Evaluation of the Australian first few X household transmission project for COVID-19.

BACKGROUND: The Australian First Few X (FFX) Household Transmission Project for COVID-19 was the first prospective, multi-jurisdictional study of its kind in Australia. The project was undertaken as a partnership between federal and state health departments and the Australian Partnership for Preparedness Research on Infectious Disease Emergencies (APPRISE) and was active from April to October 2020. METHODS: We aimed to identify and explore the challenges and strengths of the Australian FFX Project to inform future FFX study development and integration into pandemic preparedness plans. We asked key stakeholders and partners involved with implementation to identify and rank factors relating to the strengths and challenges of project implementation in two rounds of modified Delphi surveys. Key representatives from jurisdictional health departments were then interviewed to contextualise findings within public health processes and information needs to develop a final set of recommendations for FFX study development in Australia. RESULTS: Four clear recommendations emerged from the evaluation. Future preparedness planning should aim to formalise and embed partnerships between health departments and researchers to help better integrate project data collection into core public health surveillance activities. The development of functional, adaptable protocols with pre-established ethics and governance approvals and investment in national data infrastructure were additional priority areas noted by evaluation participants. CONCLUSION: The evaluation provided a great opportunity to consolidate lessons learnt from the Australian FFX Household Transmission Project. The developed recommendations should be incorporated into future pandemic preparedness plans in Australia to enable effective implementation and increase local utility and value of the FFX platform within emergency public health response.

Virology and immune dynamics reveal high household transmission of ancestral SARS-CoV-2 strain.

BACKGROUND: Household studies are crucial for understanding the transmission of SARS-CoV-2 infection, which may be underestimated from PCR testing of respiratory samples alone. We aim to combine the assessment of household mitigation measures; nasopharyngeal, saliva, and stool PCR testing; along with mucosal and systemic SARS-CoV-2-specific antibodies, to comprehensively characterize SARS-CoV-2 infection and transmission in households. METHODS: Between March and September 2020, we obtained samples from 92 participants in 26 households in Melbourne, Australia, in a 4-week period following the onset of infection with ancestral SARS-CoV-2 variants. RESULTS: The secondary attack rate was 36% (24/66) when using nasopharyngeal swab (NPS) PCR positivity alone. However, when respiratory and nonrespiratory samples were combined with antibody responses in blood and saliva, the secondary attack rate was 76% (50/66). SARS-CoV-2 viral load of the index case and household isolation measures were key factors that determine secondary transmission. In 27% (7/26) of households, all family members tested positive by NPS for SARS-CoV-2 and were characterized by lower respiratory Ct values than low transmission families (Median 22.62 vs. 32.91; IQR 17.06-28.67 vs. 30.37-34.24). High transmission families were associated with enhanced plasma antibody responses to multiple SARS-CoV-2 antigens and the presence of neutralizing antibodies. Three distinguishing saliva SARS-CoV-2 antibody features were identified according to age (IgA1 to Spike 1, IgA1 to nucleocapsid protein (NP)), suggesting that adults and children generate distinct mucosal antibody responses during the acute phase of infection. CONCLUSION: Utilizing respiratory and nonrespiratory PCR testing, along with the measurement of SARS-CoV-2-specific local and systemic antibodies, provides a more accurate assessment of infection within households and highlights some of the immunological differences in response between children and adults.

Acceptability, motivation and the prospect of cure for people living with HIV and their healthcare providers in HIV cure-focused treatment interruption studies.

BACKGROUND: Analytical treatment interruptions (ATI) are commonly used clinical endpoints to assess interventions aimed at curing HIV or achieving antiretroviral therapy (ART)-free HIV remission. Understanding the acceptability of ATI amongst people living with HIV (PLHIV) and their HIV healthcare providers (HHP) is limited. METHODS: Two online surveys for PLHIV and HHP assessed awareness and acceptability of ATI, and understanding of the prospect for HIV cure in the future. Responses were collected from July 2017-January 2018. A descriptive analysis was performed and similar questions across the two surveys were compared using χ squared test. RESULTS: 442 PLHIV and 144 HHP completed the survey. 105/400 (26%) PLHIV had ever interrupted ART, 8% of which were in a clinical trial. Altruistic motivations were drivers of participation of PLHIV in cure related research. 81/135 (60%) HHP would support their patients wishing to enrol in an HIV cure-focused trial, but fewer would promote and allow such participation (25% and 31% respectively). Compared to HHP, PLHIV were more likely to believe that an HIV cure would be achievable within 10 years (55% vs. 19%, p < 0.001), had less awareness of ATI (46% vs. 62%, p < 0.001) and were less likely to have had experience of either participation or enrolment in an ATI study (5% vs. 18%, p < 0.001) CONCLUSION: PLHIV were more optimistic about the potential for HIV cure. HHP had more direct experience with HIV cure-focused studies. Educational strategies are required for both groups to increase understanding around ATIs in HIV cure research but should be tailored specifically to each group.

