Single Mutant Viruses Research Articles

Adaptation of SIVmac to baboon primary cells results in complete absence of in vivo baboon infectivity.

While simian immunodeficiency virus (SIV) infection is non-pathogenic in naturally infected African nonhuman primate hosts, experimental or accidental infection in rhesus macaques often leads to AIDS. Baboons, widely distributed throughout Africa, do not naturally harbor SIV, and experimental infection of baboons with SIVmac results in transient low-level viral replication. Elucidation of mechanisms of natural immunity in baboons could uncover new targets of antiviral intervention. We tested the hypothesis that an SIVmac adapted to replicate in baboon primary cells will gain the capacity to establish chronic infections in vivo. Here, we generated SIVmac variants in baboon cells through serial passage in PBMC from different donors (SIVbn-PBMC s1), in PBMC from the same donors (SIVbn-PBMC s2), or in isolated CD4 cells from the same donors used for series 2 (SIVbn-CD4). While SIVbn-PBMC s1 and SIVbn-CD4 demonstrated increased replication capacity, SIVbn-PBMC s2 did not. Pharmacological blockade of CCR5 revealed SIVbn-PBMC s1 could more efficiently use available CCR5 than SIVmac, a trait we hypothesize arose to circumvent receptor occupation by chemokines. Sequencing analysis showed that all three viruses accumulated different types of mutations, and that more non-synonymous mutations became fixed in SIVbn-PBMC s1 than SIVbn-PBMC s2 and SIVbn-CD4, supporting the notion of stronger fitness pressure in PBMC from different genetic backgrounds. Testing the individual contribution of several newly fixed SIV mutations suggested that is the additive effect of these mutations in SIVbn-PBMC s1 that contributed to its enhanced fitness, as recombinant single mutant viruses showed no difference in replication capacity over the parental SIVmac239 strain. The replicative capacity of SIVbn-PBMC passage 4 (P4) s1 was tested in vivo by infecting baboons intravenously with SIVbn-PBMC P4 s1 or SIVmac251. While animals infected with SIVmac251 showed the known pattern of transient low-level viremia, animals infected with SIVbn-PBMC P4 s1 had undetectable viremia or viral DNA in lymphoid tissue. These studies suggest that adaptation of SIV to grow in baboon primary cells results in mutations that confer increased replicative capacity in the artificial environment of cell culture but make the virus unable to avoid the restrictive factors generated by a complex multicellular organism.

The genetic variability and evolution of red-spotted grouper nervous necrosis virus quasispecies can be associated with its virulence.

Nervous necrosis virus, NNV, is a neurotropic virus that causes viral nervous necrosis disease in a wide range of fish species, including European sea bass (Dicentrarchus labrax). NNV has a bisegmented (+) ssRNA genome consisting of RNA1, which encodes the RNA polymerase, and RNA2, encoding the capsid protein. The most prevalent NNV species in sea bass is red-spotted grouper nervous necrosis virus (RGNNV), causing high mortality in larvae and juveniles. Reverse genetics studies have associated amino acid 270 of the RGNNV capsid protein with RGNNV virulence in sea bass. NNV infection generates quasispecies and reassortants able to adapt to various selective pressures, such as host immune response or switching between host species. To better understand the variability of RGNNV populations and their association with RGNNV virulence, sea bass specimens were infected with two RGNNV recombinant viruses, a wild-type, rDl956, highly virulent to sea bass, and a single-mutant virus, Mut270Dl965, less virulent to this host. Both viral genome segments were quantified in brain by RT-qPCR, and genetic variability of whole-genome quasispecies was studied by Next Generation Sequencing (NGS). Copies of RNA1 and RNA2 in brains of fish infected with the low virulent virus were 1,000-fold lower than those in brains of fish infected with the virulent virus. In addition, differences between the two experimental groups in the Ts/Tv ratio, recombination frequency and genetic heterogeneity of the mutant spectra in the RNA2 segment were found. These results show that the entire quasispecies of a bisegmented RNA virus changes as a consequence of a single point mutation in the consensus sequence of one of its segments. Sea bream (Sparus aurata) is an asymptomatic carrier for RGNNV, thus rDl965 is considered a low-virulence isolate in this species. To assess whether the quasispecies characteristics of rDl965 were conserved in another host showing different susceptibility, juvenile sea bream were infected with rDl965 and analyzed as above described. Interestingly, both viral load and genetic variability of rDl965 in seabream were similar to those of Mut270Dl965 in sea bass. This result suggests that the genetic variability and evolution of RGNNV mutant spectra may be associated with its virulence.

Characterization of a large cluster of influenza A(H1N1)pdm09 viruses cross-resistant to oseltamivir and peramivir during the 2013-2014 influenza season in Japan.

