Persistence of Virus Reservoirs in ART-Treated SHIV-Infected Rhesus Macaques after Autologous Hematopoietic Stem Cell Transplant

Despite many advances in AIDS research, a cure for HIV infection remains elusive. Here, we performed autologous hematopoietic stem cell transplantation (HSCT) in three Simian/Human Immunodeficiency Virus (SHIV)-infected, antiretroviral therapy (ART)-treated rhesus macaques (RMs) using HSCs collected prior to infection and compared them to three SHIV-infected, ART-treated, untransplanted control animals to assess the effect of conditioning and autologous HSCT on viral persistence. As expected, ART drastically reduced virus replication, below 100 SHIV-RNA copies per ml of plasma in all animals. After several weeks on ART, experimental RMs received myeloablative total body irradiation (1080 cGy), which resulted in the depletion of 94–99% of circulating CD4+ T-cells, and low to undetectable SHIV-DNA levels in peripheral blood mononuclear cells. Following HSC infusion and successful engraftment, ART was interrupted (40–75 days post-transplant). Despite the observed dramatic reduction of the peripheral blood viral reservoir, rapid rebound of plasma viremia was observed in two out of three transplanted RMs. In the third transplanted animal, plasma SHIV-RNA and SHIV DNA in bulk PBMCs remained undetectable at week two post-ART interruption. No further time-points could be assessed as this animal was euthanized for clinical reasons; however, SHIV-DNA could be detected in this animal at necropsy in sorted circulating CD4+ T-cells, spleen and lymph nodes but not in the gastro-intestinal tract or tonsils. Furthermore, SIV DNA levels post-ART interruption were equivalent in several tissues in transplanted and control animals. While persistence of virus reservoir was observed despite myeloablation and HSCT in the setting of short term ART, this experiment demonstrates that autologous HSCT can be successfully performed in SIV-infected ART-treated RMs offering a new experimental in vivo platform to test innovative interventions aimed at curing HIV infection in humans.     

CD4+ CCR5+ T-cell dynamics during simian immunodeficiency virus infection of Chinese rhesus macaques

Simian immunodeficiency virus (SIV) infection of rhesus macaques (RMs) provides a reliable model to study the relationship between lentivirus replication, cellular immune responses, and CD4⁺ T-cell dynamics. Here we investigated, using SIVmac251-infected RMs of a Chinese genetic background (which experience a slower disease progression than Indian RMs), the dynamics of CD4⁺ CCR5⁺ T cells, as this subset of memory/activated CD4⁺ T cells is both a preferential target of virus replication and a marker of immune activation. As expected, we observed that the number of circulating CD4⁺ CCR5⁺ T cells decreases transiently at the time of peak viremia. However, at 60 days postinfection, i.e., when set-point viremia is established, the level of CD4⁺ CCR5⁺ T cells was increased compared to the baseline level. Interestingly, this increase correlated with faster disease progression, higher plasma viremia, and early loss of CD4⁺ T-cell function, as measured by CD4⁺ T-cell count, the fraction of memory CD4⁺ T cells, and the recall response to purified protein derivative. Taken together, these data show a key difference between the dynamics of the CD4⁺ CCR5⁺ T-cell pool (and its relationship with disease progression) in Chinese RMs and those described in previous reports for Indian SIVmac251-infected RMs. As the SIV-associated changes in the CD4⁺ CCR5⁺ T-cell pool reflect the opposing forces of SIV replication (which reduces this cellular pool) and immune activation (which increases it), our data suggest that in SIV-infected Chinese RMs the impact of immune activation is more prominent than that of virus replication in determining the size of the pool of CD4⁺ CCR5⁺ T cells in the periphery. As progression of HIV infection in humans also is associated with a relative expansion of the level of CD4⁺ CCR5⁺ T cells, we propose that SIV infection of Chinese RMs is a very valuable and important animal model for understanding the pathogenesis of human immunodeficiency virus infection.     

