Simian varicella virus infection and reactivation in rhesus macaques trigger cytokine and Aß40/42 alterations in serum and cerebrospinal fluid.

Simian varicella virus (SVV) produces peripheral inflammatory responses during varicella (primary infection) and zoster (reactivation) in rhesus macaques (RM). However, it is unclear if peripheral measures are accurate proxies for central nervous system (CNS) responses. Thus, we analyzed cytokine and Aß42/Aß40 changes in paired serum and cerebrospinal fluid (CSF) during the course of infection. During varicella and zoster, every RM had variable changes in serum and CSF cytokine and Aß42/Aß40 levels compared to pre-inoculation levels. Overall, peripheral infection appears to affect CNS cytokine and Aß42/Aß40 levels independent of serum responses, suggesting that peripheral disease may contribute to CNS disease.

Kaposi's sarcoma-associated herpesvirus T cell responses in HIV seronegative individuals from rural Uganda.

T cell responses to Kaposi's sarcoma-associated herpesvirus (KSHV) are likely essential in the control of KSHV infection and protection from associated disease, but remain poorly characterised. KSHV prevalence in rural Uganda is high at >90%. Here we investigate IFN- γ T cell responses to the KSHV proteome in HIV-negative individuals from a rural Ugandan population. We use an ex-vivo IFN- γ ELISpot assay with overlapping peptide pools spanning 83 KSHV open reading frames (ORF) on peripheral blood mononuclear cells (PBMC) from 116 individuals. KSHV-specific T cell IFN- γ responses are of low intensity and heterogeneous, with no evidence of immune dominance; by contrast, IFN- γ responses to Epstein-Barr virus, Cytomegalovirus and influenza peptides are frequent and intense. Individuals with KSHV DNA in PBMC have higher IFN- γ responses to ORF73 (p = 0.02) and lower responses to K8.1 (p = 0.004) when compared with those without KSHV DNA. In summary, we demonstrate low intensity, heterogeneous T cell responses to KSHV in immune-competent individuals.

Patterns of serum immune biomarkers during elephant endotheliotropic herpesvirus viremia in Asian and African elephants.

Hemorrhagic disease (HD) caused by a group of elephant endotheliotropic herpesviruses (EEHV) is one of the leading causes of death for young elephants in human care. These viruses are widespread and typically persist latently in adult elephants with no negative effects; however, in juvenile Asian and more recently young African elephants, the onset of disease can be rapid and the mortality rate high. Measuring biomarkers associated with the immune response could be beneficial to understanding underlying disease processes, as well as the management of infection and HD. The goal of this study was to measure acute phase proteins and cytokines in serum collected from elephants infected with EEHV (13 Asian and 1 African) and compare concentrations according to presence, severity and outcome of disease. Serum amyloid A (SAA) and haptoglobin (HP) were higher in elephants with EEHV viremia than those without; concentrations increased with increasing viral load, and were higher in fatal cases compared to those that survived. In Asian elephants, SAA was also higher during EEHV1 viremia compared to EEHV5. Cytokine concentrations were typically low, and no statistical differences existed between groups. However, in individuals with detectable levels, longitudinal profiles indicated changes in tumor necrosis factor alpha (TNF-α) and interleukin-2 (IL-2) that may reflect an immune response to EEHV infection. However, the overall low concentrations detected using previously validated assays do not support the presence of a 'cytokine storm' and suggest more work is needed to understand if sub-optimal immune responses could be involved in disease progression. These results highlight the potential benefit of measuring circulating biomarker concentrations, such as APPs and cytokines, to improve our understanding of EEHV viremia and HD, assist with monitoring the progression of disease and determining the impact of interventions.

Seroprevalence and molecular diversity of Human Herpesvirus 8 among people living with HIV in Brazzaville, Congo.

