Vesicular stomatitis virus evolution during alternation between persistent infection in insect cells and acute infection in mammalian cells is dominated by the persistence phase

Vesicular stomatitis virus has the potential for very rapid evolution in the laboratory, but like many other arboviruses, it evolves at a relatively slow rate in the natural environment. Previous work showed that alternating replication in different cell types does not promote stasis. In order to determine whether other factors promote stasis, we compared the fitness trajectories of populations evolving during acute infections in mammalian cells, populations evolving during persistent infections in insect cells, and populations evolving during alternating acute and persistent infection cycles. Populations evolving under constant conditions increased in fitness in the environment in which they replicated. An asymmetric trade-off was observed such that acute infection had no cost for persistence but persistent replication had a dramatic cost for acute infection in mammalian cells. After an initial period of increase, fitness remained approximately constant in all the populations that included persistent replication, but fitness continuously increased in populations evolving during acute infections. Determination of the consensus sequence of the genes encoding the N, P, M, and G proteins showed that the pattern of mutation accumulation was coherent with fitness changes during persistence so that once fitness reached a maximum, the rate of mutation accumulation dropped. Persistent replication dominated both the genetic and the phenotypic evolution of the populations that alternated between acute infection of mammalian cells and persistence in insect cells, and fitness loss was observed in the mammalian environment despite periodic replication in mammalian cells. These results show that stasis can be achieved without good levels of adaptation to both the mammalian and the insect environments.     

LACK,a RACK1 ortholog,facilitates cytochrome c oxidase subunit expression to promote Leishmania major fitness

Leishmania are kinetoplastid parasites that cause the sandfly‐transmitted disease leishmaniasis. To maintain fitness throughout their infectious life cycle, Leishmania must undergo rapid metabolic adaptations to the dramatically distinct environments encountered during transition between sandfly and vertebrate hosts. We performed proteomic and immunoblot analyses of attenuated L. major strains deficient for LACK, the Leishmania ortholog of the mammalian receptor for activated c kinase (RACK1), that is important for parasite thermotolerance and virulence. This approach identified cytochrome c oxidase (LmCOX) subunit IV as a LACK‐dependent fitness protein. Consistent with decreased levels of LmCOX subunit IV at mammalian temperature, and in amastigotes, LmCOX activity and mitochondrial function were also impaired in LACK‐deficient L. major under these conditions. Importantly, overexpression of LmCOX subunit IV in LACK‐deficient L. major restored thermotolerance and macrophage infectivity. Interestingly, overexpression of LmCOX subunit IV enhanced LmCOX subunit VI expression at mammalian temperature. Collectively, our data suggest LACK promotes Leishmania adaptation to the mammalian host environment by sustaining LmCOX subunit IV expression and hence energy metabolism in response to stress stimuli such as heat. These findings extend the repertoire of RACK1 protein utility to include a role in mitochondrial function.     

Lack of evolutionary stasis during alternating replication of an arbovirus in insect and mammalian cells

The evolution of vesicular stomatitis virus (VSV) in a constant environment, consisting of either mammalian or insect cells, has been compared to the evolution of the same viral population in changing environments consisting in alternating passages in mammalian and insect cells. Fitness increases were observed in all cases. An initial fitness loss of VSV passaged in insect cells was noted when fitness was measured in BHK-21 cells, but this effect could be attributed to a difference of temperature during VSV replication at 37 degrees C in BHK-21 cells. Sequencing of nucleotides 1-4717 at the 3' end of the VSV genome (N, P, M and G genes) showed that at passage 80 the number of mutations accumulated during alternated passages (seven mutations) is similar or larger than that observed in populations evolving in a constant environment (two to four mutations). Our results indicate that insect and mammalian cells can constitute similar environments for viral replication. Thus, the slow rates of evolution observed in natural populations of arboviruses are not necessarily due to the need for the virus to compromise between adaptation to both arthropod and vertebrate cell types.     