Immunological and Virological Analyses of Rhesus Macaques Immunized with Chimpanzee Adenoviruses Expressing the Simian Immunodeficiency Virus Gag/Tat Fusion Protein and Challenged Intrarectally with Repeated Low Doses of SIVmac

Human adenovirus (AdHu)-based candidate AIDS vaccine can provide protection from simian immunodeficiency virus (SIV) transmission and disease progression. However, their potential use may be limited by widespread preexisting immunity to the vector. In contrast, preexisting immunity to chimpanzee adenoviruses (AdC) is relatively rare. In this study, we utilized two regimens of prime-boost immunizations with AdC serotype SAd-V23 (also called AdC6) and SAd-V24 (also called AdC7) expressing SIV Gag/Tat to test their immunogenicity and ability to protect rhesus macaques (RMs) from a repeated low-dose SIV_mac239 challenge. Both AdC6 followed by AdC7 (AdC6/7) and AdC7 followed by AdC6 (AdC7/6) induced robust SIV Gag/Tat-specific T cell responses as measured by tetramer staining and functional assays. However, no significant protection from SIV transmission was observed in either AdC7/6- or AdC7/6-vaccinated RMs. Interestingly, in the RMs showing breakthrough infections, AdC7/6-SIV immunization was associated with a transient but significant (P = 0.035 at day 90 and P = 0.033 at day 120 postinfection) reduction in the setpoint viral load compared to unvaccinated controls. None of the measured immunological markers (i.e., number or functionality of SIV-specific CD8⁺ and CD4⁺ T cell responses and level of activated and/or CCR5⁺ CD4⁺ target cells) at the time of challenge correlated with protection from SIV transmission in the AdC-SIV-vaccinated RMs. The robust immunogenicity observed in all AdC-immunized RMs and the transient signal of protection from SIV replication exhibited by AdC7/6-vaccinated RMs even in the absence of any envelope immunogen suggest that AdC-based vectors may represent a promising platform for candidate AIDS vaccines.     

Early divergence in lymphoid tissue apoptosis between pathogenic and nonpathogenic simian immunodeficiency virus infections of nonhuman primates

The events that contribute to the progression to AIDS during the acute phase of a primate lentiviral infection are still poorly understood. In this study, we used pathogenic and nonpathogenic simian models of simian immunodeficiency virus (SIV) infection of rhesus macaques (RMs) and African green monkeys (AGMs), respectively, to investigate the relationship between apoptosis in lymph nodes and the extent of viral replication, immune activation, and disease outcome. Here, we show that, in SIVmac251-infected RMs, a marked increased in lymphocyte apoptosis is evident during primary infection at the level of lymph nodes. Interestingly, the levels of apoptosis correlated with the extent of viral replication and the rate of disease progression to AIDS, with higher apoptosis in RMs of Indian genetic background than in those of Chinese origin. In stark contrast, no changes in the levels of lymphocyte apoptosis were observed during primary infection in the nonpathogenic model of SIVagm-sab infection of AGMs, despite similarly high rates of viral replication. A further and early divergence between SIV-infected RMs and AGMs was observed in terms of the dynamics of T- and B-cell proliferation in lymph nodes, with RMs showing significantly higher levels of cycling cells (Ki67⁺) in the T-cell zones in association with relatively low levels of Ki67⁺ in the B-cell zones, whereas AGMs displayed a low frequency of Ki67⁺ in the T-cell area but a high proportion of Ki67⁺ cells in the B-cell area. As such, this study suggests that species-specific host factors determine an early immune response to SIV that predominantly involves either cellular or humoral immunity in RMs and AGMs, respectively. Taken together, these data are consistent with the hypotheses that (i) high levels of T-cell activation and lymphocyte apoptosis are key pathogenic factors during pathogenic SIV infection of RMs and (ii) low T-cell activation and apoptosis are determinants of the AIDS resistance of SIVagm-infected AGMs, despite high levels of SIVagm replication.     

Control of Simian Immunodeficiency Virus Replication by Vaccine-Induced Gag- and Vif-Specific CD8+ T Cells