Human herpesvirus 8 (HHV8) is endemic in Africa, although studies of this infection are rare in Congo. We evaluated seroprevalence and HHV-8 diversity among people living with HIV. We included 353 patients receiving highly active antiretroviral therapy. Antibodies against HHV-8 latency-associated nuclear antigen were detected by indirect immunofluorescence. In HHV-8 positive patients, we performed HHV-8 quantification in blood and saliva by real-time PCR and typing by Sanger sequencing of K1 open reading frame. HHV-8 seroprevalence was 19%, being male (odd ratio [OR] = 1.741, [95% Confidence interval {CI}, 0.97-3.07]; p = 0.0581) and having multiple sex partners before HIV diagnosis (OR = 1.682, [CI 95%, 0.97-2.92]; p = 0.0629) tended to be associated with HHV-8 seropositivity. Of the 64 HHV-8 seropositive patients, HHV-8 DNA was detected in 10 (16%) in saliva, 6 (9%) in whole-blood and in 2 (3%) in both whole-blood and saliva. Three out of 6 HHV-8 strains were subtypes A5, 2 subtype B1 and 1 subtype C. HHV-8 seroprevalence was relatively low with more frequent carriage in men, associated with asymptomatic oral excretion and a predominance of subtype A5. These data tend to support the hypothesis of horizontal transmission in people living with HIV in Brazzaville.

Serological responses to koi herpesvirus (KHV) in a non-cyprinid reservoir host.

Koi herpesvirus (KHV) is a highly contagious virus that causes KHV disease (KHVD) inducing high mortality in carp and koi (Cyprinus carpio L.). In the late stage, latency occurs with very low, often non-detectable virus concentrations, which represents a challenge for virus detection. After validation according to OIE recommendations, an antibody ELISA was established to recognize antibodies of C. carpio against KHV infection. In this study, the ELISA was modified to detect anti-KHV antibodies from a non-cyprinid fish. Experimentally infected rainbow trout (Oncorhynchus mykiss) were able to transmit KHV to naïve carp at two different temperatures, demonstrating their potential as a reservoir host. At 20°C, KHVD was induced in carp but not at 15°C. Unexpectedly, rainbow trout developed humoral response against KHV at both temperatures. In contrast to carp, at 15°C trout produced neutralizing antibodies but not at 20°C. While antibodies obtained from infected carp sera reacted in a similar way against all KHV, antibodies from rainbow trout sera reacted differently to the same isolates by ELISA. The data show that even when non-cyprinid fish species are infected with KHV, they can produce antibodies that differ from those observed in carp.

Molecular Detection of Human Herpes Viruses in Suspected Measles Serum Samples from Senegal, 2014 to 2017.

Herpesviruses are known to cause a diversity of clinical syndromes, ranging from minor cutaneous lesions to life-threatening illnesses, especially in immunocompromised hosts. Here, we investigate retrospectively the contribution of five human herpesviruses, including herpes simplex virus Cytomegalovirus (CMV), the Epstein-Barr virus (EBV), human herpesvirus 6, and varicella zoster virus (VZV) in serum samples collected from measles suspected patients with at least fever and rash. Sera specimens were first tested for serological evidence of measles and rubella virus infection by ELISA, and DNA extracted from an aliquot of each clinical specimen for molecular detection of human herpes viruses by RT-qPCR. A total of 3,358 specimens have been collected and tested for herpes viruses. Nearly half of the overall suspected cases were children younger than 5 years (49.4%). Of the 3,358 sera tested by ELISA, 227 (6.7%) were measles laboratory confirmed and 152 (4.5%) rubella laboratory confirmed. Herpes viruses were detected in 1763 (52.5%), and VZV was the most common with 44.3%, followed by EBV with 10.7%. Coinfections were found in 352 (20%) cases, and the most common co-detections were VZV/EBV or VZV/CMV (169 and 81 cases, respectively). A clear seasonal pattern of VZV, EBV, and CMV identification was observed, with the highest incidence between February and April each year. Results of this investigation provide more insights into cutaneous rash syndrome etiologies in patients sampled in the framework of measles/rubella surveillance in Senegal, which is useful for the guidance of both case definition revision and clinical practice as well as for public health policy.

Deconvoluting global cytokine signaling networks in natural killer cells.

Cytokine signaling via signal transducer and activator of transcription (STAT) proteins is crucial for optimal antiviral responses of natural killer (NK) cells. However, the pleiotropic effects of both cytokine and STAT signaling preclude the ability to precisely attribute molecular changes to specific cytokine-STAT modules. Here, we employed a multi-omics approach to deconstruct and rebuild the complex interaction of multiple cytokine signaling pathways in NK cells. Proinflammatory cytokines and homeostatic cytokines formed a cooperative axis to commonly regulate global gene expression and to further repress expression induced by type I interferon signaling. These cytokines mediated distinct modes of epigenetic regulation via STAT proteins, and collective signaling best recapitulated global antiviral responses. The most dynamically responsive genes were conserved across humans and mice, which included a cytokine-STAT-induced cross-regulatory program. Thus, an intricate crosstalk exists between cytokine signaling pathways, which governs NK cell responses.

