Prevalence of persistent SARS-CoV-2 in a large community surveillance study.

Persistent SARS-CoV-2 infections may act as viral reservoirs that could seed future outbreaks1-5, give rise to highly divergent lineages6-8 and contribute to cases with post-acute COVID-19 sequelae (long COVID)9,10. However, the population prevalence of persistent infections, their viral load kinetics and evolutionary dynamics over the course of infections remain largely unknown. Here, using viral sequence data collected as part of a national infection survey, we identified 381 individuals with SARS-CoV-2 RNA at high titre persisting for at least 30 days, of which 54 had viral RNA persisting at least 60 days. We refer to these as 'persistent infections' as available evidence suggests that they represent ongoing viral replication, although the persistence of non-replicating RNA cannot be ruled out in all. Individuals with persistent infection had more than 50% higher odds of self-reporting long COVID than individuals with non-persistent infection. We estimate that 0.1-0.5% of infections may become persistent with typically rebounding high viral loads and last for at least 60 days. In some individuals, we identified many viral amino acid substitutions, indicating periods of strong positive selection, whereas others had no consensus change in the sequences for prolonged periods, consistent with weak selection. Substitutions included mutations that are lineage defining for SARS-CoV-2 variants, at target sites for monoclonal antibodies and/or are commonly found in immunocompromised people11-14. This work has profound implications for understanding and characterizing SARS-CoV-2 infection, epidemiology and evolution.

B cell expression of E3 ubiquitin ligase Cul4b promotes chronic gammaherpesvirus infection in vivo.

IMPORTANCE: The human gammaherpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus are etiologic agents of numerous B cell lymphomas. A hallmark of gammaherpesvirus infection is their ability to establish lifelong latency in B cells. However, the specific mechanisms that mediate chronic infection in B cells in vivo remain elusive. Cellular E3 ubiquitin ligases regulate numerous biological processes by catalyzing ubiquitylation and modifying protein location, function, or half-life. Many viruses hijack host ubiquitin ligases to evade antiviral host defense and promote viral fitness. Here, we used the murine gammaherpesvirus 68 in vivo system to demonstrate that the E3 ligase Cul4b is essential for this virus to establish latency in germinal center B cells. These findings highlight an essential role for this E3 ligase in promoting chronic gammaherpesvirus infection in vivo and suggest that targeted inhibition of E3 ligases may provide a novel and effective intervention strategy against gammaherpesvirus-associated diseases.

Isolation of a virus causing a chronic infection in the archaeal model organism Haloferax volcanii reveals antiviral activities of a provirus.

Viruses are important ecological, biogeochemical, and evolutionary drivers in every environment. Upon infection, they often cause the lysis of the host cell. However, some viruses exhibit alternative life cycles, such as chronic infections without cell lysis. The nature and the impact of chronic infections in prokaryotic host organisms remains largely unknown. Here, we characterize a novel haloarchaeal virus, Haloferax volcanii pleomorphic virus 1 (HFPV-1), which is currently the only virus infecting the model haloarchaeon Haloferax volcanii DS2, and demonstrate that HFPV-1 and H. volcanii are a great model system to study virus-host interactions in archaea. HFPV-1 is a pleomorphic virus that causes a chronic infection with continuous release of virus particles, but host and virus coexist without cell lysis or the appearance of resistant cells. Despite an only minor impact of the infection on host growth, we uncovered an extensive remodeling of the transcriptional program of the host (up to 1,049 differentially expressed genes). These changes are highlighted by a down-regulation of two endogenous provirus regions in the host genome, and we show that HFPV-1 infection is strongly influenced by a cross-talk between HFPV-1 and one of the proviruses mediated by a superinfection-like exclusion mechanism. Furthermore, HFPV-1 has a surprisingly wide host range among haloarchaea, and purified virus DNA can cause an infection after transformation into the host, making HFPV-1 a candidate for being developed into a genetic tool for a range of so far inaccessible haloarchaea.

Rb-E2F-HDAC Repressor Complexes Control Interferon-Induced Repression of Adenovirus To Promote Persistent Infection.

