Natural killer cells play an important role in virus infection control: Antiviral mechanism, subset expansion and clinical application.

With the global spread of coronavirus disease 2019 (COVID-19), the important role of natural killer (NK) cells in the control of various viral infections attracted more interest, via non-specific activation, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and activating receptors, as well as specific activation, such as memory-like NK generation. In response to different viral infections, NK cells fight viruses in different ways, and different NK subsets proliferate. For instance, cytomegalovirus (CMV) induces NKG2C + CD57 + KIR+ NK cells to expand 3-6 months after hematopoietic stem cell transplantation (HSCT), but human immunodeficiency virus (HIV) induces KIR3DS1+/KIR3DL1 NK cells to expand in the acute phase of infection. However, the similarities and differences among these processes and their molecular mechanisms have not been fully discussed. In this article, we provide a summary and comparison of antiviral mechanisms, unique subset expansion and time periods in peripheral blood and tissues under different conditions of CMV, HIV, Epstein-Barr virus (EBV), COVID-19 and hepatitis B virus (HBV) infections. Accordingly, we also discuss current clinical NK-associated antiviral applications, including cell therapy and NK-related biological agents, and we state the progress and future prospects of NK cell antiviral treatment.

Nitric Oxide Circumvents Virus-Mediated Metabolic Regulation during Human Cytomegalovirus Infection.

Nitric oxide is a versatile and critical effector molecule that can modulate many cellular functions. Although recognized as a regulator of infections, the inhibitory mechanism of nitric oxide against human cytomegalovirus (HCMV) replication remains elusive. We demonstrate that nitric oxide attenuates viral replication by interfering with HCMV-mediated modulation of several cellular processes. Nitric oxide exposure reduced HCMV genome synthesis and infectious viral progeny with cell-type-dependent differences observed. Mitochondrial respiration was severely reduced in both uninfected and HCMV-infected cells during exposure with little impact on ATP levels indicating changes in cellular metabolism. Metabolomics identified significantly altered small molecules in multiple pathways during nitric oxide exposure including nucleotide biosynthesis, tricarboxylic acid (TCA) cycle, and glutamine metabolism. Glutathione metabolites were increased coinciding with a reduction in the glutathione precursor glutamine. This shift was accompanied by increased antioxidant enzymes. Glutamine deprivation mimicked defects in HCMV replication and mitochondrial respiration observed during nitric oxide exposure. These data suggest that nitric oxide limits glutaminolysis by shuttling glutamine to glutathione synthesis. In addition, lipid intermediates were severely altered, which likely contributes to the observed increase in defective viral particles. Nitric oxide disrupts multiple cellular processes, and we had limited success in rescuing replication defects by supplementing with metabolic intermediates. Our studies indicate that nitric oxide attenuation of HCMV is multifactorial with interference in viral manipulation of cellular metabolism playing a central role.IMPORTANCE Human cytomegalovirus is a prevalent pathogen that can cause serious disease in patients with compromised immune systems, including transplant patients and during congenital infection. HCMV lytic replication likely occurs in localized sites of infection with immune cells infiltrating and releasing nitric oxide with other effector molecules. This nonspecific immune response results in both uninfected and infected cells exposed to high levels of nitric oxide. The absence of nitric oxide synthase has been associated with lethal HCMV infection. We demonstrate that nitric oxide inhibition of HCMV replication is multifactorial and cell type dependent. Our results indicate that nitric oxide controls replication by interfering with viral modulation of cellular metabolism while also affecting proliferation and mitochondrial respiration of neighboring uninfected cells. These studies identify the mechanism and contribution of nitric oxide during immune control of HCMV infection and provide insight into its role in other viral infections.

Anti-Cytomegalovirus Activity in Human Milk and Colostrum From Mothers of Preterm Infants.

OBJECTIVES: This study aimed to investigate the anti-human cytomegalovirus (CMV) activity of milk from seropositive and seronegative mothers of preterm infants and to analyze its changes throughout the different stages of lactation and after Holder pasteurization, a procedure adopted by donor human milk banks. METHODS: Eighteen mothers of preterm infants were enrolled in the study. Colostrum, transitional milk, and mature milk samples were collected and tested for anti-CMV activity. Depletion of immunoglobulins A from milk samples was carried out by jacalin resin. Pools of milk samples were pasteurized according to Holder technique. RESULTS: All samples were endowed with anti-CMV activity, although to a different extent. In CMV IgG-positive mothers, colostra were significantly more active than the transitional milk and mature milk samples. Moreover, they were more potent than colostra from seronegative mothers. Immunoglobulins A depletion in colostra from IgG-positive mothers resulted in a partial loss of anti-CMV activity. Holder pasteurization significantly reduced the antiviral activity. CONCLUSIONS: Human milk is endowed with anti-CMV activity and its potency may vary depending on the stage of lactation and the serological status of the mother. This biological property could partially neutralize CMV particles excreted in the milk of CMV IgG-positive mothers thus reducing the risk of transmitting infectious viruses to the infant.