For development of an effective T cell-based AIDS vaccine, it is critical to define the antigens that elicit the most potent responses. Recent studies have suggested that Gag-specific and possibly Vif/Nef-specific CD8⁺ T cells can be important in control of the AIDS virus. Here, we tested whether induction of these CD8⁺ T cells by prophylactic vaccination can result in control of simian immunodeficiency virus (SIV) replication in Burmese rhesus macaques sharing the major histocompatibility complex class I (MHC-I) haplotype 90-010-Ie associated with dominant Nef-specific CD8⁺ T-cell responses. In the first group vaccinated with Gag-expressing vectors (n = 5 animals), three animals that showed efficient Gag-specific CD8⁺ T-cell responses in the acute phase postchallenge controlled SIV replication. In the second group vaccinated with Vif- and Nef-expressing vectors (n = 6 animals), three animals that elicited Vif-specific CD8⁺ T-cell responses in the acute phase showed SIV control, whereas the remaining three with Nef-specific but not Vif-specific CD8⁺ T-cell responses failed to control SIV replication. Analysis of 18 animals, consisting of seven unvaccinated noncontrollers and the 11 vaccinees described above, revealed that the sum of Gag- and Vif-specific CD8⁺ T-cell frequencies in the acute phase was inversely correlated with plasma viral loads in the chronic phase. Our results suggest that replication of the AIDS virus can be controlled by vaccine-induced subdominant Gag/Vif epitope-specific CD8⁺ T cells, providing a rationale for the induction of Gag- and/or Vif-specific CD8⁺ T-cell responses by prophylactic AIDS vaccines.     

Multi-dose Romidepsin Reactivates Replication Competent SIV in Post-antiretroviral Rhesus Macaque Controllers

Viruses that persist despite seemingly effective antiretroviral treatment (ART) and can reinitiate infection if treatment is stopped preclude definitive treatment of HIV-1 infected individuals, requiring lifelong ART. Among strategies proposed for targeting these viral reservoirs, the premise of the “shock and kill” strategy is to induce expression of latent proviruses [for example with histone deacetylase inhibitors (HDACis)] resulting in elimination of the affected cells through viral cytolysis or immune clearance mechanisms. Yet, ex vivo studies reported that HDACis have variable efficacy for reactivating latent proviruses, and hinder immune functions. We developed a nonhuman primate model of post-treatment control of SIV through early and prolonged administration of ART and performed in vivo reactivation experiments in controller RMs, evaluating the ability of the HDACi romidepsin (RMD) to reactivate SIV and the impact of RMD treatment on SIV-specific T cell responses. Ten RMs were IV-infected with a SIVsmmFTq transmitted-founder infectious molecular clone. Four RMs received conventional ART for >9 months, starting from 65 days post-infection. SIVsmmFTq plasma viremia was robustly controlled to <10 SIV RNA copies/mL with ART, without viral blips. At ART cessation, initial rebound viremia to ~10⁶ copies/mL was followed by a decline to < 10 copies/mL, suggesting effective immune control. Three post-treatment controller RMs received three doses of RMD every 35–50 days, followed by in vivo experimental depletion of CD8⁺ cells using monoclonal antibody M-T807R1. RMD was well-tolerated and resulted in a rapid and massive surge in T cell activation, as well as significant virus rebounds (~10⁴ copies/ml) peaking at 5–12 days post-treatment. CD8⁺ cell depletion resulted in a more robust viral rebound (10⁷ copies/ml) that was controlled upon CD8⁺ T cell recovery. Our results show that RMD can reactivate SIV in vivo in the setting of post-ART viral control. Comparison of the patterns of virus rebound after RMD administration and CD8⁺ cell depletion suggested that RMD impact on T cells is only transient and does not irreversibly alter the ability of SIV-specific T cells to control the reactivated virus.     

Conditionally Defective Helper Adenoviruses and Satellite Virus Replication

ADENO-ASSOCIATED satellite viruses (ASV) are extremely defective in that they need a helper adenovirus to complete their replication cycle in susceptible cells^1–3. Although the helper virus is usually not defective there have been reports of systems which are at least conditionally defective. Smith and Gehle⁴ found that a canine adenovirus, ICH, which did not seem to replicate in human amnion cells (essentially a non-permissive system) could be used to pass the satellite serially in these cells if the passage was reinfected each time with helper virus. Ito et al.⁵ reported that a temperature-sensitive mutant of human adenovirus type 31, ts 13, defective in viral DNA synthesis, could complement a cycle of satellite virus replication at the non-permissive temperature.     