Activation of HIV transcription with short-course vorinostat in HIV-infected patients on suppressive antiretroviral therapy.

UNLABELLED: Human immunodeficiency virus (HIV) persistence in latently infected resting memory CD4+ T-cells is the major barrier to HIV cure. Cellular histone deacetylases (HDACs) are important in maintaining HIV latency and histone deacetylase inhibitors (HDACi) may reverse latency by activating HIV transcription from latently infected CD4+ T-cells. We performed a single arm, open label, proof-of-concept study in which vorinostat, a pan-HDACi, was administered 400 mg orally once daily for 14 days to 20 HIV-infected individuals on suppressive antiretroviral therapy (ART). The primary endpoint was change in cell associated unspliced (CA-US) HIV RNA in total CD4+ T-cells from blood at day 14. The study is registered at ClinicalTrials.gov (NCT01365065). Vorinostat was safe and well tolerated and there were no dose modifications or study drug discontinuations. CA-US HIV RNA in blood increased significantly in 18/20 patients (90%) with a median fold change from baseline to peak value of 7.4 (IQR 3.4, 9.1). CA-US RNA was significantly elevated 8 hours post drug and remained elevated 70 days after last dose. Significant early changes in expression of genes associated with chromatin remodeling and activation of HIV transcription correlated with the magnitude of increased CA-US HIV RNA. There were no statistically significant changes in plasma HIV RNA, concentration of HIV DNA, integrated DNA, inducible virus in CD4+ T-cells or markers of T-cell activation. Vorinostat induced a significant and sustained increase in HIV transcription from latency in the majority of HIV-infected patients. However, additional interventions will be needed to efficiently induce virus production and ultimately eliminate latently infected cells. TRIAL REGISTRATION: ClinicalTrials.gov NCT01365065.

CD8+ T cells are activated in an antigen-independent manner in HIV-infected individuals.

Hyperactivation of T cells, particularly of CD8(+) T cells, is a hallmark of chronic HIV 1 (HIV-1) infection. Little is known about the antigenic specificities and the mechanisms by which HIV-1 causes activation of CD8(+) T cells during chronic infection. We report that CD8(+) T cells were activated during in vivo HIV-1 replication irrespective of their Ag specificity. Cytokines present during untreated HIV-1 infection, most prominently IL-15, triggered proliferation and expression of activation markers in CD8(+) T cells, but not CD4(+) T cells, in the absence of TCR stimulation. Moreover, LPS or HIV-1-activated dendritic cells (DCs) stimulated CD8(+) T cells in an IL-15-dependent but Ag-independent manner, and IL-15 expression was highly increased in DCs isolated from viremic HIV-1 patients, suggesting that CD8(+) T cells are activated by inflammatory cytokines in untreated HIV-1 patients independent of Ag specificity. This finding contrasts with CD4(+) T cells whose in vivo activation seems biased toward specificities for persistent Ags. These observations explain the higher abundance of activated CD8(+) T cells compared with CD4(+) T cells in untreated HIV-1 infection.

Learning from COVID-19: strengthening Australia's research capacity through preparedness and collaboration.

Summary: The coronavirus disease 2019 (COVID-19) pandemic has highlighted that preparedness for and responsiveness to pandemics requires public health platforms and processes which are nimble and evidence-based and a research ecosystem which is rapidly responsive to the evolving needs of society and decision-makers. The national BEAT COVID-19 research consortium was funded in 2020 by the Snow Medical Research Foundation (Snow Medical). Its Expert Advisory Committee met with the consortium post-pandemic to summarise the research undertaken and to consider lessons learned through the research response to COVID-19 in Australia. The panel observed that philanthropy offered an important 'kick-starter' funding mechanism for urgent research, which facilitated leveraging of additional funds. It further agreed that research requirements for strengthening Australia's pandemic preparedness and response include: (1) development of a national health and medical research strategy for pandemic research; (2) long-term investment in pre-established research partnerships and networks; (3) systemic procedural improvements, e.g. in ethics, governance and resource allocation; (4) responsive funding mechanisms including philanthropy; and (5) integration of research outputs into health practice and decision-making, as illustrated in Figure 1.

