Progression of herpesvirus infection remodels mitochondrial organization and metabolism.

Viruses target mitochondria to promote their replication, and infection-induced stress during the progression of infection leads to the regulation of antiviral defenses and mitochondrial metabolism which are opposed by counteracting viral factors. The precise structural and functional changes that underlie how mitochondria react to the infection remain largely unclear. Here we show extensive transcriptional remodeling of protein-encoding host genes involved in the respiratory chain, apoptosis, and structural organization of mitochondria as herpes simplex virus type 1 lytic infection proceeds from early to late stages of infection. High-resolution microscopy and interaction analyses unveiled infection-induced emergence of rough, thin, and elongated mitochondria relocalized to the perinuclear area, a significant increase in the number and clustering of endoplasmic reticulum-mitochondria contact sites, and thickening and shortening of mitochondrial cristae. Finally, metabolic analyses demonstrated that reactivation of ATP production is accompanied by increased mitochondrial Ca2+ content and proton leakage as the infection proceeds. Overall, the significant structural and functional changes in the mitochondria triggered by the viral invasion are tightly connected to the progression of the virus infection.

Equine gamma herpesvirus presence and viral load are not associated with equine glandular gastric disease.

OBJECTIVE: To investigate the role of equine herpesvirus-2 (EHV-2) and equine herpesvirus-5 (EHV-5) in equine glandular gastric disease (EGGD) by visualizing and quantifying these gamma herpesviruses in EGGD-affected and normal glandular gastric mucosa of horses. A secondary objective was to describe the histopathological abnormalities in the equine gastric glandular mucosa in horses with EGGD. ANIMALS: 29 horses (n = 21 postmortem and 8 gastroscopy) categorized as normal (11), EGGD (12), or both EGGD and equine squamous gastric disease (6). METHODS: Glandular gastric mucosal samples were collected from horses by gastroscopy or postmortem. Histopathology and in situ hybridization targeting EHV-2 and EHV-5 were performed on grossly normal and abnormal glandular gastric mucosa. The number of in situ hybridization-positive cells per millimeter squared of tissue was calculated. Evaluators were blinded to groups. RESULTS: Glandular gastric tissues from horses without EGGD had higher viral loads in the mucosa than normal or abnormal tissues from EGGD horses. There was no difference in viral loads for EHV-2 or EHV-5 between grossly or endoscopically normal to abnormal gastric tissues within horses with EGGD. Lymphocytic plasmacytic gastritis was the most common histopathological abnormality, with only 3 horses having mucosal disruption (glandular ulcer or erosion). CLINICAL RELEVANCE: Equine gamma herpesviruses are unlikely to play a role in the pathophysiology of EGGD. EGGD is frequently inflammatory with occasional mucosal disruption (ulcer or erosion).

Diagnostic Methods and Management Strategies of Herpes Simplex and Herpes Zoster Infections.

Herpesviruses are medium-sized double-stranded DNA viruses. Of more than 80 herpesviruses identified, only 9 human herpesviruses have been found to cause infection in humans. These include herpes simplex viruses 1 and 2 (HSV-1 and HSV-2), varicella-zoster virus (VZV), human cyto-megalovirus (HCMV), Epstein-Barr virus (EBV), and human herpesvirus (HHV-6A, HHV-6B, HHV-7, HHV-8). HSV-1, HSV-2, and VZV can be problematic given their characteristic neurotropism which is the ability to invade via fusion of its plasma membrane and reside within neural tissue. HSV and VZV primarily infect mucocutaneous surfaces and remain latent in the dorsal root ganglia for a host's entire life. Reactivation causes either asymptomatic shedding of virus or clinical manifestation of vesicular lesions. The clinical presentation is influenced by the portal of entry, the immune status of the host, and whether the infection is primary or recurrent. Affecting 60% to 95% of adults, herpesvirus-associated infections include gingivostomatitis, orofacial and genital herpes,and primary varicella and herpes zoster. Symptomatology, treatment, and potential complications vary based on primary and recurrent infections as well as the patient's immune status.

A Mouse Model to Study the Pathogenesis of γ-herpesviral Infections in Germinal Center B Cells.