Abdominal aortic aneurysm and virus infection: A potential causative role for cytomegalovirus infection?

An abdominal aortic aneurysm (AAA) is a multifactorial disease with a variety of genetic and environmental risk factors, but the exact mechanism of AAA formation and progression is still not well understood. The present study investigated the frequency of cytomegalovirus (CMV), Epstein-Barr virus (EBV), and papillomavirus types 6 and 11 (HPV6 and HPV11), their impact on clinical manifestations of cardiovascular diseases, and their possible association with inflammation in patients with AAA and healthy volunteers. Genotyping of CMV UL75, EBV LMP-1, and HPV6, and HPV11 E6 was performed by polymerase chain reaction (PCR), while the viral DNA loads were measured by quantitative real-time PCR. Cytokine levels were determined by enzyme-linked immunosorbent assays. The CMV UL75 was detected more frequently in the blood of patients with AAA than in the blood of healthy volunteers (32.7% vs. 6.3%, p < .0001). Neither EBV LMP-1 nor HPV6 E6 was found in blood and aortic wall biopsies, while the HPV11 E6 was detected in 36.4% of AAA walls. The CMV infection in patients with AAA was associated with an increased risk of hypertension and coronary artery disease (OR, 9.057; 95% CI, 1.141-71.862; p = .037; and OR, 2.575; 95% CI, 1.002-6.615; p = .049, respectively). Additionally, CMV-infected patients with AAA had higher tumor necrosis factor-α levels compared with noninfected subjects (p = .017). Our findings suggest that CMV infection can stimulate local inflammation in the aorta but is not a direct cause of most abdominal aortic aneurysms.

Seroprevalence of human herpesvirus 8 in Ireland among blood donors, men who have sex with men, and heterosexual genitourinary medicine and infectious diseases clinic attendees.

Human herpesvirus 8 (HHV-8) seroprevalence varies geographically and between subpopulations. High seroprevalence rates have been ascribed to men who have sex with men (MSM), African migrants, and HIV-infected individuals. The objective of this study was to determine the seroprevalence of HHV-8 in an Irish population, including specific risk groups. A cross-sectional study of 200 blood donors and 200 genitourinary medicine (GUM) and infectious diseases (ID) clinic patients was performed, with testing for Immunoglobulin G (IgG) antibodies to HHV-8 lytic antigens using a commercial indirect fluorescence assay (Scimedx Corp.). Verification was performed at the Centers for Disease Control and Prevention (CDC). All 200 blood donor samples were negative for HHV-8 IgG antibodies. 21% of GUM and ID patients were positive for HHV-8 IgG antibodies. One hundred of these patients were MSM, 35% of whom were HHV-8 seropositive (46% of HIV-positive MSM and 24% of HIV-negative MSM). Of 100 heterosexual patients, only 7% were HHV-8 seropositive. The absence of seropositivity in 200 Irish blood donors may suggest that Ireland has a low overall population HHV-8 seroprevalence. The proportion of HHV-8 seropositivity in the MSM population was significantly higher than in the heterosexual population and most marked in HIV-positive MSM.

Evaluation of IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ cytokines in HIV/HHV-8 coinfection.

Imbalance in the immune response is one of the main pathogenic mechanisms of diseases related with human immunodeficiency virus (HIV)/human gammaherpesvirus 8 (HHV-8) coinfection, such as Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), multicentric Castleman disease (MCD) and the Kaposi's sarcoma-associated herpesvirus inflammatory cytokine syndrome (KICS). However, significant changes in pro- and anti-inflammatory cytokine levels may be observed in HIV/HHV-8 individuals who are negative for KS, PEL, MCD, and/or KICS. In this study, serum levels of interleukin-2 (IL-2), IL-4, IL-6, IL-10, tumor nucrosis factor α (TNF-α) and interferon γ (IFN-γ) were assessed in 69 HIV and 48 HIV/HHV-8 individuals, all negatives for HHV-8-related diseases. The cytokines were measured by flow cytometry and analyzed by the Mann-Whitney test. The p < .05 and 95% confidence interval were considered in all analyzes. IL-4 (p = .0155), IL-6 (p = .0036), and IL-10 (p = .0036) levels were significantly higher in HIV/HHV-8 patients than in the HIV group. On the other hand, IL-2 (p = .2295), TNF-α (p = .1216) and IFN-γ (p = .1178) did not differ between the groups analyzed. To our knowledge, to date, this is the first report on significant differences in the levels of IL-4 and IL-6 in HIV versus HIV/HHV-8 individuals. Finally, these early findings are important as a prognostic tool and contribute to clarifying the HHV-8-host interaction.

