Viruses associated with measles-like illnesses in Uganda.

OBJECTIVES: In this study, we investigated the causes of measles-like illnesses (MLI) in the Uganda national surveillance program in order to inform diagnostic assay selection and vaccination strategies. METHODS: We used metagenomic next-generation sequencing (M-NGS) on the Illumina platform to identify viruses associated with MLI (defined as fever and rash in the presence of either cough, coryza or conjunctivitis) in patient samples that had tested IgM negative for measles between 2010 and 2019. RESULTS: Viral genomes were identified in 87/271 (32%) of samples, of which 44/271 (16%) contained 12 known viral pathogens. Expected viruses included rubella, human parvovirus B19, Epstein Barr virus, human herpesvirus 6B, human cytomegalovirus, varicella zoster virus and measles virus (detected within the seronegative window-period of infection) and the blood-borne hepatitis B virus. We also detected Saffold virus, human parvovirus type 4, the human adenovirus C2 and vaccine-associated poliovirus type 1. CONCLUSIONS: The study highlights the presence of undiagnosed viruses causing MLI in Uganda, including vaccine-preventable illnesses. NGS can be used to monitor common viral infections at a population level, especially in regions where such infections are prevalent, including low and middle income countries to guide vaccination policy and optimize diagnostic assays.

Cryptic proteins translated from deletion-containing viral genomes dramatically expand the influenza virus proteome.

Productive infections by RNA viruses require faithful replication of the entire genome. Yet many RNA viruses also produce deletion-containing viral genomes (DelVGs), aberrant replication products with large internal deletions. DelVGs interfere with the replication of wild-type virus and their presence in patients is associated with better clinical outcomes. The DelVG RNA itself is hypothesized to confer this interfering activity. DelVGs antagonize replication by out-competing the full-length genome and triggering innate immune responses. Here, we identify an additionally inhibitory mechanism mediated by a new class of viral proteins encoded by DelVGs. We identified hundreds of cryptic viral proteins translated from DelVGs. These DelVG-encoded proteins (DPRs) include canonical viral proteins with large internal deletions, as well as proteins with novel C-termini translated from alternative reading frames. Many DPRs retain functional domains shared with their full-length counterparts, suggesting they may have activity during infection. Mechanistic studies of DPRs derived from the influenza virus protein PB2 showed that they poison replication of wild-type virus by acting as dominant-negative inhibitors of the viral polymerase. These findings reveal that DelVGs have a dual inhibitory mechanism, acting at both the RNA and protein level. They further show that DPRs have the potential to dramatically expand the functional proteomes of diverse RNA viruses.

Cryptic proteins translated from deletion-containing viral genomes dramatically expand the influenza virus proteome.

Productive infections by RNA viruses require faithful replication of the entire genome. Yet many RNA viruses also produce deletion-containing viral genomes (DelVGs), aberrant replication products with large internal deletions. DelVGs interfere with the replication of wild-type virus and their presence in patients is associated with better clinical outcomes as they. The DelVG RNA itself is hypothesized to confer this interfering activity. DelVGs antagonize replication by out-competing the full-length genome and triggering innate immune responses. Here, we identify an additionally inhibitory mechanism mediated by a new class of viral proteins encoded by DelVGs. We identified hundreds of cryptic viral proteins translated from DelVGs. These DelVG-encoded proteins (DPRs) include canonical viral proteins with large internal deletions, as well as proteins with novel C-termini translated from alternative reading frames. Many DPRs retain functional domains shared with their full-length counterparts, suggesting they may have activity during infection. Mechanistic studies of DPRs derived from the influenza virus protein PB2 showed that they poison replication of wild-type virus by acting as dominant-negative inhibitors of the viral polymerase. These findings reveal that DelVGs have a dual inhibitory mechanism, acting at both the RNA and protein level. They further show that DPRs have the potential to dramatically expand the functional proteomes of diverse RNA viruses.

