Pathogenicity In Mice Research Articles

The R251K Substitution in Viral Protein PB2 Increases Viral Replication and Pathogenicity of Eurasian Avian-like H1N1 Swine Influenza Viruses.

The Eurasian avian-like swine (EA) H1N1 virus has affected the Chinese swine industry, and human infection cases have been reported occasionally. However, little is known about the pathogenic mechanism of EA H1N1 virus. In this study, we compared the mouse pathogenicity of A/swine/Guangdong/YJ4/2014 (YJ4) and A/swine/Guangdong/MS285/2017 (MS285) viruses, which had similar genotype to A/Hunan/42443/2015 (HuN-like). None of the mice inoculated with 106 TCID50 of YJ4 survived at 7 days post infection, while the survival rate of the MS285 group was 100%. Therefore, a series of single fragment reassortants in MS285 background and two rescued wild-type viruses were generated by using the reverse genetics method, and the pathogenicity analysis revealed that the PB2 gene contributed to the high virulence of YJ4 virus. Furthermore, there were 11 amino acid differences in PB2 between MS285 and YJ4 identified by sequence alignment, and 11 single amino acid mutant viruses were generated in the MS285 background. We found that the R251K mutation significantly increased the virulence of MS285 in mice, contributed to high polymerase activity and enhanced viral genome transcription and replication. These results indicate that PB2-R251K contributes to the virulence of the EA H1N1 virus and provide new insight into future molecular epidemiological surveillance strategies.

Influenza A virus lacking the effector and C terminal domains of NS1 protein induces cytokines associated with high pathogenicity in mice.

Non-structural NS1 protein of influenza A virus counters host antiviral defences by antagonizing the interferon response. The C-terminal effector domain suppresses the host response and is associated with the pathogenicity of the virus. To better understand the regulatory role of the C-terminal domain, we used reverse genetics system to generate NS1-truncated virus (NS80) and compared the cytokine profiles in the lungs of mice infected with the NS80 mutant and with the control virus A/WSN/33 (WSN). The NS80 virus was attenuated and the viral titer in the lungs was about 25 times lower than viral titer of control A/WSN/33. Mice infected with NS80 virus exhibited more severe clinical symptoms and 2 mice died 6 days post infection. NS80 virus activated retinoic-inducible gene (RIG)-1-like receptor signaling pathway more strongly than control WSN virus and mice infected with NS80 virus exhibited a greater abundance and more diverse cytokine profile. Infection with NS80 virus induced the expression of the following factors: pro-inflammatory cytokines (IL-1α, IL-1β, TNF-α, IL-16), interferons (IFN-α and IFN-ε), chemokines (CCL2, CCL11, CXCL1, CXCL5, CXCL10, CXCL11 and CXCL13), matrix metallopeptidase 9 (MMP-9), metallopeptidase inhibitor 1 (TIMP-1), macrophage colony-stimulating factor (M-CSF), and vascular cell adhesion protein 1 (VCAM-1). All these cytokines are associated with viral pathogenicity. Our data show that attenuation of the virus should not be directly linked with pathogenicity. Keywords: influenza virus; NS1 protein; cytokines; interferon; pathogenicity.

Identification and pathogenicity analysis of <i>Streptococcus equinus</i> FMD1, a beta-hemolytic strain isolated from forest musk deer lung

Streptococcus spp. cause a wide range of diseases in animals and humans. A Streptococcus strain (FMD1) was isolated from forest musk deer lung. To identify the bacterium at the species level and investigate its pathogenicity, whole genome sequencing and experimental infections of mice were performed. The genome had 97.63% average nucleotide identity with the S. equinus strain. Through virulence gene analysis, a beta-hemolysin/cytolysin genome island was found in the FMD1 genome, which contained 12 beta-hemolysin/cytolysin-related genes. Hemolytic reaction and histopathological analysis established the strain’s pathogenicity in mice. This is the first report of a beta-hemolytic S. equinus strain in forest musk deer identified based on phenotypic and genotypic analyzes; this strategy could be useful for analyzing pathogens affecting rare animals.

Resistance to ectromelia virus infection requires cGAS in bone marrow-derived cells which can be bypassed with cGAMP therapy.