Impact of antigen specificity on CD4+ T cell activation in chronic HIV-1 infection.

BACKGROUND: HIV infection induces chronic immune activation which is associated with accelerated disease progression; the causes of this activation, however, are incompletely understood. We investigated the activation status of CD4+ T cells specific for chronic herpes viruses and the non-persistent antigen tetanus toxoid (TT) in HIV positive and HIV negative donors to assess whether persistent infections contribute to chronic CD4+ T cell activation. METHODS: Untreated HIV+ patients and healthy, aged matched controls were recruited and activation levels assessed and compared between cells specific for persistent and non-persistent antigens. Activation levels on antigen-specific CD4+ T cells were measured by intracellular cytokine staining following in vitro stimulation with various recall antigens (CMV, EBV, HSV, VZV and TT) in conjunction with cell surface phenotyping. RESULTS: Activation levels of herpes virus-specific CD4+ T cell populations, assessed by co-expression of CD38 and HLA-DR, were significantly elevated in HIV+ individuals compared to normal controls and compared to TT-specific responses. In contrast, we found similar levels of activation of TT-specific CD4+ T cells in HIV+ and HIV- donors. CONCLUSIONS: These results show a disparate distribution of immune activation within CD4+ T cell populations depending on their specificity and suggest that the elevated level of immune activation that characterizes chronic HIV infection may be influenced by the persistence of other antigens.

The APPRISE Virtual Biobank for Infectious Diseases.

The Australian Partnership for Preparedness Research on InfectiouS disease Emergencies (APPRISE) has developed a virtual biobank to support infectious disease research in Australia. The virtual biobank (https://apprise.biogrid.org.au) integrates access to existing distributed infectious disease biospecimen collections comprising multiple specimen types, including plasma, serum, and peripheral blood mononuclear cells. Through the development of a common data model, multiple collections can be searched simultaneously via a secure web portal. The portal enhances the visibility and searchability of existing collections within their current governance and custodianship arrangements. The portal is easily scalable for integration of additional collections.

HIV reservoirs and strategies for eradication.

Combination antiretroviral therapy (cART) has led to a reduction in morbidity and mortality in HIV-infected patients but therapy is lifelong and there is no cure for HIV. The major barriers to cure include HIV latency, which has been identified in different T-cell subsets, as well as persistence of HIV in anatomical reservoirs. We review recent developments in our understanding of the major reservoirs of HIV in patients on cART as well as how latency is established and maintained in T cells. Finally, we review the scientific rationale of and clinical experience with pharmacotherapeutic strategies aimed at eliminating latently infected cells.

Value of consultation in establishing a public health research network: lessons from APPRISE.

OBJECTIVES: To understand the challenges and benefits of an extensive consultation process relating to the establishment and ongoing funding of a novel, disseminated national research network for infectious disease preparedness. METHODS: We used a two-part modified Delphi process to identify and rank factors relating to the consultation process across the different stages of setting up a new research network. RESULTS: Research priorities for the new research network remained the same following consultation with a broad range of stakeholders. Broad networking and the establishment of a nationally recognised preparedness research network were clearly identified as the consultation's key strengths. The need for ongoing management of diverse expectations, particularly between researchers and public health practitioners, are clear challenges. Clarity on the distinct roles of researchers and decision makers are necessary to integrate research into a translational pathway. Researcher expectations for investigator-driven detailed inquiry must be balanced with expectations of routine public health activities and decision making. CONCLUSIONS: Consultation had a clear benefit for the development of a complex public health network with a focus on policy translation. Ongoing challenges include managing diverse expectations and recognising the need for continuing relationship management. Understanding the strengths and limitations of consultation to enable ongoing funding should inform the development of further collaborative research networks in multidisciplinary and translational contexts in health.

Control of viremia and prevention of AIDS following immunotherapy of SIV-infected macaques with peptide-pulsed blood.

