Viral Evolution Research Articles

Dominant HPAIV H5N1 genotypes of Germany 2021/2022 are linked to high virulence in Pekin ducklings

Abstract Highly pathogenic avian influenza viruses (HPAIV) of H5 clade 2.3.4.4b pose an ongoing threat worldwide. It remains unclear whether this panzootic situation would favor low virulent phenotypes expected by the ‘avirulence hypothesis’ of viral evolution. Assessing virulence in Pekin ducklings in an intramuscular infection model revealed that the two genotypes that dominated the epidemiological situation in Germany during the period 2021 and 2022 (EU-RL:CH and EU-RL:AB) were of high virulence. In contrast, rare genotypes were of intermediate virulence. The genetic constellation of these reassortants pointed to an important role of the viral polymerase complex (RdRP), particularly the PB1 genome segment, in shaping virulence in ducklings. Occulo-nasal infection of ducklings confirmed the phenotypes for two representative viruses and indicated a more efficient replication for the high virulence strain. These observations would be in line with the ‘virulence-transmission trade-off’ model for describing HPAIV epidemiology in wild birds in Germany.

Quantifying defective and wild-type viruses from high-throughput RNA sequencing.

Defective viral genomes (DVGs) are variants of the wild-type (wt) virus that lack the ability to complete autonomously an infectious cycle. However, in the presence of their parental (helper) wt virus, DVGs can interfere with the replication, encapsidation and spread of functional genomes, acting as a significant selective force in viral evolution. DVGs also affect the host's immune responses and are linked to chronic infections and milder symptoms. Thus, identifying and characterizing DVGs is crucial for understanding infection prognosis. Quantifying DVGs is challenging due to their inability to sustain themselves, which makes it difficult to distinguish them from the helper virus, especially using high-throughput RNA sequencing (RNA-seq). An accurate quantification is essential for understanding their very dynamical interactions with the helper virus. We present a method to simultaneously estimate the abundances of DVGs and wt genomes within a sample by identifying genomic regions with significant deviations from the expected sequencing depth. Our approach involves reconstructing the depth profile through a linear system of equations, which provides an estimate of the number of wt and DVG genomes of each type. Until now, in silico methods have only estimated the DVG-to-wt ratio for localized genomic regions. This is the first method that simultaneously estimates the proportions of wt and DVGs genome wide from short-reads RNA sequencing. The MATLAB code and the synthetic datasets are freely available at https://github.com/jmusan/wtDVGquantific.

Rapid production of recombinant rotaviruses by overexpression of NSP2 and NSP5 genes with modified nucleotide sequences.

Reverse genetics systems for rotaviruses (RV) facilitate the generation of genetically engineered RVs by transfection of 11 plasmids encoding 11 genomic viral RNA segments. In addition to viral genome expression, overexpression of NSP2 and NSP5 has been used to increase the rescue efficiency of recombinant RVs. Here, we showed that the overexpression of nucleotide sequence-modified NSP2 and NSP5 enabled the rapid and efficient production of recombinant RVs. Using improved reverse genetics, we established a reverse genetics system for human and bovine RV clinical isolates, as well as laboratory strains of bovine RV (NCDV and UK) and porcine RV (Gottfried). In addition, we rescued low-replicating recombinant RVs carrying a mutant NSP4 lacking the double-layered particle-binding domain, which was deficient in the efficient production of mature virions. These advancements in reverse genetics enabled the generation of molecular clones of RV clinical isolates and recombinant RVs harboring critical amino acid mutations, offering a versatile platform for investigating RV biology and pathogenesis.IMPORTANCERecombinant rotavirus (RV) synthesis via reverse genetics relies on both the viral propagation capacity and the efficiency of the experimental system. Since the establishment of our reverse genetics system, several enhancements have been implemented to augment the rescue efficiency. Nevertheless, challenges persist in generating RV clinical strains and recombinant viruses with low replication capacities. Notably, this improved reverse genetics system successfully facilitated the establishment of molecular clones of human and bovine RV clinical isolates. Fecal samples from patients with RV typically harbor quasi-species or, occasionally, multiple genotypes of RV. In the present study, we performed the genetic sequencing of clinical viral strains during the early propagation stages in cultured cells. Subsequently, infectious viruses were synthesized, allowing the characterization of circulating viruses in nature. This approach provides valuable insights into the genetic diversity and dynamics of RV populations and contributes to a more comprehensive understanding of viral pathogenesis and evolution.