Enzyme-Linked Immunospot Assay as a Complementary Method to Assess and Monitor Cytomegalovirus Infection in Kidney Transplant Recipients on Pre-emptive Antiviral Therapy: A Single-Center Experience.

BACKGROUND: Cytomegalovirus (CMV) disease represents a major cause of post-transplantation morbidity and mortality. To estimate the risk of infection and monitor response to antiviral therapy, current guidelines suggest combination of viral load monitoring with direct assessment of CMV-specific immune response. We used enzyme-linked immunospot (ELISpot) for the evaluation of CMV-specific T-cell response in kidney transplant recipients with CMV viremia and investigated how information gained could help manage CMV infection. METHODS: Seventeen patients on pre-emptive antiviral therapy and CMV quantitative polymerase chain reaction (qPCR) ≥500 copies/mL (first episode after transplantation) were assessed using ELISpot and divided into Weak (9 patients with baseline ELISpot <25 spot-forming colonies [SFCs]/200,000 peripheral blood mononuclear cells [PBMCs]) and Strong Responders (8 patients with baseline ELISpot ≥25 SFCs/200,000 PBMCs). CMV-specific T-cell response, infection severity, viral load, and antiviral therapy were prospectively recorded and compared between groups at 1, 2, and 24 months of follow-up. RESULTS: Demographic and transplant characteristics of Weak and Strong Responders were similar. No episodes of CMV disease were observed. Weak Responders were more likely to experience CMV syndrome (56% vs 36.5%) and late virus reactivation (56% vs 25%) than Strong Responders. Weak Responders showed higher baseline median viral loads (19,700 vs 9265 copies/mL) and needed antiviral therapy for longer (179 vs 59.5 days). T-cell response showed 2 main patterns: early and delayed. CONCLUSIONS: ELISpot provides prognostic information about infection severity, risk of late reactivation, and response to therapy. Randomized trials, evaluating the need for antiviral therapy in kidney transplant recipients with asymptomatic infection and effective virus-specific T-cell immune response, are warranted.

Successful Treatment of Combined Aspergillus and Cytomegalovirus Abscess in Brain and Lung After Liver Transplant for Toxic Fulminant Hepatitis.

Invasive aspergillosis is one of the most important and fatal complications after liver transplant, especially in patients with involvement of the central nervous system. We present a case of a patient who developed cerebral and pulmonary aspergillosis, coinfected with cytomegalovirus, after liver transplant for toxic fulminant hepatitis. The patient was treated successfully with neurosurgical intervention and voriconazole. Voriconazole is considered more effective in cerebral aspergillosis than other anti-fungal agents due to the greater penetration into central nervous system and higher cerebrospinal fluid and brain tissue levels.

Consistent associations between measures of psychological stress and CMV antibody levels in a large occupational sample.

Cytomegalovirus (CMV) is a herpes virus that has been implicated in biological aging and impaired health. Evidence, largely accrued from small-scale studies involving select populations, suggests that stress may promote non-clinical reactivation of this virus. However, absent is evidence from larger studies, which allow better statistical adjustment for confounding and mediating factors, in more representative samples. The present study involved a large occupational cohort (N=887, mean age=44, 88% male). Questionnaires assessed psychological (i.e., depression, anxiety, vital exhaustion, SF-12 mental health), demographic, socioeconomic (SES), and lifestyle variables. Plasma samples were analyzed for both the presence and level of CMV-specific IgG antibodies (CMV-IgG), used as markers for infection status and viral reactivation, respectively. Also assessed were potential biological mediators of stress-induced reactivation, such as inflammation (C-reactive protein) and HPA function (awakening and diurnal cortisol). Predictors of CMV infection and CMV-IgG among the infected individuals were analyzed using logistic and linear regression analyses, respectively. Confirming prior reports, lower SES (education and job status) was positively associated with infection status. Among those infected (N=329), higher CMV-IgG were associated with increased anxiety (ß=.14, p<.05), depression (ß=.11, p=.06), vital exhaustion (ß=.14, p<.05), and decreased SF-12 mental health (ß=-.14, p<.05), adjusting for a range of potential confounders. Exploratory analyses showed that these associations were generally stronger in low SES individuals. We found no evidence that elevated inflammation or HPA-function mediated any of the associations. In the largest study to date, we established associations between CMV-IgG levels and multiple indicators of psychological stress. These results demonstrate the robustness of prior findings, and extend these to a general working population. We propose that stress-induced CMV replication warrants further research as a psychobiological mechanism linking stress, aging and health.