Interferons (IFNs) are cytokines that induce a global change in the cell to establish antiviral immunity. We previously demonstrated that human adenovirus (HAdV) exploits IFN-induced viral repression to persist in infected cells. Although this in vitro persistence model has been described, the mechanism behind how persistent HAdV infection is established is not well understood. In this study, we demonstrate that IFN signaling is essential for viral repression and promoting persistent infection. Cyclin-dependent kinase 4 (CDK4), an antagonist of retinoblastoma (Rb) family proteins, was shown to disrupt the viral repression induced by IFNs. Consistent with this result, knockout of the Rb family proteins pRb, p107, and/or p130 drastically reduced the effect of IFNs on viral replication. The pRb protein specifically contributed the greatest effect to IFN inhibition of viral replication. Interestingly, IFNs did not impact pRb through direct changes in protein or phosphorylation levels. Cells treated with IFNs continued to cycle normally, consistent with observations that persistently infected cells remain for long periods of time in the host and in our in vitro persistent infection model. Finally, we observed that histone deacetylase (HDAC) inhibitors activated productive viral replication in persistently infected cells in the presence of IFN. Thus, HDACs, specifically class I HDACs, which are commonly associated with Rb family proteins, play a major role in the maintenance of persistent HAdV infection in vitro. This study uncovers the critical role of pRb and class I HDACs in the IFN-induced formation of a repressor complex that promotes persistent HAdV infections. IMPORTANCE Adenoviruses are ubiquitous viruses infecting more than 90% of the human population. HAdVs cause persistent infections that may lead to serious complications in immunocompromised patients. Therefore, exploring how HAdVs establish persistent infections is critical for understanding viral reactivation in immunosuppressed individuals. The mechanism underlying HAdV persistence has not been fully explored. Here, we provide insight into the contributions of the host cell to IFN-mediated persistent HAdV infection. We found that HAdV-C5 productive infection is inhibited by an Rb-E2F-HDAC repressor complex. Treatment with HDAC inhibitors converted a persistent infection to a lytic infection. Our results suggest that this process involves the noncanonical regulation of Rb-E2F signaling. This study provides insight into a highly prevalent human pathogen, bringing a new level of complexity and understanding to the replicative cycle.

The Reservoir of Persistent Human Papillomavirus Infection; Strategies for Elimination Using Anti-Viral Therapies.

Human Papillomaviruses have co-evolved with their human host, with each of the over 200 known HPV types infecting distinct epithelial niches to cause diverse disease pathologies. Despite the success of prophylactic vaccines in preventing high-risk HPV infection, the development of HPV anti-viral therapies has been hampered by the lack of enzymatic viral functions, and by difficulties in translating the results of in vitro experiments into clinically useful treatment regimes. In this review, we discuss recent advances in anti-HPV drug development, and highlight the importance of understanding persistent HPV infections for future anti-viral design. In the infected epithelial basal layer, HPV genomes are maintained at a very low copy number, with only limited viral gene expression; factors which allow them to hide from the host immune system. However, HPV gene expression confers an elevated proliferative potential, a delayed commitment to differentiation, and preferential persistence of the infected cell in the epithelial basal layer, when compared to their uninfected neighbours. To a large extent, this is driven by the viral E6 protein, which functions in the HPV life cycle as a modulator of epithelial homeostasis. By targeting HPV gene products involved in the maintenance of the viral reservoir, there appears to be new opportunities for the control or elimination of chronic HPV infections.

Lack of Fetal Protection against Bovine Viral Diarrhea Virus in a Vaccinated Heifer.