Between September 2013 and July 2014, 2,482 influenza 2009 pandemic A(H1N1) [A(H1N1)pdm09] viruses were screened in Japan for the H275Y substitution in their neuraminidase (NA) protein, which confers cross-resistance to oseltamivir and peramivir. We found that a large cluster of the H275Y mutant virus was present prior to the main influenza season in Sapporo /: Hokkaido, with the detection rate for this mutant virus reaching 29% in this area. Phylogenetic analysis suggested the clonal expansion of a single mutant virus in Sapporo /: Hokkaido. To understand the reason for this large cluster, we examined the in vitro and in vivo properties of the mutant virus. We found that it grew well in cell culture, with growth comparable to that of the wild-type virus. The cluster virus also replicated well in the upper respiratory tract of ferrets and was transmitted efficiently between ferrets by way of respiratory droplets. Almost all recently circulating A(H1N1)pdm09 viruses, including the cluster virus, possessed two substitutions in NA, V241I and N369K, which are known to increase replication and transmission fitness. A structural analysis of NA predicted that a third substitution (N386K) in the NA of the cluster virus destabilized the mutant NA structure in the presence of the V241I and N369K substitutions. Our results suggest that the cluster virus retained viral fitness to spread among humans and, accordingly, caused the large cluster in Sapporo/Hokkaido. However, the mutant NA structure was less stable than that of the wild-type virus. Therefore, once the wild-type virus began to circulate in the community, the mutant virus could not compete and faded out.

Altered viral fitness and drug susceptibility in HIV-1 carrying mutations that confer resistance to nonnucleoside reverse transcriptase and integrase strand transfer inhibitors.

Nonnucleoside reverse transcriptase (RT) inhibitors (NNRTI) and integrase (IN) strand transfer inhibitors (INSTI) are key components of antiretroviral regimens. To explore potential interactions between NNRTI and INSTI resistance mutations, we investigated the combined effects of these mutations on drug susceptibility and fitness of human immunodeficiency virus type 1 (HIV-1). In the absence of drug, single-mutant viruses were less fit than the wild type; viruses carrying multiple mutations were less fit than single-mutant viruses. These findings were explained in part by the observation that mutant viruses carrying NNRTI plus INSTI resistance mutations had reduced amounts of virion-associated RT and/or IN protein. In the presence of efavirenz (EFV), a virus carrying RT-K103N together with IN-G140S and IN-Q148H (here termed IN-G140S/Q148H) mutations was fitter than a virus with a RT-K103N mutation alone. Similarly, in the presence of EFV, the RT-E138K plus IN-G140S/Q148H mutant virus was fitter than one with the RT-E138K mutation alone. No effect of INSTI resistance mutations on the fitness of RT-Y181C mutant viruses was observed. Conversely, RT-E138K and -Y181C mutations improved the fitness of the IN-G140S/Q148H mutant virus in the presence of raltegravir (RAL); the RT-K103N mutation had no effect. The NNRTI resistance mutations had no effect on RAL susceptibility. Likewise, the IN-G140S/Q148H mutations had no effect on EFV or RPV susceptibility. However, both the RT-K103N plus IN-G140S/Q148H and the RT-E138K plus IN-G140S/Q148H mutant viruses had significantly greater fold increases in 50% inhibitory concentration (IC50) of EFV than viruses carrying a single NNRTI mutation. Likewise, the RT-E138K plus IN-G140S/Q148H mutant virus had significantly greater fold increases in RAL IC50 than that of the IN-G140S/Q148H mutant virus. These results suggest that interactions between RT and IN mutations are important for NNRTI and INSTI resistance and viral fitness. Nonnucleoside reverse transcriptase inhibitors and integrase inhibitors are used to treat infection with HIV-1. Mutations that confer resistance to these drugs reduce the ability of HIV-1 to reproduce (that is, they decrease viral fitness). It is known that reverse transcriptase and integrase interact and that some mutations can disrupt their interaction, which is necessary for proper functioning of these two enzymes. To determine whether resistance mutations in these enzymes interact, we investigated their effects on drug sensitivity and viral fitness. Although individual drug resistance mutations usually reduced viral fitness, certain combinations of mutations increased fitness. When present in certain combinations, some integrase inhibitor resistance mutations increased resistance to nonnucleoside reverse transcriptase inhibitors and vice versa. Because these drugs are sometimes used together in the treatment of HIV-1 infection, these interactions could make viruses more resistant to both drugs, further limiting their clinical benefit.

Effects of vaccinia virus uracil DNA glycosylase catalytic site and deoxyuridine triphosphatase deletion mutations individually and together on replication in active and quiescent cells and pathogenesis in mice