CD30-positive germinal center (GC)-derived B cell lymphomas are frequently linked to Epstein-Barr Virus (EBV) infection. However, a suitable animal model for the investigation of the interplay between γ-herpesvirus and host cells in B cell pathogenesis is currently lacking. Here, we present a novel in vivo model enabling the analysis of genetically modified viruses in combination with genetically modified GC B cells. As a murine γ-herpesvirus, we used MHV-68 closely mirroring the biology of EBV. Our key finding was that Cre-mediated recombination can be successfully induced by an MHV-68 infection in GC B cells from Cγ1-Cre mice allowing for deletion or activation of loxP-flanked cellular genes. The implementation of PrimeFlow RNA assay for MHV-68 demonstrated the enrichment of MHV-68 in GC and isotype-switched B cells. As illustrations of virus and cellular modifications, we inserted the EBV gene LMP2A into the MHV-68 genome and induced constitutively active CD30-signaling in GC B cells through MHV-68 infections, respectively. While the LMP2A-expressing MHV-68 behaved similarly to wildtype MHV-68, virally induced constitutively active CD30-signaling in GC B cells led to the expansion of a pre-plasmablastic population. The findings underscore the potential of our novel tools to address crucial questions about the interaction between herpesviral infections and deregulated cellular gene-expression in future studies.

Association of equine gammaherpesvirus-5 with facial lymphohistiocytic interface dermatitis in seven adult horses from the United States.

Equine herpesvirus-5 (EHV-5) is commonly found in healthy asymptomatic horses worldwide. Although a cause-and-effect relationship has not been thoroughly determined, this virus has been associated with several disease conditions including equine multinodular pulmonary fibrosis (EMPF) and 1 case of interface dermatitis. The authors searched the New York State Animal Health Diagnostic Center database for cases of equine interface dermatitis between 2007 and 2022. Ten cases were identified and scrutinized for viral inclusion bodies which were present in 5 of 10 cases. Two similar cases with interface dermatitis and viral inclusion bodies, which were not part of a retrospective search, were from the Oregon Veterinary Diagnostic Laboratory. The authors describe a total of 7 horses with dermatitis characterized by crusted, alopecic, non-pruritic, non-painful, irregular to annular areas over the face, most commonly the muzzle, for up to several years duration. Histologically, there was a CD3+ T lymphocyte-dominated lymphohistiocytic interface dermatitis with hydropic degeneration, apoptotic keratinocytes, and pigmentary incontinence. Keratinocytes within the upper stratum spinosum and stratum granulosum had glassy pale basophilic intranuclear inclusion bodies consistent with herpesvirus. The presence of EHV-5 was confirmed by quantitative polymerase chain reaction (qPCR) and in situ hybridization in 7 horses and by electron microscopy in 1 horse. One horse later developed EMPF and was euthanized. EHV-5 was not detected with qPCR from 5 control horses and 5 horses with interface dermatitis without histologic evidence of viral inclusion bodies. These are the first cases of facial interface dermatitis associated with EHV-5 reported in the United States.

Modeling the effects of cigarette smoke extract on influenza B virus infections in mice.

Influenza B virus (IBV) is a major respiratory viral pathogen. Due to a lack of pandemic potential for IBV, there is a lag in research on IBV pathology and immunological responses compared to IAV. Therefore, the impact of various lifestyle and environmental factors on IBV infections, such as cigarette smoking (CS), remains elusive. Despite the increased risk and severity of IAV infections with CS, limited information exists on the impact of CS on IBV infections due to the absence of suitable animal models. To this end, we developed an animal model system by pre-treating mice for two weeks with cigarette smoke extract (CSE), then infected them with IBV and monitored the resulting pathological, immunological, and virological effects. Our results reveal that the CSE treatment decreased IBV specific IgG levels yet did not change viral replication in the upper airway/the lung, and weight recovery post infection. However, higher concentrations of CSE did result in higher mortality post infection. Together, this suggests that CS induced inflammation coupled with IBV infection resulted in exacerbated disease outcome.

Herpesvirus-Related Lesions of the Oral Mucosa.

The human herpesvirus (HHV) family is a group of enveloped DNA viruses containing 8 members known to produce oral mucosal lesions. Following initial exposure, which may result in symptomatic primary infection, the viruses establish latency within specific cells/tissues. After reactivation, herpesviruses can cause localized symptomatic or asymptomatic recurrent (secondary) infections or diseases. HHV may have a significant role in the cause of oral mucosal infectious diseases in immunocompromised patients. This article discusses the role of those herpesviruses that can induce oral mucosal lesions, with focus on the clinical features and treatment/management.

Murine herpesvirus-68-related growth factors treatment correlates with decrease of p53 and HIF-1α protein levels.