The aim of the report was to present the circulation of BVDV (bovine viral diarrhea virus) in the cattle population and determine the cause of the failure of vaccination failure leading to the birth of the PI (persistently infected) calf. The case study was carried out at the BVDV-free animal breeding center and cattle farm, where the vaccination program against BVDV was implemented in 2012, and each newly introduced animal was serologically and virologically tested for BVDV. In this case, a blood sample was taken from a 9-month-old breeding bull. Positive RT-PCR and negative ELISA serology results were obtained. The tests were repeated at 2-week intervals, and the results confirmed the presence of the virus and the absence of specific antibodies, i.e., persistent infection. Additionally, sequencing and phylogenetic analysis were performed, and the BVDV-1d subgenotype was detected. The results of this study showed that pregnant heifers and cows that are vaccinated multiple times with the killed vaccine containing BVDV-1a may not be fully protected against infection with other subgenotypes of BVDV, including their fetuses, which can become PI calves.

Prolonged SARS-CoV-2 Infection in Patients with Lymphoid Malignancies.

Coronavirus disease 2019 (COVID-19) infection results in both acute mortality and persistent and/or recurrent disease in patients with hematologic malignancies, but the drivers of persistent infection in this population are unknown. We found that B-cell lymphomas were at particularly high risk for persistent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positivity. Further analysis of these patients identified discrete risk factors for initial disease severity compared with disease chronicity. Active therapy and diminished T-cell counts were drivers of acute mortality in COVID-19-infected patients with lymphoma. Conversely, B cell-depleting therapy was the primary driver of rehospitalization for COVID-19. In patients with persistent SARS-CoV-2 positivity, we observed high levels of viral entropy consistent with intrahost viral evolution, particularly in patients with impaired CD8+ T-cell immunity. These results suggest that persistent COVID-19 infection is likely to remain a risk in patients with impaired adaptive immunity and that additional therapeutic strategies are needed to enable viral clearance in this high-risk population. SIGNIFICANCE: We describe the largest cohort of persistent symptomatic COVID-19 infection in patients with lymphoid malignancies and identify B-cell depletion as the key immunologic driver of persistent infection. Furthermore, we demonstrate ongoing intrahost viral evolution in patients with persistent COVID-19 infection, particularly in patients with impaired CD8+ T-cell immunity.This article is highlighted in the In This Issue feature, p. 1.

Peripheral blood iNKT cell activation correlates with liver damage during acute hepatitis C.

Invariant NK T (iNKT) cells are implicated in viral clearance; however, their role in hepatitis C virus (HCV) infection remains controversial. Here, iNKT cells were studied during different stages of HCV infection. iNKT cells from patients with acute HCV infection and people who inject drugs (PWID) with chronic or spontaneously resolved HCV infection were characterized by flow cytometry. In a longitudinal analysis during acute HCV infection, frequencies of activated CD38+ iNKT cells reproducibly declined in spontaneously resolving patients, whereas they were persistently elevated in patients progressing to chronic infection. During the first year of infection, the frequency of activated CD38+ or CD69+ iNKT cells strongly correlated with alanine transaminase levels with particularly pronounced correlations in spontaneously resolving patients. Increased frequencies of activated iNKT cells in chronic HCV infection were confirmed in cross-sectional analyses of PWID with chronic or spontaneously resolved HCV infection; however, no apparent functional differences were observed with various stimulation protocols. Our data suggest that iNKT cells are activated during acute hepatitis C and that activation is sustained in chronic infection. The correlation between the frequency of activated iNKT cells and alanine transaminase may point toward a role of iNKT cells in liver damage.

Characteristics of the Cervicovaginal Microenvironment in Childbearing-Age Women with Different Degrees of Cervical Lesions and HR-HPV Positivity.