[POSITIVE EFFECTS OF MODERN IMMUNOCORRECTION ACCORDING TO THE ANALYSIS OF CYTOKINES AND SLPI IN PATIENTS WITH PYELONEPHRITIS].

The peculiarities of cytokines as compounds of immunogenesis are shown in the patients having acute (A) and chronic (Ch) pyelonephritis (PN). The combination of antibacterial therapy with Nukleinat and Galavit promotes the positive changes of cytokin-producing ability of immunocompetent cells and decrease in the level of proinflammation cytokines in blood and urine, secretory leucocyte protease inhibitor (SLPI) in urine. In children with PN and adult patients with diagnostically elevated titres of antibodies (IgG) to Herpes simplex virus, Cytomegalovirus are shown the positive effects of Kanephron® H and Proteflazidum, accordingly. Clinico-immunological effects of immunomodulators testify to the expediency of this usage in complex therapy with the aim to modulate the cytokine link of immunity for improvement of the effective treatment in APN and the protection against aggravation of kidney functioning in ChPN.

Current state of clinical diagnosis and treatment of infantile cytomegaloviral hepatitis.

Cytomegaloviral hepatitis is an infantile liver disease commonly encountered in China, which could be differentiated into 4 patterns with different clinical conditions. Along with the progress of laboratory diagnostic techniques, multiple diagnostic approaches are available for this disease, but accurate diagnosis can only be made when individual patients' realities are taken into consideration. Clinical treatments are various, and the Western medicine used is mainly anti-viral agents such as Ganciclovir, and so far no unified therapeutic program has been formed. More and more ways of regarding Chinese medicine treatment of cytomegaloviral hepatitis have been published increasingly in recent years, though further research to seek preferable treatment programs is still expected.

Cytomegalovirus prostatitis: a series of 4 cases.

Cytomegalovirus (CMV) prostatitis is very rare with only 1 report of biopsy-proven CMV prostatitis in the literature. The authors report 4 cases, 3 detected on needle biopsy and 1 detected on transurethral resection. Patients were 36, 41, 48, and 71 years old. All patients were immunosuppressed, including 1 with AIDS and 3 undergoing immunosuppressive therapy following organ transplantation. CMV inclusions were seen in secretory cells of the prostatic glands, endothelial cells of small vessels, and prostatic stromal cells associated with a dense lymphoid inflammation. Only very rarely is CMV prostatitis detected on clinical specimens, typically in immunosuppressed hosts undergoing immunosuppressive therapy following organ transplantation. Patients with CMV prostatitis may harbor multiple infections or have other serious medical conditions adversely affecting their prognosis.

VCL-CB01, an injectable bivalent plasmid DNA vaccine for potential protection against CMV disease and infection.

Vaccines for the prevention of human CMV (hCMV) infection and disease are a major public health priority. Immunization with DNA vaccines encoding key proteins involved in the immune response to hCMV has emerged as a major focus of hcmv vaccine research. Validation of the protective effect of DNA vaccination in animal models has provided support for clinical trials. VCL-CB01, under development by Vical Inc for the prevention of hCMV infection and disease, is a poloxamer-formulated, bivalent DNA vaccine that contains plasmids encoding hCMV tegument phosphoprotein 65 and the major hCMV surface glycoprotein B. In a phase I trial in healthy adults, VCL-CB01 was well tolerated. In interim results from a phase II trial in hCMV-seropositive hematopoietic cell transplant recipients, VCL-CB01 increased T-cell responses compared with placebo. The final results from the phase II trial will be of value for developing strategies to prevent hCMV disease in hCMV-seropositive transplant recipients, and may lead to other trials of VCL-CB01 or related vaccines for the prevention of congenital hCMV infection.

Macrocyclic lathyrane diterpenes as antitumor promoters.