Borna disease virus nucleoprotein interacts with the CDC2-cyclin B1 complex

Transition from G(2) to M phase, a cell cycle checkpoint, is regulated by the Cdc2-cyclin B1 complex. Here, we report that persistent infection with Borna disease virus (BDV), a noncytolytic RNA virus infecting the central nervous system, results in decelerated proliferation of infected host cells due to a delayed G(2)-to-M transition. Persistent BDV-infected rat fibroblast cells showed reduced proliferation compared to uninfected cells. In pull-down assays we observed an interaction of the viral nucleoprotein with the Cdc2-cyclin B1 complex. Transfection of the viral nucleoprotein but not of the phosphoprotein also results in decelerated proliferation. This phenomenon was found in BDV-susceptible primary rat fibroblast cells and also in primary mouse cells, which are not susceptible to BDV infection. This is the first evidence that the noncytolytic Borna disease virus can manipulate host cell functions via interaction of the viral nucleoprotein with mitotic entry regulators. BDV preferentially infects and persists in nondividing neurons. The present report could give an explanation for this selective choice of host cell by BDV.     

Plasmidal maintenance of composite DNA derived from polyoma related plasmid, L factor.

Recently, we reported a multicopy mammalian plasmid with a structure related to polyoma. The plasmid, named L factor, was found at a high copy number (5,000 or more per cell) in a subclone derived from mouse L cells. We attempted to utilize L factor as a plasmid vector for mammalian cells. A series of composite DNA consisting of L factor and a foreign (herpes simplex virus tk) were constructed. These DNA could be established as plasmids after transfection to several mouse cell lines, although the copy number of the re-established plasmids was considerably less than that observed for the original subclone. The composite DNA maintained the structure of the original DNA after prolonged culture and the copy number remained constant even with no selective pressure. A composite DNA, with no DNA sequence corresponding to polyoma T antigen, could also be established as a plasmid in a mouse L cell line in which polyoma T antigen is expressed. The potential use of the plasmid is discussed.     

Prevalence of Toscana sandfly fever virus antibodies in neurological patients and control subjects in Sicily

Toscana sandfly fever virus (TOSV) is an arthropod-borne virus transmitted to humans by sandfly vectors. It has been associated with human cases of meningitis and meningo-encephalitis mainly occurring during the warm season. We performed a retrospective serological study to evaluate TOSV circulation in Palermo, Sicily, and to compare TOSV seroprevalence in patients with neurological symptoms and in a control group of patients without neurological symptoms. When sera from 155 patients with and without neurological symptoms were evaluated, the rate of overall TOSV IgG reactivity was 17.4%. Patients with neurological symptoms showed a higher percentage of TOSV IgG positivity than control patients (25% versus 10.8%). TOSV exposure was confirmed by virus neutralization tests which also detected a Naples virus (SFNV) infection. TOSV should be considered as an etiologic agent in the differential diagnosis of fever and meningo-encephalitis in Sicily.     

Early virus-associated bystander events affect the fitness of the CD8 T cell response to persistent virus infection

Chronic Ag exposure during persistent viral infection erodes virus-specific CD8 T cell numbers and effector function, with a concomitant loss of pathogen control. Less clear are the respective contributions of Ag-specific and Ag-nonspecific (bystander) events on the quantity, quality, and maintenance of antiviral CD8 T cells responding to persistent virus infection. In this study, we show that low-dose inoculation with mouse polyomavirus (PyV) elicits a delayed, but numerically equivalent, antiviral CD8 T cell response compared with high-dose inoculation. Low-dose infection generated virus-specific CD8 T cells endowed with multicytokine functionality and a superior per cell capacity to produce IFN-gamma. PyV-specific CD8 T cells primed by low-dose inoculation also expressed higher levels of IL-7Ralpha and bcl-2 and possessed enhanced Ag-independent survival. Importantly, the quantity and quality of the antiviral CD8 T cell response elicited by dendritic cell-mediated immunization were mitigated by infection with a mutant PyV lacking the dominant CD8 T cell viral epitope. These findings suggest that the fitness of the CD8 T cell response to persistent virus infection is programmed in large part by early virus-associated Ag-nonspecific factors, and imply that limiting bystander inflammation at the time of inoculation, independent of Ag load, may optimize adaptive immunity to persistent viral infection.     

Emergence of mammalian cell-adapted vesicular stomatitis virus from persistent infections of insect vector cells

Arboviruses (arthropod-borne viruses) represent quintessential generalists, with the ability to infect and perform well in multiple hosts. However, antagonistic pleiotropy imposed a cost during the adaptation to persistent replication of vesicular stomatitis virus in sand fly cells and resulted in strains that initially replicated poorly in hamster cells, even when the virus was allowed to replicate periodically in the latter. Once a debilitated strain started replicating continuously in mammalian cells, fitness increased significantly. Fitness recovery did not entail back mutations or compensatory mutations, but instead, we observed the replacement of persistence-adapted genomes by mammalian cell-adapted strains with a full set of new, unrelated sequence changes. These mammalian cell-adapted genomes were present at low frequencies in the populations with a history of persistence for up to a year and quickly became dominant during mammalian infection, but coexistence was not stable in the long term. Periodic acute replication in mammalian cells likely contributed to extending the survival of minority genomes, but these genomes were also found in strictly persistent populations.     