Persistent infection with high-risk human papillomavirus (HR-HPV) is the most important determinate in the development of cervical cancer, and cervical microecology can modulate cervical viral infection. However, few studies have been conducted on the microecological analysis of cervical diseases using strict physiological factors. This study investigated the characteristics and dynamics of cervical microecology in childbearing-age Chinese women with different degrees of HR-HPV-positive cervical lesions. A total of 168 subjects were selected according to the selection criteria, including healthy HPV-negative individuals (n = 29), HR-HPV-infected individuals (n = 29), low-grade squamous intraepithelial lesion individuals (LSIL, n = 32), high-grade squamous intraepithelial lesion individuals (HSIL, n = 40), and cervical cancer individuals (n = 38). We sampled cervical secretions from each subject and performed comparative analysis using the 16S rRNA sequencing method. Comparison analysis showed that Lactobacillus and Ignatzschineria were the dominant genera in the healthy group, while Gardnerella and Prevotella were more enriched in the disease groups. Based on the taxa composition, we roughly divided the development of cervical cancer into two phases: phase I was from healthy status to HR-HPV infection and LSIL; phase II was from LSIL to HSIL and cervical cancer. Different interactions among different genera were observed in different groups. Prevotella inhibited the abundance of Lactobacillus in the healthy group, while Prevotella inhabited the abundance of Gardnerella in the other groups. In the HR-HPV infection group, Ignatzschineria and Enterococcus showed a positive interaction but dissociated with the increase in cervical lesions, which might eventually lead to a continuous decrease in the abundances of Lactobacillus and Ignatzschineria.

Persistent SARS-CoV-2 infection in patients with secondary antibody deficiency: successful clearance following combination casirivimab and imdevimab (REGN-COV2) monoclonal antibody therapy.

BACKGROUND: There is growing evidence that antibody responses play a role in the resolution of SARS-CoV-2 infection. Patients with primary or secondary antibody deficiency are at increased risk of persistent infection. This challenging clinical scenario is associated with adverse patient outcome and potentially creates an ecological niche for the evolution of novel SARS-CoV-2 variants with immune evasion capacity. Case reports and/or series have implied a therapeutic role for convalescent plasma (CP) to secure virological clearance, although concerns have been raised about the effectiveness of CP and its potential to drive viral evolution, and it has largely been withdrawn from clinical use in the UK. CASE PRESENTATION: We report two cases in which persistent SARS-CoV-2 infection was cleared following administration of the monoclonal antibody combination casirivimab and imdevimab (REGN-COV2, Ronapreve). A 55-year-old male with follicular lymphoma, treated with B cell depleting therapy, developed SARS-CoV-2 infection in September 2020 which then persisted for over 200 days. He was hospitalised on four occasions with COVID-19 and suffered debilitating fatigue and malaise throughout. There was no clinical response to antiviral therapy with remdesivir or CP, and SARS-CoV-2 was consistently detected in nasopharyngeal swabs. Intrahost evolution of several spike variants of uncertain significance was identified by viral sequence analysis. Delivery of REGN-COV2, in combination with remdesivir, was associated with clinical improvement and viral clearance within 6 days, which was sustained for over 150 days despite immunotherapy for relapsed follicular lymphoma. The second case, a 68-year-old female with chronic lymphocytic leukaemia on ibrutinib, also developed persistent SARS-CoV-2 infection. Despite a lack of response to remdesivir, infection promptly cleared following REGN-COV2 in combination with remdesivir, accompanied by resolution of inflammation and full clinical recovery that has been maintained for over 290 days. CONCLUSIONS: These cases highlight the potential benefit of REGN-COV2 as therapy for persistent SARS-CoV-2 infection in antibody deficient individuals, including after failure of CP treatment. Formal clinical studies are warranted to assess the effectiveness of REGN-COV2 in antibody-deficient patients, especially in light of the emergence of variants of concern, such as Omicron, that appear to evade REGN-COV2 neutralisation.

Preferential and persistent impact of acute HIV-1 infection on CD4+ iNKT cells in colonic mucosa.

Acute HIV-1 infection (AHI) results in the widespread depletion of CD4+ T cells in peripheral blood and gut mucosal tissue. However, the impact on the predominantly CD4+ immunoregulatory invariant natural killer T (iNKT) cells during AHI remains unknown. Here, iNKT cells from peripheral blood and colonic mucosa were investigated during treated and untreated AHI. iNKT cells in blood were activated and rapidly depleted in untreated AHI. At the time of peak HIV-1 viral load, these cells showed the elevated expression of cell death-associated transcripts compared to preinfection. Residual peripheral iNKT cells suffered a diminished responsiveness to in vitro stimulation early into chronic infection. Additionally, HIV-1 DNA, as well as spliced and unspliced viral RNA, were detected in iNKT cells isolated from blood, indicating the active infection of these cells in vivo. The loss of iNKT cells occurred from Fiebig stage III in the colonic mucosa, and these cells were not restored to normal levels after initiation of ART during AHI. CD4+ iNKT cells were depleted faster and more profoundly than conventional CD4+ T cells, and the preferential infection of CD4+ iNKT cells over conventional CD4+ T cells was confirmed by in vitro infection experiments. In vitro data also provided evidence of latent infection in iNKT cells. Strikingly, preinfection levels of peripheral blood CD4+ iNKT cells correlated directly with the peak HIV-1 load. These findings support a model in which iNKT cells are early targets for HIV-1 infection, driving their rapid loss from circulation and colonic mucosa.

