced time. This method combines the conventional homologous recombination with fluorescent-activated cells sorting (FACS), to isolate and purify viruses expressing fluorescent reporter genes. With three rounds of single cell isolation via FACS and two rounds of limiting dilution, we deleted two additional genes, EP153R and EP402R, from Benin 97/1 ASFV lacking the DP148R gene. By combining different fluorescent markers, this method has the potential to greatly facilitate studies on understanding ASFV gene functions and develop candidate live-attenuated vaccines. Full article

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a member of the betacoronavirus family, which causes COVID-19 disease. SARS-CoV-2 pathogenicity in humans leads to increased mortality rates due to alterations of significant pathways, including some resulting in exacerbated inflammatory responses linked to the “cytokine storm” and extensive lung pathology, as well as being linked to a number of comorbidities. Our current study compared five SARS-CoV-2 sequences from different geographical regions to those from SARS, MERS and two cold viruses, OC43 and 229E, to identify the presence of miR-like sequences. We identified seven key miRs, which highlight considerable differences between the SARS-CoV-2 sequences, compared with the other viruses. The level of conservation between the five SARS-CoV-2 sequences was identical but poor compared with the other sequences, with SARS showing the highest degree of conservation. This decrease in similarity could result in reduced levels of transcriptional control, as well as a change in the physiological effect of the virus and associated host-pathogen responses. MERS and the milder symptom viruses showed greater differences and even significant sequence gaps. This divergence away from the SARS-CoV-2 sequences broadly mirrors the phylogenetic relationships obtained from the whole-genome alignments. Therefore, patterns of mutation, occurring during sequence divergence from the longer established human viruses to the more recent ones, may have led to the emergence of sequence motifs that can be related directly to the pathogenicity of SARS-CoV-2. Importantly, we identified 7 key-microRNAs (miRs 8066, 5197, 3611, 3934-3p, 1307-3p, 3691-3p, 1468-5p) with significant links to KEGG pathways linked to viral pathogenicity and host responses. According to Bioproject data (PRJNA615032), SARS-CoV-2 mediated transcriptomic alterations were similar to the target pathways of the selected 7 miRs identified in our study. This mechanism could have considerable significance in determining the symptom spectrum of future potential pandemics. KEGG pathway analysis revealed a number of critical pathways linked to the seven identified miRs that may provide insight into the interplay between the virus and comorbidities. Based on our reported findings, miRNAs may constitute potential and effective therapeutic approaches in COVID-19 and its pathological consequences. Full article

The discovery of highly divergent RNA viruses is compromised by their limited sequence similarity to known viruses. Evolutionary information obtained from protein structural modelling offers a powerful approach to detect distantly related viruses based on the conservation of tertiary structures in key proteins such as the RNA-dependent RNA polymerase (RdRp). We utilised a template-based approach for protein structure prediction from amino acid sequences to identify distant evolutionary relationships among viruses detected in meta-transcriptomic sequencing data from Australian wildlife. The best predicted protein structural model was compared with the results of similarity searches against protein databases. Using this combination of meta-transcriptomics and protein structure prediction we identified the RdRp (PB1) gene segment of a divergent negative-sense RNA virus, denoted Lauta virus (LTAV), in a native Australian gecko (Gehyra lauta). The presence of this virus was confirmed by PCR and Sanger sequencing. Phylogenetic analysis revealed that Lauta virus likely represents a newly described genus within the family Amnoonviridae, order Articulavirales, that is most closely related to the fish virus Tilapia tilapinevirus (TiLV). These findings provide important insights into the evolution of negative-sense RNA viruses and structural conservation of the viral replicase among members of the order Articulavirales. Full article

Members of Picornaviridae and of the Hepacivirus, Pegivirus and Pestivirus genera of Flaviviridae all contain an internal ribosomal entry site (IRES) in the 5′-untranslated region (5′UTR) of their genomes. Each class of IRES has a conserved structure and promotes 5′-end-independent initiation of translation by a different mechanism. Picornavirus 5′UTRs, including the IRES, evolve independently of other parts of the genome and can move between genomes, most commonly by intratypic recombination. We review accumulating evidence that IRESs are genetic entities that can also move between members of different genera and even between families. Type IV IRESs, first identified in the Hepacivirus genus, have subsequently been identified in over 25 genera of Picornaviridae, juxtaposed against diverse coding sequences. In several genera, members have either type IV IRES or an IRES of type I, II or III. Similarly, in the genus Pegivirus, members contain either a type IV IRES or an unrelated type; both classes of IRES also occur in members of the genus Hepacivirus. IRESs utilize different mechanisms, have different factor requirements and contain determinants of viral growth, pathogenesis and cell type specificity. Their dissemination between viruses by horizontal gene transfer has unexpectedly emerged as an important facet of viral evolution. Full article