No evidence that mutations in SARS-CoV-2 variants of concern derive from homologous fragments in gut microbiota.

Understanding the origin and evolution of mutations in SARS-CoV-2 variants of concern (VOCs) is a critical area of research. B. Cao, X. Wang, W. Yin, Z. Gao, and B. Xia (mBio 15:e03187-23, 2024, https://doi.org/10.1128/mbio.03187-23) proposed that these mutations originated from bacterial sequences incorporated into the viral genome through stochastic template-switching by the viral RNA-dependent RNA polymerase (RdRp). Their analysis suggested that 62% of the viral mutation fragments (VMFs) in key SARS-CoV-2 proteins were identical to bacterial protein sequences. Given the implications of this finding, we re-examined the methods employed and argue that they resulted in false-positive findings. Specifically, the short query length of VMFs, seven amino acids, leads to spurious matches in large protein databases, as indicated by high BLAST Expect values. Furthermore, we analyzed the nucleotide sequence of VMFs, revealing no unique homology between SARS-CoV-2 and bacterial sequences. Consequently, the evidence does not support the hypothesis that bacterial sequences contribute to the evolution of SARS-CoV-2 VOCs. Instead, the emergence of these variants is more plausibly attributed to factors intrinsic to viral replication and evolution, such as the error-prone nature of RdRp, intrahost diversity, and recombination of related viral sublineages.

Population dynamics of defective viral genomes of tomato black ring virus during host-to-host transmission.

Defective viral genomes (DVGs) have been identified in vivo and in vitro for different virus species infecting humans, animals, and plants. The ability to form DVGs during the passaging of virus in one host has been demonstrated, i.e., for tomato black ring virus (TBRV). In our research, RNA-Seq data obtained after TBRV passaging through a combination of four distinct host species were analyzed. Our results indicate that the level of DVG abundance varied across host plant combinations. Deletions were the most prevalent class of DVGs, with the domination of longer species. Additionally, the conserved junction sites in the TBRV genome were identified, resulting in the generation of identical deletions in independently evolved viral lineages. In summary, our findings provide significant insights into the origin and structure of DVGs of plant viruses. The obtained results will help in understanding viral evolution and host-virus interactions.

Perspective for Drug Discovery Targeting SARS Coronavirus Methyltransferases: Function, Structure and Inhibition.

Severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), is highly contagious and caused a catastrophic pandemic. It has infected billions of people worldwide with >6 million deaths. With expedited development of effective vaccines and antiviral drugs, there have been significantly reduced SARS-CoV-2 infections and associated mortalities and morbidities. The virus is closely related to SARS-CoV, which emerged in 2003 and infected several thousand people with a higher mortality rate of ∼10%. Because of continued viral evolution and drug-induced resistance, as well as the possibility of a new coronavirus in the future, studies for new therapies are needed. The viral methyltransferases play critical roles in SARS coronavirus replication and are therefore promising drug targets. This review summarizes the function, structure and inhibition of methyltransferases of SARS-CoV-2 and SARS-CoV. Challenges and perspectives of targeting the viral methyltransferases to treat viral infections are discussed.