Cells sensing infection produce Type I interferons (IFN-I) to stimulate Interferon Stimulated Genes (ISGs) that confer resistance to viruses. During lympho-hematogenous spread of the mouse pathogen ectromelia virus (ECTV), the adaptor STING and the transcription factor IRF7 are required for IFN-I and ISG induction and resistance to ECTV. However, it is unknown which cells sense ECTV and which pathogen recognition receptor (PRR) upstream of STING is required for IFN-I and ISG induction. We found that cyclic-GMP-AMP (cGAMP) synthase (cGAS), a DNA-sensing PRR, is required in bone marrow-derived (BMD) but not in other cells for IFN-I and ISG induction and for resistance to lethal mousepox. Also, local administration of cGAMP, the product of cGAS that activates STING, rescues cGAS but not IRF7 or IFN-I receptor deficient mice from mousepox. Thus, sensing of infection by BMD cells via cGAS and IRF7 is critical for resistance to a lethal viral disease in a natural host.

Structure of galactarate dehydratase, a new fold in an enolase involved in bacterial fitness after antibiotic treatment.

Galactarate dehydratase (GarD) is the first enzyme in the galactarate/glucarate pathway and catalyzes the dehydration of galactarate to 3-keto-5-dehydroxygalactarate. This protein is known to increase colonization fitness of intestinal pathogens in antibiotic-treated mice and to promote bacterial survival during stress. The galactarate/glucarate pathway is widespread in bacteria, but not in humans, and thus could be a target to develop new inhibitors for use in combination therapy to combat antibiotic resistance. The structure of almost all the enzymes of the galactarate/glucarate pathway were solved previously, except for GarD, for which only the structure of the N-terminal domain was determined previously. Herein, we report the first crystal structure of full-length GarD solved using a seleno-methoionine derivative revealing a new protein fold. The protein consists of three domains, each presenting a novel twist as compared to their distant homologs. GarD in the crystal structure forms dimers and each monomer consists of three domains. The N-terminal domain is comprised of a β-clip fold, connected to the second domain by a long unstructured linker. The second domain serves as a dimerization interface between two monomers. The C-terminal domain forms an unusual variant of a Rossmann fold with a crossover and is built around a seven-stranded parallel β-sheet supported by nine α-helices. A metal binding site in the C-terminal domain is occupied by Ca2+ . The activity of GarD was corroborated by the production of 5-keto-4-deoxy-D-glucarate under reducing conditions and in the presence of iron. Thus, GarD is an unusual enolase with a novel protein fold never previously seen in this class of enzymes.

Genetic characterization of Toxoplasma gondii in Iranian HIV positive patients using multilocus nested-PCR-RFLP method.

The aim of the present study was to investigate the prevalence and genotyping of Toxoplasma gondii in Iranian human immunodeficiency virus (HIV)-positive patients using multilocus-nested polymerase chain reaction restriction fragment length polymorphism (Mn-PCR-RFLP). A total of 102 serum samples obtained from infected patients were collected from the laboratory centres in northern Iran. Anti-T. gondii antibodies and deoxyribonucleic acid (DNA) detection were accomplished by an enzyme-linked immunosorbent assay and PCR. The Mn-PCR-RFLP method was used for the genotyping of T. gondii. Overall, 68.6% (70/102) and 11.7% (12/102) of the individuals were tested positive for anti-T. gondii immunoglobulin G and T. gondii DNA, respectively. Complete genotyping was performed on 10/12 (83.3%) PCR-positive samples. Accordingly, the samples were classified as genotype #1 (type II clonal; n = 3, 30%), genotype #2 (type III clonal; n = 2, 20%), genotype #10 (type I clonal; n = 2, 20%), genotype #27 (type I variant; n = 1, 10%), genotype #35 (type I variant; n = 1, 10%) and genotype #48 (type III variant; n = 1, 10%). The results were indicative of the high frequency of the type I and type I variant of T. gondii strains in HIV-positive patients in northern Iran. Given the high prevalence of T. gondii and frequency of pathogenic types (pathogen in laboratory mice) in the patients, special measures should be taken to prevent the possible increased incidence of encephalitis by T. gondii.

