Viral Genetic Background Research Articles

Whole-Genome Sequence Approach and Phylogenomic Stratification Improve the Association Analysis of Mutations With Patient Data in Influenza Surveillance.

Each year, seasonal influenza results in high mortality and morbidity. The current classification of circulating influenza viruses is mainly focused on the hemagglutinin gene. Whole-genome sequencing (WGS) enables tracking mutations across all influenza segments allowing a better understanding of the epidemiological effects of intra- and inter-seasonal evolutionary dynamics, and exploring potential associations between mutations across the viral genome and patient’s clinical data. In this study, mutations were identified in 253 Influenza A (H3N2) clinical isolates from the 2016-2017 influenza season in Belgium. As a proof of concept, available patient data were integrated with this genomic data, resulting in statistically significant associations that could be relevant to improve the vaccine and clinical management of infected patients. Several mutations were significantly associated with the sampling period. A new approach was proposed for exploring mutational effects in highly diverse Influenza A (H3N2) strains through considering the viral genetic background by using phylogenetic classification to stratify the samples. This resulted in several mutations that were significantly associated with patients suffering from renal insufficiency. This study demonstrates the usefulness of using WGS data for tracking mutations across the complete genome and linking these to patient data, and illustrates the importance of accounting for the viral genetic background in association studies. A limitation of this association study, especially when analyzing stratified groups, relates to the number of samples, especially in the context of national surveillance of small countries. Therefore, we investigated if international databases like GISAID may help to verify whether observed associations in the Belgium A (H3N2) samples, could be extrapolated to a global level. This work highlights the need to construct international databases with both information of viral genome sequences and patient data.

Influenza A virus segments five and six can harbor artificial introns allowing expanded coding capacity.

Influenza A viruses encode their genomes across eight, negative sense RNA segments. The six largest segments produce mRNA transcripts that do not generally splice; however, the two smallest segments are actively spliced to produce the essential viral proteins NEP and M2. Thus, viral utilization of RNA splicing effectively expands the viral coding capacity without increasing the number of genomic segments. As a first step towards understanding why splicing is not more broadly utilized across genomic segments, we designed and inserted an artificial intron into the normally nonsplicing NA segment. This insertion was tolerated and, although viral mRNAs were incompletely spliced, we observed only minor effects on viral fitness. To take advantage of the unspliced viral RNAs, we encoded a reporter luciferase gene in frame with the viral ORF such that when the intron was not removed the reporter protein would be produced. This approach, which we also show can be applied to the NP encoding segment and in different viral genetic backgrounds, led to high levels of reporter protein expression with minimal effects on the kinetics of viral replication or the ability to cause disease in experimentally infected animals. These data together show that the influenza viral genome is more tolerant of splicing than previously appreciated and this knowledge can be leveraged to develop viral genetic platforms with utility for biotechnology applications.

Reversion to ancestral Zika virus NS1 residues increases competence of Aedes albopictus.

Both mosquito species-specific differences and virus strain -specific differences impact vector competence. Previous results in our laboratory with individual populations of N. American mosquitoes support studies suggesting Aedes aegypti are more competent than Ae. albopictus for American Zika virus (ZIKV) strains and demonstrate that U.S. Ae. albopictus have higher competence for an ancestral Asian ZIKV strain. A982V, an amino acid substitution in the NS1 gene acquired prior to the American outbreak, has been shown to increase competence in Ae. aegypti. We hypothesized that variability in the NS1 could therefore contribute to species-specific differences and developed a reverse genetics system based on a 2016 ZIKV isolate from Honduras (ZIKV-WTic) to evaluate the phenotypic correlates of individual amino acid substitutions. In addition to A982V, we evaluated G894A, which was acquired during circulation in the Americas. Reversion of 982 and 894 to ancestral residues increased infectivity, transmissibility and viral loads in Ae. albopictus but had no effect on competence or replication in Ae. aegypti. In addition, while host cell-specific differences in NS1 secretion were measured, with significantly higher secretion in mammalian cells relative to mosquito cells, strain-specific differences in secretion were not detected, despite previous reports. These results demonstrate that individual mutations in NS1 can influence competence in a species-specific manner independent of differences in NS1 secretion and further indicate that ancestral NS1 residues confer increased competence in Ae. albopictus. Lastly, experimental infections of Ifnar1-/- mice demonstrated that these NS1 substitutions can influence viral replication in the host and, specifically, that G894A could represent a compensatory change following a fitness loss from A982V with some viral genetic backgrounds. Together these data suggest a possible role for epistatic interactions in ZIKV fitness in invertebrate and vertebrate hosts and demonstrate that strains with increased transmission potential in U.S. Ae. albopictus could emerge.