Evolution shapes and conserves genomic signatures in viruses

The genomic signature of an organism captures the characteristics of repeated oligonucleotide patterns in its genome 1, such as oligomer frequencies, GC content, and differences in codon usage. Viruses, however, are obligate intracellular parasites that are dependent on their host cells for replication, and information about genomic signatures in viruses has hitherto been sparse.Here, we investigate the presence and specificity of genomic signatures in 2,768 eukaryotic viral species from 105 viral families, aiming to illuminate dependencies and selective pressures in viral genome evolution. We demonstrate that most viruses have highly specific genomic signatures that often also differ significantly between species within the same family. The species-specificity is most prominent among dsDNA viruses and viruses with large genomes. We also reveal consistent dissimilarities between viral genomic signatures and those of their host cells, although some viruses present slight similarities, which may be explained by genetic adaptation to their native hosts. Our results suggest that significant evolutionary selection pressures act upon viral genomes to shape and preserve their genomic signatures, which may have implications for the field of synthetic biology in the construction of live attenuated vaccines and viral vectors.

Recombinant SARS-CoV-2 Delta/Omicron BA.5 emerging in an immunocompromised long-term infected COVID-19 patient.

The emergence of the SARS-CoV-2 virus led to a global pandemic, prompting extensive research efforts to understand its molecular biology, transmission dynamics, and pathogenesis. Recombination events have been increasingly recognized as significant contributor to the virus's diversity and evolution, potentially leading to the emergence of novel strains with altered biological properties. Indeed, recombinant lineages such as the XBB variant and its descendants have subsequently dominated globally. Therefore, continued surveillance and monitoring of viral genome diversity are crucial to identify and understand the emergence and spread of novel strains. Through routine genomic surveillance of SARS-CoV-2 cases in Norway, we discovered a SARS-CoV-2 recombination event in a long-term infected immunocompromised COVID-19 (coronavirus disease) patient. A deeper investigation showed several recombination events between two distinct lineages of the virus, namely AY.98.1 and BA.5, that resulted in a single novel recombinant viral strain with a unique genetic signature. Our data is consistent with the presence of several concomitant recombinants in the patient, suggesting that these events occur frequently in vivo. This study underscores the importance of continued tracking of viral diversity and the potential impact of recombination events on the evolution of the SARS-CoV-2 virus.

Characterization of the viral genome of Omicron variants of SARS-CoV-2 circulating in Tripura, a remote frontier state in Northeastern India.

From 2020, with advent of COVID-19 pandemic, Tripura has experienced SARS-CoV-2 viral evolution in accordance with other parts of India. Since January 2022, the Omicron variant of SARS-CoV-2 virus became the predominant lineage circulating in India and neighboring countries. This study characterizes the viral genome of the omicron variant circulating in the state since its inception to June, 2023. The current study was performed on nasopharyngeal and oropharyngeal samples received from the various departments of AGMC, as well as eight district hospitals from Tripura. The positive samples with a cycle threshold value of 25 or less for the E and/or N gene were considered for whole genome sequencing using Illumina Miseq NGS platform. Majority of the sequences belonged to Clade 21L, with BA.5.2 being the major sub variant detected during the study period. Majority of the mutations were detected in the Spike protein region, including L24-, P25-, P26-, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, S477N, T478K, Q498R, Y505H, Q954H and N969K. All the sequences uniquely showed the mutations A27S and G142D in N terminal domain in Spike protein, not being reported from other Indian sequences like BA.5 variants. T9I, A63T and P13L were major substitutions in E, M and N protein regions respectively. Escape of mutants from vaccine induced immunity was mostly observed in BA.2 sub variants, majority endowed with the triplet mutation of K417N + E484K + N501Y. The current study indicates that Omicron variants circulating in the state of Tripura is comparable to other regions of India and the neighbouring country of Bangladesh. Genetic mutations increasing viral transmissibility have been identified in the circulating viral genomes.

