Classification of viral zoonosis through receptor pattern analysis

Background  

Viral zoonosis, the transmission of a virus from its primary vertebrate reservoir species to humans, requires ubiquitous cellular proteins known as receptor proteins. Zoonosis can occur not only through direct transmission from vertebrates to humans, but also through intermediate reservoirs or other environmental factors. Viruses can be categorized according to genotype (ssDNA, dsDNA, ssRNA and dsRNA viruses). Among them, the RNA viruses exhibit particularly high mutation rates and are especially problematic for this reason. Most zoonotic viruses are RNA viruses that change their envelope proteins to facilitate binding to various receptors of host species. In this study, we sought to predict zoonotic propensity through the analysis of receptor characteristics. We hypothesized that the major barrier to interspecies virus transmission is that receptor sequences vary among species--in other words, that the specific amino acid sequence of the receptor determines the ability of the viral envelope protein to attach to the cell.     

Multi-Gene Detection and Identification of Mosquito-Borne RNA Viruses Using an Oligonucleotide Microarray

Background

Arthropod-borne viruses are important emerging pathogens world-wide. Viruses transmitted by mosquitoes, such as dengue, yellow fever, and Japanese encephalitis viruses, infect hundreds of millions of people and animals each year. Global surveillance of these viruses in mosquito vectors using molecular based assays is critical for prevention and control of the associated diseases. Here, we report an oligonucleotide DNA microarray design, termed ArboChip5.1, for multi-gene detection and identification of mosquito-borne RNA viruses from the genera Flavivirus (family Flaviviridae), Alphavirus (Togaviridae), Orthobunyavirus (Bunyaviridae), and Phlebovirus (Bunyaviridae).

Methodology/Principal Findings

The assay utilizes targeted PCR amplification of three genes from each virus genus for electrochemical detection on a portable, field-tested microarray platform. Fifty-two viruses propagated in cell-culture were used to evaluate the specificity of the PCR primer sets and the ArboChip5.1 microarray capture probes. The microarray detected all of the tested viruses and differentiated between many closely related viruses such as members of the dengue, Japanese encephalitis, and Semliki Forest virus clades. Laboratory infected mosquitoes were used to simulate field samples and to determine the limits of detection. Additionally, we identified dengue virus type 3, Japanese encephalitis virus, Tembusu virus, Culex flavivirus, and a Quang Binh-like virus from mosquitoes collected in Thailand in 2011 and 2012.

Conclusions/Significance

We demonstrated that the described assay can be utilized in a comprehensive field surveillance program by the broad-range amplification and specific identification of arboviruses from infected mosquitoes. Furthermore, the microarray platform can be deployed in the field and viral RNA extraction to data analysis can occur in as little as 12 h. The information derived from the ArboChip5.1 microarray can help to establish public health priorities, detect disease outbreaks, and evaluate control programs.     

Analysis of HIV-1 drug resistant mutations by line probe assay and direct sequencing in a cohort of therapy naive HIV-1 infected Italian patients

Background  

Recent studies of viral entry proteins from influenza, measles, human immunodeficiency virus, type 1 (HIV-1), and Ebola virus have shown, first with molecular modeling, and then X-ray crystallographic or other biophysical studies, that these disparate viruses share a coiled-coil type of entry protein.     

Use of a multi-virus array for the study of human viral and retroviral pathogens: gene expression studies and ChIP-chip analysis

Background

Since the discovery of human immunodeficiency virus (HIV-1) twenty years ago, AIDS has become one of the most studied diseases. A number of viruses have subsequently been identified to contribute to the pathogenesis of HIV and its opportunistic infections and cancers. Therefore, a multi-virus array containing eight human viruses implicated in AIDS pathogenesis was developed and its efficacy in various applications was characterized.

Results

The amplified open reading frames (ORFs) of human immunodeficiency virus type 1, human T cell leukemia virus types 1 and 2, hepatitis C virus, Epstein-Barr virus, human herpesvirus 6A and 6B, and Kaposi's sarcoma-associated herpesvirus were spotted on glass slides and hybridized to DNA and RNA samples. Using a random priming method for labeling genomic DNA or cDNA probes, we show specific detection of genomic viral DNA from cells infected with the human herpesviruses, and effectively demonstrate the inhibitory effects of a cellular cyclin dependent kinase inhibitor on viral gene expression in HIV-1 and KSHV latently infected cells. In addition, we coupled chromatin immunoprecipitation with the virus chip (ChIP-chip) to study cellular protein and DNA binding.

