Barmah Forest virus (BFV) is a medically important mosquito-borne alphavirus endemic to Australia. Symptomatic disease can be a major cause of morbidity, associated with fever, rash, and debilitating arthralgia. BFV disease is similar to that caused by Ross River virus (RRV), the other major Australian alphavirus. Currently, just four BFV whole-genome sequences are available with no genome-scale phylogeny in existence to robustly characterise genetic diversity. Thirty novel genome sequences were derived for this study, for a final 34-taxon dataset sampled over a 44 year period. Three distinct BFV genotypes were characterised (G1-3) that have circulated in Australia and Papua New Guinea (PNG). Evidence of spatio-temporal co-circulation of G2 and G3 within regions of Australia was noted, including in the South West region of Western Australia (WA) during the first reported disease outbreaks in the state's history. Compared with RRV, the BFV population appeared more stable with less frequent emergence of novel lineages. Preliminary in vitro assessment of RRV and BFV replication kinetics found that RRV replicates at a significantly faster rate and to a higher, more persistent titre compared with BFV, perhaps indicating mosquitoes may be infectious with RRV for longer than with BFV. This investigation resolved a greater diversity of BFV, and a greater understanding of the evolutionary dynamics and history was attained.

Virus host range, i.e., the number and diversity of host species of viruses, is an important determinant of disease emergence and of the efficiency of disease control strategies. However, for plant viruses, little is known about the genetic or ecological factors involved in the evolution of host range. Using available genome sequences and host range data, we performed a phylogenetic analysis of host range evolution in the genus Potyvirus, a large group of plant RNA viruses that has undergone a radiative evolution circa 7000 years ago, contemporaneously with agriculture intensification in mid Holocene. Maximum likelihood inference based on a set of 59 potyviruses and 38 plant species showed frequent host range changes during potyvirus evolution, with 4.6 changes per plant species on average, including 3.1 host gains and 1.5 host loss. These changes were quite recent, 74% of them being inferred on the terminal branches of the potyvirus tree. The most striking result was the high frequency of correlated host gains occurring repeatedly in different branches of the potyvirus tree, which raises the question of the dependence of the molecular and/or ecological mechanisms involved in adaptation to different plant species.

In the last two decades, molecular surveys of arboviruses have enabled the identification of several new viruses, contributing to the knowledge of viral diversity and providing important epidemiological data regarding possible new emerging viruses. A combination of diagnostic assays, Illumina sequencing and phylogenetic inference are here used to characterize two new Massilia phlebovirus strains isolated from sandflies collected in the Arrábida region, Portugal. Whole genome sequence analysis enabled their identification as reassortants and the recognition of genomic variants co-circulating in Portugal. Much is still unknown about the life cycle, geographic range, evolutionary forces and public health importance of these viruses in Portugal and elsewhere, and more studies are needed.

Complete genome sequences of two Australian isolates of H. armigera single nucleopolyhedrovirus (HaSNPV) and nine strains isolated by plaque selection in tissue culture identified multiple polymorphisms in tissue culture-derived strains compared to the consensus sequence of the parent isolate. Nine open reading frames (ORFs) in all tissue culture-derived strains contained changes in nucleotide sequences that resulted in changes in predicted amino acid sequence compared to the parent isolate. Of these, changes in predicted amino acid sequence of six ORFs were identical in all nine derived strains. Comparison of sequences and maximum likelihood estimation (MLE) of specific ORFs and whole genome sequences were used to compare the isolates and derived strains to published sequence data from other HaSNPV isolates. The Australian isolates and derived strains had greater sequence similarity to New World SNPV isolates from H. zea than to Old World isolates from H. armigera, but with characteristics associated with both. Three distinct geographic clusters within HaSNPV genome sequences were identified: Australia/Americas, Europe/Africa/India, and China. Comparison of sequences and fragmentation of ORFs suggest that geographic movement and passage in vitro result in distinct patterns of baculovirus strain selection and evolution.