Phenotyping the virulence of SARS-CoV-2 variants in hamsters by digital pathology and machine learning.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continued to evolve throughout the coronavirus disease-19 (COVID-19) pandemic, giving rise to multiple variants of concern (VOCs) with different biological properties. As the pandemic progresses, it will be essential to test in near real time the potential of any new emerging variant to cause severe disease. BA.1 (Omicron) was shown to be attenuated compared to the previous VOCs like Delta, but it is possible that newly emerging variants may regain a virulent phenotype. Hamsters have been proven to be an exceedingly good model for SARS-CoV-2 pathogenesis. Here, we aimed to develop robust quantitative pipelines to assess the virulence of SARS-CoV-2 variants in hamsters. We used various approaches including RNAseq, RNA in situ hybridization, immunohistochemistry, and digital pathology, including software assisted whole section imaging and downstream automatic analyses enhanced by machine learning, to develop methods to assess and quantify virus-induced pulmonary lesions in an unbiased manner. Initially, we used Delta and Omicron to develop our experimental pipelines. We then assessed the virulence of recent Omicron sub-lineages including BA.5, XBB, BQ.1.18, BA.2, BA.2.75 and EG.5.1. We show that in experimentally infected hamsters, accurate quantification of alveolar epithelial hyperplasia and macrophage infiltrates represent robust markers for assessing the extent of virus-induced pulmonary pathology, and hence virus virulence. In addition, using these pipelines, we could reveal how some Omicron sub-lineages (e.g., BA.2.75 and EG.5.1) have regained virulence compared to the original BA.1. Finally, to maximise the utility of the digital pathology pipelines reported in our study, we developed an online repository containing representative whole organ histopathology sections that can be visualised at variable magnifications (https://covid-atlas.cvr.gla.ac.uk). Overall, this pipeline can provide unbiased and invaluable data for rapidly assessing newly emerging variants and their potential to cause severe disease.

SARS-CoV-2 in Domestic UK Cats from Alpha to Omicron: Swab Surveillance and Case Reports.

Although domestic cats are susceptible to infection with SARS-CoV-2, the role of the virus in causing feline disease is less well defined. We conducted a large-scale study to identify SARS-CoV-2 infections in UK pet cats, using active and passive surveillance. Remnant feline respiratory swab samples, submitted for other pathogen testing between May 2021 and February 2023, were screened using RT-qPCR. In addition, we appealed to veterinarians for swab samples from cats suspected of having clinical SARS-CoV-2 infections. Bespoke testing for SARS-CoV-2 neutralising antibodies was also performed, on request, in suspected cases. One RT-qPCR-positive cat was identified by active surveillance (1/549, 0.18%), during the Delta wave (1/175, 0.57%). Passive surveillance detected one cat infected with the Alpha variant, and two of ten cats tested RT-qPCR-positive during the Delta wave. No cats tested RT-qPCR-positive after the emergence of Omicron BA.1 and its descendants although 374 were tested by active and eleven by passive surveillance. We describe four cases of SARS-CoV-2 infection in pet cats, identified by RT-qPCR and/or serology, that presented with a range of clinical signs, as well as their SARS-CoV-2 genome sequences. These cases demonstrate that, although uncommon in cats, a variety of clinical signs can occur.

Isolation and genome sequencing of cytomegaloviruses from Natal multimammate mice (Mastomys natalensis).