Phenotypes of cell hybrids formed by fusion of normal and Rous sarcoma virus transformed cells

Untransformed mouse cells were fused with rat cells transformed by a temperature sensitive mutant of avian sarcoma virus, and cell hybrids were isolated in the absence and in the presence of selective medium. None of the hybrids isolated were as transformed as the parent rat cells. All hybrids isolated in the absence of selective medium showed a phenotype similar to that of the untransformed mouse cell parent. Cell hybrids isolated on selective media, however, were more heterogenous. Some showed a phenotype that were intermediate between that of the two parental cells, while others were more like the untransformed mouse cells.     

Heterologous T cells can help restore function in dysfunctional hepatitis C virus nonstructural 3/4A-specific T cells during therapeutic vaccination

The hepatitis C virus (HCV)-specific T cell response in patients with chronic HCV is dysfunctional. In this study, we aimed at restoring immunological function through therapeutic vaccination in a transgenic mouse model with impaired HCV-specific T cell responses due to a persistent presence of hepatic HCV nonstructural (NS)3/4A Ags. The HCV-specific T cells have an actively maintained dysfunction reflected in reduced frequency, impaired cytokine production, and impaired effector function in vivo, which can be partially restored by blocking regulatory T cells or programmed cell death ligand 1. We hypothesized that the impairment could be corrected by including sequences that created a normal priming environment by recruiting "healthy" heterologous T cells and by activating innate signaling. Endogenously expressed hepatitis B core Ag (HBcAg) can recruit heterologous T cells and activate TLR (TLR7) signaling. Hence, by combining HCV NS3/4A with different forms of HBcAg we found that heterologous sequences somewhat improved activation and expansion of NS3/4A-specific T cells in a wild-type host. Importantly, the signals provided by HBcAg effectively restored the activation of HCV-specific T cells in a tolerant NS3/4A-transgenic mouse model. The adjuvant effect could also be transferred to the priming of dysfunctional HLA-A2-restricted NS3-specific T cells in vivo. Thus, recruiting healthy heterologous T cells to the site of priming may also help restore HCV-specific responses present in a chronically infected host.     

Clonal analysis of mammalian cell cultures persistently infected with Japanese encephalitis virus.

More than 200 cells were cloned from populations of mammalian cells persistently infected with Japanese encephalitis virus. Only four cloned cultures contained cells that had viral antigen measurable by immunofluorescence and that released infectious virus, yet all clones harbored virus-specific RNA. Superinfection of cloned cells with wild-type Japanese encephalitis virus did not produce cytopathic effects, but resulted in production of viral antigen and infectious virus in formerly nonproducing clones. Cocultivation of nonproducer clone cells with normally permissive cells did not induce virus production, nor did treatment of nonproducer clones with various inhibitors of DNA, RNA, or protein synthesis. It is suggested that the cloning procedure may have selected for a particular subpopulation of cells and that defective virus is also involved in establishment and maintenance of persistent infection.     

Identification and characterization of a ross river virus variant that grows persistently in macrophages, shows altered disease kinetics in a mouse model, and exhibits resistance to type I interferon

Alphaviruses, such as chikungunya virus, o'nyong-nyong virus, and Ross River virus (RRV), cause outbreaks of human rheumatic disease worldwide. RRV is a positive-sense single-stranded RNA virus endemic to Australia and Papua New Guinea. In this study, we sought to establish an in vitro model of RRV evolution in response to cellular antiviral defense mechanisms. RRV was able to establish persistent infection in activated macrophages, and a small-plaque variant (RRV(PERS)) was isolated after several weeks of culture. Nucleotide sequence analysis of RRV(PERS) found several nucleotide differences in the nonstructural protein (nsP) region of the RRV(PERS) genome. A point mutation was also detected in the E2 gene. Compared to the parent virus (RRV-T48), RRV(PERS) showed significantly enhanced resistance to beta interferon (IFN-β)-stimulated antiviral activity. RRV(PERS) infection of RAW 264.7 macrophages induced lower levels of IFN-β expression and production than infection with RRV-T48. RRV(PERS) was also able to inhibit type I IFN signaling. Mice infected with RRV(PERS) exhibited significantly enhanced disease severity and mortality compared to mice infected with RRV-T48. These results provide strong evidence that the cellular antiviral response can direct selective pressure for viral sequence evolution that impacts on virus fitness and sensitivity to alpha/beta IFN (IFN-α/β).     