Synergistic PA and HA mutations confer mouse adaptation of a contemporary A/H3N2 influenza virus

The mouse is the most widely used animal model for influenza virus research. However, the susceptibility of mice to seasonal influenza virus depends on the strain of mouse and on the strain of the influenza virus. Seasonal A/H3N2 influenza viruses do not replicate well in mice and therefore they need to be adapted to this animal model. In this study, we generated a mouse-adapted A/H3N2 virus (A/Switzerland/9715293/2013 [MA-H3N2]) by serial passaging in mouse lungs that exhibited greater virulence compared to the wild-type virus (P0-H3N2). Seven mutations were found in the genome of MA-H3N2: PA(K615E), NP(G384R), NA(G320E) and HA(N122D, N144E, N246K, and A304T). Using reverse genetics, two synergistically acting genes were found as determinants of the pathogenicity in mice. First, the HA substitutions were shown to enhanced viral replication in vitro and, second, the PA-K615E substitution increased polymerase activity, although did not alter virus replication in vitro or in mice. Notably, single mutations had only limited effects on virulence in vitro. In conclusion, a co-contribution of HA and PA mutations resulted in a lethal mouse model of seasonal A/H3N2 virus. Such adapted virus is an excellent tool for evaluation of novel drugs or vaccines and for study of influenza pathogenesis.

Phylogeny, Pathogenicity, Transmission, and Host Immune Responses of Four H5N6 Avian Influenza Viruses in Chickens and Mice.

H5Nx viruses have continuously emerged in the world, causing poultry industry losses and posing a potential public health risk. Here, we studied the phylogeny, pathogenicity, transmission, and immune response of four H5N6 avian influenza viruses in chickens and mice, which were isolated from waterfowl between 2013 and 2014. Their HA genes belong to Clade 2.3.4.4, circulated in China since 2008. Their NA genes fall into N6-like/Eurasian sublineage. Their internal genes originated from different H5N1 viruses. The results suggested that the four H5N6 viruses were reassortants of the H5N1 and H6N6 viruses. They cause lethal infection with high transmission capability in chickens. They also cause mild to severe pathogenicity in mice and can spread to the brain through the blood–brain barrier. During the infection, the viruses result in the up-regulation of PRRs and cytokine in brains and lungs of chickens and mice. Our results suggested that the high viral loads of several organs may result in disease severity in chickens and mice; there were varying levels of cytokines induced by the H5N6 viruses with different pathogenicity in chickens and mice.

E and prM proteins of genotype V Japanese encephalitis virus are required for its increased virulence in mice

We previously showed that the Japanese encephalitis virus (JEV) genotype V (GV) strain Muar exhibits significantly higher virulence in mice than the genotype I (GI) JEV strain Mie/41/2002. In this study, we attempted to identify the region responsible for the increased virulence of GV JEV using recombinant intertypic and single mutant JEVs. Intertypic viruses containing the GV E region in the Mie/41/2002 backbone showed increased pathogenicity in mice. The amino acid at position 123 in the E protein (E123) of the Mie/41/2002 and GV JEVs was serine and histidine, respectively. A serine-to-histidine substitution at E123 of the Mie/41/2002 increased its virulence. However, histidine-to-serine changes at E123 in the intertypic mutants with the GV E region remained highly virulent. GV Muar prM-bearing mutants were also highly pathogenic in mice. Our results suggest that the E and prM proteins of GV JEV are responsible for the highly virulent characteristics of GV JEV.

Here, there, and everywhere: How pathogenic Escherichia coli sense and respond to gastrointestinal biogeography.

Gastrointestinal (GI) pathogens enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC), and related mouse pathogen Citrobacter rodentium, are referred to as attaching and effacing (AE) pathogens for the lesions they form upon colonisation of the host epithelium. EPEC, EHEC, and C. rodentium are well known to use a type III secretion system to intimately attach to intestinal cells and secrete bacterial effectors to manipulate host cell processes. Less well known is the ability of AE pathogens to overcome significant physiological and microbial barriers and target specific gut niches for initial colonisation of the host epithelium. This review considers recent work highlighting the biogeography of the GI tract as it applies to colonisation by enteric pathogens, including environmental barriers to enteric infection, signals sensed by AE pathogens for navigation of the GI tract, and the tools AE pathogens use to respond to the changing host environment.

Citrobacter rodentium induces rapid and unique metabolic and inflammatory responses in mice suffering from severe disease.

