Liver X Receptor-Inducible Host E3 Ligase IDOL Targets a Human Cytomegalovirus Reactivation Determinant.

Liver X receptor (LXR) signaling broadly restricts virus replication; however, the mechanisms of restriction are poorly defined. Here, we demonstrate that the cellular E3 ligase LXR-inducible degrader of low-density lipoprotein receptor (IDOL) targets the human cytomegalovirus (HMCV) UL136p33 protein for turnover. UL136 encodes multiple proteins that differentially impact latency and reactivation. UL136p33 is a determinant of reactivation. UL136p33 is targeted for rapid turnover by the proteasome, and its stabilization by mutation of lysine residues to arginine results in a failure to quiet replication for latency. We show that IDOL targets UL136p33 for turnover but not the stabilized variant. IDOL is highly expressed in undifferentiated hematopoietic cells where HCMV establishes latency but is sharply downregulated upon differentiation, a stimulus for reactivation. We hypothesize that IDOL maintains low levels of UL136p33 for the establishment of latency. Consistent with this hypothesis, knockdown of IDOL impacts viral gene expression in wild-type (WT) HCMV infection but not in infection where UL136p33 has been stabilized. Furthermore, the induction of LXR signaling restricts WT HCMV reactivation from latency but does not affect the replication of a recombinant virus expressing a stabilized variant of UL136p33. This work establishes the UL136p33-IDOL interaction as a key regulator of the bistable switch between latency and reactivation. It further suggests a model whereby a key viral determinant of HCMV reactivation is regulated by a host E3 ligase and acts as a sensor at the tipping point between the decision to maintain the latent state or exit latency for reactivation. IMPORTANCE Herpesviruses establish lifelong latent infections, which pose an important risk for disease particularly in the immunocompromised. Our work is focused on the betaherpesvirus human cytomegalovirus (HCMV) that latently infects the majority of the population worldwide. Defining the mechanisms by which HCMV establishes latency or reactivates from latency is important for controlling viral disease. Here, we demonstrate that the cellular inducible degrader of low-density lipoprotein receptor (IDOL) targets a HCMV determinant of reactivation for degradation. The instability of this determinant is important for the establishment of latency. This work defines a pivotal virus-host interaction that allows HCMV to sense changes in host biology to navigate decisions to establish latency or to replicate.

The liver X receptor agonist LXR 623 restricts flavivirus replication.

The vector-borne flaviviruses (VBFVs) are well known for causing great misery and death in humans worldwide. The VBFVs include those transmitted by mosquitos, such as Zika virus (ZIKV), dengue virus; and those transmitted by ticks including the tick-borne flavivirus serocomplex and Powassan virus (POWV). Two of our recent reports showed that intracranial POWV infection in the reservoir host, Peromyscus leucopus, was restricted and caused no overt clinical disease. Several modes of analyses suggested activation of the LXR pathway. Activation of the LXR pathway leads to increased efflux of cholesterol from cells and consequent disturbances in membrane biogenesis. Because VBFV replication is dependent on membrane biogenesis, we evaluated the effect of an LXR agonist (LXR623) on POWV and ZIKV infection and observed that the compound impaired permissive replication of both viruses in a human neuroblastoma SK-N-SH cell line. The LXR agonist resulted in failure of the viruses to induce ER expansion and elaborate vesicle formation, suggesting that the efflux of cholesterol was part of the antiviral mechanism. We also observed that the LXR agonist contributed to the mechanism of virus suppression by increased expression of mRNAs encoding for the antiviral cytokines CXCL10, RANTES and IFN1ß. In sharp contrast, a LXR antagonist (GSK2033) had no significant effect on VBFV replication. We conclude that LXR623 impairs flavivirus replication by stimulating cellular antiviral factors.

Cell type-specific biogenesis of novel vesicles containing viral products in human cytomegalovirus infection.