A 5-year molecular epidemiology survey of human enterovirus 71 before vaccine application in Yunnan Province, China.

Enterovirus A71 (EV-A71) infection is known to cause hand, foot, and mouth disease (HFMD). Last year, an inactivated EV-A71 whole virus vaccine was used to prevent this disease in Yunnan, China. To obtain a viral genetic background for evaluating vaccine protection and monitor the adaptive evolution of the virus after the vaccination, a 5-year molecular epidemiology survey was performed before the vaccination. Twenty-six EV-A71 strains were separated from 561 stool specimens of patients with serious HFMD. The whole-genomic sequences of these strains were sequenced. Phylogenetic trees were constructed, and the mutation spectra were analyzed based on these viral sequences. There was no obvious mutation for the circular EV-A71 strains of the same year. Pathogenic EV-A71 strains may arise from a "subgroup" randomly each year. Whole-genomic analyses showed that a hotspot nonsynonymous substitution potentially affecting the immunogenicity of vaccines was found in the 2A gene, but not in genes of the viral capsid proteins, and the genetic diversity of whole viral genomes associated with the incidence of HFMD. Therefore, it will be valuable to monitor the genome-wide changes of EV-A71 to detect the adaptive mutations affecting immunogenicity or perform investigations using genetic diversity as a parameter.

A complete map of potential pathogenicity markers of avian influenza virus subtype H5 predicted from 11 expressed proteins.

BackgroundPolybasic cleavage sites of the hemagglutinin (HA) proteins are considered to be the most important determinants indicating virulence of the avian influenza viruses (AIV). However, evidence is accumulating that these sites alone are not sufficient to establish high pathogenicity. There need to exist other sites located on the HA protein outside the cleavage site or on the other proteins expressed by AIV that contribute to the pathogenicity.ResultsWe employed rule-based computational modeling to construct a map, with high statistical significance, of amino acid (AA) residues associated to pathogenicity in 11 proteins of the H5 type viruses. We found potential markers of pathogenicity in all of the 11 proteins expressed by the H5 type of AIV. AA mutations S-43HA1-D, D-83HA1-A in HA; S-269-D, E-41-H in NA; S-48-N, K-212-N in NS1; V-166-A in M1; G-14-E in M2; K-77-R, S-377-N in NP; and Q-48-P in PB1-F2 were identified as having a potential to shift the pathogenicity from low to high. Our results suggest that the low pathogenicity is common to most of the subtypes of the H5 AIV while the high pathogenicity is specific to each subtype. The models were developed using public data and validated on new, unseen sequences.ConclusionsOur models explicitly define a viral genetic background required for the virus to be highly pathogenic and thus confirm the hypothesis of the presence of pathogenicity markers beyond the cleavage site.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0465-x) contains supplementary material, which is available to authorized users.

Differences in Viral Load Testing Methodology May Play an Important Role in Optimizing the Initial Management of HIV-Infected Pregnant Women