Distinct cytomegaloviruses (CMVs) are widely distributed across their mammalian hosts in a highly host species-restricted pattern. To date, evidence demonstrating this has been limited largely to PCR-based approaches targeting small, conserved genomic regions, and only a few complete genomes of isolated viruses representing distinct CMV species have been sequenced. We have now combined direct isolation of infectious viruses from tissues with complete genome sequencing to provide a view of CMV diversity in a wild animal population. We targeted Natal multimammate mice (Mastomys natalensis), which are common in sub-Saharan Africa, are known to carry a variety of zoonotic pathogens, and are regarded as the primary source of Lassa virus (LASV) spillover into humans. Using transformed epithelial cells prepared from M. natalensis kidneys, we isolated CMVs from the salivary gland tissue of 14 of 37 (36 %) animals from a field study site in Mali. Genome sequencing showed that these primary isolates represent three different M. natalensis CMVs (MnatCMVs: MnatCMV1, MnatCMV2 and MnatCMV3), with some animals carrying multiple MnatCMVs or multiple strains of a single MnatCMV presumably as a result of coinfection or superinfection. Including primary isolates and plaque-purified isolates, we sequenced and annotated the genomes of two MnatCMV1 strains (derived from sequencing 14 viruses), six MnatCMV2 strains (25 viruses) and ten MnatCMV3 strains (21 viruses), totalling 18 MnatCMV strains isolated as 60 infectious viruses. Phylogenetic analysis showed that these MnatCMVs group with other murid viruses in the genus Muromegalovirus (subfamily Betaherpesvirinae, family Orthoherpesviridae), and that MnatCMV1 and MnatCMV2 are more closely related to each other than to MnatCMV3. The availability of MnatCMV isolates and the characterization of their genomes will serve as the prelude to the generation of a MnatCMV-based vaccine to target LASV in the M. natalensis reservoir.

Directions of change in intrinsic case severity across successive SARS-CoV-2 variant waves have been inconsistent.

OBJECTIVES: To determine how the intrinsic severity of successively dominant SARS-CoV-2 variants changed over the course of the pandemic. METHODS: A retrospective cohort analysis in the NHS Greater Glasgow and Clyde (NHS GGC) Health Board. All sequenced non-nosocomial adult COVID-19 cases in NHS GGC with relevant SARS-CoV-2 lineages (B.1.177/Alpha, Alpha/Delta, AY.4.2 Delta/non-AY.4.2 Delta, non-AY.4.2 Delta/Omicron, and BA.1 Omicron/BA.2 Omicron) during analysis periods were included. Outcome measures were hospital admission, ICU admission, or death within 28 days of positive COVID-19 test. We report the cumulative odds ratio; the ratio of the odds that an individual experiences a severity event of a given level vs all lower severity levels for the resident and the replacement variant after adjustment. RESULTS: After adjustment for covariates, the cumulative odds ratio was 1.51 (95% CI: 1.08-2.11) for Alpha versus B.1.177, 2.09 (95% CI: 1.42-3.08) for Delta versus Alpha, 0.99 (95% CI: 0.76-1.27) for AY.4.2 Delta versus non-AY.4.2 Delta, 0.49 (95% CI: 0.22-1.06) for Omicron versus non-AY.4.2 Delta, and 0.86 (95% CI: 0.68-1.09) for BA.2 Omicron versus BA.1 Omicron. CONCLUSIONS: The direction of change in intrinsic severity between successively emerging SARS-CoV-2 variants was inconsistent, reminding us that the intrinsic severity of future SARS-CoV-2 variants remains uncertain.

The Timing and Magnitude of the Type I Interferon Response Are Correlated with Disease Tolerance in Arbovirus Infection.