Conclusions

An amplicon based virus chip representing eight human viruses was successfully used to identify each virus with little cross hybridization. Furthermore, the identity of both viruses was correctly determined in co-infected cells. The utility of the virus chip was demonstrated by a variety of expression studies. Additionally, this is the first demonstrated use of ChIP-chip analysis to show specific binding of proteins to viral DNA, which, importantly, did not require further amplification for detection.     

The Use of NanoTrap Particles as a Sample Enrichment Method to Enhance the Detection of Rift Valley Fever Virus

Background

Rift Valley Fever Virus (RVFV) is a zoonotic virus that is not only an emerging pathogen but is also considered a biodefense pathogen due to the threat it may cause to public health and national security. The current state of diagnosis has led to misdiagnosis early on in infection. Here we describe the use of a novel sample preparation technology, NanoTrap particles, to enhance the detection of RVFV. Previous studies demonstrated that NanoTrap particles lead to both 100 percent capture of protein analytes as well as an improvement of more than 100-fold in sensitivity compared to existing methods. Here we extend these findings by demonstrating the capture and enrichment of viruses.

Results

Screening of NanoTrap particles indicated that one particle, NT53, was the most efficient at RVFV capture as demonstrated by both qRT-PCR and plaque assays. Importantly, NT53 capture of RVFV resulted in greater than 100-fold enrichment from low viral titers when other diagnostics assays may produce false negatives. NT53 was also capable of capturing and enhancing RVFV detection from serum samples. RVFV that was inactivated through either detergent or heat treatment was still found bound to NT53, indicating the ability to use NanoTrap particles for viral capture prior to transport to a BSL-2 environment. Furthermore, both NP-40-lysed virus and purified RVFV RNA were bound by NT53. Importantly, NT53 protected viral RNA from RNase A degradation, which was not observed with other commercially available beads. Incubation of RVFV samples with NT53 also resulted in increased viral stability as demonstrated through preservation of infectivity at elevated temperatures. Finally, NanoTrap particles were capable of capturing VEEV and HIV, demonstrating the broad applicability of NanoTrap particles for viral diagnostics.

Conclusion

This study demonstrates NanoTrap particles are capable of capturing, enriching, and protecting RVFV virions. Furthermore, the use of NanoTrap particles can be extended to a variety of viruses, including VEEV and HIV.     

Reliability of Pseudotyped Influenza Viral Particles in Neutralizing Antibody Detection

Background

Current influenza control strategies require an active surveillance system. Pseudotyped viral particles (pp) together with the evaluation of pre-existing immunity in a population might satisfy this requirement. However, the reliability of using pp in neutralizing antibody (nAb) detection are undefined.

Methodology/Principal Findings

Pseudotyped particles of A(H1N1)pmd09 (A/California/7/2009) and HPAI H5N1 (A/Anhui/1/2005), as well as their reassortants, were generated. The reliability of using these pp in nAb detection were compared concurrently with the corresponding viruses by a hemagglutination inhibition test, as well as ELISA-, cytopathic effect-, and fluorescence-based microneutralization assays. In the qualitative detection on nAbs, the pp and their corresponding viruses were in complete agreement, with an R² value equal to or near 1 in two different populations. In the quantitative detection on nAbs, although the geometric mean titers (95% confidence interval) differed between the pp and viruses, no significant difference was observed. Furthermore, humoral immunity against the reassortants was evaluated; our results indicated strong consistency between the nAbs against reassortant pp and those against naïve pp harboring the same hemagglutinin.

Conclusion/Significance

The pp displayed high reliability in influenza virus nAb detection. The use of reassortant pp is a safe and convenient strategy for characterizing emerging influenza viruses and surveying the disease burden.     