Surface Alterations of Cells Carrying RNA Tumour Virus Genetic Information

CELLS transformed by the DNA tumour viruses, polyoma virus and SV40, are agglutinated by lectins such as wheat germ agglutinin¹, concanavalin A (Con A)² and soybean agglutinin³. Agglutination in these cases presumably reflects changes in the cell surface related to the transformed properties of the cell; studies with a temperature-dependent mutant of polyoma virus has shown that cell surface changes are controlled by viral genes⁴. Here we describe experiments in which we investigated the agglutinability of cells transformed by RNA tumour viruses. One recent report had suggested that cells transformed by RNA tumour viruses were not specifically agglutinated⁵, whereas a second more recent report claimed the specific agglutination of cells transformed by RSV⁶. We find that transformed rat, mouse and cat cells that replicate the sarcoma-leukaemia virus complex of murine (MSV) and feline (FeSV) origin are strongly agglutinated by Con A, but mouse and human cells that replicate the murine and feline leukaemia virus components alone are not agglutinated. The ability to agglutinate is rapidly acquired by normal mouse cells on infection with the murine sarcoma virus at a rate that parallels virus replication. In contrast to the results obtained with cells producing virus, non-virus-producing transformed hamster and mouse cells that synthesize virus-specific RNA are either not agglutinated or are agglutinated to a lesser degree. These results suggest that the cell surface alterations responsible for agglutination are not necessarily associated with the transformed state of the cell, but rather with the possession of sarcoma virus-specific information.     

Enhanced control of pathogenic Simian immunodeficiency virus SIVmac239 replication in macaques immunized with an interleukin-12 plasmid and a DNA prime-viral vector boost vaccine regimen

DNA priming has previously been shown to elicit augmented immune responses when administered by electroporation (EP) or codelivered with a plasmid encoding interleukin-12 (pIL-12). We hypothesized that the efficacy of a DNA prime and recombinant adenovirus 5 boost vaccination regimen (DNA/rAd5) would be improved when incorporating these vaccination strategies into the DNA priming phase, as determined by pathogenic simian immunodeficiency virus SIVmac239 challenge outcome. The whole SIVmac239 proteome was delivered in 5 separate DNA plasmids (pDNA-SIV) by EP with or without pIL-12, followed by boosting 4 months later with corresponding rAd5-SIV vaccine vectors. Remarkably, after repeated low-dose SIVmac239 mucosal challenge, we demonstrate 2.6 and 4.4 log reductions of the median SIV peak and set point viral loads in rhesus macaques (RMs) that received pDNA-SIV by EP with pIL-12 compared to the median peak and set point viral loads in mock-immunized controls (P < 0.01). In 5 out of 6 infected RMs, strong suppression of viremia was observed, with intermittent "blips" in virus replication. In 2 RMs, we could not detect the presence of SIV RNA in tissue and lymph nodes, even after 13 viral challenges. RMs immunized without pIL-12 demonstrated a typical maximum of 1.5 log reduction in virus load. There was no significant difference in the overall magnitude of SIV-specific antibodies or CD8 T-cell responses between groups; however, pDNA delivery by EP with pIL-12 induced a greater magnitude of SIV-specific CD4 T cells that produced multiple cytokines. This vaccine strategy is relevant for existing vaccine candidates entering clinical evaluation, and this model may provide insights into control of retrovirus replication.     

Similar Impact of CD8+ T Cell Responses on Early Virus Dynamics during SIV Infections of Rhesus Macaques and Sooty Mangabeys

Despite comparable levels of virus replication, simian immunodeficiency viruses (SIV) infection is non-pathogenic in natural hosts, such as sooty mangabeys (SM), whereas it is pathogenic in non-natural hosts, such as rhesus macaques (RM). Comparative studies of pathogenic and non-pathogenic SIV infection can thus shed light on the role of specific factors in SIV pathogenesis. Here, we determine the impact of target-cell limitation, CD8+ T cells, and Natural Killer (NK) cells on virus replication in the early SIV infection. To this end, we fit previously published data of experimental SIV infections in SMs and RMs with mathematical models incorporating these factors and assess to what extent the inclusion of individual factors determines the quality of the fits. We find that for both rhesus macaques and sooty mangabeys, target-cell limitation alone cannot explain the control of early virus replication, whereas including CD8+ T cells into the models significantly improves the fits. By contrast, including NK cells does only significantly improve the fits in SMs. These findings have important implications for our understanding of SIV pathogenesis as they suggest that the level of early CD8+ T cell responses is not the key difference between pathogenic and non-pathogenic SIV infection.     

