Wildebeest-Derived Malignant Catarrhal Fever: A Bovine Peripheral T Cell Lymphoma Caused by Cross-Species Transmission of Alcelaphine Gammaherpesvirus 1.

Gammaherpesviruses (γHVs) include viruses that can induce lymphoproliferative diseases and tumors. These viruses can persist in the long term in the absence of any pathological manifestation in their natural host. Alcelaphine gammaherpesvirus 1 (AlHV-1) belongs to the genus Macavirus and asymptomatically infects its natural host, the wildebeest (Connochaetes spp.). However, when transmitted to several susceptible species belonging to the order Artiodactyla, AlHV-1 is responsible for the induction of a lethal lymphoproliferative disease, named wildebeest-derived malignant catarrhal fever (WD-MCF). Understanding the pathogenic mechanisms responsible for the induction of WD-MCF is important to better control the risks of transmission and disease development in susceptible species. The aim of this review is to synthesize the current knowledge on WD-MCF with a particular focus on the mechanisms by which AlHV-1 induces the disease. We discuss the potential mechanisms of pathogenesis from viral entry into the host to the maintenance of viral genomes in infected CD8+ T lymphocytes, and we present current hypotheses to explain how AlHV-1 infection induces a peripheral T cell lymphoma-like disease.

Hemorrhagic bowel syndrome in dairy cattle: Gross, histological, and microbiological characterization.

Hemorrhagic bowel syndrome (HBS) is a sporadic and fatal disease of predominantly lactating dairy cattle, characterized by segmental hemorrhage and luminal clot formation in the small intestine. Although, Clostridium perfringens and Aspergillus fumigatus have been associated with HBS, the pathogenesis and cause are currently unknown. In this study, 18 naturally occurring cases of HBS (7 necropsied immediately following euthanasia, 11 with 12-48 hour postmortem intervals) were investigated to characterize the pathology and the intestinal microbiome. Hemorrhagic bowel syndrome was characterized by a single small-intestinal, intramucosal hematoma with dissection of the lamina muscularis mucosae. In most cases necropsied immediately after euthanasia (4/7), the intestinal mucosa proximal to the hematoma contained 9 to 14, dispersed, solitary or clustered, erosions or lacerations measuring 4 to 45 mm. In 77% (37/48) of these mucosal lesions, microscopic splitting of the lamina muscularis mucosae comparable to the hematoma was present. These findings suggest the intramucosal hematoma to originate from small mucosal erosions through dissecting hemorrhage within the lamina muscularis mucosae. No invasive fungal growth was observed in any tissue. Bacteriological cultivation and nanopore sequencing showed a polymicrobial population at the hematoma and unaffected intestine, with mostly mild presence of C perfringens at selective culture. Gross and microscopic lesions, as well as the culture and sequencing results, were not in support of involvement of C perfringens or A fumigatus in the pathogenesis of HBS.

Epidemiology of Bovine Hemorrhagic Bowel Syndrome in Belgium and The Netherlands.

Hemorrhagic bowel syndrome (HBS) is a poorly understood, sporadic and often fatal disease in cattle. Although, HBS is considered an important disease in dairy cattle, epidemiological data is largely lacking. This study describes the epidemiology of HBS in Belgium and the Netherlands, based on necropsy records from 2009 to 2022, and reports characteristics from 27 cows and 35 dairy operations with HBS, gathered through a survey. The annual incidence of HBS has a significantly increasing trend both at cow and herd level, with incidence above 3.2% in necropsied mature dairy cattle in the most recent years. Estimated herd-level incidence in the Netherlands was double the estimated incidence in Belgium, which might be explained by higher herd size in the Netherlands. Occurrence of HBS was most prevalent in fall, being 40.1% higher than the average of the other seasons. In 35 Flemish (Belgian) surveyed dairy herds with HBS, manifestation of HBS was mostly as solitary cases, and if multiple cases occurred, the time interval was highly variable. In addition, the majority of cows with HBS (61.1%; 16/26) were in more than 100 days lactation. In conclusion, HBS is an important and possibly emerging disease in dairy cattle in Belgium and the Netherlands.

Species-Specific Humoral Immune Responses in Sheep and Goats upon Small Ruminant Lentivirus Infections Inversely Correlate with Protection against Virus Replication and Pathological Lesions.

Maedi-Visna-like genotype A strains and Caprine arthritis encephaltis-like genotype B strains are small ruminant lentiviruses (SRLV) which, for incompletely understood reasons, appear to be more virulent in sheep and goats, respectively. A 9-month in vivo infection experiment using Belgian genotype A and B SRLV strains showed that almost all homologous (genotype A in sheep; genotype B in goats) and heterologous (genotype A in goats; genotype B in sheep) intratracheal inoculations resulted in productive infection. No differences in viremia and time to seroconversion were observed between homologous and heterologous infections. Higher viral loads and more severe lesions in the mammary gland and lung were however detected at 9 months post homologous compared to heterologous infection which coincided with strongly increased IFN-γ mRNA expression levels upon homologous infection. Pepscan analysis revealed a strong antibody response against immune-dominant regions of the capsid and surface proteins upon homologous infection, which was absent after heterologous infection. These results inversely correlated with protection against virus replication in target organs and observed histopathological lesions, and thus require an in-depth evaluation of a potential role of antibody dependent enhancement in SRLV infection. Finally, no horizontal intra- and cross-species SRLV transmission to contact animals was detected.

Nationwide Screening for Bee Viruses and Parasites in Belgian Honey Bees.