Effective immunotherapies for HIV are needed. Drug therapies are life-long with significant toxicities. Dendritic-cell based immunotherapy approaches are promising but impractical for widespread use. A simple immunotherapy, reinfusing fresh autologous blood cells exposed to overlapping SIV peptides for 1 hour ex vivo, was assessed for the control of SIV(mac251) replication in 36 pigtail macaques. An initial set of four immunizations was administered under antiretroviral cover and a booster set of three immunizations administered 6 months later. Vaccinated animals were randomized to receive Gag peptides alone or peptides spanning all nine SIV proteins. High-level, SIV-specific CD4 and CD8 T-cell immunity was induced following immunization, both during antiretroviral cover and without. Virus levels were durably approximately 10-fold lower for 1 year in immunized animals compared to controls, and a significant delay in AIDS-related mortality resulted. Broader immunity resulted following immunizations with peptides spanning all nine SIV proteins, but the responses to Gag were weaker in comparison to animals only immunized with Gag. No difference in viral outcome occurred in animals immunized with all SIV proteins compared to animals immunized against Gag alone. Peptide-pulsed blood cells are an immunogenic and effective immunotherapy in SIV-infected macaques. Our results suggest Gag alone is an effective antigen for T-cell immunotherapy. Fresh blood cells pulsed with overlapping Gag peptides is proceeding into trials in HIV-infected humans.

Perspectives on Analytical Treatment Interruptions in People Living with HIV and Their Health Care Providers in the Landscape of HIV Cure-Focused Studies.

Analytical treatment interruptions (ATIs) aim to assess effects of HIV cure-focused interventions, but poses potential risks. Understanding of ATI acceptability among people living with HIV (PLHIV) and their HIV health care providers (HHP) is limited. Two international online surveys for PLHIV and HHP assessed understanding and acceptability of monitoring strategies during ATI. Survey items included the following: frequency of CD4, viral load (VL) and clinical assessment, CD4 and VL threshold to restart antiretroviral therapy (ART), acceptability of detectable viremia during ATI, and potential risks of ATI. Responses were collected from July 2017 to January 2018, and a descriptive analysis was performed. Responses to questions asked in both surveys were compared by χ2 test. Four hundred forty-two completed the PLHIV survey: 22% identified as female, 39% older than 50 years of age, and 64% identified as gay/homosexual/lesbian. Ninety-five percent were on ART, of which 83% reported an undetectable VL. The preferred frequency of CD4, VL, and clinical monitoring during ATI was monthly. Thirty-five percent of respondents preferred VL to remain undetectable during ATI and would not accept any sustained period of viremia, compared to 18% of 144 HHP (p < .01). Having previously interrupted ART predicted preference for VL to remain undetectable during ATI (odds ratio 0.6, p = .05). Both clinicians and PLHIV were concerned about HIV transmission during ATI. Our work demonstrates that PLHIV expectations of ATI in cure-focused clinical trials do not align with current practices, with PLHIV less accepting of viremia during ATI and preferring less frequent monitoring. Clear education messages and careful consent processes need to be developed in relationship to ATIs in HIV cure research.

Time for revolution? Enhancing meaningful involvement of people living with HIV and affected communities in HIV cure-focused science.

INTRODUCTION: Involving affected communities and people living with HIV (PLHIV) in HIV cure-focused clinical trials has ethical and practical benefits. However, there can be barriers to meaningful involvement of 'lay people' in scientific research meaning community consultation is often limited or tokenistic. This paper reports on an Australian project, the INSPIRE project (Improve, Nurture and Strengthen education, collaboration, and communication between PLHIV and Researchers), which aimed to explore barriers and enablers to enactment of the principles of meaningful involvement of PLHIV (MIPA) and affected communities in HIV cure-focused research. METHODS: The project involved a workshop attended by 40 stakeholders involved in HIV care, research or advocacy including PLHIV, community organizations, basic scientists, and clinicians. The workshop involved a facilitated discussion about community involvement in a hypothetical HIV cure-focused clinical trial. Data were collected through notetaking and video recordings. Qualitative, thematic analysis was undertaken to organize the data and identify core themes related to MIPA. RESULTS: Workshop discussions revealed community stakeholders often feel their involvement in HIV clinical research is undervalued, evidenced by limited financial remuneration and minimal capacity to influence the research design or processes. Building long-term, formal and informal relationships between community organizations, PLHIV, researchers and research teams or laboratories was identified as a strategy to support MIPA at all stages of a clinical trial, from design to dissemination of findings. CONCLUSIONS: Enacting MIPA principles in HIV cure-focused research requires a better understanding of the potential to improve research outcomes and ensure quality in the research process.