Innate Immune Antagonism of Mosquito-Borne Flaviviruses in Humans and Mosquitoes.

Mosquito-borne viruses of the Flavivirus genus (Flaviviridae family) pose an ongoing threat to global public health. For example, dengue, Japanese encephalitis, West Nile, yellow fever, and Zika viruses are transmitted by infected mosquitoes and cause severe and fatal diseases in humans. The means by which mosquito-borne flaviviruses establish persistent infection in mosquitoes and cause disease in humans are complex and depend upon a myriad of virus-host interactions, such as those of the innate immune system, which are the main focus of our review. This review also covers the different strategies utilized by mosquito-borne flaviviruses to antagonize the innate immune response in humans and mosquitoes. Given the lack of antiviral therapeutics for mosquito-borne flaviviruses, improving our understanding of these virus-immune interactions could lead to new antiviral therapies and strategies for developing refractory vectors incapable of transmitting these viruses, and can also provide insights into determinants of viral tropism that influence virus emergence into new species.

Host defence and persistent human papillomavirus infection.

The ability to establish long term persistent infection is a feature of human papillomaviruses. The available evidence is that this ability is a consequence of a complex local immune milieu whereby innate immune receptors and signalling pathway cascades are inhibited by HPV early proteins resulting in failure of dendritic cell maturation, antigen processing and presentation and activation of cytotoxic antigen specific T cell responses. The development of cutaneous and mucosal infection models with the mouse papillomavirus MmuPV1 and the access to multiple gene deficient strains is providing the frame work to dissect the mechanisms underlying these complex host virus interactions.

Early Effects of HTLV-1 Infection on the Activation, Exhaustion, and Differentiation of T-Cells in Humanized NSG Mice.

Adult T-cell leukemia/lymphoma (ATLL) is an aggressive malignancy of CD4+ T-cells associated with HTLV-1 infection. In this study, we used the model of immunodeficient NSG mice reconstituted with a functional human immune system (HIS) to investigate early events in HTLV-1 pathogenesis. Upon infection, human T-cells rapidly increased in the blood and lymphoid tissues, particularly CD4+CD25+ T-cells. Proliferation of CD4+ T-cells in the spleen and mesenteric lymph nodes (MLN) correlated with HTLV-1 proviral load and CD25 expression. In addition, splenomegaly, a common feature of ATLL in humans, was also observed. CD4+ and CD8+ T-cells predominantly displayed an effector memory phenotype (CD45RA-CCR7-) and expressed CXCR3 and CCR5 chemokine receptors, suggesting the polarization into a Th1 phenotype. Activated CD8+ T-cells expressed granzyme B and perforin; however, the interferon-γ response by these cells was limited, possibly due to elevated PD-1 expression and increased frequency of CD4+FoxP3+ regulatory T-cells in MLN. Thus, HTLV-1-infected HIS-NSG mice reproduced several characteristics of infection in humans, and it may be helpful to investigate ATLL-related events and to perform preclinical studies. Moreover, aspects of chronic infection were already present at early stages in this experimental model. Collectively, we suggest that HTLV-1 infection modulates host immune responses to favor viral persistence.

Chronic LCMV Infection Is Fortified with Versatile Tactics to Suppress Host T Cell Immunity and Establish Viral Persistence.