The mouse pathogen Citrobacter rodentium is used to model infections with enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC). Pathogenesis is commonly modelled in mice developing mild disease (e.g., C57BL/6). However, little is known about host responses in mice exhibiting severe colitis (e.g., C3H/HeN), which arguably provide a more clinically relevant model for human paediatric enteric infection. Infection of C3H/HeN mice with C. rodentium results in rapid colonic colonisation, coinciding with induction of key inflammatory signatures and colonic crypt hyperplasia. Infection also induces dramatic changes to bioenergetics in intestinal epithelial cells, with transition from oxidative phosphorylation (OXPHOS) to aerobic glycolysis and higher abundance of SGLT4, LDHA, and MCT4. Concomitantly, mitochondrial proteins involved in the TCA cycle and OXPHOS were in lower abundance. Similar to observations in C57BL/6 mice, we detected simultaneous activation of cholesterol biogenesis, import, and efflux. Distinctly, however, the pattern recognition receptors NLRP3 and ALPK1 were specifically induced in C3H/HeN. Using cell‐based assays revealed that C. rodentium activates the ALPK1/TIFA axis, which is dependent on the ADP‐heptose biosynthesis pathway but independent of the Type III secretion system. This study reveals for the first time the unfolding intestinal epithelial cells' responses during severe infectious colitis, which resemble EPEC human infections.

Novel Mutations Evading Avian Immunity around the Receptor Binding Site of the Clade 2.3.2.1c Hemagglutinin Gene Reduce Viral Thermostability and Mammalian Pathogenicity.

Since 2007, highly pathogenic clade 2.3.2 H5N1 avian influenza A (A(H5N1)) viruses have evolved to clade 2.3.2.1a, b, and c; currently only 2.3.2.1c A(H5N1) viruses circulate in wild birds and poultry. During antigenic evolution, clade 2.3.2.1a and c A(H5N1) viruses acquired both S144N and V223I mutations around the receptor binding site of hemagglutinin (HA), with S144N generating an N-glycosylation sequon. We introduced single or combined reverse mutations, N144S and/or I223V, into the HA gene of the clade 2.3.2.1c A(H5N1) virus and generated PR8-derived, 2 + 6 recombinant A(H5N1) viruses. When we compared replication efficiency in embryonated chicken eggs, mammalian cells, and mice, the recombinant virus containing both N144S and I223V mutations showed increased replication efficiency in avian and mammalian hosts and pathogenicity in mice. The N144S mutation significantly decreased avian receptor affinity and egg white inhibition, but not all mutations increased mammalian receptor affinity. Interestingly, the combined reverse mutations dramatically increased the thermostability of HA. Therefore, the adaptive mutations possibly acquired to evade avian immunity may decrease viral thermostability as well as mammalian pathogenicity.

Safety of yellow fever vaccine strain (Tiantan strain) in China and WHO vaccine strain 17D-213

Objective To study the intracerebral pathogenicity of the yellow fever vaccine strains of Tiantan strain used in China and WHO vaccine strain 17D-213 in mice. Methods Mice of different ages and strains were intracerebrally injected with same amount of Tiantan strain and 17D-213 strain. The death and survival of mice were observed and recorded. The LD50/ml and half survival time of the two vaccine strains were compared and analyzed. Results There was no difference in LD50/ml between the Tiantan strain and 17D-213 strain when used through intracerebral injection in one-day-old suckling mice, 7-9 g mice or 12-14 g mice. Moreover, no significant difference in survival trend was found in 7-9 g mice or 12-14 g mice injected with the two vaccine strains. However, the two strains had statistically different influences on the survival trend of one-day-old suckling mice. The half survival time of the Tiantan strain was 11 d, while that of the WHO vaccine strain 17D-213 was 6 d. Excepting in NIH mice, no significant differences in LD50/ml were detected between the same amount of two strains in BALB/c, KM, ICR or C57 mice. Conclusions The yellow fever Tiantan vaccine strain and WHO vaccine strain 17D-213 have no significant difference in the intracerebral pathogenicity in mice of different ages and strains with good safety. Key words: Yellow fever; Vaccine strain; Intracerebral pathogenicity; Safety

Effects of the PA-X and PB1-F2 Proteins on the Virulence of the 2009 Pandemic H1N1 Influenza A Virus in Mice.

There have been several previous reports showing that PA-X and PB1-F2 proteins can regulate innate immune responses and may play roles in the adaptation of influenza viruses to new hosts. In this research, we investigated, for the first time, the combined effects of PA-X and PB1-F2 proteins on viral virulence in mice. Based on the 2009 pH1N1 A/Guangdong/1057/2010 virus backbone, four viruses encoding different combinations of full-length or truncated PA-X and PB1-F2 proteins were rescued by a reverse genetic engineering system. We analyzed viral replication, host-shutoff activity, in vitro viral pathogenicity and in vivo host immune response. We found that simultaneously expressing the full-length PA-X and PB1-F2 proteins enhanced viral replication in vitro through increasing the accumulation of the RNP complex protein and enhanced viral pathogenicity in mice during the early stage of infection. Furthermore, PA-X and PB1-F2 simultaneously regulated the host innate response, and different forms of PB1-F2 proteins may have impacts on the host shutoff activity induced by the PA-X protein. Our results provide a better understanding of the mechanisms of PA-X and PB1-F2 proteins during viral replication, pathogenicity and host immune response.