Human cytomegalovirus (HCMV), while highly restricted for the human species, infects an diverse array of cell types in the host. Patterns of infection are dictated by the cell type infected, but cell type-specific factors and how they impact tropism for specific cell types is poorly understood. Previous studies in primary endothelial cells showed that HCMV infection induces large multivesicular-like bodies (MVBs) that incorporate viral products, including dense bodies (DBs) and virions. Here we define the nature of these large vesicles using a recombinant virus where UL32, encoding the pp150 tegument protein, is fused in frame with green fluorescent protein (GFP, TB40/E-UL32-GFP). In fibroblasts, UL32-GFP-positive vesicles were marked with classical markers of MVBs, including CD63 and lysobisphosphatidic acid (LBPA), both classical MVB markers, as well as the clathrin and LAMP1. Unexpectedly, UL32-GFP-positive vesicles in primary human microvascular endothelial cells (HMVECs) were not labeled by CD63, and LBPA was completely lost from infected cells. We defined these UL32-positive vesicles in endothelial cells using markers for the cis-Golgi (GM130), lysosome (LAMP1), and autophagy (LC3B). These findings suggest that UL32-GFP containing MVBs in fibroblasts are derived from the canonical endocytic pathway and takeover classical exosomal release pathway. However, UL32-GFP containing MVBs in HMVECs are derived from the early biosynthetic pathway and exploit a less characterized early Golgi-LAMP1-associated non- canonical secretory autophagy pathway. These results reveal striking cell-type specific membrane trafficking differences in host pathways that are exploited by HCMV, which may reflect distinct pathways for virus egress.ImportanceHuman cytomegalovirus (HCMV) is a herpesvirus that, like all herpesvirus, that establishes a life-long infection. HCMV remains a significant cause of morbidity and mortality in the immunocompromised and HCMV seropositivity is associated with age-related pathology. HCMV infects many cells in the human host and the biology underlying the different patterns of infection in different cell types is poorly understood. Endothelial cells are important target of infection that contribute to hematogenous spread of the virus to tissues. Here we define striking differences in the biogenesis of large vesicles that incorporate virions in fibroblasts and endothelial cells. In fibroblasts, HCMV is incorporated into canonical MVBs derived from an endocytic pathway, whereas HCMV matures through vesicles derived from the biosynthetic pathway in endothelial cells. This work defines basic biological differences between these cell types that may impact how progeny virus is trafficked out of infected cells.

Tick-Borne Flaviviruses Depress AKT Activity during Acute Infection by Modulating AKT1/2.

Tick-borne flaviviruses (TBFVs) are reemerging public health threats. To develop therapeutics against these pathogens, increased understanding of their interactions with the mammalian host is required. The PI3K-AKT pathway has been implicated in TBFV persistence, but its role during acute virus infection remains poorly understood. Previously, we showed that Langat virus (LGTV)-infected HEK 293T cells undergo a lytic crisis with a few surviving cells that become persistently infected. We also observed that AKT2 mRNA is upregulated in cells persistently infected with TBFV. Here, we investigated the virus-induced effects on AKT expression over the course of acute LGTV infection and found that total phosphorylated AKT (pAKT), AKT1, and AKT2 decrease over time, but AKT3 increases dramatically. Furthermore, cells lacking AKT1 or AKT2 were more resistant to LGTV-induced cell death than wild-type cells because they expressed higher levels of pAKT and antiapoptotic proteins, such as XIAP and survivin. The differential modulation of AKT by LGTV may be a mechanism by which viral persistence is initiated, and our results demonstrate a complicated manipulation of host pathways by TBFVs.

The Role of the Human Cytomegalovirus UL133-UL138 Gene Locus in Latency and Reactivation.

Human cytomegalovirus (HCMV) latency, the means by which the virus persists indefinitely in an infected individual, is a major frontier of current research efforts in the field. Towards developing a comprehensive understanding of HCMV latency and its reactivation from latency, viral determinants of latency and reactivation and their host interactions that govern the latent state and reactivation from latency have been identified. The polycistronic UL133-UL138 locus encodes determinants of both latency and reactivation. In this review, we survey the model systems used to investigate latency and new findings from these systems. Particular focus is given to the roles of the UL133, UL135, UL136 and UL138 proteins in regulating viral latency and how their known host interactions contribute to regulating host signaling pathways towards the establishment of or exit from latency. Understanding the mechanisms underlying viral latency and reactivation is important in developing strategies to block reactivation and prevent CMV disease in immunocompromised individuals, such as transplant patients.