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory illness in humans; the second-largest and most deadly outbreak to date occurred in Saudi Arabia. The dromedary camel is considered a possible host of the virus and also to act as a reservoir, transmitting the virus to humans. Here, we studied evolutionary relationships for 31 complete genomes of betacoronaviruses, including eight newly sequenced MERS-CoV genomes isolated from dromedary camels in Saudi Arabia. Through bioinformatics tools, we also used available sequences and 3D structure of MERS-CoV spike glycoprotein to predict MERS-CoV epitopes and assess antibody binding affinity. Phylogenetic analysis showed the eight new sequences have close relationships with existing strains detected in camels and humans in Arabian Gulf countries. The 2019-nCov strain appears to have higher homology to both bat coronavirus and SARS-CoV than to MERS-CoV strains. The spike protein tree exhibited clustering of MERS-CoV sequences similar to the complete genome tree, except for one sequence from Qatar (KF961222). B cell epitope analysis determined that the MERS-CoV spike protein has 24 total discontinuous regions from which just six epitopes were selected with score values of >80%. Our results suggest that the virus circulates by way of camels crossing the borders of Arabian Gulf countries. This study contributes to finding more effective vaccines in order to provide long-term protection against MERS-CoV and identifying neutralizing antibodies. Full article

Viruses alter a multitude of host-cell processes to create a more optimal environment for viral replication. This includes altering metabolism to provide adequate substrates and energy required for replication. Typically, viral infections induce a metabolic phenotype resembling the Warburg effect, with an upregulation of glycolysis and a concurrent decrease in cellular respiration. Human adenovirus (HAdV) has been observed to induce the Warburg effect, which can be partially attributed to the adenovirus protein early region 4, open reading frame 1 (E4orf1). E4orf1 regulates a multitude of host-cell processes to benefit viral replication and can influence cellular metabolism through the transcription factor avian myelocytomatosis viral oncogene homolog (MYC). However, E4orf1 does not explain the full extent of Warburg-like HAdV metabolic reprogramming, especially the accompanying decrease in cellular respiration. The HAdV protein early region 1A (E1A) also modulates the function of the infected cell to promote viral replication. E1A can interact with a wide variety of host-cell proteins, some of which have been shown to interact with metabolic enzymes independently of an interaction with E1A. To determine if the HAdV E1A proteins are responsible for reprogramming cell metabolism, we measured the extracellular acidification rate and oxygen consumption rate of A549 human lung epithelial cells with constitutive endogenous expression of either of the two major E1A isoforms. This was followed by the characterization of transcript levels for genes involved in glycolysis and cellular respiration, and related metabolic pathways. Cells expressing the 13S encoded E1A isoform had drastically increased baseline glycolysis and lower maximal cellular respiration than cells expressing the 12S encoded E1A isoform. Cells expressing the 13S encoded E1A isoform exhibited upregulated expression of glycolysis genes and downregulated expression of cellular respiration genes. However, tricarboxylic acid cycle genes were upregulated, resembling anaplerotic metabolism employed by certain cancers. Upregulation of glycolysis and tricarboxylic acid cycle genes was also apparent in IMR-90 human primary lung fibroblast cells infected with a HAdV-5 mutant virus that expressed the 13S, but not the 12S encoded E1A isoform. In conclusion, it appears that the two major isoforms of E1A differentially influence cellular glycolysis and oxidative phosphorylation and this is at least partially due to the altered regulation of mRNA expression for the genes in these pathways. Full article