Murine herpesvirus 68 (MHV-68) belongs to the subfamily Gammaherpesvirinae of the family Herpesviridae. This exceptional murine herpesvirus is an excellent model for the study of human gammaherpesvirus infections. Cells infected with MHV-68 under nonpermissive conditions for viral replication produce substances designated as MHV-68 growth factors (MHGF-68), that can cause transformation of the cells, or on the other side, turn transformed cells into normal. It was already proposed, that the MHGF-68 fractions cause transformation, disruption of the cytoskeleton and slower growth of the tumors in nude mice. Here, we examined newly extracted fractions of MHGF-68 designated as F5 and F8. Both fractions proved to inhibit the growth of the spheroids and also tumours induced in nude mice. What more, the fractions caused the decrease of the protein levels of wt p53 and HIF-1α. Decreased levels of p53 and HIF-1α activity leads to decreased vascularization, slower tumour growth, and lower adaptation to hypoxic conditions. This would propose MHGF-68 fractions, or their human herpesvirus equivalents, as a potential anticancer drugs in combined chemotherapy.

Family Herpesviridae and neuroinfections: current status and research in progress.

This article addresses the relationship between human herpesviruses (HHVs) and neuroinfections. Alphaherpesviruses, betaherpesviruses and gammaherpesviruses are neurotropic viruses that establish latency and exhibit reactivation capacity. Encephalitis and meningitis are common in cases of HHV. The condition promoted by HHV infection is a purported trigger for certain neurodegenerative diseases. Ongoing studies have identified an association between HSV-1 and the occurrence of Alzheimer's disease, multiple sclerosis and infections by HHV-6 and Epstein-Barr virus. In this review, we highlight the importance of research investigating the role of herpesviruses in the pathogenesis of diseases that affect the nervous system and describe other studies in progress.

Gammaherpesvirus Alters Alveolar Macrophages According to the Host Genetic Background and Promotes Beneficial Inflammatory Control over Pneumovirus Infection.

Human respiratory syncytial virus (hRSV) infection brings a wide spectrum of clinical outcomes, from a mild cold to severe bronchiolitis or even acute interstitial pneumonia. Among the known factors influencing this clinical diversity, genetic background has often been mentioned. In parallel, recent evidence has also pointed out that an early infectious experience affects heterologous infections severity. Here, we analyzed the importance of these two host-related factors in shaping the immune response in pneumoviral disease. We show that a prior gammaherpesvirus infection improves, in a genetic background-dependent manner, the immune system response against a subsequent lethal dose of pneumovirus primary infection notably by inducing a systematic expansion of the CD8+ bystander cell pool and by modifying the resident alveolar macrophages (AMs) phenotype to induce immediate cyto/chemokinic responses upon pneumovirus exposure, thereby drastically attenuating the host inflammatory response without affecting viral replication. Moreover, we show that these AMs present similar rapid and increased production of neutrophil chemokines both in front of pneumoviral or bacterial challenge, confirming recent studies attributing a critical antibacterial role of primed AMs. These results corroborate other recent studies suggesting that the innate immunity cells are themselves capable of memory, a capacity hitherto reserved for acquired immunity.

Novel Mouse Model of Murine Cytomegalovirus-Induced Adaptive NK Cells.

NK cells are important mediators of viral control with the capacity to form adaptive immune features following infection. However, studies of infection-induced adaptive NK cells require adoptive cell transfer to lower the precursor frequency of "Ag-specific" NK cells, potentially limiting the diversity of the NK cell response. In seeking an unmanipulated model to probe the adaptive NK cells, we interrogated a wide range of Collaborative Cross (CC) inbred mice, inbred mouse strains that exhibit broad genetic diversity across strains. Our assessment identified and validated a putative "ideal" CC strain, CC006, which does not require manipulation to generate and maintain adaptive NK cells. Critically, CC006 mice, in contrast to C57BL/6 mice, are capable of developing enhanced NK cell-mediated protective responses to murine CMV infection following m157-mediated vaccination. This work both furthers our understanding of adaptive NK cells and demonstrates the utility of CC mice in the development and interrogation of immunologic models.

HHV-8 and EBV Positive Lymphoproliferative Disease: A Challenging Case.