One of the major success stories of HIV management is the significant drop in perinatal transmission rates. Much effort has been made in achieving a rate of 1100 cells/mm, by her report. Unfortunately, previous medical records were not available to verify her history. Testing was repeated at our center, with the HIV enzyme-linked immunosorbent assay (ELISA) as well as all Western blot bands returning positive, VL <75 copies/mL, and CD4 count 690 cells/mm. Viral load was performed using Versant HIV kPCR (Siemens Diagnostics, Malvern, PA, USA). We found it surprising that her VL was undetectable without therapy. After discussion with the National Clinician’s Consultation Center Perinatal HIV Hotline (1-888-448-8765), additional VL testing was sent to a laboratory that used real-time HIV-1 PCR (Abbott Molecular Diagnostics, Des Plaines, IL, USA) which has better sensitivity for groups N and O. The result from this assay returned with a VL of 108 copies/mL. Testing for HIV-1 DNA PCR was positive and HIV-2 DNA/RNA PCR was negative. An empiric regimen of abacavir (ABC)/lamivudine (3TC) and lopinavir/ ritonavir (LPV/r) was started in order to avoid hemosuppression that can be seen with zidovudine (ZDV). Repeat VL was undetectable thereafter using real time PCR. She delivered a healthy full-term infant whose HIV DNA PCRs remained negative. Numerous HIV-infected individuals with diverse viral genetic backgrounds are living in the United States. Choosing the correct testing method to determine antiretroviral therapy is essential in providing optimal care. Are current standards for HIV testing adequate to best manage these individuals? For the majority of infected pregnant patients, the answer is likely yes. Presently, 7 commercially available assays (only 4 with US Food and Drug Administration [FDA] approval) provide results with high sensitivity, specificity, and reproducibility. All are equipped to detect group M along with most of its 9 subtypes. Only the real-time PCR and Versant HIV kPCR can detect group O. Group N, however, can only be detected using the real-time PCR assay. Groups N and O are more commonly found in West-Central Africa, particularly Cameroon. Our patient came from a region that has higher rates of group O and may have been infected by someone from that region. In this situation, real-time PCR would be the correct method to determine the VL. Even though we believe the correct assay was used, our patient’s VL remained low, indicating that she was either an elite controller or the original PCR testing was inadequate. We suspect the former. As expected, commercial genotype testing failed due to a low VL. A blood sample sent to a research laboratory for further genotyping (using RT-PCR) determined that our patient had a pansensitive group M, subtype C virus (94.9% similarity to protease gene and 94.3% similarity to the reverse transcriptase gene) using the Stanford University HIV Drug Resistance Database. Health care providers should be mindful that there are subtle differences in VL testing methods. It is imperative that the type of assay chosen be based on the geographic location from where the infection was likely acquired. Although this does not

In vitro HIV-1 evolution in response to triple reverse transcriptase inhibitors & in silico phenotypic analysis.

BackgroundEffectiveness of ART regimens strongly depends upon complex interactions between the selective pressure of drugs and the evolution of mutations that allow or restrict drug resistance.MethodsFour clinical isolates from NRTI-exposed, NNRTI-naive subjects were passaged in increasing concentrations of NVP in combination with 1 µM 3 TC and 2 µM ADV to assess selective pressures of multi-drug treatment. A novel parameter inference procedure, based on a stochastic viral growth model, was used to estimate phenotypic resistance and fitness from in vitro combination passage experiments.ResultsNewly developed mathematical methods estimated key phenotypic parameters of mutations arising through selective pressure exerted by 3 TC and NVP. Concentrations of 1 µM 3 TC maintained the M184V mutation, which was associated with intrinsic fitness deficits. Increasing NVP concentrations selected major NNRTI resistance mutations. The evolutionary pathway of NVP resistance was highly dependent on the viral genetic background, epistasis as well as stochasticity. Parameter estimation indicated that the previously unrecognized mutation L228Q was associated with NVP resistance in some isolates.ConclusionSerial passage of viruses in the presence of multiple drugs may resemble the selection of mutations observed among treated individuals and populations in vivo and indicate evolutionary preferences and restrictions. Phenotypic resistance estimated here “in silico” from in vitro passage experiments agreed well with previous knowledge, suggesting that the unique combination of “wet-” and “dry-lab” experimentation may improve our understanding of HIV-1 resistance evolution in the future.