Infected hosts possess two alternative strategies to protect themselves against the negative impact of virus infections: resistance, used to abrogate virus replication, and disease tolerance, used to avoid tissue damage without controlling viral burden. The principles governing pathogen resistance are well understood, while less is known about those involved in disease tolerance. Here, we studied bluetongue virus (BTV), the cause of bluetongue disease of ruminants, as a model system to investigate the mechanisms of virus-host interactions correlating with disease tolerance. BTV induces clinical disease mainly in sheep, while cattle are considered reservoirs of infection, rarely exhibiting clinical symptoms despite sustained viremia. Using primary cells from multiple donors, we show that BTV consistently reaches higher titers in ovine cells than cells from cattle. The variable replication kinetics of BTV in sheep and cow cells were mostly abolished by abrogating the cell type I interferon (IFN) response. We identified restriction factors blocking BTV replication, but both the sheep and cow orthologues of these antiviral genes possess anti-BTV properties. Importantly, we demonstrate that BTV induces a faster host cell protein synthesis shutoff in primary sheep cells than cow cells, which results in an earlier downregulation of antiviral proteins. Moreover, by using RNA sequencing (RNA-seq), we also show a more pronounced expression of interferon-stimulated genes (ISGs) in BTV-infected cow cells than sheep cells. Our data provide a new perspective on how the type I IFN response in reservoir species can have overall positive effects on both virus and host evolution. IMPORTANCE The host immune response usually aims to inhibit virus replication in order to avoid cell damage and disease. In some cases, however, the infected host avoids the deleterious effects of infection despite high levels of viral replication. This strategy is known as disease tolerance, and it is used by animal reservoirs of some zoonotic viruses. Here, using a virus of ruminants (bluetongue virus [BTV]) as an experimental system, we dissected virus-host interactions in cells collected from species that are susceptible (sheep) or tolerant (cow) to disease. We show that (i) virus modulation of the host antiviral type I interferon (IFN) responses, (ii) viral replication kinetics, and (iii) virus-induced cell damage differ in tolerant and susceptible BTV-infected cells. Understanding the complex virus-host interactions in disease tolerance can allow us to disentangle the critical balance between protective and damaging host immune responses.

Streams in the Mediterranean Region are not for mussels: Predicting extinctions and range contractions under future climate change.

Climate change is becoming the leading driver of biodiversity loss. The Mediterranean region, particularly southwestern Europe, is already confronting the consequences of ongoing global warming. Unprecedented biodiversity declines have been recorded, particularly within freshwater ecosystems. Freshwater mussels contribute to essential ecosystem services but are among the most threatened faunal groups on Earth. Their poor conservation status is related to the dependence on fish hosts to complete the life cycle, which also makes them particularly vulnerable to climate change. Species Distribution Models (SDMs) are commonly used to predict species distributions, but often disregard the potential effect of biotic interactions. This study investigated the potential impact of future climate on the distribution of freshwater mussel species while considering their obligatory interaction with fish hosts. Specifically, ensemble models were used to forecast the current and future distribution of six mussel species in the Iberian Peninsula, including environmental conditions and the distribution of fish hosts as predictors. We found that climate change is expected to severely impact the future distribution of Iberian mussels. Species with narrow ranges, namely Margaritifera margaritifera and Unio tumidiformis, were predicted to have their suitable habitats nearly lost and could potentially be facing regional and global extinctions, respectively. Anodonta anatina, Potomida littoralis, and particularly Unio delphinus and Unio mancus, are expected to suffer distributional losses but may gain new suitable habitats. A shift in their distribution to new suitable areas is only possible if fish hosts are able to disperse while carrying larvae. We also found that including the distribution of fish hosts in the mussels' models avoided the underprediction of habitat loss under climate change. This study warns of the imminent loss of mussel species and populations and the urgent need of management actions to reverse current trends and mitigate irreversible damage to species and ecosystems in Mediterranean regions.

The SARS-CoV-2 Alpha variant was associated with increased clinical severity of COVID-19 in Scotland: A genomics-based retrospective cohort analysis.

OBJECTIVES: The SARS-CoV-2 Alpha variant was associated with increased transmission relative to other variants present at the time of its emergence and several studies have shown an association between Alpha variant infection and increased hospitalisation and 28-day mortality. However, none have addressed the impact on maximum severity of illness in the general population classified by the level of respiratory support required, or death. We aimed to do this. METHODS: In this retrospective multi-centre clinical cohort sub-study of the COG-UK consortium, 1475 samples from Scottish hospitalised and community cases collected between 1st November 2020 and 30th January 2021 were sequenced. We matched sequence data to clinical outcomes as the Alpha variant became dominant in Scotland and modelled the association between Alpha variant infection and severe disease using a 4-point scale of maximum severity by 28 days: 1. no respiratory support, 2. supplemental oxygen, 3. ventilation and 4. death. RESULTS: Our cumulative generalised linear mixed model analyses found evidence (cumulative odds ratio: 1.40, 95% CI: 1.02, 1.93) of a positive association between increased clinical severity and lineage (Alpha variant versus pre-Alpha variants). CONCLUSIONS: The Alpha variant was associated with more severe clinical disease in the Scottish population than co-circulating lineages.