A molecular mechanism for probabilistic bet hedging and its role in viral latency.

Probabilistic bet hedging, a strategy to maximize fitness in unpredictable environments by matching phenotypic variability to environmental variability, is theorized to account for the evolution of various fate-specification decisions, including viral latency. However, the molecular mechanisms underlying bet hedging remain unclear. Here, we report that large variability in protein abundance within individual herpesvirus virion particles enables probabilistic bet hedging between viral replication and latency. Superresolution imaging of individual virions of the human herpesvirus cytomegalovirus (CMV) showed that virion-to-virion levels of pp71 tegument protein-the major viral transactivator protein-exhibit extreme variability. This super-Poissonian tegument variability promoted alternate replicative strategies: high virion pp71 levels enhance viral replicative fitness but, strikingly, impede silencing, whereas low virion pp71 levels reduce fitness but promote silencing. Overall, the results indicate that stochastic tegument packaging provides a mechanism enabling probabilistic bet hedging between viral replication and latency.

PERK-Mediated Unfolded Protein Response Signaling Restricts Replication of the Tick-Borne Flavivirus Langat Virus.

The unfolded protein response (UPR) maintains protein-folding homeostasis in the endoplasmic reticulum (ER) and has been implicated as both beneficial and detrimental to flavivirus infection. Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), a sensor of the UPR, is commonly associated with antiviral effects during mosquito-borne flavivirus (MBFV) infection, but its relation to tick-borne flavivirus (TBFV) infection remains largely unexplored. In this study, we identified changes in UPR and autophagic activity during Langat virus (LGTV) infection. LGTV robustly activated UPR and altered autophagic flux. Knockdown of endogenous PERK in human cells resulted in increased LGTV replication, but not that of closely related Powassan virus (POWV). Finally, on examining changes in protein levels of components associated with UPR and autophagy in the absence of PERK, we could show that LGTV-infected cells induced UPR but did not lead to expression of C/EBP homologous protein (CHOP), an important downstream transcription factor of multiple stress pathways. From these data, we hypothesize that LGTV can antagonize other kinases that target eukaryotic initiation factor 2α (eIF2α), but not PERK, implicating PERK as a potential mediator of intrinsic immunity. This effect was not apparent for POWV, a more pathogenic TBFV, suggesting it may be better equipped to mitigate the antiviral effects of PERK.

An RNA-centric dissection of host complexes controlling flavivirus infection.

Flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), cause severe human disease. Co-opting cellular factors for viral translation and viral genome replication at the endoplasmic reticulum is a shared replication strategy, despite different clinical outcomes. Although the protein products of these viruses have been studied in depth, how the RNA genomes operate inside human cells is poorly understood. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we took an RNA-centric viewpoint of flaviviral infection and identified several hundred proteins associated with both DENV and ZIKV genomic RNA in human cells. Genome-scale knockout screens assigned putative functional relevance to the RNA-protein interactions observed by ChIRP-MS. The endoplasmic-reticulum-localized RNA-binding proteins vigilin and ribosome-binding protein 1 directly bound viral RNA and each acted at distinct stages in the life cycle of flaviviruses. Thus, this versatile strategy can elucidate features of human biology that control the pathogenesis of clinically relevant viruses.

Differential Zika Virus Infection of Testicular Cell Lines.