Seoul Virus-Infected Rat Lung Endothelial Cells and Alveolar Macrophages Differ in Their Ability To Support Virus Replication and Induce Regulatory T Cell Phenotypes

Hantaviruses cause a persistent infection in reservoir hosts that is attributed to the upregulation of regulatory responses and downregulation of proinflammatory responses. To determine whether rat alveolar macrophages (AMs) and lung microvascular endothelial cells (LMVECs) support Seoul virus (SEOV) replication and contribute to the induction of an environment that polarizes CD4⁺ T cell differentiation toward a regulatory T (Treg) cell phenotype, cultured primary rat AMs and LMVECs were mock infected or infected with SEOV and analyzed for viral replication, cytokine and chemokine responses, and expression of cell surface markers that are related to T cell activation. Allogeneic CD4⁺ T cells were cocultured with SEOV-infected or mock-infected AMs or LMVECs and analyzed for helper T cell (i.e., Treg, Th17, Th1, and Th2) marker expression and Treg cell frequency. SEOV RNA and infectious particles in culture media were detected in both cell types, but at higher levels in LMVECs than in AMs postinfection. Expression of Ifnβ, Ccl5, and Cxcl10 and surface major histocompatibility complex class II (MHC-II) and MHC-I was not altered by SEOV infection in either cell type. SEOV infection significantly increased Tgfβ mRNA in AMs and the amount of programmed cell death 1 ligand 1 (PD-L1) in LMVECs. SEOV-infected LMVECs, but not AMs, induced a significant increase in Foxp3 expression and Treg cell frequency in allogeneic CD4⁺ T cells, which was virus replication and cell contact dependent. These data suggest that in addition to supporting viral replication, AMs and LMVECs play distinct roles in hantavirus persistence by creating a regulatory environment through increased Tgfβ, PD-L1, and Treg cell activity.     

Growth of Pseudotypes of Vesicular Stomatitis Virus with N-Tropic Murine Leukemia Virus Coats in Cells Resistant to N-Tropic Viruses

Formation of pseudotypes between murine RNA tumor viruses and vesicular stomatitis virus (VSV) has been confirmed. Pseudotypes of VSV genomes coated by the surface envelope from an N-tropic tumor virus grew equally well in cells homozygous for either the Fv-1ⁿ or Fv-1^b alleles. Therefore, the product of the Fv-1 locus, which restricts growth of murine RNA tumor viruses, must act on an intracellular aspect of tumor virus replication, a step after attachment and penetration.     

Infection of Tumour Cells by Fowl Plague Virus in the Presence of Actinomycin D

INFLUENZA virus is one of the few viruses in which replication is inhibited by the antimetabolite actinomycin D (AM-D)^1–4, which inhibits DNA-dependent RNA synthesis in mammalian cells⁵. It has been reported that the growth of fowl plague virus (FPV) in virus-transformed hamster cells is less sensitive to AM-D^6,7. We have examined the sensitivity of FPV to AM-D to see whether it is related to differences in the oncogenic properties of tumour cells. We found that in cells transformed by polyoma virus (PV) and also in cells transformed by methylcholanthrene, although no infectious virus was produced the cells synthesized viral haemagglutinin (HA). It was only the cell-associated HA, however, that was affected by AM-D and not that released by the cells.     

Damaged Intestinal Epithelial Integrity Linked to Microbial Translocation in Pathogenic Simian Immunodeficiency Virus Infections

The chronic phase of HIV infection is marked by pathological activation of the immune system, the extent of which better predicts disease progression than either plasma viral load or CD4⁺ T cell count. Recently, translocation of microbial products from the gastrointestinal tract has been proposed as an underlying cause of this immune activation, based on indirect evidence including the detection of microbial products and specific immune responses in the plasma of chronically HIV-infected humans or SIV-infected Asian macaques. We analyzed tissues from SIV-infected rhesus macaques (RMs) to provide direct in situ evidence for translocation of microbial constituents from the lumen of the intestine into the lamina propria and to draining and peripheral lymph nodes and liver, accompanied by local immune responses in affected tissues. In chronically SIV-infected RMs this translocation is associated with breakdown of the integrity of the epithelial barrier of the gastrointestinal (GI) tract and apparent inability of lamina propria macrophages to effectively phagocytose translocated microbial constituents. By contrast, in the chronic phase of SIV infection in sooty mangabeys, we found no evidence of epithelial barrier breakdown, no increased microbial translocation and no pathological immune activation. Because immune activation is characteristic of the chronic phase of progressive HIV/SIV infections, these findings suggest that increased microbial translocation from the GI tract, in excess of capacity to clear the translocated microbial constituents, helps drive pathological immune activation. Novel therapeutic approaches to inhibit microbial translocation and/or attenuate chronic immune activation in HIV-infected individuals may complement treatments aimed at direct suppression of viral replication.     