Combination antiretroviral therapy (cART) is successful in maintaining undetectable levels of HIV in the blood; however, the persistence of latent HIV reservoirs has become the major barrier for a HIV cure. Substantial efforts are underway in finding the best latency-reversing agents (LRAs) to purge the latent viruses from the reservoirs. We hypothesize that identifying the right combination of LRAs will be the key to accomplishing that goal. In this study, we evaluated the effect of combinations of three protein kinase C activators (prostratin, (-)-indolactam V, and TPPB) with four histone deacetylase inhibitors (AR-42, PCI-24781, givinostat, and belinostat) on reversing HIV latency in different cell lines including in a primary CD4+ T-cell model. Combinations including indolactam and TPPB with AR-42 and PCI produced a strong synergistic effect in reactivating latent virus as indicated by higher p24 production and envelope gp120 expression. Furthermore, treatment with TPPB and indolactam greatly downregulated the cellular receptor CD4. Indolactam/AR-42 combination emerged from this study as the best combination that showed a strong synergistic effect in reactivating latent virus. Although AR-42 alone did not downregulate CD4 expression, indolactam/AR-42 showed the most efficient downregulation. Our results suggest that indolactam/AR-42 is the most effective combination, showing a strong synergistic effect in reversing HIV latency combined with the most efficient CD4 downregulation. Full article

Here, we used a mouse model with defective autophagy to further decipher the role of Beclin1 in the infection and disease of Zika virus (ZIKV)-R103451. Hemizygous (Becn1^+/−) and wild-type (Becn1^+/+) pregnant mice were transiently immunocompromised using the anti-interferon alpha/beta receptor subunit 1 monoclonal antibody MAR1-5A3. Despite a low mortality rate among the infected dams, 25% of Becn1^+/− offspring were smaller in size and had smaller, underdeveloped brains. This phenotype became apparent after 2-to 3-weeks post-birth. Furthermore, the smaller-sized pups showed a decrease in the mRNA expression levels of insulin-like growth factor (IGF)-1 and the expression levels of several microcephaly associated genes, when compared to their typical-sized siblings. Neuronal loss was also noticeable in brain tissues that were removed postmortem. Further analysis with murine mixed glia, derived from ZIKV-infected Becn1^+/− and Becn1^+/+ pups, showed greater infectivity in glia derived from the Becn1^+/− genotype, along with a significant increase in pro-inflammatory molecules. In the present study, we identified a link by which defective autophagy is causally related to increased inflammatory molecules, reduced growth factor, decreased expression of microcephaly-associated genes, and increased neuronal loss. Specifically, we showed that a reduced expression of Beclin1 aggravated the consequences of ZIKV infection on brain development and qualifies Becn1 as a susceptibility gene of ZIKV congenital syndrome. Full article

Transmission of honey bee viruses to other insects, and vice versa, has previously been reported and the true ecological importance of this phenomenon is still being realized. Members of the family Vespidae interact with honey bees via predation or through the robbing of brood or honey from colonies, and these activities could result in virus transfer. In this study we screened Vespa velutina and Vespa crabro collected from Europe and China and also honey bees and Vespula vulgaris from the UK for Moku virus (MV), an Iflavirus first discovered in the predatory social wasp Vespula pensylvanica in Hawaii. MV was found in 71% of Vespula vulgaris screened and was also detected in UK Vespa crabro. Only seven percent of Vespa velutina individuals screened were MV-positive and these were exclusively samples from Jersey. Of 69 honey bee colonies screened, 43% tested positive for MV. MV replication was confirmed in Apis mellifera and Vespidae species, being most frequently detected in Vespula vulgaris. MV sequences from the UK were most similar to MV from Vespula pensylvanica compared to MV from Vespa velutina in Belgium. The implications of the transfer of viruses between the Vespidae and honey bees are discussed. Full article

As part of research and wildlife disease surveillance efforts, we performed necropsy examinations of 125 free-ranging (n = 114) and captive (n = 11) prairie dogs in Colorado from 2009 to 2017. From these cases, we identified three cases of thymic lymphoma in free-ranging Gunnison’s prairie dogs (Cynomys gunnisoni), and we identified a novel retroviral sequence associated with these tumors. The viral sequence is 7700 nucleotides in length and exhibits a genetic organization that is consistent with the characteristics of a type D betaretrovirus. The proposed name of this virus is Gunnison’s prairie dog retrovirus (GPDRV). We screened all 125 prairie dogs for the presence of GPDRV using PCR with envelope-specific primers and DNA extracted from spleen samples. Samples were from Gunnison’s prairie dogs (n = 59), black-tailed prairie dogs (Cynomys ludovicianus) (n = 40), and white-tailed prairie dogs (Cynomys leucurus) (n = 26). We identified GPDRV in a total of 7/125 (5.6%) samples including all three of the prairie dogs with thymic lymphoma, as well as spleen from an additional four Gunnison’s prairie dogs with no tumors recognized at necropsy. None of the GPDRV-negative Gunnison’s prairie dogs had thymic lymphomas. We also identified a related, apparently endogenous retroviral sequence in all prairie dog samples. These results suggest that GPDRV infection may lead to development of thymic lymphoma in Gunnison’s prairie dogs. Full article