Isolation and characterization of Bordetella pseudohinzii in mice in China.

We report on the first detection and isolation of B. pseudohinzii (Bordetella pseudohinzii) in laboratory mice in China. Forty‐one B. pseudohinzii strains were isolated from 3094 mice in 33 different laboratory animal facilities in southern China. The isolates were identified through culture and genome sequenceing. Phylogenetic analysis based on the sequences of 16S rRNA and OmpA genes demonstrated that these strains were on the same clade as other B. pseudohinzii strains isolated from mice. Experimental infected mice presented an asymptomatic infection. B. pseudohinzii replicated in both the respiratory tract and the digestive tract. Most importantly B. pseudohinzii shed via feces and infected a group of sentinel mice in a separate cage via cage padding contaminated with B. pseudohinzii‐positive feces, indicating that B. pseudohinzii could transmit efficiently among mice and contaminate environmental facilities. Our study highlights the importance of routine monitoring of the pathogen in laboratory mice and provides vital insights into the transmission of Brodetellae in rodents and human.

Effects of Corynebacterium bovis on Engraftment of Patient-derived Chronic-Myelomonocytic Leukemia Cells in NSGS Mice.

Modeling chronic myelomonocytic leukemia (CMML) in immunodeficient NSGS mice relies on unique human CMML specimens and consistent murine engraftment. Only anecdotal comments have thus far supported the notion that research data may be altered by Corynebacterium bovis, an opportunistic cutaneous pathogen of immunodeficient mice. C. bovis disseminated by asymptomatic and clinically affected mice with hyperkeratotic dermatitis, resulting in resilient facility contamination and infectious recurrence. Herein we report that, compared with C. bovis PCR-negative counterparts, C. bovis PCR-positive NSGS mice developed periocular and facial hyperkeratosis and alopecia and had reduced metrics indicative of ineffective human CMML engraftment, including less thrombocytopenia, less splenomegaly, fewer CMML infiltrates in histopathologic sections of murine organs, and fewer human CD45+ cells in samples from murine spleen, bone marrow, and peripheral blood that were analyzed by flow cytometry. All CMML model metrics of engraftment were significantly reduced in the C. bovis PCR-positive cohort compared with the - negative cohort. In addition, a survey of comprehensive cancer center practices revealed that most murine facilities do not routinely test for C. bovis or broadly decontaminate the facility or its equipment after a C. bovis outbreak, thus increasing the likelihood of recurrence of invalidated studies. Our findings document that CMML engraftment of NSGS mice is diminished-and the integrity of murine research data jeopardized-by C. bovis infection of immunodeficient mice. In addition, our results indicate that C. bovis should be excluded from and not tolerated in murine facilities housing immunodeficient strains.

Caspase-8 restricts antiviral CD8 T cell hyperaccumulation

The magnitude of CD8 T cell responses against viruses is checked by the balance of proliferation and death. Caspase-8 (CASP8) has the potential to influence response characteristics through initiation of apoptosis, suppression of necroptosis, and modulation of cell death-independent signal transduction. Mice deficient in CASP8 and RIPK3 (Casp8-/-Ripk3-/- ) mount enhanced peak CD8 T cell levels against the natural mouse pathogen murine cytomegalovirus (MCMV) or the human pathogen herpes simplex virus-1 compared with littermate control RIPK3-deficient or WT C57BL/6 mice, suggesting an impact of CASP8 on the magnitude of antiviral CD8 T cell expansion and not on contraction. The higher peak response to MCMV in Casp8-/-Ripk3-/- mice resulted from accumulation of greater numbers of terminally differentiated KLRG1hi effector CD8 T cell subsets. Antiviral Casp8-/-Ripk3-/- T cells exhibited enhanced proliferation when splenocytes were transferred into WT recipient mice. Thus, cell-autonomous CASP8 normally restricts CD8 T cell proliferation following T cell receptor activation in response to foreign antigen. Memory inflation is a hallmark quality of the T cell response to cytomegalovirus infection. Surprisingly, MCMV-specific memory inflation was not sustained long-term in Casp8-/-Ripk3-/- mice even though these mice retained immunity to secondary challenge. In addition, the accumulation of abnormal B220+CD3+ T cells in these viable CASP8-deficient mice was reduced by chronic MCMV infection. Combined, these data brings to light the cell death-independent role of CASP8 during CD8 T cell expansion in mice lacking the confounding impact of RIPK3-mediated necroptosis.