This study delves into the complex landscape of viral infections in tomatoes (Solanum lycopersicum) using available transcriptome data. We conducted a virome analysis, revealing 219 viral contigs linked to four distinct viruses: tomato chlorosis virus (ToCV), southern tomato virus (STV), tomato yellow leaf curl virus (TYLCV), and cucumber mosaic virus (CMV). Among these, ToCV predominated in contig count, followed by STV, TYLCV, and CMV. A notable finding was the prevalence of coinfections, emphasizing the concurrent presence of multiple viruses in tomato plants. Despite generally low viral levels in fruit transcriptomes, STV emerged as the primary virus based on viral read count. We delved deeper into viral abundance and the contributions of RNA segments to replication. While initially focused on studying the impact of sound treatment on tomato fruit transcriptomes, the unexpected viral presence underscores the importance of considering viruses in plant research. Geographical variations in virome communities hint at potential forensic applications. Phylogenetic analysis provided insights into viral origins and genetic diversity, enhancing our understanding of the Korean tomato virome. In conclusion, this study advances our knowledge of the tomato virome, stressing the need for robust pest control in greenhouse-grown tomatoes and offering insights into virus management and crop protection.

We report a major improvement to the assembly of published short read sequencing data from an ancient variola virus (VARV) genome by the removal of contig-capping sequencing tags and manual searches for gap-spanning reads. The new assembly, together with camelpox and taterapox genomes, permitted new dates to be calculated for the last common ancestor of all VARV genomes. The analysis of recently sequenced VARV-like cowpox virus genomes showed that single nucleotide polymorphisms (SNPs) and amino acid changes in the vaccinia virus (VACV)-Cop-O1L ortholog, predicted to be associated with VARV host specificity and virulence, were introduced into the lineage before the divergence of these viruses. A comparison of the ancient and modern VARV genome sequences also revealed a measurable drift towards adenine + thymine (A + T) richness.

Metagenomic analysis of Aedes aegypti and Ae. albopictus mosquitoes from diverse geographical regions of India revealed the presence of several insect viruses of human interest. Most abundant reads found in Ae. aegypti mosquitoes were of Phasi Charoen-like virus (PCLV), Choristoneura fumiferana granulovirus (CfGV), Cell fusing agent virus (CFAV), and Wenzhou sobemo-like virus 4 (WSLV4), whereas WSLV4 and CfGV constituted the highest percentage of reads in Ae. albopictus viromes. Other reads that were of low percentage included Hubei mosquito virus 2 (HMV2), Porcine astrovirus 4 (PAstV4), and Wild Boar astrovirus (WBAstV). PCLV and CFAV, which were found to be abundant in Ae. aegypti viromes were absent in Ae. albopictus viromes. Among the viromes analyzed, Ae. aegypti sampled from Pune showed the highest percentage (79.82%) of viral reads, while Ae. aegypti mosquitoes sampled from Dibrugarh showed the lowest percentage (3.47%). Shamonda orthobunyavirus (SHAV), African swine fever virus (ASFV), Aroa virus (AROAV), and Ilheus virus (ILHV), having the potential to infect vertebrates, including humans, were also detected in both mosquito species, albeit with low read numbers. Reads of gemykibivirus, avian retrovirus, bacteriophages, herpesviruses, and viruses infecting protozoans, algae, etc., were also detected in the mosquitoes. A high percentage of reads in the Ae. albopictus mosquito samples belonged to unclassified viruses and warrant further investigation. The data generated in the present work may not only lead to studies to explain the influence of these viruses on the replication and transmission of viruses of clinical importance but also to find applications as biocontrol agents against pathogenic viruses.

Porcine parvovirus 1 (PPV1) is recognized as a major cause of reproductive failure in pigs, leading to several clinical outcomes globally known as SMEDI. Despite being known since the late 1960s its circulation is still of relevance to swine producers. Additionally, the emergence of variants such as the virulent 27a strain, for which lower protection induced by vaccines has been demonstrated, is of increasing concern. Even though constant monitoring of PPV1 using molecular epidemiological approaches is of pivotal importance, viral sequence data are scarce especially in low-income countries. To fill this gap, a collection of 71 partial VP2 sequences originating from eight African countries (Burkina Faso, Côte d'Ivoire, Kenya, Mozambique, Namibia, Nigeria, Senegal, and Tanzania) during the period 2011-2021 were analyzed within the context of global PPV1 variability. The observed pattern largely reflected what has been observed in high-income regions, i.e., 27a-like strains were more frequently detected than less virulent NADL-8-like strains. A phylogeographic analysis supported this observation, highlighting that the African scenario has been largely shaped by multiple PPV1 importation events from other continents, especially Europe and Asia. The existence of such an international movement coupled with the circulation of potential vaccine-escape variants requires the careful evaluation of the control strategies to prevent new strain introduction and persistence.