Identifying and prioritizing potential human-infecting viruses from their genome sequences

Determining which animal viruses may be capable of infecting humans is currently intractable at the time of their discovery, precluding prioritization of high-risk viruses for early investigation and outbreak preparedness. Given the increasing use of genomics in virus discovery and the otherwise sparse knowledge of the biology of newly discovered viruses, we developed machine learning models that identify candidate zoonoses solely using signatures of host range encoded in viral genomes. Within a dataset of 861 viral species with known zoonotic status, our approach outperformed models based on the phylogenetic relatedness of viruses to known human-infecting viruses (area under the receiver operating characteristic curve [AUC] = 0.773), distinguishing high-risk viruses within families that contain a minority of human-infecting species and identifying putatively undetected or so far unrealized zoonoses. Analyses of the underpinnings of model predictions suggested the existence of generalizable features of viral genomes that are independent of virus taxonomic relationships and that may preadapt viruses to infect humans. Our model reduced a second set of 645 animal-associated viruses that were excluded from training to 272 high and 41 very high-risk candidate zoonoses and showed significantly elevated predicted zoonotic risk in viruses from nonhuman primates, but not other mammalian or avian host groups. A second application showed that our models could have identified Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) as a relatively high-risk coronavirus strain and that this prediction required no prior knowledge of zoonotic Severe Acute Respiratory Syndrome (SARS)-related coronaviruses. Genome-based zoonotic risk assessment provides a rapid, low-cost approach to enable evidence-driven virus surveillance and increases the feasibility of downstream biological and ecological characterization of viruses.

Surveillance of emerging viruses is one of the first steps to avoid the next pandemic. This study uses machine learning to identify many zoonotic viruses directly from their genomes. This allows rapid assessment of research priorities as soon as new viruses are discovered, focusing research and surveillance efforts on the viruses most likely to infect humans.     

Characterization of a synthetic bacterial self-destruction device for programmed cell death and for recombinant proteins release

Background

In a globalized word, prevention of infectious diseases is a major challenge. Rapid detection of viable virus particles in water and other environmental samples is essential to public health risk assessment, homeland security and environmental protection. Current virus detection methods, especially assessing viral infectivity, are complex and time-consuming, making point-of-care detection a challenge. Faster, more sensitive, highly specific methods are needed to quantify potentially hazardous viral pathogens and to determine if suspected materials contain viable viral particles. Fourier transform infrared (FTIR) spectroscopy combined with cellular-based sensing, may offer a precise way to detect specific viruses. This approach utilizes infrared light to monitor changes in molecular components of cells by tracking changes in absorbance patterns produced following virus infection. In this work poliovirus (PV1) was used to evaluate the utility of FTIR spectroscopy with cell culture for rapid detection of infective virus particles.

Results

Buffalo green monkey kidney (BGMK) cells infected with different virus titers were studied at 1 - 12 hours post-infection (h.p.i.). A partial least squares (PLS) regression method was used to analyze and model cellular responses to different infection titers and times post-infection. The model performs best at 8 h.p.i., resulting in an estimated root mean square error of cross validation (RMSECV) of 17 plaque forming units (PFU)/ml when using low titers of infection of 10 and 100 PFU/ml. Higher titers, from 10³ to 10⁶ PFU/ml, could also be reliably detected.

Conclusions

This approach to poliovirus detection and quantification using FTIR spectroscopy and cell culture could potentially be extended to compare biochemical cell responses to infection with different viruses. This virus detection method could feasibly be adapted to an automated scheme for use in areas such as water safety monitoring and medical diagnostics.     

Salmonella Typhimurium-specific bacteriophage ΦSH19 and the origins of species specificity in the Vi01-like phage family

Background

PHYVV and PepGMV are plant viruses reported in Mexico and Southern US as causal agents of an important pepper disease known as "rizado amarillo". Mixed infections with PHYVV and PepGMV have been reported in several hosts over a wide geographic area. Previous work suggested that these viruses might interact at the replication and/or movement level in a complex manner. The aim of present report was to study some aspects of a synergistic interaction between PHYVV and PepGMV in pepper plants. These include analyses of symptom severity, viral DNA concentration and tissue localization of both viruses in single and mixed infections.