Human cytomegalovirus (CMV) preferentially infects tumor tissues and the accumulated CMV immediate-early (IE) antigen may lead to tumor promotion and progression. The development of strategies to inhibit human CMV IE antigen expression and/or function is an important goal to prevent and treat certain forms of cancers associated with human CMV. The aim of this study was to search for antitumor promoters from plant sources. The effect of six macrocyclic lathyrane-type diterpenoids, latilagascenes A-E (1-5) and jolkinol B (6), isolated from the methanol extract of Euphorbia lagascae, on the expression of IE antigen in lung cancer cells (A549) infected by CMV was studied. All the compounds, except latilagascene D (4), decreased IE antigen expression of CMV.

I. Poloxamer-formulated plasmid DNA-based human cytomegalovirus vaccine: evaluation of plasmid DNA biodistribution/persistence and integration.

Preclinical studies were conducted in mice and rabbits to evaluate biodistribution/persistence and potential integration of plasmid DNA (pDNA) after intramuscular administration of a poloxamer-formulated pDNAbased vaccine, VCL-CT01, encoding gB, pp65, and IE1 human cytomegalovirus (hCMV) immunogens. Tissue distribution in mice vaccinated with VCL-CT01 was compared with that in mice vaccinated with a phosphate- buffered saline (PBS)-formulated control pDNA vaccine. Residual pDNA copy number (PCN), in selected tissues collected on days 3, 30, and 60 after vaccination, was measured by quantitative polymerase chain reaction. In VCL-CT01-vaccinated mice and in control pDNA-vaccinated mice, pDNA was below the limit of detection by day 60 in all tissues except the injection site. Clearance of pDNA from the injection site was slower in VCL-CT01-vaccinated mice compared with PBS-pDNA-vaccinated mice. An integration study was conducted in rabbits to determine whether pDNA integration into the genome of the vaccinated animal contributed to pDNA persistence. Residual pDNA in VCL-CT01-injected rabbit muscle collected 60 days after vaccination (geometric mean of 1085 PCN/microg total DNA) was comparable to that observed in VCL-CT01- injected mouse muscle (geometric mean of 1471 PCN/microg total DNA) collected at the same time point. pDNA integration was not detectable by column agarose gel electrophoresis despite the persistence of pDNA at the injection site 60 days after vaccination. Therefore the risk of genomic integration of hCMV pDNA formulated with poloxamer was considered negligible.

Progress toward an elusive goal: current status of cytomegalovirus vaccines.

Although infection with human cytomegalovirus (CMV) is ubiquitous and generally asymptomatic in most individuals, certain patient populations are at high risk for CMV-associated disease. These include HIV-infected individuals with AIDS, transplant patients, and newborn infants with congenital CMV infection. Immunity to CMV infection, both in the transplant setting and among women of childbearing age, plays a vital role in the control of CMV-induced injury and disease. Although immunity induced by CMV infection is not completely protective against reinfection, there is nevertheless a sound basis on which to believe that vaccination could help control CMV disease in high-risk patient populations. Evidence from several animal models of CMV infection indicates that a variety of vaccine strategies are capable of inducing immune responses sufficient to protect against CMV-associated illness following viral challenge. Vaccination has also proven effective in improving pregnancy outcomes following CMV challenge of pregnant guinea pigs, providing a 'proof-of-principle' relevant to human clinical trials of CMV vaccines. Although there are no licensed vaccines currently available for human CMV, progress toward this goal has been made, as evidenced by ongoing clinical trial testing of a number of immunization strategies. CMV vaccines currently in various stages of preclinical and clinical testing include: protein subunit vaccines; DNA vaccines; vectored vaccines using viral vectors, such as attenuated pox- and alphaviruses; peptide vaccines; and live attenuated vaccines. This review summarizes some of the obstacles that must be overcome in development of a CMV vaccine, and provides an overview of the current state of preclinical and clinical trial evaluation of vaccines for this important public health problem.

Protective effect of black seed oil from Nigella sativa against murine cytomegalovirus infection.

In this study, antiviral effect of black seed oil (BSO) from Nigella sativa was investigated using murine cytomegalovirus (MCMV) as a model. The viral load and innate immunity mediated by NK cells and Mφ during early stage of the infection were analyzed. Intraperitoneal (i.p.) administration of BSO to BALB/c mice, a susceptible strain of MCMV infection, strikingly inhibited the virus titers in spleen and liver on day 3 of infection with 1x10(5) PFU MCMV. This effect coincided with an increase in serum level of IFN-gamma. Although BSO treatment decreased both number and cytolytic function of NK cells on day 3 of infection, it increased numbers of Mφ and CD4(+) T cells. On day 10 of infection, the virus titer was undetectable in spleen and liver of BSO-treated mice, while it was detectable in control mice. Although spleen of both control and BSO-treated mice showed similar CTL activities on day 10 after infection, serum level of IFN-gamma in BSO-treated mice was higher. Furthermore, BSO treatment upregulated suppressor function of Mφ in spleen. These results show that BSO exhibited a striking antiviral effect against MCMV infection which may be mediated by increasing of Mφ number and function, and IFN-gamma production.