Canid alphaherpesvirus 1 (CHV-1) is a widespread pathogen of dogs with multiple associated clinical signs. There has been limited prior investigation into the genomics and phylogeny of this virus using whole viral genome analysis. Fifteen CHV-1 isolates were collected from animals with ocular disease based in the USA. Viral DNA was extracted for Illumina MiSeq full genome sequencing from each isolate. These data were combined with genomes of previously sequenced CHV-1 isolates obtained from hosts in the UK, Australia and Brazil. Genomic, recombinational and phylogenetic analysis were performed using multiple programs. Two isolates were separated into a clade apart from the remaining isolates and accounted for the majority of genomic distance (0.09%): one was obtained in 2019 from a USA-based host (ELAL-1) and the other in 2012 from a host in Brazil (BTU-1). ELAL-1 was found to contain variants previously reported in BTU-1 but also novel variants in the V57 gene region. Multiple non-synonymous variants were found in USA-based isolates in regions associated with antiviral resistance. Evidence of recombination was detected between ELAL-1 and BTU-1. Collectively, this represents evidence of trans-boundary transmission of a novel form of CHV-1, which highlights the importance of surveillance for this pathogen in domestic dog populations.

We generated genome sequences from 218 cases of Ebola virus disease (EVD) in Sierra Leone (SLE) during 2014⁻2015 to complement available datasets, particularly by including cases from a period of low sequence coverage during peak transmission of Ebola virus (EBOV) in the highly-affected Western Area division of SLE. The combined dataset was utilized to produce phylogenetic and phylodynamic inferences, to study sink⁻source dynamics and virus dispersal from highly-populated transmission hotspots. We identified four districts in SLE where EBOV was introduced and transmission occurred without onward exportation to other districts. We also identified six districts that substantially contributed to the dispersal of the virus and prolonged the EVD outbreak: five of these served as major hubs, with lots of movement in and out, and one acted primarily as a source, exporting the virus to other areas of the country. Positive correlations between case numbers, inter-district transition events, and district population sizes reaffirm that population size was a driver of EBOV transmission dynamics in SLE. The data presented here confirm the role of urban hubs in virus dispersal and of a delayed laboratory response in the expansion and perpetuation of the EVD outbreak in SLE.

The hepatitis delta virus (HDV) exhibits high genetic and evolutionary variability and is classified into eight genotypes (HDV-1 to -8). HDV-1 is the most widespread genotype worldwide and includes several subtypes. It predominates mainly in Europe, the Middle East, North America, and Northern Africa, and is associated with both severe and mild forms of liver disease. In this study, we performed phylogenetic and phylodynamic analyses of HDV strains circulating in Regione Lazio, Italy, to understand when these strains were introduced into the Lazio region and to define their genetic variability in Italy. Fifty HDV RNA positive patient samples were amplified using a nested RT-PCR approach targeting the HDV R0 region and sequenced. A phylogenetic tree of patient-derived sequences and reference sequences representing HDV-1 to -8 was constructed using the GTRGAMMA model in RAxML v8. The results indicated that HDV-1 was the predominant genotype with HDV-1d being the most frequently inferred subtype. HDV-1 sequences clustering with subtypes 1b and 1e were also identified. A phylodynamic analysis of HDV-1 sequences employing a Bayesian birth-death model inferred a clock rate of 3.04 × 10-4 substitutions per site per million years, with a 95% Highest Posterior Density (HPD) interval of 3.45 × 10-5 to 5.72 × 10-4. A Bayesian birth-death analysis with tree calibration based on a sample dating approach indicated multiple original sources of infection (from the late 1950s to late 1980s). Overall, these results suggest that HDV sequences from the native Italian and non-Italian patients analyzed in this study represent multiple lineages introduced across a wide period. A common ancestral origin should be excluded.