Analysis of control measures inducing the evolution of infectious viral diseases

With the continuation of the infectious disease, pathogens mutate and interact with the resident pathogen. In this paper, we develop an SIR model that considers the evolution of viral virulence under the regulation of different defense strengths. By using infectious disease dynamics and evolutionary adaptive dynamics, we obtain the evolutionary criteria allowing for evolutionary branching and continuously stable strategy across varying intensities of prevention and control measures. Our study finds that robust prevention and control measures can contribute to a rapid evolutionary trend toward higher virulence. We perform a critical function analysis to reveal the evolutionary consequences of trade-offs of arbitrary shape and analyze a diverse range of evolutionary dynamics. Furthermore, we investigate the coexistence of two different strains and construct an evolutionary model to obtain their invasion fitness functions and the evolutionarily singular coalition of the dimorphism. We contemplate the presence of secondary branching on a significantly extended evolutionary time scale. Last, we conduct the numerical simulations to prove our theoretical findings. This reveals the law that the evolutionary state of pathogens is affected by the intensity of prevention and control strategies. The results and methods can be used as references for studying the effects of other factors on virus evolution.

Unprecedented spread and genetic evolution of the Oropouche virus.

Unprecedented spread and genetic evolution of the Oropouche virus.

First Description of Loreto Virus in Three Culicidae Species from the Atlantic Forest, Bahia, Brazil

Loreto virus (LORV) is an insect-specific virus classified into the proposed taxon Negevirus. It was originally described in Iquitos, Peru, in 1977. Here, we describe three novel LORV genomes obtained from the isolates of three pooled samples of Trichoprosopon digitatum, Aedes (Ochlerotatus) fulvus, and Limatus durhamii collected in Ilhéus—Bahia, 2014. Samples were submitted to RNA sequencing on the Illumina platform to recover the LORV genome. The genomes presented, on average, 81.5% nucleotide identity and 92.6% global amino acid identity with the LORV reference genome (NC_034158). Subsequently, phylogenetic analysis was performed based on a multiple sequence alignment of the concatenated amino acid sequences predicted for the three ORFs of the Negevirus genomes, and the target sequences were clustered within the LORV clade. The taxon Negevirus is in constant expansion of its species content and host range. New data about insect specific negeviruses are important for virus evolution studies, along with those approaching interactions with the hosts and their influence in the transmission of arboviruses. Also, the assessment of these data may allow the development of biologic control strategies for arboviral vectors. This is the original report of the identification of LORV in Brazil, infecting three Culicidae species hosts native to the Atlantic Forest biome.

Clade I mpox virus genomic diversity in the Democratic Republic of the Congo, 2018–2024: Predominance of zoonotic transmission

Recent reports raise concerns on the changing epidemiology of mpox in the Democratic Republic of the Congo (DRC). High-quality genomes were generated for 337 patients from 14/26 provinces to document whether the increase in number of cases is due to zoonotic spillover events or viral evolution, with enrichment of APOBEC3 mutations linked to human adaptation. Our study highlights two patterns of transmission contributing to the source of human cases. All new sequences from the eastern South Kivu province (n= 17; 4.8%) corresponded to the recently described clade Ib, associated with sexual contact and sustainedhuman-to-human transmission. By contrast, all other genomes are clade Ia, which exhibits high genetic diversity with low numbers of APOBEC3 mutations compared with clade Ib, suggesting multiplezoonotic introductions. The presence of multiple clade I variants in urban areas highlights the need for coordinated international response efforts and more studies on the transmission and the reservoir of mpox.

Sequencing RNA from old, dried specimens reveals past viromes and properties of long-surviving RNA.