Antiviral and immunomodulatory effects of desferrioxamine in cytomegalovirus-infected rat liver allografts with rejection.

BACKGROUND: Cytomegalovirus (CMV) infection is associated with acute and chronic allograft rejection. We have recently shown that rat CMV increases portal inflammation and bile duct destruction in a model of rat liver allograft rejection. Desferrioxamine (DFO), an iron chelator and antioxidant, has recently been demonstrated to have antiviral as well as immunomodulatory effects in vitro. We therefore investigated whether DFO inhibits (a) CMV infection and (b) graft destruction in our rat model. METHOD: One day after liver transplantation, PVG (RT1c) into BN(RT1n), the rats were infected with rat CMV (RCMV, Maastricht strain; 10(5) plaque-forming units i.p.). The effects of 100 mg/kg body weight and 200 mg/kg body weight DFO were examined. RESULTS: In the untreated group, the grafts were uniformly RCMV culture-positive. In the group receiving 200 mg/kg DFO, RCMV replication was effectively inhibited. Inflammatory response in the graft, and especially the number of macrophages, was significantly reduced by DFO. Portal inflammation and bile duct destruction were also significantly reduced. In the untreated group, the bile duct epithelial cells were found to be strongly positive for tumor necrosis factor-alpha and this expression was clearly decreased by DFO. In addition, DFO significantly inhibited vascular cell adhesion molecule-1 expression on sinusoidal endothelial cells. CONCLUSIONS: Our in vivo transplant study strongly supports the inhibitory effects of metal chelators on CMV infection and their possible usefulness in the treatment of CMV-induced pathogenic changes.

Human cytomegalovirus infection of breast milk.

Human cytomegalovirus is the most common cause of congenital and perinatal infections throughout the world. Primary infection with human cytomegalovirus usually follows a benign course, but the virus remains latent or persistent in the host cell thereafter. Understanding the epidemiology of human cytomegalovirus is a key element in the development of strategies for prevention of infection. Although the actual sites of latency or persistence of human cytomegalovirus infections are still controversial, peripheral blood mononuclear cells and endothelial cells appear to be major sites of infection. Persistent infections caused by human cytomegalovirus could be augmented by a decrease in major histocompatibility complex expression as well as by virus-mediated immune dysfunction. It is possible that specific cellular interactions as well as production of several cytokines are necessary for the reactivation of human cytomegalovirus. Breast-fed infants are susceptible to human cytomegalovirus infection from breast milk. Human cytomegalovirus was isolated more frequently from breast milk at more than 1 month after delivery than from colostrum or early breast milk. Human cytomegalovirus DNA was also not detected in colostrum, but was found in breast milk samples 1 month after delivery. To clarify the role of milk cells and whey in vertical infection by breast feeding, we separated breast milk into milk cells and whey and examined each fraction. Human cytomegalovirus was isolated more frequently from milk whey samples than from cell samples. Human cytomegalovirus particle shedding into whey may be more important in vertical infection by breast milk than cell-to-cell transmission. The supernatant of colostrum did not exert an inhibitory effect on human cytomegalovirus-infected cells. Serum levels of cell free soluble interleukin-2 receptor of mothers with DNA-positive milk at 1 month after delivery were significantly higher than those of mothers with DNA-negative milk. It is likely that levels of factors such as soluble interleukin-2 receptor in serum are related to the reactivation of human cytomegalovirus which occurs locally in the mammary gland of the lactating mother after delivery. This minireview focuses on recent advances in the study of human cytomegalovirus infection of breast milk.

Prophylactic treatment of cytomegalovirus infection with traditional herbs.