Results

Mixed infections with PepGMV and PHYVV induced symptoms more severe than those observed in single viral infections. Whereas plants infected with either virus (single infection) presented a remission stage with a corresponding decrease in viral DNA levels, double-infected plants did not present symptom remission and both viral DNA concentrations dramatically increased. In situ hybridization experiments revealed that both viruses are restricted to the vascular tissue. Interestingly, the amount of viral DNA detected was higher in plants inoculated with PepGMV than that observed in PHYVV-infected plants. During mixed infections, the location of both viruses remained similar to the one observed in single infections, although the number of infected cells increases. Infections with the tripartite mixture PHYVV (A+B) + PepGMV A produced a similar synergistic infection to the one observed after inoculation with both full viruses. On the contrary, tripartite mixture PepGMV (A+B) + PHYVV A did not produce a synergistic interaction. In an attempt to study the contribution of individual genes to the synergism, several mutants of PHYVV or PepGMV were inoculated in combination with the corresponding wild type, second virus (wt PepGMV or wt PHYVV). All combinations tested resulted in synergistic infections, with exception of the TrAP mutant of PepGMV (PepGMV TrAP-) + PHYVV.

Conclusion

In this report, we have demonstrated that synergistic interaction between PHYVV and PepGMV during a mixed infection is mainly due to an increased DNA concentration of both viruses, without any noticeable effect on the localization of either virus on infected plant tissue. Our results have shown that the viral component A from PepGMV is important for synergism during PHYVV-PepGMV mixed infections.     

Donor variability in HIV binding to peripheral blood mononuclear cells

Background

Currently applied indicator organism systems, such as coliforms, are not fully protective of public health from enteric viruses in water sources. Waterborne disease outbreaks have occurred in systems that tested negative for coliforms, and positive coliform results do not necessarily correlate with viral risk. It is widely recognized that bacterial indicators do not co-occur exclusively with infectious viruses, nor do they respond in the same manner to environmental or engineered stressors. Thus, a more appropriate indicator of health risks from infectious enteric viruses is needed.

Presentation of the hypothesis

Torque teno virus is a small, non-enveloped DNA virus that likely exhibits similar transport characteristics to pathogenic enteric viruses. Torque teno virus is unique among enteric viral pathogens in that it appears to be ubiquitous in humans, elicits seemingly innocuous infections, and does not exhibit seasonal fluctuations or epidemic spikes. Torque teno virus is transmitted primarily via the fecal-oral route and can be assayed using rapid molecular techniques. We hypothesize that Torque teno virus is a more appropriate indicator of viral pathogens in drinking waters than currently used indicator systems based solely on bacteria.

Testing the hypothesis

To test the hypothesis, a multi-phased research approach is needed. First, a reliable Torque teno virus assay must be developed. A rapid, sensitive, and specific PCR method using established nested primer sets would be most appropriate for routine monitoring of waters. Because PCR detects both infectious and inactivated virus, an in vitro method to assess infectivity also is needed. The density and occurrence of Torque teno virus in feces, wastewater, and source waters must be established to define spatial and temporal stability of this potential indicator. Finally, Torque teno virus behavior through drinking water treatment plants must be determined with co-assessment of traditional indicators and enteric viral pathogens to assess whether correlations exist.

Implications of the hypothesis

If substantiated, Torque teno virus could provide a completely new, reliable, and efficient indicator system for viral pathogen risk. This indicator would have broad application to drinking water utilities, watershed managers, and protection agencies and would provide a better means to assess viral risk and protect public health.     

Human Immunodeficiency Virus Type-1 (HIV-1) Continues to Evolve in Presence of Broadly Neutralizing Antibodies More than Ten Years after Infection

Background

The evolution of HIV-1 and its immune escape to autologous neutralizing antibodies (Nabs) during the acute/early phases of infection have been analyzed in depth in many studies. In contrast, little is known about neither the long-term evolution of the virus in patients who developed broadly Nabs (bNabs) or the mechanism of escape in presence of these bNabs.

Results

We have studied the viral population infecting a long term non progressor HIV-1 infected patient who had developed broadly neutralizing antibodies toward all tier 2/3 viruses (6 clades) tested, 9 years after infection, and was then followed up over 7 years. The autologous neutralization titers of the sequential sera toward env variants representative of the viral population significantly increased during the follow-up period. The most resistant pseudotyped virus was identified at the last visit suggesting that it represented a late emerging escape variant. We identified 5 amino acids substitutions that appeared associated with escape to broadly neutralizing antibodies. They were V319I/S, R/K355T, R/W429G, Q460E and G/T463E, in V3, C3 and V5 regions.

Conclusion

This study showed that HIV-1 may continue to evolve in presence of both broadly neutralizing antibodies and increasing autologous neutralizing activity more than 10 years post-infection.     