Recovery of virus sequences from old samples provides an opportunity to study virus evolution and reconstruct historic virus-host interactions. Studies of old virus sequences have mainly relied on DNA or on RNA from fixed or frozen samples. The millions of specimens in natural history museums represent a potential treasure trove of old virus sequences, but it is not clear how well RNA survives in old samples. We experimentally assessed the stability of RNA in insects stored dry at room temperature over 72 weeks. Although RNA molecules grew fragmented, RNA yields remained surprisingly constant. RT-qPCR of host and virus RNA showed minimal differences between dried and frozen specimens. To assess RNA survival in much older samples we acquired Drosophila specimens from North American entomological collections. We recovered sequences from known and novel viruses including several coding complete virus genomes from a fly collected in 1908. We found that the virome of D. melanogaster has changed little over the past century. Galbut virus, the most prevalent virus infection in contemporary D. melanogaster , was also the most common in historic samples. Finally, we investigated the genomic and physical features of surviving RNA. RNA that survived was fragmented, chemically damaged, and preferentially double stranded or contained in ribonucleoprotein complexes. This showed that RNA - especially certain types of RNA - can survive in biological specimens over extended periods in the absence of fixation or freezing and confirms the utility of dried specimens to provide a clearer understanding of virus evolution.

Epidemiology of human metapneumovirus in Taiwan from 2013 to 2023

Human metapneumovirus (HMPV) is a member of the genus Metapneumovirus in the family Pneumoviridae of the order Mononegavirales that can cause upper and lower respiratory tract disease. This retrospective study describes the epidemiology of hMPV based on community viral surveillance results from sentinel sites across Taiwan from 2013 to 2023. A total of 114 hMPV strains were isolated and analyzed to assess viral evolution through sequencing of their fusion protein genes. This study revealed that hMPV cases occur almost year-round in Taiwan, with a peak occurring during spring (March to May). Of the 114 infected patients, 68.4% were children under 4 years old. The geographical distribution of hMPV positivity was highest in Penghu County, followed by Changhua County and Hsinchu County. The clinical symptoms of hMPV infection are nonspecific, with fever (56.1%), cough (44.7%), rhinorrhea (21.1%), and sore throat (14.9%) being the most common. However, a few patients also developed severe central nervous system symptoms (1.8%) or dyspnea (0.9%). Phylogenetic analysis revealed genetic diversity among the 114 isolated hMPV strains, with the A2 lineage (57.9%) being the most frequently observed, followed by the B2 lineage (33.3%), in the Taiwanese community from 2013 to 2023. In conclusion, hMPV causes a serious acute respiratory disease in Taiwan that should not be neglected. Further epidemiological surveillance and investigations of the clinical characteristics of hMPV should be performed continually for prevention and control of this virus.

The 2024 Public Health Emergency of International Concern: A Global Failure to Control Mpox.

On August 14, 2024, following a regional declaration by the Africa Centres for Disease Control and Prevention, the World Health Organization declared mpox a Public Health Emergency of International Concern, marking the second such declaration in two years. A series of outbreaks involving the more virulent clade I virus (compared to clade II, which caused a global outbreak in 2022), has now spread in 13 African countries, exposing the inadequacies of the public health infrastructure in these settings. There was significant investment during the 2022 global outbreak, but these efforts failed to address vaccine access and treatment in the Global South. Regulatory delays, unequal access to vaccines, and a lack of compassionate use treatments for severe cases have resulted in preventable cases and deaths, especially among vulnerable populations such as pregnant women, children, and the immunocompromised. The current outbreak also underscores critical knowledge gaps in our understanding of mpox, including its transmission, pathogenesis, and viral evolution. We join intensified calls for global solidarity and action to control mpox, emphasizing immediate containment measures and long-term local and international investment in African public health systems, to prevent future epidemics.

Deep mining reveals the diversity of endogenous viral elements in vertebrate genomes

Integration of viruses into host genomes can give rise to endogenous viral elements (EVEs), which provide insights into viral diversity, host range and evolution. A systematic search for EVEs is becoming computationally challenging given the available genomic data. We used a cloud-computing approach to perform a comprehensive search for EVEs in the kingdoms Shotokuvirae and Orthornavirae across vertebrates. We identified 2,040 EVEs in 295 vertebrate genomes and provide evidence for EVEs belonging to the families Chuviridae, Paramyxoviridae, Nairoviridae and Benyviridae. We also find an EVE from the Hepacivirus genus of flaviviruses with orthology across murine rodents. In addition, our analyses revealed that reptarenaviruses and filoviruses probably acquired their glycoprotein ectodomains three times independently from retroviral elements. Taken together, these findings encourage the addition of 4 virus families and the Hepacivirus genus to the growing virus fossil record of vertebrates, providing key insights into their natural history and evolution.