Hot water extracts of four traditional herbs, Geum japonicum, Syzygium aromaticum, Terminalia chebula and Rhus javanica, which have been shown to have anti-herpes simplex virus (HSV) activity in vivo, were examined for anti-cytomegalovirus (CMV) activity in vitro and in vivo in this study. They inhibited replication of human CMV and murine CMV (MCMV) in vitro. These anti-CMV activities in vivo were examined in an MCMV infection model using immunosuppressed mice. Mice were subcutaneously treated with various doses of cyclosporine, and immunosuppression and MCMV infection were monitored by suppression of antibody production and virus yield in the lung, respectively. Each herbal extract was orally administered to mice treated with 50 mg/kg of cyclosporine from a day before intraperitoneal infection, and the efficacy of herbs was evaluated by the reduction in the virus yield in the lung. Among them Geum japonicum, Syzygium aromaticum, and Terminalia chebula significantly suppressed MCMV yields in lungs of treated mice compared with water treatment. Efficacy of oral treatment with 750 mg/kg per day of Geum japonicum extract was similar to that of the intraperitoneal administration of 2 mg/kg per day of ganciclovir in increasing the body weight of infected mice and reducing the virus yield in the lungs. These herbs may be beneficial for the prophylaxis of CMV diseases in immunocompromised patients.

The influence of age on transfer factor treatment of cellular immunodeficiency, chronic fatigue syndrome and/or chronic viral infections.

A group of 222 patients suffering from cellular immunodeficiency (CID), frequently combined with chronic fatigue syndrome (CFS) and/or chronic viral infections by Epstein-Barr virus (EBV) and/or cytomegalovirus (CMV), were immunologically investigated and treated with transfer factor (TF). The age range was 17-77 years. In order to elucidate the influence of aging on the course of the disease and on treatment, 3 subgroups were formed: 17-43 years, 44-53 years, and 54-77 years. Six injections of Immodin (commercial preparation of TF by SEVAC, Prague) were given in the course of 8 weeks. When active viral infection was present, IgG injections and vitamins were added. Immunological investigation was performed before the start of therapy, and subsequently according to need, but not later than after 3 months. The percentages of failures to improve clinical status of patients were in the individual subgroups, respectively: 10.6%, 11.5% and 28.9%. The influence of increasing age on the percentage of failures to normalize low numbers of T cells was very evident: 10.6%, 21.2% and 59.6%. In individuals uneffected by therapy, persistent absolute lymphocyte numbers below 1,200 cells were found in 23.1%, 54.5% and 89.3% in the oldest group. Statistical analysis by Pearson's Chi-square test, and the test for linear trend proved that the differences among the individual age groups were significant. Neither sex, nor other factors seemed to influence the results. The results of this pilot study show that age substantially influences the failure rate of CID treatment using TF. In older people, it is easier to improve the clinical condition than CID: this may be related to the diminished number of lymphocytes, however, a placebo effect cannot be totally excluded.

Prevention of bacteraemia by systemic ciprofloxacin treatment in rat cytomegalovirus-infected immunocompromised rats.

After total body irradiation (TBI) and subsequent rat cytomegalovirus (RCMV) infection rats developed severe neutropenia and in 90% of these animals Gram-negative bacteraemia was observed. Neutropenia was less severe (P < 0.01) and bacteraemia was not recorded (P < 0.01) in uninfected irradiated control groups. The effect of selective bowel decontamination (SBD) on the bowel flora and the incidence of bacteraemia in RCMV-infected irradiated rats was studied. Animals received ciprofloxacin 2 mg/kg/day, administered by either the oral or the subcutaneous route. After either treatment regimens the numbers of Enterobacteriaceae in the caecum and colon were reduced significantly (from 10(7) to < 10(2) g of faeces), while the total numbers of facultatively aerobic and obligatory anaerobic bacteria and the number of enterococci in the faeces was unaffected when measured 1, 3 and 7 days after the onset of treatment. The effect of ciprofloxacin treatment on the incidence of bacteraemia was recorded in RCMV-infected irradiated groups. Blood and organ cultures were taken 10 days after virus inoculation. The rate of bacteraemia decreased from 80% in untreated RCMV-infected irradiated rats affected by severe RCMV disease to < 10% in the ciprofloxacin treated groups (P < 0.01). The route of treatment did not influence these results. The uninfected irradiated and infected non-irradiated control groups showed no bacteraemia. It is concluded that symptomatic RCMV infection induces severe neutropenia, which predisposes to a high rate of Gram-negative bacteraemia, which can be prevented by eradication of the Gram-negative aerobic bacteria by means of SBD with ciprofloxacin. Systemic treatment seems to be superior for the control of Enterobacteriaceae. This may be especially useful for patients, who cannot be treated effectively by the oral route because of intestinal stasis.