Progressive Multifocal Leukoencephalopathy (PML) Development Is Associated With Mutations in JC Virus Capsid Protein VP1 That Change Its Receptor Specificity

Progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease caused by JC virus (JCV) infection of oligodendrocytes, may develop in patients with immune disorders following reactivation of chronic benign infection. Mutations of JCV capsid viral protein 1 (VP1), the capsid protein involved in binding to sialic acid cell receptors, might favor PML onset. Cerebrospinal fluid sequences from 37/40 PML patients contained one of several JCV VP1 amino acid mutations, which were also present in paired plasma but not urine sequences despite the same viral genetic background. VP1-derived virus-like particles (VLPs) carrying these mutations lost hemagglutination ability, showed different ganglioside specificity, and abolished binding to different peripheral cell types compared with wild-type VLPs. However, mutants still bound brain-derived cells, and binding was not affected by sialic acid removal by neuraminidase. JCV VP1 substitutions are acquired intrapatient and might favor JCV brain invasion through abrogation of sialic acid binding with peripheral cells, while maintaining sialic acid-independent binding with brain cells.

Genetic background matters: a plant–virus gene‐for‐gene interaction is strongly influenced by genetic contexts

Evolutionary processes responsible for parasite adaptation to their hosts determine our capacity to manage sustainably resistant plant crops. Most plant-parasite interactions studied so far correspond to gene-for-gene models in which the nature of the alleles present at a plant resistance locus and at a pathogen pathogenicity locus determine entirely the outcome of their confrontation. The interaction between the pepper pvr2 resistance locus and Potato virus Y (PVY) genome-linked protein VPg locus obeys this kind of model. Using synthetic chimeras between two parental PVY cDNA clones, we showed that the viral genetic background surrounding the VPg pathogenicity locus had a strong impact on the resistance breakdown capacity of the virus. Indeed, recombination of the cylindrical inclusion (CI) coding region between two PVY cDNA clones multiplied by six the virus capacity to break down the pvr2(3) -mediated resistance. High-throughput sequencing allowed the exploration of the diversity of PVY populations in response to the selection pressure of the pvr2(3) resistance. The CI chimera, which possessed an increased resistance breakdown capacity, did not show an increased mutation accumulation rate. Instead, selection of the most frequent resistance-breaking mutation seemed to be more efficient for the CI chimera than for the parental virus clone. These results echoed previous observations, which showed that the plant genetic background in which the pvr2(3) resistance gene was introduced modified strongly the efficiency of selection of resistance-breaking mutations by PVY. In a broader context, the PVY CI coding region is one of the first identified genetic factors to determine the evolvability of a plant virus.

A Post-entry Step in the Mammalian Orthoreovirus Replication Cycle Is a Determinant of Cell Tropism

Mammalian reoviruses replicate in a broad range of hosts, cells, and tissues. These viruses display strain-dependent variation in tropism for different types of cells in vivo and ex vivo. Early steps in the reovirus life cycle, attachment, entry, and disassembly, have been identified as pivotal points of virus-cell interaction that determine the fate of infection, either productive or abortive. However, in studies of the differential capacity of reovirus strains type 1 Lang and type 3 Dearing to replicate in Madin-Darby canine kidney (MDCK) cells, we found that replication efficiency is regulated at a late point in the viral life cycle following primary transcription and translation. Results of genetic studies using recombinant virus strains show that reovirus tropism for MDCK cells is primarily regulated by replication protein μ2 and further influenced by the viral RNA-dependent RNA polymerase protein, λ3, depending on the viral genetic background. Furthermore, μ2 residue 347 is a critical determinant of replication efficiency in MDCK cells. These findings indicate that components of the reovirus replication complex are mediators of cell-selective viral replication capacity at a post-entry step. Thus, reovirus cell tropism may be determined at early and late points in the viral replication program.

Virologic Characterization of HIV Type 1 With a Codon 70 Deletion in Reverse Transcriptase

We identified a deletion at codon 70 (Delta70) of HIV-1 reverse transcriptase (RT) occurring together with L74V and Q151M mutations in a sample from a tenofovir (TFV)- and abacavir (ABC)-treated patient with extensive prior antiretroviral treatment. To investigate the characteristics of this mutant, we studied the drug susceptibility, relative infectivity, and fitness of viruses carrying Delta70 and associated RT mutations. The Delta70, L74V, and Q151M mutations were introduced into Hxb2 RT by site-directed mutagenesis and expressed in HIV-1 recombinants. The Delta70 mutation increased resistance to lamivudine and emtricitabine alone and in combination with various resistance mutations and augmented resistance to ABC and didanosine when present together with L74V. A recombinant virus expressing RT from the original clinical viral sample (Delta70-PRT) exhibited greater fitness than one in which the deletion had been repaired (K70-PRT). The Delta70 mutation also increased fitness of Hxb2 wild-type and 74V and Q151M mutants. Recombinants carrying Delta70-PRT showed greater relative infectivity in the presence of ABC (but not TFV) compared with K70-PRT recombinants. These results show that Delta70 enhances resistance to certain purine and pyrimidine analogues and contributes to multinucleoside resistance in the appropriate viral genetic background.