Human herpes virus-8 (HHV-8) is linked to four lymphoproliferative diseases: primary effusion lymphoma, HHV-8 positive multicentric Castleman disease (MCD), HHV-8 positive diffuse large B cell lymphoma and HHV-8 positive germinotropic lymphoproliferative disorder (GLPD). The diagnosis of HHV-8 associated lymphoproliferative diseases is quite challenging because each entity is rare and has a wide morphological spectrum. Our aim is to emphasize the overlapping histopathological features of MCD and GLPD as well as to underline the importance of clinicopathological correlation in case these two entities cannot be distinguished by pathological examination. We present here a case of an 82-year-old male patient who was examined for weight loss and multiple lymphadenopathy. Histopathological examination of the axillary lymph node revealed lymphoid follicle structures of varying shapes and sizes, including some atrophic germinal centers. Plasmablast-like cells were notable in some of these areas. HHV-8 and Epstein Barr Virus (EBV) positivity were noted in some of these cells and in a small number of cells in the mantle zone. Based on these findings; a diagnosis of "HHV-8 and EBV positive lymphoproliferative disease" was established. Since HHV-8 positive MCD and GLPD have similar histopathological features, it may not be possible to distinguish these two entities by histopathological examination only. At this point, the importance of clinicopathological correlation becomes more evident, especially in the determination of the treatment protocol to be applied to the patient.

Kaposi's sarcoma-associated herpesvirus vFLIP promotes MEndT to generate hybrid M/E state for tumorigenesis.

Kaposi's sarcoma (KS) is an angioproliferative and invasive tumor caused by Kaposi's sarcoma-associated herpesvirus (KSHV). The cellular origin of KS tumor cells remains contentious. Recently, evidence has accrued indicating that KS may arise from KSHV-infected mesenchymal stem cells (MSCs) through mesenchymal-to-endothelial transition (MEndT), but the transformation process has been largely unknown. In this study, we investigated the KSHV-mediated MEndT process and found that KSHV infection rendered MSCs incomplete endothelial lineage differentiation and formed hybrid mesenchymal/endothelial (M/E) state cells characterized by simultaneous expression of mesenchymal markers Nestin/PDGFRA/α-SAM and endothelial markers CD31/PDPN/VEGFR2. The hybrid M/E cells have acquired tumorigenic phenotypes in vitro and the potential to form KS-like lesions after being transplanted in mice under renal capsules. These results suggest a homology of KSHV-infected MSCs with Kaposi's sarcoma where proliferating KS spindle-shaped cells and the cells that line KS-specific aberrant vessels were also found to exhibit the hybrid M/E state. Furthermore, the genetic analysis identified KSHV-encoded FLICE inhibitory protein (vFLIP) as a crucial regulator controlling KSHV-induced MEndT and generating hybrid M/E state cells for tumorigenesis. Overall, KSHV-mediated MEndT that transforms MSCs to tumorigenic hybrid M/E state cells driven by vFLIP is an essential event in Kaposi's sarcomagenesis.

Nanotechnology Frontiers in γ-Herpesviruses Treatments.

Epstein-Barr Virus (EBV) and Kaposi's sarcoma associated-herpesvirus (KSHV) are γ-herpesviruses that belong to the Herpesviridae family. EBV infections are linked to the onset and progression of several diseases, such as Burkitt lymphoma (BL), nasopharyngeal carcinoma (NPC), and lymphoproliferative malignancies arising in post-transplanted patients (PTDLs). KSHV, an etiologic agent of Kaposi's sarcoma (KS), displays primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). Many therapeutics, such as bortezomib, CHOP cocktail medications, and natural compounds (e.g., quercetin or curcumin), are administrated to patients affected by γ-herpesvirus infections. These drugs induce apoptosis and autophagy, inhibiting the proliferative and cell cycle progression in these malignancies. In the last decade, many studies conducted by scientists and clinicians have indicated that nanotechnology and nanomedicine could improve the outcome of several treatments in γ-herpesvirus-associated diseases. Some drugs are entrapped in nanoparticles (NPs) expressed on the surface area of polyethylene glycol (PEG). These NPs move to specific tissues and exert their properties, releasing therapeutics in the cell target. To treat EBV- and KSHV-associated diseases, many studies have been performed in vivo and in vitro using virus-like particles (VPLs) engineered to maximize antigen and epitope presentations during immune response. NPs are designed to improve therapeutic delivery, avoiding dissolving the drugs in toxic solvents. They reduce the dose-limiting toxicity and reach specific tissue areas. Several attempts are ongoing to synthesize and produce EBV vaccines using nanosystems.

Susceptibility of Goldfish to Cyprinid Herpesvirus 2 (CyHV-2) SH01 Isolated from Cultured Crucian Carp.