Between 2017 and 2018, several farms across Bulgaria reported outbreaks of H5 highly-pathogenic avian influenza (HPAI) viruses. In this study we used genomic and traditional epidemiological analyses to trace the origin and subsequent spread of these outbreaks within Bulgaria. Both methods indicate two separate incursions, one restricted to the northeastern region of Dobrich, and another largely restricted to Central and Eastern Bulgaria including places such as Plovdiv, Sliven and Stara Zagora, as well as one virus from the Western region of Vidin. Both outbreaks likely originate from different European 2.3.4.4b virus ancestors circulating in 2017. The viruses were likely introduced by wild birds or poultry trade links in 2017 and have continued to circulate, but due to lack of contemporaneous sampling and sequences from wild bird viruses in Bulgaria, the precise route and timing of introduction cannot be determined. Analysis of whole genomes indicates a complete lack of reassortment in all segments but the matrix protein gene (MP), which presents as multiple smaller clusters associated with different European 2.3.4.4b viruses. Ancestral reconstruction of host states of the hemagglutinin (HA) gene of viruses involved in the outbreaks suggests that transmission is driven by domestic ducks into galliform poultry. Thus, according to present evidence, we suggest the surveillance of domestic ducks as they are an epidemiologically relevant species for subclinical infection. Monitoring the spread due to movement between farms within regions and links to poultry production systems in European countries can help to predict and prevent future outbreaks. The 2.3.4.4b lineage which caused the largest recorded poultry epidemic in Europe continues to circulate, and the risk of further transmission by wild birds during migration remains. Full article

Acinetobacter baumannii is an opportunistic pathogen that presents a serious clinical challenge due to its increasing resistance to all available antibiotics. Phage therapy has been introduced recently to treat antibiotic-incurable A. baumannii infections. In search for new A. baumannii specific bacteriophages, 20 clinical A. baumannii strains were used in two pools in an attempt to enrich phages from sewage. The enrichment resulted in induction of resident prophage(s) and three temperate bacteriophages, named vB_AbaS_fEg-Aba01, vB_AbaS_fLi-Aba02 and vB_AbaS_fLi-Aba03, all able to infect only one strain (#6597) of the 20 clinical strains, were isolated. Morphological characteristics obtained by transmission electron microscopy together with the genomic information revealed that the phages belong to the family Siphoviridae. The ca. 35 kb genomic sequences of the phages were >99% identical to each other. The linear ds DNA genomes of the phages contained 10 nt cohesive end termini, 52-54 predicted genes, an attP site and one tRNA gene each. A database search revealed an >99% identical prophage in the genome of A. baumannii strain AbPK1 (acc. no. CP024576.1). Over 99% identical prophages were also identified from two of the original 20 clinical strains (#5707 and #5920) and both were shown to be spontaneously inducible, thus very likely being the origins of the isolated phages. The phage vB_AbaS_fEg-Aba01 was also able to lysogenize the susceptible strain #6597 demonstrating that it was fully functional. The phages showed a very narrow host range infecting only two A. baumannii strains. In conclusion, we have isolated and characterized three novel temperate Siphoviridae phages that infect A. baumannii. Full article

Dengue is an arboviral disease caused by dengue virus (DENV), which is transmitted to humans by Aedes aegypti mosquitoes. Infection by DENV most commonly results in a mild flu-like illness; however, the disease has been increasingly associated with neurological symptomatology. This association draws attention to further investigations on the impact of DENV infection in the host’s central nervous system. Here, we analyzed brain samples of three fatal dengue cases that occurred in 2002 during an outbreak in Rio de Janeiro, Brazil. Brain tissues of these cases were marked by histopathological alterations, such as degenerated neurons, demyelination, hemorrhage, edema, and increased numbers of astrocytes and microglial cells. Samples were also characterized by lymphocytic infiltrates mainly composed of CD8 T cells. DENV replication was evidenced in neurons, microglia and endothelial cells through immunohistochemistry and in situ hybridization techniques. Pro-inflammatory cytokines, such as TNF-α and IFN-γ were detected in microglia, while endothelial cells were marked by the expression of RANTES/CCL5. Cytoplasmic HMGB1 and the production of nitric oxide were also found in neurons and microglial cells. This work highlights the possible participation of several local pro-inflammatory mediators in the establishment of dengue neuropathogenesis. Full article