The viral genetic background determines the outcome of coxsackievirus B3 infection in outbred NMRI mice

The reasons for the different outcome of coxsackievirus B3 (CVB3)-induced heart disease in humans are not well understood. Since there are no experimental data on the course of disease after infection with genetically different CVB3 in a natural variable population until now, we studied the outcome of virus infection in outbred NMRI mice after inoculation of genetically different CVB3 variants. Adult male mice were inoculated with seven closely related CVB3 variants. The histopathological changes of heart and pancreas tissue, antibody induction, virus titers, and persistence of viral positive- as well as negative-strand RNA in spleen and heart tissue were compared at day 7 or day 28 after infection to detect prerequisites and predictive factors for chronic myocarditis. Six CVB3 variants infected NMRI mice. CVB3 infection (i) did not induce detectable myocardial injury, (ii) caused signs of healing up acute myocarditis or (iii) ongoing chronic myocarditis. Neither IgG antibody responses nor the extent of destruction of exocrine pancreatic tissue or viral RNA load in spleen did correlate with myocardial histopathology. In contrast, a high persistent viral RNA load in heart tissue specimens was characteristic for mice developing chronic myocarditis. The results of the present study corroborate high viral load in the acute stage of myocarditis and high amounts of persisting CVB3 RNA in heart tissue as predictive marker of chronic myocarditis. The outcome of CVB3-induced heart disease in outbred NMRI mice depends strongly on the viral genetic background. In particular an important role of viral capsid proteins is suggested.

Nuclear Import Defect of Human Immunodeficiency Virus Type 1 DNA Flap Mutants Is Not Dependent on the Viral Strain or Target Cell Type

We have previously established, using human immunodeficiency virus type 1 (HIV-1) strain LAI, that the HIV-1 central DNA Flap acts as a cis determinant of viral genome nuclear import. Although the impact of the DNA Flap on nuclear import has already found numerous independent confirmations in the context of lentivirus vectors, it has been claimed that it may be nonessential for infectious virus strains LAI, YU-2 (J. D. Dvorin et al., J. Virol. 76:12087-12096, 2002), HXB2, and NL4-3 (A. Limon et al., J. Virol. 76:12078-12086, 2002). We conducted a detailed analysis of virus infectivity using the provirus clones provided by the authors and analogous target cells. In contrast to published data, our results show that all cPPT mutant viruses exhibit reduced infectivity corresponding to a nuclear import defect irrespective of the viral genetic background or target cell.

Use of a novel assay based on intact recombinant viruses expressing green (EGFP) or red (DsRed2) fluorescent proteins to examine the contribution of pol and env genes to overall HIV-1 replicative fitness

Multiple studies have described a reduction in the replicative fitness of HIV-1 isolates harboring mutations that confer resistance to antiretroviral drugs. Contradictory results, however, have been obtained depending on the methodology used in each study (Quinones-Mateu, M.E., Arts, E.J., 2002. Fitness of drug resistant HIV-I: methodology and clinical implications. Drug Resist. Update 5, 224–233), affecting our understanding of the potential relationship of viral replicative fitness with HIV-1 disease. It has been demonstrated previously that both pol and env genes play a major role in HIV-1 replicative fitness of clinical isolates. Therefore, measuring clinically relevant replicative fitness using recombinant viruses where a single mutation and/or viral gene have been introduced does not seem like a reasonable approach in this era of multi-target antiretroviral therapy. A novel method was developed to measure HIV-1 replicative fitness based on recombinant viruses expressing the enhanced green fluorescent (EGFP) or the Discosoma sp. red fluorescent (DsRed2) proteins in a HIV-1 NL4-3 backbone. Contrary to previous designs to analyze HIV-1 fitness, these replication competent viruses were created in an intact viral genetic background (without deleting or affecting the expression of any viral gene). This new system was used to evaluate the contribution of drug-resistance mutations in the pol and env genes to overall viral replicative fitness (in the presence and absence of drug pressure) using direct growth competition experiments. Mutations in pol showed a stronger effect on HIV-1 replicative fitness than mutations in the env gene associated with resistance to enfuvirtide, corroborating the plasticity of the later gene to accept mutations and the sensibility of the protease and reverse transcriptase enzymes to drug-associated primary mutations. In conclusion, a new protocol was used to measure HIV-1 replicative fitness in either the presence or absence of antiretroviral drugs, which may be used as a high-throughput assay to help us understand the clinical significance of viral fitness.