Genetic and fitness changes accompanying adaptation of an arbovirus to vertebrate and invertebrate cells

The alternating host cycle and persistent vector infection may constrain the evolution of arboviruses. To test this hypothesis, eastern equine encephalitis virus was passaged in BHK or mosquito cells, as well as in alternating (both) host cell passages. High and low multiplicities were used to examine the effect of defective interfering particles. Clonal BHK and persistent mosquito cell infections were also evaluated. Fitness was measured with one-step growth curves and competition assays, and mutations were evaluated by nucleotide sequencing and RNA fingerprinting. All passages and assays were done at 32 degrees C to eliminate temperature as a selection factor. Viruses passaged in either cell type alone exhibited fitness declines in the bypassed cells, while high-multiplicity and clonal passages caused fitness declines in both types of cells. Bypassed cell fitness losses were mosquito and vertebrate specific and were not restricted to individual cell lines. Fitness increases occurred in the cell line used for single-host-adaptation passages and in both cells for alternately passaged viruses. Surprisingly, single-host-cell passage increased fitness in that cell type no more than alternating passages. However, single-host-cell adaptation resulted in more mutations than alternating cell passages. Mosquito cell adaptation invariably resulted in replacement of the stop codon in nsP3 with arginine or cysteine. In one case, BHK cell adaptation resulted in a 238-nucleotide deletion in the 3' untranslated region. Many nonsynonymous substitutions were shared among more than one BHK or mosquito cell passage series, suggesting positive Darwinian selection. Our results suggest that alternating host transmission cycles constrain the evolutionary rates of arboviruses but not their fitness for either host alone.     

Defective interfering influenza viruses and host cells: establishment and maintenance of persistent influenza virus infection in MDBK and HeLa cells.

WSN (H0N1) influenza virus upon undiluted passages in different species of cells, namely, bovine kidney (MDBK), chicken embryo (CEF), and HeLa cells, produced a varying amount of defective interfering (DI) virus which correlated well with the ability of the species of cell to produce infectious virus. However, the nature of the influenza DI viral RNA produced from a single clonal stock was essentially identical in all three cells types, suggesting that these cells do not exert a great selective pressure in the amplification of specific DI viral RNAs either at early or late passages. DI viruses produced from one subtype (H0N1) could interfere with the replication of infectious viruses belonging to other subtypes (H1N1, H3N2). DI viral RNAs could also replicate with the helper function of other subtype viruses. The persistent infection of MDBK and HeLa cells could be initiated by coinfecting cells with both temperature-sensitive mutants (ts-) and DI influenza viruses. Persistently infected cultures cultures at early passages (up to passage 7) showed a cyclical pattern of cell lysis and virus production (crisis), whereas, at later passages (after passage 20), they produced little or no virus and were resistant to infection by homologous virus but not by heterologous virus. The majority of persistently infected cells, however, contained the complete viral genome since they expressed viral antigens and produced infectious centers. Selection of a slow-growing temperature-sensitive variant rather than the presence of DI virus or interferon appears to be critical in maintaining persistent influenza infection in these cells.     

Quantitation, with a new assay, of Theiler's virus capsid protein in the central nervous system of mice.

We developed a quantitative assay for antigens at the single-cell level. Tissue sections were reacted with a primary antibody, a biotinylated secondary antibody, or 35S-streptavidin. Binding of streptavidin to cells was quantitated by microscopic autoradiography. We showed that the number of autoradiographic grains was proportional to the amount of antigen per cell. With this assay, we studied the synthesis of Theiler's virus capsid proteins VP1, VP2, and VP3 in permissive BHK cells grown in vitro and in mouse central nervous system (CNS) cells during a persistent infection. We found that synthesis of the three capsid proteins was restricted in mouse CNS cells. Restricted virus replication could play a major role in the persistence of Theiler's virus in mouse CNS cells.