Adeno-associated virus 2 infection in children with non-A-E hepatitis.

An outbreak of acute hepatitis of unknown aetiology in children was reported in Scotland1 in April 2022 and has now been identified in 35 countries2. Several recent studies have suggested an association with human adenovirus with this outbreak, a virus not commonly associated with hepatitis. Here we report a detailed case-control investigation and find an association between adeno-associated virus 2 (AAV2) infection and host genetics in disease susceptibility. Using next-generation sequencing, PCR with reverse transcription, serology and in situ hybridization, we detected recent infection with AAV2 in plasma and liver samples in 26 out of 32 (81%) cases of hepatitis compared with 5 out of 74 (7%) of samples from unaffected individuals. Furthermore, AAV2 was detected within ballooned hepatocytes alongside a prominent T cell infiltrate in liver biopsy samples. In keeping with a CD4+ T-cell-mediated immune pathology, the human leukocyte antigen (HLA) class II HLA-DRB1*04:01 allele was identified in 25 out of 27 cases (93%) compared with a background frequency of 10 out of 64 (16%; P = 5.49 × 10-12). In summary, we report an outbreak of acute paediatric hepatitis associated with AAV2 infection (most likely acquired as a co-infection with human adenovirus that is usually required as a 'helper virus' to support AAV2 replication) and disease susceptibility related to HLA class II status.

Effects of culling vampire bats on the spatial spread and spillover of rabies virus.

Controlling pathogen circulation in wildlife reservoirs is notoriously challenging. In Latin America, vampire bats have been culled for decades in hopes of mitigating lethal rabies infections in humans and livestock. Whether culls reduce or exacerbate rabies transmission remains controversial. Using Bayesian state-space models, we show that a 2-year, spatially extensive bat cull in an area of exceptional rabies incidence in Peru failed to reduce spillover to livestock, despite reducing bat population density. Viral whole genome sequencing and phylogeographic analyses further demonstrated that culling before virus arrival slowed viral spatial spread, but reactive culling accelerated spread, suggesting that culling-induced changes in bat dispersal promoted viral invasions. Our findings question the core assumptions of density-dependent transmission and localized viral maintenance that underlie culling bats as a rabies prevention strategy and provide an epidemiological and evolutionary framework to understand the outcomes of interventions in complex wildlife disease systems.

Ledipasvir/sofosbuvir and ribavirin for the treatment of ribavirin-refractory persistent hepatitis E virus infection.

Persistent Hepatitis E Virus infection (HEV) is a rare but increasingly recognised condition in immunocompromised individuals. Untreated, this infection can rapidly progress to cirrhosis. Ribavirin is recommended as the first line treatment and the majority achieve sustained viral clearance. However, treatment options are limited for those who fail ribavirin. We report a case of a patients with ribavirin-refractory persistent HEV who responded to ledipasvir/sofosbuvir and ribavirin treatment. This patients had failed 2 course of ribavirin and 1 course of PEG-Interferon and ribavirin and he was known to harbour ribavirin-associated mutations (G1634R, D1384G and K1383N) in the RNA dependent RNA polymerase. He was treated with ledipasvir/sofosbuvir (LDV/SOF; Harvoni 90/400 mg) and ribavirin (R) 400 mg twice daily for 32 weeks. At treatment initiation his HEV RNA was 1.1 × 106 IU/ML and reduced to 1.8 × 104 IU/ML and 43 IU/ML at one and four weeks of treatment, respectively, becoming not detected in blood and stool by week eight. His blood HEV RNA remained undetectable for seven months after treatment completion. Unfortunately, at eight months post-treatment, his blood HEV RNA became detectable at a low level (35 IU/ML). His stool HEV RNA was also detectable at 620 IU/ML consistent with a late relapse. He restarted LDV/SOF+R and by week four of treatment HEV RNA was not detected in blood and stool. He remains on treatment. In conclusion, this is the first report demonstrating the antiviral activity of LDV/SOF+R in the treatment of persistent HEV infection.