Feline morbillivirus (FeMV) was isolated for the first time in 2012 with an association with chronic kidney disease (CKD) suggested. This study aimed at investigating in cats from southern Italy FeMV prevalence and risk factors for exposure to FeMV, including the relationship with CKD; sequencing amplicons and analyzing phylogeny of PCR positive samples. Blood serum, K3EDTA blood and urine samples from 223 cats were investigated. Ten carcasses were also evaluated. FeMV RNA was detected in 2.4% (5/211) blood and 16.1% (36/223) urine samples. One carcass tested positive by qPCRFeMV from kidney, urinary bladder, and submandibular lymph nodes. Antibodies against FeMV were detected in 14.5% (28/193) cats. We followed up 27 cats (13 FeMV positive cats) and documented in some cases urine shedding after up to 360 days. Older and foundling cats and cats living in rescue catteries, were more frequently infected with FeMV. A significant correlation between FeMV and higher serum creatinine values or low urine specific gravity was found. FeMV positivity was significantly associated with retroviral infection, and the presence of some clinical signs apart from CKD clinicopathological markers. Our study highlights the possibility of a link between FeMV exposure and CKD and a general impairment of feline health.

Monkeypox is an infectious zoonotic disease caused by an Orthopoxvirus and results in symptoms similar to smallpox. In a recent outbreak, monkeypox virus (MPXV) cases have been reported globally since May 2022, and the numbers are increasing. Monkeypox was first diagnosed in humans in the Democratic Republic of Congo and has now spread to throughout Europe, the USA, and Africa. In this study, we analyzed the whole genome sequences of MPXV sequences from recent outbreaks in various countries and performed phylogenomic analysis. Our analysis of the available human MPXV strains showed the highest mutations per sample in 2022 with the average number of mutations per sample being the highest in South America and the European continents in 2022. We analyzed specific mutations in 11 Indian MPXV strains occurring in the variable end regions of the MPXV genome, where the mutation number was as high as 10 mutations per gene. Among these, envelope glycoproteins, the B2R protein, the Ankyrin repeat protein, DNA polymerase, and the INF alpha receptor-like secreted glycoprotein were seen to have a relatively high number of mutations. We discussed the stabilizing effects of the mutations in some of the highly mutating proteins. Our results showed that the proteins involved in binding to the host receptors were mutating at a faster rate, which empowered the virus for active selection towards increased disease transmissibility and severity.

Characterizing the spatial transmission pattern is critical for better surveillance and control of human influenza. Here, we propose a mutation network framework that utilizes network theory to study the transmission of human influenza H3N2. On the basis of the mutation network, the transmission analysis captured the circulation pattern from a global simulation of human influenza H3N2. Furthermore, this method was applied to explore, in detail, the transmission patterns within Europe, the United States, and China, revealing the regional spread of human influenza H3N2. The mutation network framework proposed here could facilitate the understanding, surveillance, and control of other infectious diseases.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the ongoing global COVID-19 pandemic that began in late December 2019. The rapid spread of SARS-CoV-2 is primarily due to person-to-person transmission. To understand the epidemiological traits of SARS-CoV-2 transmission, we conducted phylogenetic analysis on genome sequences from >54K SARS-CoV-2 cases obtained from two public databases. Hierarchical clustering analysis on geographic patterns in the resulting phylogenetic trees revealed a co-expansion tendency of the virus among neighboring countries with diverse sources and transmission routes for SARS-CoV-2. Pairwise sequence similarity analysis demonstrated that SARS-CoV-2 is transmitted locally and evolves during transmission. However, no significant differences were seen among SARS-CoV-2 genomes grouped by host age or sex. Here, our identified epidemiological traits provide information to better prevent transmission of SARS-CoV-2 and to facilitate the development of effective vaccines and therapeutics against the virus.

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.