BackgroundLow levels of uracil in DNA result from misincorporation of dUMP or cytosine deamination. Vaccinia virus (VACV), the prototype poxvirus, encodes two enzymes that can potentially reduce the amount of uracil in DNA. Deoxyuridine triphosphatase (dUTPase) hydrolyzes dUTP, generating dUMP for biosynthesis of thymidine nucleotides while decreasing the availability of dUTP for misincorporation; uracil DNA glycosylase (UNG) cleaves uracil N-glycosylic bonds in DNA initiating base excision repair. Studies with actively dividing cells showed that the VACV UNG protein is required for DNA replication but the UNG catalytic site is not, whereas the dUTPase gene can be deleted without impairing virus replication. Recombinant VACV with an UNG catalytic site mutation was attenuated in vivo, while a dUTPase deletion mutant was not. However, the importance of the two enzymes for replication in quiescent cells, their possible synergy and roles in virulence have not been fully assessed.ResultsVACV mutants lacking the gene encoding dUTPase or with catalytic site mutations in UNG and double UNG/dUTPase mutants were constructed. Replication of UNG and UNG/dUTPase mutants were slightly reduced compared to wild type or the dUTPase mutant in actively dividing cells. Viral DNA replication was reduced about one-third under these conditions. After high multiplicity infection of quiescent fibroblasts, yields of wild type and mutant viruses were decreased by 2-logs with relative differences similar to those observed in active fibroblasts. However, under low multiplicity multi-step growth conditions in quiescent fibroblasts, replication of the dUTPase/UNG mutant was delayed and 5-fold lower than that of either single mutant or parental virus. This difference was exacerbated by 1-day serial passages on quiescent fibroblasts, resulting in 2- to 3-logs lower titer of the double mutant compared to the parental and single mutant viruses. Each mutant was more attenuated than a revertant virus upon intranasal infection of mice.ConclusionVACV UNG and dUTPase activities are more important for replication in quiescent cells, which have low levels of endogenous UNG and dUTPase, than in more metabolically active cells and the loss of both is more detrimental than either alone. Both UNG and dUTPase activities are required for full virulence in mice.

Attenuating Mutations of the Matrix Gene of Influenza A/WSN/33 Virus

The matrix protein (M1) of influenza virus plays an essential role in viral replication. Our previous studies have shown that basic amino acids 101RKLKR105 of M1 are involved in RNP binding and nuclear localization. For the present work, the functions of 101RKLKR105 were studied by introducing mutations into the M gene of influenza virus A/WSN/33 by reverse genetic methods. Individual substitution, R101S or R105S, had a minimal effect on viral replication. In contrast, the double mutation R101S-R105S was synergistic and resulted in temperature sensitivity reflected by reduced viral replication at a restrictive temperature. To investigate the in vivo effect on infection, BALB/c mice were infected with either A/WSN/33 wild-type (Wt) or mutant viruses and assessed for signs of illness, viral replication in the lungs, and survival rates. The results from mouse studies indicated that the R101S-R105S double mutant virus was strongly attenuated, while single mutant viruses R101S and R105S were minimally attenuated compared to A/WSN33 Wt under the same conditions. In challenge studies, mice immunized by infection with R101S-R105S were fully protected from lethal challenge with A/WSN/33. The replication and attenuating properties of R101S-R105S suggest its potential in development of live influenza virus vaccines.

Construction, phenotypic analysis, and immunogenicity of a UL5/UL29 double deletion mutant of herpes simplex virus 2.

A number of studies have shown that replication-defective mutant strains of herpes simplex virus (HSV) can induce protective immunity in animal systems against wild-type HSV challenge. However, all of those studies used viruses with single mutations. Because multiple, stable mutations provide optimal levels of safety for live vaccines, we felt that additional mutations needed to be engineered into a candidate vaccine strain for HSV-2 and genital herpes. We therefore isolated an HSV-2 strain with deletion mutations in two viral DNA replication protein genes, UL5 and UL29. The resulting double deletion mutant virus strain, dl5-29, fails to form plaques or to give any detectable single cycle yields in normal monkey or human cells. Nevertheless, dl5-29 expresses nearly the same pattern of gene products as the wild-type virus or the single mutant viruses and induces antibody titers in mice that are equivalent to those induced by single deletion mutant viruses. Therefore, it is feasible to isolate a mutant HSV strain with two mutations in essential genes and with an increased level of safety but which is still highly immunogenic.

Development of herpes simplex virus replication-defective multigene vectors for combination gene therapy applications.

Some gene therapy applications will require simultaneous expression of multiple gene products to achieve a therapeutic effect. In this study we describe the generation and characterization of replication incompetent herpes simplex virus type 1 (HSV-1) vectors (HX86Z or HX86G) carrying distinct and independently regulated expression cassettes for five transgenes (hIL-2, hGM-CSF, hB7.1, HSV-tk and lacZ or hIFN gamma). The transgenes, representing 12 kb of DNA sequence, were recombined into separate loci of a single mutant virus vector deleted for 11.6 kb of vector sequences representing portions of nine viral genes, ICP4, ICP22, ICP27, ICP47, UL24, UL41, UL44, US10 and US11. Deletion of the immediate--early genes ICP4, ICP22 and ICP27 substantially reduced vector cytotoxicity, prevented early and late viral gene expression and left intact MHC class I antigen expression. Simultaneous expression of multiple transgenes was obtained for up to 7 days in primary human melanoma cells with peak expression at 2-3 days after infection. The transgenes were chosen for their potential to function synergistically in tumor destruction and vaccine gene therapy applications, but the method and vector employed could be applied to other multigene therapy strategies. This study demonstrates the potential for engineering large transgene capacity DNA viruses such as HSV-1 for expression of multiple transgenes.