BACKGROUND: Zika virus is a mosquito-borne flavivirus responsible for recent outbreaks of epidemic proportions in Latin America. Sexual transmission of the virus has been reported in 13 countries and may be an important route of infection. Sexual transmission of ZIKV has mostly been male-to-female, and persistence of viral RNA in semen for up to 370 days has been recorded. The susceptibility to ZIKV of different testicular cell types merits investigation. METHODS: We infected primary Sertoli cells, a primary testicular fibroblast Hs1.Tes, and 2 seminoma cell lines SEM-1 and TCam-2 cells with ZIKV Paraiba and the prototype ZIKV MR766 to evaluate their susceptibility and to look for viral persistence. A human neuroblastoma cell line SK-N-SH served as a control cell type. RESULTS: Both virus strains were able to replicate in all cell lines tested, but ZIKV MR766 attained higher titers. Initiation of viral persistence by ZIKV Paraiba was observed in Sertoli, Hs1.Tes, SEM-1 and TCam-2 cells, but was of limited duration due to delayed cell death. ZIKV MR766 persisted only in Hs1.Tes and Sertoli cells, and persistence was also limited. In contrast, SK-N-SH cells were killed by both ZIKV MR766 and ZIKV Paraiba and persistence could not be established in these cells. CONCLUSIONS: ZIKV prototype strain MR766 and the clinically relevant Paraiba strain replicated in several testicular cell types. Persistence of ZIKV MR766 was only observed in Hs1.Tes and Sertoli cells, but the persistence did not last more than 3 or 4 passages, respectively. ZIKV Paraiba persisted in TCam-2, Hs1.Tes, Sertoli and SEM-1 cells for up to 5 passages, depending on cell type. TCam-2 cells appeared to clear persistent infection by ZIKV Paraiba.

Routes of Zika virus dissemination in the testis and epididymis of immunodeficient mice.

Sexual transmission and persistence of Zika virus (ZIKV) in the male reproductive tract (MRT) poses new challenges for controlling virus outbreaks and developing live-attenuated vaccines. To elucidate routes of ZIKV dissemination in the MRT, we here generate microRNA-targeted ZIKV clones that lose the infectivity for (1) the cells inside seminiferous tubules of the testis, or (2) epithelial cells of the epididymis. We trace ZIKV dissemination in the MRT using an established mouse model of ZIKV pathogenesis. Our results support a model in which ZIKV infects the testis via a hematogenous route, while infection of the epididymis can occur via two routes: (1) hematogenous/lymphogenous and (2) excurrent testicular. Co-targeting of the ZIKV genome with brain-, testis-, and epididymis-specific microRNAs restricts virus infection of these organs, but does not affect virus-induced protective immunity in mice and monkeys. These defined alterations of ZIKV tropism represent a rational design of a safe live-attenuated ZIKV vaccine.

The Role of Mammalian Reservoir Hosts in Tick-Borne Flavivirus Biology.

Small-to-medium sized mammals and large animals are lucrative sources of blood meals for ixodid ticks that transmit life-threatening tick-borne flaviviruses (TBFVs). TBFVs have been isolated from various organs obtained from wild-caught Myodes and Apodemus species in Europe and Asia. Thus, these rodents are well-established reservoirs of TBFVs. Wild-caught Peromyscus species have demonstrated seropositivity against Powassan virus, the only TBFV known to circulate in North America, suggesting that they may play an important role in the biology of the virus in this geographic region. However, virus isolation from Peromyscus species is yet to be demonstrated. Wild-caught medium-sized mammals, such as woodchucks (Marmota monax) and skunks (Mephitis mephitis) have also demonstrated seropositivity against POWV, and virus was isolated from apparently healthy animals. Despite the well-established knowledge that small-to-medium sized animals are TBFV reservoirs, specific molecular biology addressing host-pathogen interactions remains poorly understood. Elucidating these interactions will be critical for gaining insight into the mechanism(s) of viral pathogenesis and/or resistance.

Peromyscus leucopus mouse brain transcriptome response to Powassan virus infection.