Limited maintenance of vaccine-induced simian immunodeficiency virus-specific CD8 T-cell receptor clonotypes after virus challenge.

T-cell receptors (TCRs) govern the specificity, efficacy, and cross-reactivity of CD8 T cells. Here, we studied CD8 T-cell clonotypes from Mane-A*10(+) pigtail macaques responding to the simian immunodeficiency virus (SIV) Gag KP9 epitope in a setting of vaccination and subsequent viral challenge. We observed a diverse TCR repertoire after DNA, recombinant poxvirus, and live attenuated virus vaccination, with none of 59 vaccine-induced KP9-specific TCRs being identical between macaques. The KP9-specific TCR repertoires remained diverse after SIV or simian-human immunodeficiency virus challenge but, remarkably, exhibited substantially different clonotypic compositions compared to the corresponding populations prechallenge. Within serial samples from individual pigtail macaques, only a small subset (33.9%) of TCRs induced by vaccination were maintained or expanded after challenge. Most (66.1%) of the TCRs induced by vaccination were not detectable after challenge. Our results suggest that some CD8 T cells induced by vaccination are more efficient than others at responding to a viral challenge. These findings have implications for future AIDS virus vaccine studies, which should consider the "fitness" of vaccine-induced T cells in order to generate robust responses in the face of virus exposure.

Clinical trials of antiretroviral treatment interruption in HIV-infected individuals.

: Despite the benefits of antiretroviral therapy (ART) for people living with HIV, there has been a long-standing research interest in interrupting ART as a strategy to minimize adverse effects of ART as well as to test interventions aiming to achieve a degree of virological control without ART. We performed a systematic review of HIV clinical studies involving treatment interruption from 2000 to 2017 to describe the differences between treatment interruption in studies that contained and didn't contain an intervention. We assessed differences in monitoring strategies, threshold to restart ART, duration and adverse outcomes of treatment interruption, and factors aimed at minimizing transmission. We found that treatment interruption has been incorporated into 159 clinical studies since 2000 and is increasingly being included in trials to assess the efficacy of interventions to achieve sustained virological remission off ART. Great heterogeneity was noted in immunological, virological and clinical monitoring strategies, as well as in thresholds to recommence ART. Treatment interruption in recent intervention studies were more closely monitored, had more conservative thresholds to restart ART and had a shorter treatment interruption duration, compared with older treatment interruption studies that didn't include an intervention.

Persistence of integrated HIV DNA in CXCR3 + CCR6 + memory CD4+ T cells in HIV-infected individuals on antiretroviral therapy.

BACKGROUND: HIV latent infection can be established in vitro by treating resting CD4 T cells with chemokines that bind to chemokine receptors (CKR), CCR7, CXCR3, and CCR6, highly expressed on T cells. OBJECTIVE: To determine if CKR identify CD4 T cells enriched for HIV in HIV-infected individuals receiving suppressive antiretroviral therapy (ART). DESIGN: A cross-sectional study of CKR expression and HIV persistence in blood from HIV-infected individuals on suppressive ART for more than 3 years (n = 48). A subset of 20 individuals underwent leukapheresis and sorting of specific CD4 T-cell subsets. METHODS: We used flow cytometry to quantify CCR5, CCR6, CXCR3, and CXCR5 expression on CD4 T cells. HIV persistence was quantified using real-time Polymerase Chain Reaction to detect total, integrated HIV DNA, 2-long terminal repeat circles and cell-associated unspliced (CA-US) HIV RNA in total CD4 T cells from blood or sorted T-cell subsets. Associations between CKR and HIV persistence in CD4 T cells in blood were determined using regression models and adjusted for current and nadir CD4 T-cell counts. RESULTS: The frequency of cells harbouring integrated HIV DNA was inversely associated with current CD4 T-cell count and positively associated with CCR5+ CD4 T cells, CXCR3+CCR6+ and CXCR3+CCR6- expression on total memory CD4 T cells (P < 0.001, 0.048, 0.015, and 0.016, respectively). CXCR3+CCR6+ CM CD4 T cells contained the highest amount of integrated HIV DNA and lowest ratio of CA-US HIV RNA to DNA compared to all T-cell subsets examined. CONCLUSION: CXCR3 and CCR6 coexpression defines a subset of CD4 T cells that are preferentially enriched for HIV DNA in HIV-infected individuals on ART.