During recent decades West Nile Virus (WNV) outbreaks have continuously occurred in the Mediterranean area. In August 2020 a new WNV outbreak affected 71 people with meningoencephalitis in Andalusia and six more cases were detected in Extremadura (south-west of Spain), causing a total of eight deaths. The whole genomes of four viruses were obtained and phylogenetically analyzed in the context of recent outbreaks. The Andalusian viral samples belonged to lineage 1 and were relatively similar to those of previous outbreaks which occurred in the Mediterranean region. Here we present a detailed analysis of the outbreak, including an extensive phylogenetic study. As part on this effort, we implemented a local Nextstrain server, which has become a constituent piece of regional epidemiological surveillance, wherein forthcoming genomes of environmental samples or, eventually, future outbreaks, will be included.

Lyssavirus is a diverse genus of viruses that infect a variety of mammalian hosts, typically causing encephalitis. The evolution of this lineage, particularly the rabies virus, has been a focus of research because of the extensive occurrence of cross-species transmission, and the distinctive geographical patterns present throughout the diversification of these viruses. Although numerous studies have examined pattern-related questions concerning Lyssavirus evolution, analyses of the evolutionary processes acting on Lyssavirus diversification are scarce. To clarify the relevance of positive natural selection in Lyssavirus diversification, we conducted a comprehensive scan for episodic diversifying selection across all lineages and codon sites of the five coding regions in lyssavirus genomes. Although the genomes of these viruses are generally conserved, the glycoprotein (G), RNA-dependent RNA polymerase (L) and polymerase (P) genes were frequently targets of adaptive evolution during the diversification of the genus. Adaptive evolution is particularly manifest in the glycoprotein gene, which was inferred to have experienced the highest density of positively selected codon sites along branches. Substitutions in the L gene were found to be associated with the early diversification of phylogroups. A comparison between the number of positively selected sites inferred along the branches of RABV population branches and Lyssavirus intespecies branches suggested that the occurrence of positive selection was similar on the five coding regions of the genome in both groups.

Infectious Bronchitis (IB) is a respiratory disease caused by a highly variable Gammacoronavirus, which generates a negative impact on poultry health worldwide. GI-11 and GI-16 lineages have been identified in South America based on Infectious Bronchitis virus (IBV) partial S1 sequences. However, full genome sequence information is limited. In this study we report, for the first time, the whole-genome sequence of IBV from Colombia. Seven IBV isolates obtained during 2012 and 2013 from farms with respiratory disease compatible with IB were selected and the complete genome sequence was obtained by NGS. According to S1 sequence phylogenetic analysis, six isolates belong to lineage GI-1 and one to lineage GVI-1. When whole genome was analyzed, five isolates were related to the vaccine strain Ma5 2016 and two showed mosaic genomes. Results from complete S1 sequence analysis provides further support for the hypothesis that GVI-1, considered a geographically confined lineage in Asia, could have originated in Colombia. Complete genome information reported in this research allow a deeper understanding of the phylogenetic evolution of variants and the recombination events between strains that are circulating worldwide, contributing to the knowledge of coronavirus in Latin America and the world.

Tomato is an important vegetable in the United States and around the world. Recently, tomato brown rugose fruit virus (ToBRFV), an emerging tobamovirus, has impacted tomato crops worldwide and can result in fruit loss. ToBRFV causes severe symptoms, such as mosaic, puckering, and necrotic lesions on leaves; other symptoms include brown rugose and marbling on fruits. More importantly, ToBRFV can overcome resistance in tomato cultivars carrying the Tm-22 locus. In this study, we recovered ToBRFV sequences from tomato seeds, leaves, and fruits from the U.S., Mexico, and Peru. Samples were pre-screened using a real-time RT-PCR assay prior to high-throughput sequencing. Virus draft genomes from 22 samples were assembled and analyzed against more than 120 publicly available genomes. Overall, most sequenced isolates were similar to each other and did not form a distinct population. Phylogenetic analysis revealed three clades within the ToBRFV population. Most of the isolates (95%) clustered in clade 3. Genetic analysis revealed differentiation between the three clades indicating minor divergence occurring. Overall, pairwise identity showed limited genetic diversity among the isolates in this study with worldwide isolates, with a pairwise identity ranging from 99.36% and 99.97%. The overall population is undergoing high gene flow and population expansion with strong negative selection pressure at all ToBRFV genes. Based on the results of this study, it is likely that the limited ToBRFV diversity is associated with the rapid movement and eradication of ToBRFV-infected material between countries.