Cyprinid herpesvirus 2 (CyHV-2), a member of the Alloherpesviridae family belonging to the genus Cyprinivirus, is a fatal contagious aquatic pathogen that affects goldfish (Carassius auratus) and crucian carp (Carassius carassius). Although crucian carp and goldfish belong to the genus Carassius, it is unclear whether they are susceptible to the same CyHV-2 isolate. In addition, the origin of the crucian carp-derived CyHV-2 virus isolate remains unclear. CyHV-2 SH01 was isolated during herpesviral hematopoietic necrosis disease (HVHN) outbreaks in crucian carp at a local fish farm near Shanghai. CyHV-2 SH01 was confirmed by PCR and Western blot analysis of kidney, spleen, muscle, and blood tissue from the diseased crucian carp. Moreover, histopathological and ultra-pathological analyses revealed pathological changes characteristic of CyHV-2 SH01 infection in the tissues of the diseased crucian carp. In the present study, goldfish and crucian carp were challenged with CyHV-2 SH01 to elucidate viral virulence. We found that CyHV-2 SH01 could cause rapid and fatal disease progression in goldfish and crucian carp 24 h post-injection at 28 °C. Experimental infection of goldfish by injection indicated that the average virus titer in the kidney of the goldfish was 103.47 to 103.59 copies/mg. In addition, tissues exhibited the most prominent histopathological changes (cellular wrinkling and shrinkage, cytoplasmic vacuolation, fusion of the gill lamellae, and hepatic congestion) in CyHV-2 SH01-infected goldfish and crucian carp. Thus, crucian carp and goldfish showed a high sensitivity, with typical symptoms, to HVHN disease caused by CyHV-2 SH01.

Olfactory Entry Promotes Herpesvirus Recombination.

Herpesvirus genomes show abundant evidence of past recombination. Its functional importance is unknown. A key question is whether recombinant viruses can outpace the immunity induced by their parents to reach higher loads. We tested this by coinfecting mice with attenuated mutants of murid herpesvirus 4 (MuHV-4). Infection by the natural olfactory route routinely allowed mutant viruses to reconstitute wild-type genotypes and reach normal viral loads. Lung coinfections rescued much less well. Attenuated murine cytomegalovirus mutants similarly showed recombinational rescue via the nose but not the lungs. These infections spread similarly, so route-specific rescue implied that recombination occurred close to the olfactory entry site. Rescue of replication-deficient MuHV-4 confirmed this, showing that coinfection occurred in the first encountered olfactory cells. This worked even with asynchronous inoculation, implying that a defective virus can wait here for later rescue. Virions entering the nose get caught on respiratory mucus, which the respiratory epithelial cilia push back toward the olfactory surface. Early infection was correspondingly focused on the anterior olfactory edge. Thus, by concentrating incoming infection into a small area, olfactory entry seems to promote functionally significant recombination. IMPORTANCE All organisms depend on genetic diversity to cope with environmental change. Small viruses rely on frequent point mutations. This is harder for herpesviruses because they have larger genomes. Recombination provides another means of genetic optimization. Human herpesviruses often coinfect, and they show evidence of past recombination, but whether this is rare and incidental or functionally important is unknown. We showed that herpesviruses entering mice via the natural olfactory route meet reliably enough for recombination routinely to repair crippling mutations and restore normal viral loads. It appeared to occur in the first encountered olfactory cells and reflected a concentration of infection at the anterior olfactory edge. Thus, natural host entry incorporates a significant capacity for herpesvirus recombination.

Histopathological Analysis of Adrenal Glands after Simian Varicella Virus Infection.

Latent varicella zoster virus (VZV) has been detected in human adrenal glands, raising the possibility of virus-induced adrenal damage and dysfunction during primary infection or reactivation. Rare cases of bilateral adrenal hemorrhage and insufficiency associated with VZV reactivation have been reported. Since there is no animal model for VZV infection of adrenal glands, we obtained adrenal glands from two non-human primates (NHPs) that spontaneously developed varicella from primary simian varicella virus (SVV) infection, the NHP VZV homolog. Histological and immunohistochemical analysis revealed SVV antigen and DNA in the adrenal medulla and cortex of both animals. Adrenal glands were observed to have Cowdry A inclusion bodies, cellular necrosis, multiple areas of hemorrhage, and varying amounts of polymorphonuclear cells. No specific association of SVV antigen with ßIII-tubulin-positive nerve fibers was found. Overall, we found that SVV can productively infect NHP adrenal glands, and is associated with inflammation, hemorrhage, and cell death. These findings suggest that further studies are warranted to examine the contribution of VZV infection to human adrenal disease. This study also suggests that VZV infection may present itself as acute adrenal dysfunction with "long-hauler" symptoms of fatigue, weakness, myalgias/arthralgias, and hypotension.