Powassan virus (POWV) is a tick-borne Flavivirus responsible for life-threatening encephalitis in North America and some regions of Russia. The ticks that have been reported to transmit the virus belong to the Ixodes species, and they feed on small-to-medium-sized mammals, such as Peromyscus leucopus mice, skunks, and woodchucks. We previously developed a P. leucopus mouse model of POWV infection, and the model is characterized by a lack of clinical signs of disease following intraperitoneal or intracranial inoculation. However, intracranial inoculation results in mild subclinical encephalitis from 5 days post infection (dpi), but the encephalitis resolves by 28 dpi. We used RNA sequencing to profile the P. leucopus mouse brain transcriptome at different time points after intracranial challenge with POWV. At 24 h post infection, 42 genes were significantly differentially expressed and the number peaked to 232 at 7 dpi before declining to 31 at 28 dpi. Using Ingenuity Pathway Analysis, we determined that the genes that were significantly expressed from 1 to 15 dpi were mainly associated with interferon signaling. As a result, many interferon-stimulated genes (ISGs) were upregulated. Some of the ISGs include an array of TRIMs (genes encoding tripartite motif proteins). These results will be useful for the identification of POWV restriction factors.

Flavivirus Infection of Ixodes scapularis (Black-Legged Tick) Ex Vivo Organotypic Cultures and Applications for Disease Control.

Ixodes scapularis ticks transmit many infectious agents that cause disease, including tick-borne flaviviruses (TBFVs). TBFV infections cause thousands of human encephalitis cases worldwide annually. In the United States, human TBFV infections with Powassan virus (POWV) are increasing and have a fatality rate of 10 to 30%. Additionally, Langat virus (LGTV) is a TBFV of low neurovirulence and is used as a model TBFV. TBFV replication and dissemination within I. scapularis organs are poorly characterized, and a deeper understanding of virus biology in this vector may inform effective countermeasures to reduce TBFV transmission. Here, we describe short-term, I. scapularis organ culture models of TBFV infection. Ex vivo organs were metabolically active for 9 to 10 days and were permissive to LGTV and POWV replication. Imaging and videography demonstrated replication and spread of green fluorescent protein-expressing LGTV in the organs. Immunohistochemical staining confirmed LGTV envelope and POWV protein synthesis within the infected organs. LGTV- and POWV-infected organs produced infectious LGTV and POWV; thus, the ex vivo cultures were suitable for study of virus replication in individual organs. LGTV- and POWV-infected midgut and salivary glands were subjected to double-stranded RNA (dsRNA) transfection with dsRNA to the LGTV 3' untranslated region (UTR), which reduced infectious LGTV and POWV replication, providing a proof-of-concept use of RNA interference in I. scapularis organ cultures to study the effects on TBFV replication. The results contribute important information on TBFV localization within ex vivo I. scapularis organs and provide a significant translational tool for evaluating recombinant, live vaccine candidates and potential tick transcripts and proteins for possible therapeutic use and vaccine development to reduce TBFV transmission.IMPORTANCE Tick-borne flavivirus (TBFV) infections cause neurological and/or hemorrhagic disease in humans worldwide. There are currently no licensed therapeutics or vaccines against Powassan virus (POWV), the only TBFV known to circulate in North America. Evaluating tick vector targets for antitick vaccines directed at reducing TBFV infection within the arthropod vector is a critical step in identifying efficient approaches to controlling TBFV transmission. This study characterized infection of female Ixodes scapularis tick organ cultures of midgut, salivary glands, and synganglion with the low-neurovirulence Langat virus (LGTV) and the more pathogenic POWV. Cell types of specific organs were susceptible to TBFV infection, and a difference in LGTV and POWV replication was noted in TBFV-infected organs. This tick organ culture model of TBFV infection will be useful for various applications, such as screening of tick endogenous dsRNA corresponding to potential control targets within midgut and salivary glands to confirm restriction of TBFV infection.

Modeling Powassan virus infection in Peromyscus leucopus, a natural host.