Comparison of Virulence between Seoul Virus Strain SR-11 and Hantaan Virus Strain 76–118 of Hantaviruses in Newborn Mice

Virulence of hantavirus strain of SR-11 Seoul virus and Hantaan 76–118 (HTN) of Hantaan virus were compared. Infections of both strains were lethal in newborn mice. However, inoculum required to cause lethal infection was about 4,000 times higher for strain HTN (1.65 × 10³ PFU/mouse/LD₅₀) than for strain SR-11 (0.36 PFU). Thus, both strains were considered pathogenic to newborn mice but they possessed different levels of virulence. The assay system used for these strains in newborn mice proved to be useful in the study of hantavirus vilurence. Growth curves of the two strains in CV-7 cell cultures were compared. Strain SR-11 was shown to have higher activity of virus replication and virus release into the culture fluids than strain HTN. The possibility of a relationship between replication activity and high levels of virulence in mice was suggested.     

In Vivo Pathogenesis of a Human Immunodeficiency Virus Type 1 Reporter Virus

Our understanding of human immunodeficiency virus type 1 (HIV-1)-induced pathogenesis is hampered by the inability to detect HIV-1 gene expression in infected viable cells. In this report, we describe two HIV-1 reporter constructs that are replication competent and cytopathic in vivo. These constructs contain DNA regions of two different lengths that bear the cDNA for the murine heat-stable antigen in the vpr region of a CXCR4-tropic virus. We used the SCID-hu mouse model and these reporter viruses to perform detailed kinetic studies of HIV-1 infection of human thymocytes in vivo. We document that the CD4⁺/CD8⁺ thymocytes are the first to express virus and that this subset demonstrates the most rapid and extensive HIV-1-induced cell depletion. Following depletion of this subset, subsequent virus expression occurs predominantly in phenotypically CD4⁻ cells, suggesting that CD4 down-regulation occurs in HIV-1-infected thymocytes in vivo. These results demonstrate the utility of these HIV-1 reporter constructs to monitor HIV pathogenesis in vitro and in vivo.     

Foodborne Transmission of Nipah Virus in Syrian Hamsters

Since 2001, outbreaks of Nipah virus have occurred almost every year in Bangladesh with high case-fatality rates. Epidemiological data suggest that in Bangladesh, Nipah virus is transmitted from the natural reservoir, fruit bats, to humans via consumption of date palm sap contaminated by bats, with subsequent human-to-human transmission. To experimentally investigate this epidemiological association between drinking of date palm sap and human cases of Nipah virus infection, we determined the viability of Nipah virus (strain Bangladesh/200401066) in artificial palm sap. At 22°C virus titers remained stable for at least 7 days, thus potentially allowing food-borne transmission. Next, we modeled food-borne Nipah virus infection by supplying Syrian hamsters with artificial palm sap containing Nipah virus. Drinking of 5×10⁸ TCID₅₀ of Nipah virus resulted in neurological disease in 5 out of 8 hamsters, indicating that food-borne transmission of Nipah virus can indeed occur. In comparison, intranasal (i.n.) inoculation with the same dose of Nipah virus resulted in lethal respiratory disease in all animals. In animals infected with Nipah virus via drinking, virus was detected in respiratory tissues rather than in the intestinal tract. Using fluorescently labeled Nipah virus particles, we showed that during drinking, a substantial amount of virus is deposited in the lungs, explaining the replication of Nipah virus in the respiratory tract of these hamsters. Besides the ability of Nipah virus to infect hamsters via the drinking route, Syrian hamsters infected via that route transmitted the virus through direct contact with naïve hamsters in 2 out of 24 transmission pairs. Although these findings do not directly prove that date palm sap contaminated with Nipah virus by bats is the origin of Nipah virus outbreaks in Bangladesh, they provide the first experimental support for this hypothesis. Understanding the Nipah virus transmission cycle is essential for preventing and mitigating future outbreaks.