Detection of infectious laryngotracheitis virus (ILTV) in tissues and blood fractions from experimentally infected chickens using PCR and immunostaining analyses.

The ability of infectious laryngotracheitis virus (ILTV) to replicate in organs outside of the upper respiratory tract and conjunctiva associated-lymphoid tissues is still not well understood. This study investigated the tissue distribution of an Australian field strain of ILTV (class 9) on birds experimentally inoculated via eye-drop at 7 days of age by using quantitative PCR (qPCR) and immunohistochemistry. Tissues including conjunctiva, caecal tonsil, kidney, liver, lung, spleen, thymus, trachea and blood were collected from sham-inoculated (control group; n = 2) and ILTV-inoculated (n = 8) birds at 7 days post-inoculation (dpi). Blood was collected from 13 infected birds at 14 dpi and fractionated using ficoll-paque. At 7 dpi, the highest detection rate and genomic copies (GC) were in conjunctiva (8/8; 8.08 ± 0.48 log10 GC/mg) followed by trachea (8/8; 4.64 ± 0.48) and thymus (8/8; 4.52 ± 0.48), kidney (8/8; 3.97 ± 0.48), lung (8/8; 3.65 ± 0.48), spleen (8/8; 3.55 ± 0.48), liver (8/8; 3.51 ± 0.48), caecal tonsil (7/8; 3.76 ± 0.48) and plasma (4/8; 2.40 ± 0.48 log10 GC/ml). ILTV antigen was only detected in conjunctiva (7/8), trachea (6/8) and lung (4/8) samples. At 14 dpi, ILTV detection rate and genomic copies in buffy coat cells were 12/13 and 2.86 ± 0.39 log10 GC/mg, respectively while those of plasma were 11/13 and 4.29 ± 0.39 log10 GC/ml and red blood cell were 3/13 and 0.36 ± 0.39 log10 GC/mg. In conclusion, ILTV DNA was detected in a wide range of tissues and blood fractions but ILTV antigen was only detected in respiratory organs and conjunctiva.

Gammaherpesvirus in Cervid Species from Norway: Characterization of a New Virus in Wild and Semi-Domesticated Eurasian Tundra Reindeer (Rangifer tarandus tarandus).

Gammaherpesvirus infections have been described in cervids worldwide, mainly the genera Macavirus or Rhadinovirus. However, little is known about the gammaherpesviruses species infecting cervids in Norway and Fennoscandia. Blood samples from semi-domesticated (n = 39) and wild (n = 35) Eurasian tundra reindeer (Rangifer tarandus tarandus), moose (Alces alces, n = 51), and red deer (Cervus elaphus, n = 41) were tested using a panherpesvirus DNA polymerase (DPOL) PCR. DPOL-PCR-positive samples were subsequently tested for the presence of glycoprotein B (gB) gene. The viral DPOL gene was amplified in 28.2% (11/39) of the semi-domesticated reindeer and in 48.6% (17/35) of the wild reindeer. All moose and red deer tested negative. Additionally, gB gene was amplified in 4 of 11 semi-domesticated and 15 of 17 wild Eurasian reindeer DPOL-PCR-positive samples. All the obtained DPOL and gB sequences were highly similar among them, and corresponded to a novel gammaherpesvirus species, tentatively named Rangiferine gammaherpesvirus 1, that seemed to belong to a genus different from Macavirus and Rhadinovirus. This is the first report of a likely host-specific gammaherpesvirus in semi-domesticated reindeer, an economic and cultural important animal, and in wild tundra reindeer, the lastpopulation in Europe. Future studies are required to clarify the potential impact of this gammaherpesvirus on reindeer health.

Influence of corticosteroid therapy on viral reactivation in drug-induced hypersensitivity syndrome/drug reaction with eosinophilia and systemic symptoms.