The tick-borne flavivirus, Powassan virus (POWV) causes life-threatening encephalitis in humans in North America and Europe. POWV is transmitted by ixodid tick vectors that feed on small to medium-sized mammals, such as Peromyscus leucopus mice, which may serve as either reservoir, bridge or amplification hosts. Intraperitoneal and intracranial inoculation of 4-week old Peromyscus leucopus mice with 103 PFU of POWV did not result in overt clinical signs of disease. However, following intracranial inoculation, infected mice seroconverted to POWV and histopathological examinations revealed that the mice uniformly developed mild lymphocytic perivascular cuffing and microgliosis in the brain and spinal cord from 5 to 15 days post infection (dpi), suggesting an early inflammatory response. In contrast, intracranial inoculation of 4-week old C57BL/6 and BALB/c mice was lethal by 5 dpi. Intraperitoneal inoculation was lethal in BALB/c mice, but 40% (2/5) of C57BL/6 mice survived. We concluded that Peromyscus leucopus mice infected i.c. with a lethal dose of POWV support a limited infection, restricted to the central nervous system and mount an antibody response to the virus. However, they fail to develop clinical signs of disease and are able to control the infection. These results suggest the involvement of restriction factors, and the mechanism by which Peromyscus leucopus mice restrict POWV infection remains under study.

Analysis of the Langat Virus Genome in Persistent Infection of an Ixodes scapularis Cell Line.

Tick-borne flaviviruses (TBFVs) cause a broad spectrum of disease manifestations ranging from asymptomatic to mild febrile illness and life threatening encephalitis. These single-stranded positive-sense (ss(+)) RNA viruses are naturally maintained in a persistent infection of ixodid ticks and small-medium sized mammals. The development of cell lines from the ixodid ticks has provided a valuable surrogate system for studying the biology of TBFVs in vitro. When we infected ISE6 cells, an Ixodes scapularis embryonic cell line, with Langat virus (LGTV) we observed that the infection proceeded directly into persistence without any cytopathic effect. Analysis of the viral genome at selected time points showed that no defective genomes were generated during LGTV persistence by 10 weeks of cell passage. This was in contrast to LGTV persistence in 293T cells in which defective viral genomes are detectable by five weeks of serial cell passage. We identified two synonymous nucleotide changes i.e., 1893A→C (29% of 5978 reads at 12 h post infection (hpi)) and 2284T→A (34% of 4191 reads at 12 hpi) in the region encoding for the viral protein E. These results suggested that the mechanisms supporting LGTV persistence are different between tick and mammalian cells.

Transcriptome Analysis Reveals a Signature Profile for Tick-Borne Flavivirus Persistence in HEK 293T Cells.

UNLABELLED: Tick-borne flaviviruses (TBFVs) cause febrile illnesses, which may progress to severe encephalitis and/or death in humans globally. Most people who recover from severe acute disease suffer from debilitating neurological sequelae, which may be due to viral persistence, infection-induced neurological cell damage, host response, or some combination of these. Acute TBFV infection of human embryonic kidney (HEK) 293T cells in vitro results in the death of >95% of infected cells by day 5. However, replacing cell growth medium allows surviving cells to repopulate and become persistently infected for extended periods of time. The mechanisms responsible for initiation and maintenance of viral persistence remain vague. We subjected the HEK 293T cell transcriptome to deep sequencing to identify genes differentially expressed during acute infection and persistent infection. A total of 451 genes showed unique significant differential expression levels in persistently infected cells relative to the acute phase of infection. Ingenuity Pathway Analysis results suggested that the expression of prosurvival oncogenes AKT2 and ERBB2 was upregulated in persistently infected cells, whereas proapoptotic genes, such as Bad and the beta interferon 1 (IFN-ß1) gene, were downregulated. Genes encoding antiviral cytokines such as the CCL5, tumor necrosis factor alpha (TNF-α), and CXCL10 genes were upregulated during the acute phase, but the same genes were relatively quiescent in persistently infected cells. Exogenous induction of apoptosis demonstrated that persistently infected cells were resistant to apoptosis in a dose-dependent manner. In summary, the differential transcriptome profiles of acute-phase compared to persistently infected HEK 293T cells demonstrated an evasion of apoptosis, which may be critical for a chronic TBFV infection state. These results provide a basis for further study of the mechanisms of TBFV persistence. IMPORTANCE: Tick-borne flaviviruses (TBFVs) cause life-threatening encephalitic disease in humans worldwide. Some people who recover from severe disease may suffer prolonged neurological symptoms due to either virus- or host response-induced cell damage or a combination of the two that are linked to viral persistence. By examining the genes that are significantly differentially expressed in acute TBFV infection versus persistent TBFV infection, we may be able to find the molecular basis of viral persistence. Here we used deep sequencing of the host cell transcriptome to discover that the expression levels of prosurvival genes were upregulated in persistently infected cells relative to acute TBFV infections whereas the expression levels of genes that promote programmed cell death were downregulated. In addition, persistently infected cells were also resistant to exogenous chemical induction of cell death, in a dose-dependent manner, compared to uninfected cells. Our results pave the way for further studies aimed at understanding the precise mechanisms of TBFV persistence.