Scale of population synchrony confirms macroecological estimates of minimum viable range size.

Global ecosystems are facing a deepening biodiversity crisis, necessitating robust approaches to quantifying species extinction risk. The lower limit of the macroecological relationship between species range and body size has long been hypothesized as an estimate of the relationship between the minimum viable range size (MVRS) needed for species persistence and the organismal traits that affect space and resource requirements. Here, we perform the first explicit test of this assumption by confronting the MVRS predicted by the range-body size relationship with an independent estimate based on the scale of synchrony in abundance among spatially separated populations of riverine fish. We provide clear evidence of a positive relationship between the scale of synchrony and species body size, and strong support for the MVRS set by the lower limit of the range-body size macroecological relationship. This MVRS may help prioritize first evaluations for unassessed or data-deficient taxa in global conservation assessments.

Identifying freshwater priority areas for cross-taxa interactions.

Information about biotic interactions (e.g. competition, predation, parasitism, diseases, mutualism, allelopathy) is fundamental to better understand species distribution and abundance, ecosystem functioning, and ultimately guide conservation efforts. However, conservation planning often overlooks these important interactions. Here, we aim to demonstrate a new framework to include biotic interactions into Marxan. For that, we use freshwater mussels and fish interaction (as mussels rely on fishes to complete their life cycle) in the Douro River basin (Iberian Peninsula) as a case study. While doing that, we also test the importance of including biotic interactions into conservation planning exercises, by running spatial prioritisation analysis considering either: 1) only the target species (freshwater mussels); 2) freshwater mussels and their obligatory hosts (freshwater fishes); 3) freshwater mussels, fishes and their interactions. With this framework we found that biotic interactions tend to be underrepresented when the data on both freshwater mussels and fishes is not simultaneously included in the spatial prioritisation. Overall, the priority areas selected across all scenarios are mostly located in the western part of the Douro River basin, where most freshwater mussels and fishes still occur. Given the low overlap of priority areas identified here and the current Natura 2000 network, our approach may be useful for establishing (or enlarging) protected areas, especially in light of the EU Biodiversity Strategy for 2030. Also, this work may provide guidance for future habitat restoration and management of main threats to freshwater biodiversity.

The apparent interferon resistance of transmitted HIV-1 is possibly a consequence of enhanced replicative fitness.

HIV-1 transmission via sexual exposure is an inefficient process. When transmission does occur, newly infected individuals are colonized by the descendants of either a single virion or a very small number of establishing virions. These transmitted founder (TF) viruses are more interferon (IFN)-resistant than chronic control (CC) viruses present 6 months after transmission. To identify the specific molecular defences that make CC viruses more susceptible to the IFN-induced 'antiviral state', we established a single pair of fluorescent TF and CC viruses and used arrayed interferon-stimulated gene (ISG) expression screening to identify candidate antiviral effectors. However, we observed a relatively uniform ISG resistance of transmitted HIV-1, and this directed us to investigate possible underlying mechanisms. Simple simulations, where we varied a single parameter, illustrated that reduced growth rate could possibly underly apparent interferon sensitivity. To examine this possibility, we closely monitored in vitro propagation of a model TF/CC pair (closely matched in replicative fitness) over a targeted range of IFN concentrations. Fitting standard four-parameter logistic growth models, in which experimental variables were regressed against growth rate and carrying capacity, to our in vitro growth curves, further highlighted that small differences in replicative growth rates could recapitulate our in vitro observations. We reasoned that if growth rate underlies apparent interferon resistance, transmitted HIV-1 would be similarly resistant to any growth rate inhibitor. Accordingly, we show that two transmitted founder HIV-1 viruses are relatively resistant to antiretroviral drugs, while their matched chronic control viruses were more sensitive. We propose that, when present, the apparent IFN resistance of transmitted HIV-1 could possibly be explained by enhanced replicative fitness, as opposed to specific resistance to individual IFN-induced defences. However, further work is required to establish how generalisable this mechanism of relative IFN resistance might be.