In vitro resistance profile of the human immunodeficiency virus type 1 protease inhibitor BMS-232632.

BMS-232632 is an azapeptide human immunodeficiency virus (HIV) type 1 (HIV-1) protease inhibitor that displays potent anti-HIV-1 activity (50% effective concentration [EC(50)], 2.6 to 5.3 nM; EC(90), 9 to 15 nM). In vitro passage of HIV-1 RF in the presence of inhibitors showed that BMS-232632 selected for resistant variants more slowly than nelfinavir or ritonavir did. Genotypic and phenotypic analysis of three different HIV strains resistant to BMS-232632 indicated that an N88S substitution in the viral protease appeared first during the selection process in two of the three strains. An I84V change appeared to be an important substitution in the third strain used. Mutations were also observed at the protease cleavage sites following drug selection. The evolution to resistance seemed distinct for each of the three strains used, suggesting multiple pathways to resistance and the importance of the viral genetic background. A cross-resistance study involving five other protease inhibitors indicated that BMS-232632-resistant virus remained sensitive to saquinavir, while it showed various levels (0. 1- to 71-fold decrease in sensitivity)-of cross-resistance to nelfinavir, indinavir, ritonavir, and amprenavir. In reciprocal experiments, the BMS-232632 susceptibility of HIV-1 variants selected in the presence of each of the other HIV-1 protease inhibitors showed that the nelfinavir-, saquinavir-, and amprenavir-resistant strains of HIV-1 remained sensitive to BMS-232632, while indinavir- and ritonavir-resistant viruses displayed six- to ninefold changes in BMS-232632 sensitivity. Taken together, our data suggest that BMS-232632 may be a valuable protease inhibitor for use in combination therapy.

Host and Viral Factors Determine the Dispensability of Coat Protein for Bipartite Geminivirus Systemic Movement

Geminiviruses have unique, twinned icosahedral particles which encapsidate circular single-stranded DNA. Their genomes are composed of either one or two DNA segments. Monopartite geminiviruses absolutely require a functional coat protein (CP) for infectivity, whereas bipartite geminivirus CP null mutants can infect plants systemically. However, we show here that a CP mutant of the bipartite tomato golden mosaic virus (TGMV), which can infectNicotiana benthamianasystemically, is confined to the inoculated leaves ofNicotiana tabacumorDatura stramonium.We also show that a CP mutant of the related bean golden mosaic virus (BGMV), which can infect beans systemically, is confined to the inoculated leaves ofN. benthamiana.In each case, the extent of viral DNA accumulation in inoculated leaves was unaffected by the absence of CP, which suggests that CP is required specifically for systemic movement. The dispensability of CP is correlated with the degree of virus–host adaptation. TGMV is well adapted toN. benthamianaand does not require CP to infect this host systemically, whereas BGMV is poorly adapted toN. benthamianaand requires CP. Analysis of TGMV–BGMV hybrid viruses revealed that the viral genetic background can also affect the dispensability of CP for systemic movement inN. benthamiana.Thus, bipartite geminivirus movementin plantacan be resolved genetically into three components: (i) local, cell-to-cell movement, which does not require CP; (ii) CP-dependent systemic movement, which occurs in all hosts tested; and (iii) CP-independent systemic movement, which occurs in hosts to which a given virus is well adapted.