Development of a Model System for Tick-Borne Flavivirus Persistence in HEK 293T Cells.

UNLABELLED: We devised a model system to study persistent infection by the tick-borne flavivirus Langat virus (LGTV) in 293T cells. Infection with a molecularly cloned LGTV strain produced an acute lytic crisis that left few surviving cells. The culture was repopulated by cells that were ~90% positive for LGTV E protein, thus initiating a persistent infection that was maintained for at least 35 weeks without additional lytic crises. Staining of cells for viral proteins and ultrastructural analysis revealed only minor differences from the acute phase of infection. Infectious LGTV decreased markedly over the study period, but the number of viral genomes remained relatively constant, suggesting the development of defective interfering particles (DIPs). Viral genome changes were investigated by RNA deep sequencing. At the initiation of persistent infection, levels of DIPs were below the limit of detection at a coverage depth of 11,288-fold, implying that DIPs are not required for initiation of persistence. However, after 15 passages, DIPs constituted approximately 34% of the total LGTV population (coverage of 1,293-fold). Furthermore, at this point, one specific DIP population predominated in which nucleotides 1058 to 2881 had been deleted. This defective genome specified an intact polyprotein that coded for a truncated fusion protein containing 28 N-terminal residues of E and 134 C-terminal residues of NS1. Such a fusion protein has not previously been described, and a possible function in persistent infection is uncertain. DIPs are not required for the initiation of persistent LGTV infection but may play a role in the maintenance of viral persistence. IMPORTANCE: Tick-borne flaviviruses are significant infectious agents that cause serious disease and death in humans worldwide. Infections are characterized by severe neurological symptoms, such as meningitis and encephalitis. A high percentage of people who get infected and recuperate from the acute phase of infection continue to suffer from chronic debilitating neurological sequelae, most likely as a result of nervous tissue damage, viral persistence, or both. However, little is known about mechanisms of viral persistence. Therefore, we undertook studies to investigate the persistence of Langat virus, a member of the tick-borne flavivirus group, in a mammalian cell line. Using next-generation sequencing, we determined that defective viral genomes do not play a role in the initiation of persistence, but their occurrence seems to be nonstochastic and could play a role in the maintenance of viral persistence via the expression of a novel envelope-NS1 fusion protein.

The role of viral persistence in flavivirus biology.

In nature, vector-borne flaviviruses are persistently cycled between either the tick or mosquito vector and small mammals such as rodents, skunks, and swine. These viruses account for considerable human morbidity and mortality worldwide. Increasing and substantial evidence of viral persistence in humans, which includes the isolation of RNA by RT-PCR and infectious virus by culture, continues to be reported. Viral persistence can also be established in vitro in various human, animal, arachnid, and insect cell lines in culture. Although some research has focused on the potential roles of defective virus particles, evasion of the immune response through the manipulation of autophagy and/or apoptosis, the precise mechanism of flavivirus persistence is still not well understood. We propose additional research for further understanding of how viral persistence is established in different systems. Avenues for additional studies include determining whether the multifunctional flavivirus protein NS5 has a role in viral persistence, the development of relevant animal models of viral persistence, and investigating the host responses that allow vector-borne flavivirus replication without detrimental effects on infected cells. Such studies might shed more light on the viral-host relationships and could be used to unravel the mechanisms for establishment of persistence.