Drug-induced hypersensitivity syndrome (DIHS)/drug reaction with eosinophilia and systemic symptoms (DRESS) is a severe adverse drug reaction characteristically associated with sequential reactivation of herpesviruses, such as human herpesvirus 6 (HHV-6), Epstein-Barr virus (EBV), and cytomegalovirus (CMV). Since systemic corticosteroids are thought to result in viral reactivation due to their immunosuppressive effects, we clarified the influence of systemic corticosteroid therapy on viral reactivation in DIHS/DRESS. Viral DNA in peripheral whole blood and serum sIL-2R level were measured during the disease course in twenty DIHS/DRESS patients. Six of seven patients treated without corticosteroids experienced HHV-6 viremia associated with elevated serum sIL-2R levels. In contrast, high-dose corticosteroids started within 1 week after onset tended to inhibit the occurrence of HHV-6 reactivation with remarkable suppression of serum sIL-2R level. Low-dose corticosteroids or late-start high-dose corticosteroids did not suppress occurrence of HHV-6 viremia and the increase of sIL-2R levels. HHV-6 load in the blood was clearly correlated with the serum sIL-2R level. On the other hand, increased CMV load were found in patients treated with corticosteroids regardless of the start time. The frequency of detection of EBV DNA in peripheral blood was similarly observed in all groups. In conclusion, high-dose corticosteroids started within 1 week tended to suppress HHV-6 reactivation through suppression of T cell activation. However, CMV proliferation was promoted by corticosteroids regardless of the start time. These observations suggested that careful consideration should be given to the dose and timing of administration of systemic corticosteroids in the treatment of DIHS/DRESS.

Human Herpesvirus 8 infection may contribute to oxidative stress in diabetes type 2 patients.

OBJECTIVE: To investigate the link between Human Herpesvirus 8 (HHV8) infection and plasma oxidative stress in patients with diabetes mellitus type 2 (DM2). RESULTS: Blood samples collected from DM2 and control subjects were screened for the presence of antibodies against HHV8 and for biomarkers of oxidative stress. We determined the products of radical damage on the plasma lipid fraction, such as malondialdehyde (MDA), fatty acid hydroperoxides (HP) and 7-ketocholesterol (7-keto), the oxidation products of unsaturated fatty acids (UFA) and cholesterol, respectively. The level of plasma antioxidant α-tocopherol (α-toc) was also assessed. Relevant differences were observed in the redox status in DM2 and either HHV8-positive or -negative control subjects. The level of α-toc significantly decreased in both DM2 and HHV8-positive subjects. Levels of MDA, HP and 7-keto were much higher in HHV8-positive and DM2 subjects, indicating that plasma oxidative stress is a common feature in both DM2 and HHV8-infection. In addition, 7-keto was further increased in HHV8-positive DM2 patients. We hypothesized that the HHV8-infection may contribute to the production of ROS, and hence to the oxidative stress closely related to the pathogenesis and development of DM2.

α2ß1 Integrin Is Required for Optimal NK Cell Proliferation during Viral Infection but Not for Acquisition of Effector Functions or NK Cell-Mediated Virus Control.

NK cells play an important role in antiviral resistance. The integrin α2, which dimerizes with integrin ß1, distinguishes NK cells from innate lymphoid cells 1 and other leukocytes. Despite its use as an NK cell marker, little is known about the role of α2ß1 in NK cell biology. In this study, we show that in mice α2ß1 deficiency does not alter the balance of NK cell/ innate lymphoid cell 1 generation and slightly decreases the number of NK cells in the bone marrow and spleen without affecting NK cell maturation. NK cells deficient in α2ß1 had no impairment at entering or distributing within the draining lymph node of ectromelia virus (ECTV)-infected mice or at becoming effectors but proliferated poorly in response to ECTV and did not increase in numbers following infection with mouse CMV (MCMV). Still, α2ß1-deficient NK cells efficiently protected from lethal mousepox and controlled MCMV titers in the spleen. Thus, α2ß1 is required for optimal NK cell proliferation but is dispensable for protection against ECTV and MCMV, two well-established models of viral infection in which NK cells are known to be important.

Exercise as a countermeasure for latent viral reactivation during long duration space flight.