Glial HMOX1 expression promotes central and peripheral α-synuclein dysregulation and pathogenicity in parkinsonian mice.

α-Synuclein is a key player in the pathogenesis of Parkinson disease (PD). Expression of human heme oxygenase-1 (HO-1) in astrocytes of GFAP.HMOX1 transgenic (TG) mice between 8.5 and 19 months of age results in a parkinsonian phenotype characterized by neural oxidative stress, nigrostriatal hypodopaminergia associated with locomotor incoordination, and overproduction of α-synuclein. We identified two microRNAs (miR-), miR-153 and miR-223, that negatively regulate α-synuclein in the basal ganglia of male and female GFAP.HMOX1 mice. Serum concentrations of both miRNAs progressively declined in the wild-type (WT) and GFAP.HMOX1 mice between 11 and 19 months of age. Moreover, at each time point surveyed, circulating levels of miR-153 were significantly lower in the TG animals compared to WT controls, while α-synuclein protein concentrations were elevated in erythrocytes of the GFAP.HMOX1 mice at 19 months of age relative to WT values. Primary WT neurons co-cultured with GFAP.HMOX1 astrocytes exhibited enhanced protein oxidation, mitophagy and apoptosis, aberrant expression of genes regulating the dopaminergic phenotype, and an imbalance in gene expression profiles governing mitochondrial fission and fusion. Many, but not all, of these neuronal abnormalities were abrogated by small interfering RNA (siRNA) knockdown of α-synuclein, implicating α-synuclein as a potent, albeit partial, mediator of HO-1's neurodystrophic effects in these parkinsonian mice. Overexpression of HO-1 in stressed astroglia has previously been documented in the substantia nigra of idiopathic PD and may promote α-synuclein production and toxicity by downmodulating miR-153 and/or miR-223 both within the CNS and in peripheral tissues.

Seeing Is Believing: Vitamin A Promotes Skin Health through a Host-Derived Antibiotic

In this issue of Cell Host & Microbe, Harris etal. (2019) show that members of the resistin-like molecule (RELM) family are bactericidal, antimicrobial peptides that promote skin defenses in a vitamin-A-dependent manner. Administration of vitamin A analogs triggers RELM, which improves host resistance against skin pathogens in mice.

Caspase-8 restricts natural killer cell accumulation during MCMV Infection.

Natural killer (NK) cells provide important host defense against herpesvirus infections and influence subsequent T cell control of replication and maintenance of latency. NK cells exhibit phases of expansion, contraction and memory formation in response to the natural mouse pathogen murine cytomegalovirus (MCMV). Innate and adaptive immune responses are tightly regulated in mammals to avoid excess tissue damage while preventing acute and chronic viral disease and assuring resistance to reinfection. Caspase (CASP)8 is an autoactivating aspartate-specific cysteine protease that initiates extrinsic apoptosis and prevents receptor interacting protein (RIP) kinase (RIPK)1-RIPK3-driven necroptosis. CASP8 also promotes death-independent signal transduction. All of these activities make contributions to inflammation. Here, we demonstrate that CASP8 restricts NK cell expansion during MCMV infection but does not influence NK memory. Casp8-/-Ripk3-/- mice mount higher NK response levels than Casp8+/-Ripk3-/- littermate controls or WT C57BL/6J mice, indicating that RIPK3 deficiency alone does not contribute to NK response patterns. MCMV m157-responsive Ly49H+ NK cells support increased expansion of both Ly49H- NK cells and CD8 T cells in Casp8-/-Ripk3-/- mice. Surprisingly, hyperaccumulation of NK cells depends on the pronecrotic kinase RIPK1. Ripk1-/-Casp8-/-Ripk3-/- mice fail to show the enhanced expansion of lymphocytes observed in Casp8-/-Ripk3-/- mice even though development and homeostasis are preserved in uninfected Ripk1-/-Casp8-/-Ripk3-/- mice. Thus, CASP8 naturally regulates the magnitude of NK cell responses in response to infection where strong activation signals depend on another key regulator of death signaling, RIPK1. In addition, the strong NK cell response promotes survival of effector CD8 T cells during their expansion. Thus, hyperaccumulation of NK cells and crosstalk with T cells becomes amplified in the absence of extrinsic cell death machinery.