Bispidine-Amino Acid Conjugates Act as a Novel Scaffold for the Design of Antivirals That Block Japanese Encephalitis Virus Replication

Background

Japanese encephalitis virus (JEV) is a major cause of viral encephalitis in South and South-East Asia. Lack of antivirals and non-availability of affordable vaccines in these endemic areas are a major setback in combating JEV and other closely related viruses such as West Nile virus and dengue virus. Protein secondary structure mimetics are excellent candidates for inhibiting the protein-protein interactions and therefore serve as an attractive tool in drug development. We synthesized derivatives containing the backbone of naturally occurring lupin alkaloid, sparteine, which act as protein secondary structure mimetics and show that these compounds exhibit antiviral properties.

Methodology/Principal Findings

In this study we have identified 3,7-diazabicyclo[3.3.1]nonane, commonly called bispidine, as a privileged scaffold to synthesize effective antiviral agents. We have synthesized derivatives of bispidine conjugated with amino acids and found that hydrophobic amino acid residues showed antiviral properties against JEV. We identified a tryptophan derivative, Bisp-W, which at 5 µM concentration inhibited JEV infection in neuroblastoma cells by more than 100-fold. Viral inhibition was at a stage post-entry and prior to viral protein translation possibly at viral RNA replication. We show that similar concentration of Bisp-W was capable of inhibiting viral infection of two other encephalitic viruses namely, West Nile virus and Chandipura virus.

Conclusions/Significance

We have demonstrated that the amino-acid conjugates of 3,7-diazabicyclo[3.3.1]nonane can serve as a molecular scaffold for development of potent antivirals against encephalitic viruses. Our findings will provide a novel platform to develop effective inhibitors of JEV and perhaps other RNA viruses causing encephalitis.     

Highly pathogenic influenza A(H5N1) virus survival in complex artificial aquatic biotopes

Background

Very little is known regarding the persistence of Highly Pathogenic Avian Influenza (HPAI) H5N1 viruses in aquatic environments in tropical countries, although environmental materials have been suggested to play a role as reservoirs and sources of transmission for H5N1 viruses.

Methodology/Principal Findings

The survival of HPAI H5N1 viruses in experimental aquatic biotopes (water, mud, aquatic flora and fauna) relevant to field conditions in Cambodia was investigated. Artificial aquatic biotopes, including simple ones containing only mud and water, and complex biotopes involving the presence of aquatic flora and fauna, were set up. They were experimentally contaminated with H5N1 virus. The persistence of HPAI H5N1 virus (local avian and human isolates) was determined by virus isolation in embryonated chicken eggs and by real-time reverse-polymerase chain reaction. Persistence of infectious virus did not exceed 4 days, and was only identified in rain water. No infectious virus particles were detected in pond and lake water or mud even when high inoculum doses were used. However, viral RNA persisted up to 20 days in rain water and 7 days in pond or lake water. Viral RNA was also detected in mud samples, up to 14 days post-contamination in several cases. Infectious virus and viral RNA was detected in few cases in the aquatic fauna and flora, especially in bivalves and labyrinth fish, although these organisms seemed to be mostly passive carriers of the virus rather than host allowing virus replication.

Conclusions/Significance

Although several factors for the survival and persistence of HPAI viruses in the environment are still to be elucidated, and are particularly hard to control in laboratory conditions, our results, along with previous data, support the idea that environmental surveillance is of major relevance for avian influenza control programs.     

An antiviral defense role of AGO2 in plants

Background

Argonaute (AGO) proteins bind to small-interfering (si)RNAs and micro (mi)RNAs to target RNA silencing against viruses, transgenes and in regulation of mRNAs. Plants encode multiple AGO proteins but, in Arabidopsis, only AGO1 is known to have an antiviral role.

Methodology/Principal Findings

To uncover the roles of specific AGOs in limiting virus accumulation we inoculated turnip crinkle virus (TCV) to Arabidopsis plants that were mutant for each of the ten AGO genes. The viral symptoms on most of the plants were the same as on wild type plants although the ago2 mutants were markedly hyper-susceptible to this virus. ago2 plants were also hyper-susceptible to cucumber mosaic virus (CMV), confirming that the antiviral role of AGO2 is not specific to a single virus. For both viruses, this phenotype was associated with transient increase in virus accumulation. In wild type plants the AGO2 protein was induced by TCV and CMV infection.