Ever since the immune regulatory strains of lymphocytic choriomeningitis virus (LCMV), such as Clone 13, were isolated, LCMV infection of mice has served as a valuable model for the mechanistic study of viral immune suppression and virus persistence. The exhaustion of virus-specific T cells was demonstrated during LCMV infection, and the underlying mechanisms have been extensively investigated using LCMV infection in mouse models. In particular, the mechanism for gradual CD8+ T cell exhaustion at molecular and transcriptional levels has been investigated. These studies revealed crucial roles for inhibitory receptors, surface markers, regulatory cytokines, and transcription factors, including PD-1, PSGL-1, CXCR5, and TOX in the regulation of T cells. However, the action mode for CD4+ T cell suppression is largely unknown. Recently, sphingosine kinase 2 was proven to specifically repress CD4+ T cell proliferation and lead to LCMV persistence. As CD4+ T cell regulation was also known to be important for viral persistence, research to uncover the mechanism for CD4+ T cell repression could help us better understand how viruses launch and prolong their persistence. This review summarizes discoveries derived from the study of LCMV in regard to the mechanisms for T cell suppression and approaches for the termination of viral persistence with special emphasis on CD8+ T cells.

A Cell Culture Model of BK Polyomavirus Persistence, Genome Recombination, and Reactivation.

BK polyomavirus (BKPyV) is a small nonenveloped DNA virus that establishes a ubiquitous, asymptomatic, and lifelong persistent infection in at least 80% of the world's population. In some immunosuppressed transplant recipients, BKPyV reactivation causes polyomavirus-associated nephropathy and hemorrhagic cystitis. We report a novel in vitro model of BKPyV persistence and reactivation using a BKPyV natural host cell line. In this system, viral genome loads remain constant for various times after establishment of persistent infection, during which BKPyV undergoes extensive random genome recombination. Certain recombination events result in viral DNA amplification and protein expression, resulting in production of viruses with enhanced replication ability. IMPORTANCE BK polyomavirus (BKPyV) generally establishes a persistent subclinical infection in healthy individuals but can cause severe disease in transplant recipients. While an in vitro model to study acute replication exists, no practical model with which to study BKPyV persistence is currently available. We established a BKPyV persistence model in cell culture. Our model reveals that the virus can persist for various periods of time before random recombination of the viral genome leads to enhanced replication.

Simultaneous and Staggered Foot-and-Mouth Disease Virus Coinfection of Cattle.

Foot-and-mouth disease (FMD) field studies have suggested the occurrence of simultaneous infection of individual hosts by multiple virus strains; however, the pathogenesis of foot-and-mouth disease virus (FMDV) coinfections is largely unknown. In the current study, cattle were experimentally exposed to two FMDV strains of different serotypes (O and A). One cohort was simultaneously infected with both viruses, while additional cohorts were initially infected with FMDV A and subsequently superinfected with FMDV O after 21 or 35 days. Coinfections were confirmed during acute infection, with both viruses concurrently detected in blood, lesions, and secretions. Staggered exposures resulted in overlapping infections as convalescent animals with persistent subclinical FMDV infection were superinfected with a heterologous virus. Staggering virus exposure by 21 days conferred clinical protection in six of eight cattle, which were subclinically infected following the heterologous virus exposure. This effect was transient, as all animals superinfected at 35 days post-initial infection developed fulminant FMD. The majority of cattle maintained persistent infection with one of the two viruses while clearing the other. Analysis of viral genomes confirmed interserotypic recombination events within 10 days in the upper respiratory tract of five superinfected animals from which the dominant genomes contained the capsid coding regions of the O virus and nonstructural coding regions of the A virus. In contrast, there were no dominant recombinant genomes detected in samples from simultaneously coinfected cattle. These findings inculpate persistently infected carriers as potential FMDV mixing vessels in which novel strains may rapidly emerge through superinfection and recombination. IMPORTANCE Foot-and-mouth disease (FMD) is a viral infection of livestock of critical socioeconomic importance. Field studies from areas of endemic FMD suggest that animals can be simultaneously infected by more than one distinct variant of FMD virus (FMDV), potentially resulting in emergence of novel viral strains through recombination. However, there has been limited investigation of the mechanisms of in vivo FMDV coinfections under controlled experimental conditions. Our findings confirmed that cattle could be simultaneously infected by two distinct serotypes of FMDV, with different outcomes associated with the timing of exposure to the two different viruses. Additionally, dominant interserotypic recombinant FMDVs were discovered in multiple samples from the upper respiratory tracts of five superinfected animals, emphasizing the potential importance of persistently infected FMDV carriers as sources of novel FMDV strains.