Latent viral reactivation is a commonly reported manifestation of immune system dysregulation during spaceflight. As physical fitness and exercise training have been shown to benefit multiple arms of the immune system, we hypothesized that higher levels of preflight physical fitness and/or maintaining fitness during a mission would protect astronauts from latent viral reactivation. Standardized tests of maximal strength, muscular endurance, flexibility, and cardiorespiratory fitness (CRF) were performed in 22 international space station (ISS) crewmembers before and after a ~6-month mission. Reactivation of cytomegalovirus (CMV), Epstein-Barr virus (EBV), and varicella zoster virus (VZV) was determined in crewmembers and ground-based controls before, during, and after spaceflight. Crewmembers with higher CRF before spaceflight had a 29% reduced risk of latent viral reactivation compared to crew with lower CRF. Higher preflight upper body muscular endurance was associated with a 39% reduced risk of viral reactivation, a longer time to viral reactivation, and lower peak viral DNA concentrations, particularly for EBV and VZV. Latent viral reactivation rates were highest in crew with lower preflight CRF and higher levels of CRF deconditioning on return to Earth. We conclude that physical fitness may protect astronauts from latent viral reactivation during long duration spaceflight missions.

Impact assessment and investigation of factors associated with herpesviruses viremia in the first year of renal transplantation.

The increased risk for opportunistic infections after a renal transplant requires monitoring of viral infections to avoid future complications. Our goal was to investigate the impact and factors associated with Epstein-Barr virus (EBV), human cytomegalovirus (HCMV) and human herpesvirus type 6 (HHV-6) viremia in renal transplant recipients. Whole blood samples were collected monthly from 82 patients during the first semester and then quarterly up to 1 year after transplantation. EBV, HCMV, and HHV-6 were detected and quantified by TaqMan real-time polymerase chain reaction. The results showed that EBV and HCMV viremia were detected in 32 patients (39% each), while HHV-6 viremia in only 3 patients (3.7%). EBV was significantly associated with age (P = .050), thymoglobuline induction (P = .019), mTOR inhibitor-based therapy (P = .003), and female gender (P = .044). HCMV was significantly associated with basiliximab induction (P = .015), mycophenolate mofetil (MMF)-based therapy (P = .003) and allograft acute rejection (P = .033). Moreover, HCMV-disease was correlated with MMF-based therapy (P = .021) and female gender (P = .003). In conclusion, EBV and HCMV viremia were associated with different immunosuppressive induction and maintenance strategies. Additionally, higher HCMV viremia (> 10 4 copies/mL) was related to acute allograft rejection.

PECULIARITIES OF CLINICAL AND IMMUNOLOGICAL CHARACTERISTICS OF HERPESVIRUS INFECTIONS IN CHILDREN.

Aimed at optimization of herpes viral infections diagnosis, 186 babies who had momo-or mixed herpes viral infections were examined. Their blood, liquor, urine, saliva and materials that were scrapped off in the damaged focus (skin, mucous membrane of the mouth) were studied by methods of IFA, RIF,PCR. Clinical and immunologic parallels were studied. Levels of TNF-α, IL-4 and IFN-α in blood serum and in liquor were assessed. Generalized form of mixed infection was characterized by low contents of TNF-α, IL-4 and IFN-α in blood serum and higher of TNF-α in the liquor which in the setting of increased antigen load pointed to the disruption of mechanisms of adaptation and compensation and bad prognosis.

Monitoring of the shedding and serological dynamics of Bovine gammaherpesvirus type 4 in a dairy cattle herd.

Bovine gammaherpesvirus type 4 (BoHV-4) is increasingly related with reproductive disease in cattle, but its epidemiology is not fully understood. We monitored the serological response and shedding of BoHV-4 in a positive dairy cattle farm with metritis. First, we performed an ELISA to detect BoHV-4 antibodies in all the animals (n = 104). Afterwards, ten seronegative heifers introduced in the production lot and sera samples were monthly taken for four months and then 6-10 months after introduction to detect BoHV-4 antibodies by ELISA. Moreover, a vaginal swab was taken after calving to detect BoHV-4 by PCR. Concurrently, a weekly collection of vaginal and nasal swabs and milk was performed during the first month post-partum in multiparous cows with metritis (n = 14), heifers with metritis (n = 4), heifers without metritis but positive to BoHV-4 (ELISA or PCR) (n = 2) and multiparous cows without metritis (n = 3). Seropositivity was higher in older animals and in the production lot. Three heifers which shed BoHV-4 after parturition resulted seronegative at first but eventually seroconverted. In the same vein, most heifers seroconverted after 6-10 months in the production lot (8/10). Multiparous cows shed virus by various routes: 13/14 (93 %) in vaginal secretions, 7/14 (50 %) in nasal exudates and 7/14 (50 %) in milk. However, in the other groups, shedding was only detected in vaginal swabs from the first week post-partum. Our study describes BoHV-4 shedding in field conditions. Seronegative animals may become horizontally infected when moved to a contaminated environment.