Revealing SARS-CoV-2 Mpro mutation cold and hot spots: Dynamic residue network analysis meets machine learning

Deciphering the effect of evolutionary mutations of viruses and predicting future mutations is crucial for designing long-lasting and effective drugs. While understanding the impact of current mutations on protein drug targets is feasible, predicting future mutations due to natural evolution of viruses and environmental pressures remains challenging. Here, we leveraged existing mutation data during the evolution of the SARS-CoV-2 protein drug target main protease (Mpro) to test the predictive power of dynamic residue network (DRN) analysis in identifying mutation cold and hot spots. We conducted molecular dynamics simulations on the Mpro of SARS-CoV-2 (Wuhan strain) and calculated eight DRN metrics (averaged BC, CC, DC, EC, ECC, KC, L, PR), each of which identifies a unique network feature within the protein. The sets of residues with the highest and lowest values for each metric, comprising potential cold and hot spots, were compared to published biochemical analyses and per residue mutation frequencies observed across five SARS-CoV-2 lineages, encompassing a total of 191,878 sequences. Individual DRN metrics displayed only modest power to predict the mutation frequency of individual residues. However, integrating the eight DRN metrics with additional structural and sequence-derived metrics allowed us to develop machine learning models which significantly improved the prediction of residue mutation frequency. While further refinements should enhance accuracy, we demonstrated a robust method to understand pathogen evolution. This approach can also guide the development of long-lasting drugs by targeting functional residues located in and near active site, and allosteric sites, that are less prone to mutations.

Intra-host genomic diversity and integration landscape of human tissue-resident DNA virome.

The viral intra-host genetic diversitiesand interactions with the human genome during decades of persistence remain poorly characterized. In this study, we analyzed the variability and integration sites of persisting viruses in nine organs from thirteen individuals who died suddenly from non-viralcauses. The viruses studiedincluded parvovirus B19, six herpesviruses, Merkel cell (MCPyV) and JC polyomaviruses, totaling 127 genomes. The viral sequences across organs were remarkably conserved within each individual, suggesting that persistence stems from single dominant strains. This indicates that intra-host viral evolution, thus far inferred primarily from immunocompromised patients, is likely overestimated in healthy subjects. Indeed, we detected increased viral subpopulations in two individuals with putative reactivations, suggesting that replication status influences diversity. Furthermore, we identified asymmetrical mutationpatternsreflectingselective pressures exerted by the host. Strikingly, our analysis revealed non-clonal viral integrations even in individuals without cancer. These included MCPyV integrations and truncations resembling clonally expanded variants inMerkel cell carcinomas, as well as novel junctions between herpesvirus 6B and mitochondrial sequences, the significance of which remains to be evaluated. Our work systematically characterizes the genomic landscape of the tissue-resident virome, highlighting potential deviations occurring during disease.

Trim7 does not have a role in the restriction of murine norovirus infection in vivo.

Intrinsic antiviral molecules that restrict viral replication are important drivers of viral evolution and viral tropism. Recently, Trim7 was shown to provide cell intrinsic protection against RNA viruses, including murine norovirus. Biochemically, Trim7 recognizes the cleavage product of viral proteases, suggesting a novel and broad mechanism to restrict viral replication. Here, we tested whether Trim7 had a physiological role in restricting murine norovirus replication in mice. Unexpectedly, we found no impact of viral replication or innate immune responses during murine norovirus infection. Our findings urge caution in defining Trim7 as a broad antiviral factor in the absence of in vivo evidence.