Sequence analysis of the whole genomes of five African human G9 rotavirus strains.

The G9 rotaviruses are amongst the most common global rotavirus strains causing severe childhood diarrhoea. However, the whole genomes of only a few G9 rotaviruses have been fully sequenced and characterised of which only one G9P[6] and one G9P[8] are from Africa. We determined the consensus sequence of the whole genomes of five African human group A G9 rotavirus strains, four G9P[8] strains and one G9P[6] strain collected in Cameroon (central Africa), Kenya (eastern Africa), South Africa and Zimbabwe (southern Africa) in 1999, 2009 and 2010. Strain RVA/Human-wt/ZWE/MRC-DPRU1723/2009/G9P[8] from Zimbabwe, RVA/Human-wt/ZAF/MRC-DPRU4677/2010/G9P[8] from South Africa, RVA/Human-wt/CMR/1424/2009/G9P[8] from Cameroon and RVA/Human-wt/KEN/MRC-DPRU2427/2010/G9P[8] from Kenya were on a Wa-like genetic backbone and were genotyped as G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. Strain RVA/Human-wt/ZAF/MRC-DPRU9317/1999/G9P[6] from South Africa was genotyped as G9-P[6]-I2-R2-C2-M2-A2-N1-T2-E2-H2. Rotavirus A strain MRC-DPRU9317 is the second G9 strain to be reported on a DS-1-like genetic backbone, the other being RVA/Human-wt/ZAF/GR10924/1999/G9P[6]. MRC-DPRU9317 was found to be a reassortant between DS-1-like (I2, R2, C2, M2, A2, T2, E2 and H2) and Wa-like (N1) genome segments. All the genome segments of the five strains grouped strictly according to their genotype Wa- or DS-1-like clusters. Within their respective genotypes, the genome segments of the three G9 study strains from southern Africa clustered most closely with rotaviruses from the same geographical origin and with those with the same G and P types. The highest nucleotide identity of genome segments of the study strains from eastern and central Africa regions on a Wa-like backbone was not limited to rotaviruses with G9P[8] genotypes only, they were also closely related to G12P[6], G8P[8], G1P[8] and G11P[25] rotaviruses, indicating a close inter-genotype relationship between the G9 and other rotavirus genotypes. Rotavirus strain MRC-DPRU9317 is the first G9P[6] to be characterised on a DS-1-like genetic backbone with a reassortant segment 8 (NSP2) and fourth G9P[6] to be fully sequenced globally.

Whole-genome consensus sequence analysis of a South African rotavirus SA11 sample reveals a mixed infection with two close derivatives of the SA11-H96 strain.

Whole-genome, sequence-independent amplification and 454(®) pyrosequencing of a rotavirus SA11 cell culture sample with an unknown passage history yielded consensus sequences of twelve complete genome segments. Two distinct sequences for genome segment 8 (encoding NSP2) were present, indicating a mixed infection with two rotavirus SA11 strains. The genotypes of the viruses were G3-P[2]-I2-R2-C5-M5-A5-Nx-T5-E2-H5, where x was either 5 or 2. The strains were named RVA/Simian-tc/ZAF/SA11-N5/1958/G3P[2] and RVA/Simian-tc/ZAF/SA11-N2/1958/G3P[2]. The genotype (N2) and sequence of genome segment 8 of RVA/Simian-tc/ZAF/SA11-N2/1958/G3P[2] were identical to that of the bovine rotavirus O agent. Five novel amino acids were detected in minor population variants of three genome segments. Genome segment 1 (VP1) has a high nucleotide substitution rate, but the substitutions are synonymous. Distance matrices and Bayesian molecular clock phylogenetics showed that SA11-N2 is a reassortant containing genome segment 8 from the O agent, whereas SA11-N5 is a very close derivative of the prototype SA11-H96.