Natto extract, a Japanese fermented soybean food, directly inhibits viral infections including SARS-CoV-2 in vitro.

Natto, a traditional Japanese fermented soybean food, is well known to be nutritious and beneficial for health. In this study, we examined whether natto impairs infection by viruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as well as bovine herpesvirus 1 (BHV-1). Interestingly, our results show that both SARS-CoV-2 and BHV-1 treated with a natto extract were fully inhibited infection to the cells. We also found that the glycoprotein D of BHV-1 was shown to be degraded by Western blot analysis and that a recombinant SARS-CoV-2 receptor-binding domain (RBD) was proteolytically degraded when incubated with the natto extract. In addition, RBD protein carrying a point mutation (UK variant N501Y) was also degraded by the natto extract. When the natto extract was heated at 100 °C for 10 min, the ability of both SARS-CoV-2 and BHV-1 to infect to the cells was restored. Consistent with the results of the heat inactivation, a serine protease inhibitor inhibited anti-BHV-1 activity caused by the natto extract. Thus, our findings provide the first evidence that the natto extract contains a protease(s) that inhibits viral infection through the proteolysis of the viral proteins.

Retrospective review of 27 European cases of fatal elephant endotheliotropic herpesvirus-haemorrhagic disease reveals evidence of disseminated intravascular coagulation.

Elephant endotheliotropic herpesvirus haemorrhagic disease (EEHV-HD) is widely acknowledged as the most common cause of mortality in young Asian elephants (Elephas maximus) in captivity. The objective of the current study was to perform a blinded, retrospective pathology review of European EEHV-HD fatalities, constituting the largest systematic assessment of EEHV-HD pathology to date. Findings between viral genotypes were compared with the aim to investigate if disseminated intravascular coagulation (DIC) could be substantiated as a significant complicating factor, thereby increasing the understanding of disease pathophysiology. Immunohistochemical staining confirmed endothelial cell (EC) damage and the presence of EC intranuclear inclusion bodies, demonstrating a direct viral cytopathic effect. Microthrombi were observed in 63% of cases in several organs, including lungs, which, together with widespread haemorrhage and thrombocytopenia reported in EEHV-HD case reports, supports the presence of overt DIC as a serious haemostatic complication of active EEHV infection. Death was attributed to widespread vascular damage with multi-organ dysfunction, including severe acute myocardial haemorrhage and subsequent cardiac failure. Systemic inflammation observed in the absence of bacterial infection may be caused by cytokine release syndrome. Findings reinforce the necessity to investigate cytokine responses and haemostatic status during symptomatic and asymptomatic EEHV viraemia, to potentially support the use of anti-inflammatory treatment in conjunction with anti-viral therapy and cardiovascular support.

Integrated evaluation of lung disease in single animals.

During infectious disease, pathogen load drives inflammation and immune response that together contribute to tissue injury often resulting in organ dysfunction. Pulmonary failure in SARS-CoV2-infected hospitalized COVID-19 patients is one such prominent example. Intervention strategies require characterization of the host-pathogen interaction by accurately assessing all of the above-mentioned disease parameters. To study infection in intact mammals, mice are often used as essential genetic models. Due to humane concerns, there is a constant unmet demand to develop studies that reduce the number of mice utilized while generating objective data. Here, we describe an integrated method of evaluating lung inflammation in mice infected with Pseudomonas aeruginosa or murine gammaherpesvirus (MHV)-68. This method conserves animal resources while permitting evaluation of disease mechanisms in both infection settings. Lungs from a single euthanized mouse were used for two purposes-biological assays to determine inflammation and infection load, as well as histology to evaluate tissue architecture. For this concurrent assessment of multiple parameters from a single euthanized mouse, we limit in-situ formalin fixation to the right lung of the cadaver. The unfixed left lung is collected immediately and divided into several segments for biological assays including determination of pathogen titer, assessment of infection-driven cytokine levels and appearance of cell death markers. In situ fixed right lung was then processed for histological determination of tissue injury and confirmation of infection-driven cell death patterns. This method reduces overall animal use and minimizes inter-animal variability that results from sacrificing different animals for different types of assays. The technique can be applied to any lung disease study in mice or other mammals.