Conclusions/Significance

Based on these results we propose that there are multiple layers to RNA-mediated defense and counter-defense in the interactions between plants and their viruses. AGO1 represents a first layer. With some viruses, including TCV and CMV, this layer is overcome by viral suppressors of silencing that can target AGO1 and a second layer involving AGO2 limits virus accumulation. The second layer is activated when the first layer is suppressed because AGO2 is repressed by AGO1 via miR403. The activation of the second layer is therefore a direct consequence of the loss of the first layer of defense.     

Differential Protein Modulation in Midguts of Aedes aegypti Infected with Chikungunya and Dengue 2 Viruses

Background

Arthropod borne virus infections cause several emerging and resurgent infectious diseases. Among the diseases caused by arboviruses, dengue and chikungunya are responsible for a high rate of severe human diseases worldwide. The midgut of mosquitoes is the first barrier for pathogen transmission and is a target organ where arboviruses must replicate prior to infecting other organs. A proteomic approach was undertaken to characterize the key virus/vector interactions and host protein modifications that happen in the midgut for viral transmission to eventually take place.

Methodology and Principal Findings

Using a proteomics differential approach with two-Dimensional Differential in-Gel Electrophoresis (2D-DIGE), we defined the protein modulations in the midgut of Aedes aegypti that were triggered seven days after an oral infection (7 DPI) with dengue 2 (DENV-2) and chikungunya (CHIKV) viruses. Gel profile comparisons showed that the level of 18 proteins was modulated by DENV-2 only and 12 proteins were modulated by CHIKV only. Twenty proteins were regulated by both viruses in either similar or different ways. Both viruses caused an increase of proteins involved in the generation of reactive oxygen species, energy production, and carbohydrate and lipid metabolism. Midgut infection by DENV-2 and CHIKV triggered an antioxidant response. CHIKV infection produced an increase of proteins involved in detoxification.

Conclusion/Significance

Our study constitutes the first analysis of the protein response of Aedes aegypti''s midgut infected with viruses belonging to different families. It shows that the differentially regulated proteins in response to viral infection include structural, redox, regulatory proteins, and enzymes for several metabolic pathways. Some of these proteins like antioxidant are probably involved in cell protection. On the other hand, we propose that the modulation of other proteins like transferrin, hsp60 and alpha glucosidase, may favour virus survival, replication and transmission, suggesting a subversion of the insect cell metabolism by the arboviruses.     

Mutagenesis-mediated virus extinction: virus-dependent effect of viral load on sensitivity to lethal defection

Background

Lethal mutagenesis is a transition towards virus extinction mediated by enhanced mutation rates during viral genome replication, and it is currently under investigation as a potential new antiviral strategy. Viral load and virus fitness are known to influence virus extinction. Here we examine the effect or the multiplicity of infection (MOI) on progeny production of several RNA viruses under enhanced mutagenesis.

Results

The effect of the mutagenic base analogue 5-fluorouracil (FU) on the replication of the arenavirus lymphocytic choriomeningitis virus (LCMV) can result either in inhibition of progeny production and virus extinction in infections carried out at low multiplicity of infection (MOI), or in a moderate titer decrease without extinction at high MOI. The effect of the MOI is similar for LCMV and vesicular stomatitis virus (VSV), but minimal or absent for the picornaviruses foot-and-mouth disease virus (FMDV) and encephalomyocarditis virus (EMCV). The increase in mutation frequency and Shannon entropy (mutant spectrum complexity) as a result of virus passage in the presence of FU was more accentuated at low MOI for LCMV and VSV, and at high MOI for FMDV and EMCV. We present an extension of the lethal defection model that agrees with the experimental results.

Conclusions

(i) Low infecting load favoured the extinction of negative strand viruses, LCMV or VSV, with an increase of mutant spectrum complexity. (ii) This behaviour is not observed in RNA positive strand viruses, FMDV or EMCV. (iii) The accumulation of defector genomes may underlie the MOI-dependent behaviour. (iv) LCMV coinfections are allowed but superinfection is strongly restricted in BHK-21 cells. (v) The dissimilar effects of the MOI on the efficiency of mutagenic-based extinction of different RNA viruses can have implications for the design of antiviral protocols based on lethal mutagenesis, presently under development.