Resurgence of Ebola virus in 2021 in Guinea suggests a new paradigm for outbreaks.

Seven years after the declaration of the first epidemic of Ebola virus disease in Guinea, the country faced a new outbreak-between 14 February and 19 June 2021-near the epicentre of the previous epidemic1,2. Here we use next-generation sequencing to generate complete or near-complete genomes of Zaire ebolavirus from samples obtained from 12 different patients. These genomes form a well-supported phylogenetic cluster with genomes from the previous outbreak, which indicates that the new outbreak was not the result of a new spillover event from an animal reservoir. The 2021 lineage shows considerably lower divergence than would be expected during sustained human-to-human transmission, which suggests a persistent infection with reduced replication or a period of latency. The resurgence of Zaire ebolavirus from humans five years after the end of the previous outbreak of Ebola virus disease reinforces the need for long-term medical and social care for patients who survive the disease, to reduce the risk of re-emergence and to prevent further stigmatization.

Distribution and Pathogenicity of Two Cutthroat Trout Virus (CTV) Genotypes in Canada.

The sole member of the Piscihepevirus genus (family Hepeviridae) is cutthroat trout virus (CTV) but recent metatranscriptomic studies have identified numerous fish hepevirus sequences including CTV-2. In the current study, viruses with sequences resembling both CTV and CTV-2 were isolated from salmonids in eastern and western Canada. Phylogenetic analysis of eight full genomes delineated the Canadian CTV isolates into two genotypes (CTV-1 and CTV-2) within the Piscihepevirus genus. Hepevirus genomes typically have three open reading frames but an ORF3 counterpart was not predicted in the Canadian CTV isolates. In vitro replication of a CTV-2 isolate produced cytopathic effects in the CHSE-214 cell line with similar amplification efficiency as CTV. Likewise, the morphology of the CTV-2 isolate resembled CTV, yet viral replication caused dilation of the endoplasmic reticulum lumen which was not previously observed. Controlled laboratory studies exposing sockeye (Oncorhynchus nerka), pink (O. gorbuscha), and chinook salmon (O. tshawytscha) to CTV-2 resulted in persistent infections without disease and mortality. Infected Atlantic salmon (Salmo salar) and chinook salmon served as hosts and potential reservoirs of CTV-2. The data presented herein provides the first in vitro and in vivo characterization of CTV-2 and reveals greater diversity of piscihepeviruses extending the known host range and geographic distribution of CTV viruses.

PD-1+ Tcf1+ CD8+ T cells from established chronic infection can form memory while retaining a stableimprint of persistent antigen exposure.

Virus-specific PD1+ Tcf1+ memory-like CD8+ T cells (TMLs) maintain the CD8+ T cell response during chronic viral infection. However, the fate of these cells following cessation of persistent antigen exposure has been unclear. Here, we find that TMLs persist upon transfer into antigen-free hosts and form memory following recall stimulation. Phenotypic, functional, and transcriptome analyses show that TML-derived memory cells resemble those arising in response to acute, resolved infection, but they retain features of chronically stimulated cells, including elevated PD-1 and Tox and reduced cytokine expression. This chronic infection imprint is largely accounted for by constitutive Tox expression. Virus-specific Tcf1+ CD8+ T cells that persist after clearance of systemic infection also display a chronic infection imprint. Notwithstanding, renewed virus exposure induces a recall response, which controls virus infection in part. Thus, cessation of chronic antigen exposure yields a memory CD8+ T cell compartment that reflects prior stimulation.