Safety, immunogenicity and efficacy of peptide-pulsed cellular immunotherapy in macaques.

BACKGROUND: Simple and effective delivery methods for cellular immunotherapies are needed. We recently published on the effectiveness of using ex vivo pulsing of overlapping SIV Gag 15mer peptides onto fresh peripheral blood cells in 32 SIV(mac251)-infected pigtail macaques. METHODS: We now report on the safety of this approach, analysis of a novel assay for immunogenicity, the effect of an MHC allele, Mane-A*10, on CD8 T cell escape occurring and disease outcome. RESULTS: The vaccine strategy was safe, with no perturbations in weight or hematological profiles in comparison to controls. The high levels of SIV-specific T cell immunogenicity of this approach was confirmed using a novel assay measuring upregulation of surface CD134 of CD4 T cells. A substantial effect of the Mane-A*10 allele in reducing SIV viral load of pigtail macaques was observed in both vaccinees and controls; the virologic efficacy of the immunotherapy in comparison to controls was greatest in Mane-A*10- animals. Escape mutations at several new CD8 T cell epitopes throughout the SIV proteome were observed, primarily in animals with poorer virologic control. CONCLUSIONS: In summary, we provide further information that peptide-pulsed PBMC are a safe, immunogenic and effective immunotherapy. The observed influence of MHC alleles and immune escape allows us to design more insightful future immunotherapy studies.

Killing kinetics of simian immunodeficiency virus-specific CD8+ T cells: implications for HIV vaccine strategies.

Both the magnitude and function of vaccine-induced HIV-specific CD8+ CTLs are likely to be important in the outcome of infection. We hypothesized that rapid cytolysis by CTLs may facilitate control of viral challenge. Release kinetics of the cytolytic effector molecules granzyme B and perforin, as well as the expression of the degranulation marker CD107a and IFN-gamma were simultaneously studied in SIV Gag(164-172) KP9-specific CD8+ T cells from Mane-A*10+ pigtail macaques. Macaques were vaccinated with either prime-boost poxvirus vector vaccines or live-attenuated SIV vaccines. Prime-boost vaccination induced Gag-specific CTLs capable of only slow (after 3 h) production of IFN-gamma and with limited (<5%) degranulation and granzyme B release. Vaccination with live-attenuated SIV resulted in a rapid cytolytic profile of SIV-specific CTLs with rapid (<0.5 h) and robust (>50% of tetramer-positive CD8+ T cells) degranulation and granzyme B release. The cytolytic phenotype following live-attenuated SIV vaccinations were similar to that associated with the partial resolution of viremia following SIV(mac251) challenge of prime-boost-vaccinated macaques, albeit with less IFN-gamma expression. High proportions of KP9-specific T cells expressed the costimulatory molecule CD28 when they exhibited a rapid cytolytic phenotype. The delayed cytolytic phenotype exhibited by standard vector-based vaccine-induced CTLs may limit the ability of T cell-based HIV vaccines to rapidly control acute infection following a pathogenic lentiviral exposure.

Vaccine-induced T cells control reversion of AIDS virus immune escape mutants.

Many current-generation human immunodeficiency virus (HIV) vaccines induce specific T cells to control acute viremia, but their utility following infection with escape mutant virus is unclear. We studied reversion to wild type of an escape mutant simian-HIV in major histocompatibility complex-matched vaccinated pigtail macaques. High levels of vaccine-induced CD8+ T cells strongly correlated with maintenance of escape mutant virus during acute infection. Interestingly, in animals with lower CD8+ T-cell levels, transient reversion to wild-type virus resulted in better postacute control of viremia. Killing of wild-type virus facilitated by transient reversion outweighs the benefit of a larger CD8+ T-cell response that only maintains the less fit escape mutant virus. These findings have important implications for the further development of T-cell-based HIV vaccines where exposure to escape mutant viruses is common.