Genomic and Phenotypic Comparisons Reveal Distinct Variants of Wolbachia Strain wAlbB.

The intracellular bacterium Wolbachia inhibits virus replication and is being harnessed around the world to fight mosquito-borne diseases through releases of mosquitoes carrying the symbiont. Wolbachia strains vary in their ability to invade mosquito populations and suppress viruses in part due to differences in their density within the insect and associated fitness costs. Using whole-genome sequencing, we demonstrate the existence of two variants in wAlbB, a Wolbachia strain being released in natural populations of Aedes aegypti mosquitoes. The two variants display striking differences in genome architecture and gene content. Differences in the presence/absence of 52 genes between variants include genes located in prophage regions and others potentially involved in controlling the symbiont's density. Importantly, we show that these genetic differences correlate with variation in wAlbB density and its tolerance to heat stress, suggesting that different wAlbB variants may be better suited for field deployment depending on local environmental conditions. Finally, we found that the wAlbB genome remained stable following its introduction in a Malaysian mosquito population. Our results highlight the need for further genomic and phenotypic characterization of Wolbachia strains in order to inform ongoing Wolbachia-based programs and improve the selection of optimal strains in future field interventions. IMPORTANCE Dengue is a viral disease transmitted by Aedes mosquitoes that threatens around half of the world population. Recent advances in dengue control involve the introduction of Wolbachia bacterial symbionts with antiviral properties into mosquito populations, which can lead to dramatic decreases in the incidence of the disease. In light of these promising results, there is a crucial need to better understand the factors affecting the success of such strategies, in particular the choice of Wolbachia strain for field releases and the potential for evolutionary changes. Here, we characterized two variants of a Wolbachia strain used for dengue control that differ at the genomic level and in their ability to replicate within the mosquito. We also found no evidence for the evolution of the symbiont within the 2 years following its deployment in Malaysia. Our results have implications for current and future Wolbachia-based health interventions.

Genome sequencing and comparative analysis of Wolbachia strain wAlbA reveals Wolbachia-associated plasmids are common.

Wolbachia are widespread maternally-transmitted bacteria of arthropods that often spread by manipulating their host's reproduction through cytoplasmic incompatibility (CI). Their invasive potential is currently being harnessed in field trials aiming to control mosquito-borne diseases. Wolbachia genomes commonly harbour prophage regions encoding the cif genes which confer their ability to induce CI. Recently, a plasmid-like element was discovered in wPip, a Wolbachia strain infecting Culex mosquitoes; however, it is unclear how common such extra-chromosomal elements are in Wolbachia. Here we sequenced the complete genome of wAlbA, a strain of the symbiont found in Aedes albopictus, after eliminating the co-infecting and higher density wAlbB strain that previously made sequencing of wAlbA challenging. We show that wAlbA is associated with two new plasmids and identified additional Wolbachia plasmids and related chromosomal islands in over 20% of publicly available Wolbachia genome datasets. These plasmids encode a variety of accessory genes, including several phage-like DNA packaging genes as well as genes potentially contributing to host-symbiont interactions. In particular, we recovered divergent homologues of the cif genes in both Wolbachia- and Rickettsia-associated plasmids. Our results indicate that plasmids are common in Wolbachia and raise fundamental questions around their role in symbiosis. In addition, our comparative analysis provides useful information for the future development of genetic tools to manipulate and study Wolbachia symbionts.