The health of honey bees is threatened by multiple factors, including viruses and parasites. We screened 557 honey bee (Apis mellifera) colonies from 155 beekeepers distributed all over Belgium to determine the prevalence of seven widespread viruses and two parasites (Varroa sp. and Nosema sp.). Deformed wing virus B (DWV-B), black queen cell virus (BQCV), and sacbrood virus (SBV) were highly prevalent and detected by real-time RT-PCR in more than 95% of the colonies. Acute bee paralysis virus (ABPV), chronic bee paralysis virus (CBPV) and deformed wing virus A (DWV-A) were prevalent to a lower extent (between 18 and 29%). Most viruses were only present at low or moderate viral loads. Nevertheless, about 50% of the colonies harbored at least one virus at high viral load (>107 genome copies/bee). Varroa mites and Nosema sp. were found in 81.5% and 59.7% of the honey bee colonies, respectively, and all Nosema were identified as Nosema ceranae by real time PCR. Interestingly, we found a significant correlation between the number of Varroa mites and DWV-B viral load. To determine the combined effect of these and other factors on honey bee health in Belgium, a follow up of colonies over multiple years is necessary.

Evaluation of mycobacteria-specific gamma interferon and antibody responses before and after a single intradermal skin test in cattle naturally exposed to M. avium subsp. paratuberculosis and experimentally infected with M. bovis.

This study reports on the diagnostic potential of IFN-γ release assays and serology for Mycobacterium bovis in six naturally M. avium subsp. paratuberculosis (Map) exposed bulls of which four were intratracheally infected with a Belgian field strain of M. bovis. Heparinized blood, serum and fecal samples were collected at regular time intervals for mycobacteria-specific IFN-γ release assays, antibody analysis and for Map culture respectively. Single intradermal skin test (SIT) with bovine tuberculin (PPD-B) was performed on day 115 and animals were sacrificed on day 133 after M. bovis infection. Organs were collected and stored for histopathological examination, modified Ziehl-Neelsen staining and bacteriological analysis of M. bovis and Map by culture and RT-PCR. Prior to infection five animals showed positive IFN-γ responses to avian PPD (PPD-A) and four were positive in Map PCR (IS900) on faeces. Three M. bovis infected animals reacted as early as day 14 with sustained higher PPD-B than PPD-A specific IFN-γ responses, whereas the fourth animal (with the strongest PPD-A response prior to infection) showed sustained higher PPD-B specific IFN-γ levels only a day 56 after infection. Two of the infected animals had a sustained positive IFN-γ response to the ESAT-6/CFP-10/TB7.7 (QuantiFERON®-TB Gold) peptide cocktail as early as day 14, among which the animal with the initial high PPD-A response. Later during infection, positive responses were found to ESAT-6 peptides in three infected bulls and to CFP-10 peptides in all four infected bulls. One of the control animals reacted intermittently to the ESAT-6/CFP10/TB7.7 cocktail. Prior to SIT, weak but positive MPB83/MBP70 specific antibody responses were detected in two of the infected bulls. All four M. bovis infected bulls reacted with a positive skin test and showed, as reported by others, increased mycobacteria specific IFN-γ production and increased positive responses in MPB83/MBP70 specific serology after SIT. At autopsy, M. bovis lesions were detected in all four experimentally infected bulls. Our results indicate that in Map exposed cattle, M. bovis diagnosis using IFN-γ assays needs a combination of PPD-B/A and ESAT-6/CFP10 for early and optimal sensitivity and that sensitivity of MPB83/MBP70 serodiagnosis is dramatically increased by prior skin testing. Map exposure did not interfere with the development of SIT in M. bovis infected animals, but resulted in a false positive M. bovis specific IFN-γ and antibody response after SIT in one of the two control animals (which remained negative in skin-test).

Short-term biodistribution and clearance of intravenously administered silica nanoparticles.

Recently, concerns have been raised about potential adverse effects of synthetic amorphous silica, commonly used as food additive (E551), since silica nanoparticles have been detected in food containing E551. We examined the biodistribution and excretion in female Sprague-Dawley rats of NM-200, a well characterized nanostructured silica representative for food applications. A single intravenous injection of NM-200 was applied at a dose of 20 mg/kgbw, followed by autopsy after 6 and 24 h. The main organs where silicon accumulated were liver and spleen. The silicon concentration significantly decreased in spleen between 6 and 24 h. In liver the tendency was the same but the effect was not significant. This could be due to clearance of the spleen to the liver via the splenic vein, while liver clearance takes more time due to hepatic processing and biliary excretion. In treated animals the liver showed in addition a prominent increase of macrophages between both evaluation moments. Within the first 24 h, silicon was mainly excreted through urine. Further studies are necessary to evaluate the toxicokinetics of different types of silica nanomaterials at lower exposure doses in order to be able to predict kinetics and toxicity of silica nanoparticles depending on their physicochemical characteristics.

First TBEV serological screening in Flemish wild boar.

In the frame of a Flemish wildlife surveillance in 2013, a serological screening was performed on sera from wild boar (Sus scrofa; n=238) in order to detect tick-borne encephalitis virus (TBEV)-specific antibodies. Neutralising antibodies were titrated with a seroneutralisation test (SNT), using two cut-off titres (1/10-1/15). Seven wild boars were found TBEV-seropositive and showed moderate (>1/15) to high (>1/125) SNT-titres; three individuals had borderline results (1/10-1/15). This study demonstrated the presence of TBEV-specific antibodies in wild boar and highlighted potential TBEV-foci in Flanders. Additional surveillance including direct virus testing is now recommended.

Serologic screening for 13 infectious agents in roe deer (Capreolus capreolus) in Flanders.

INTRODUCTION: In order to investigate the role of roe deer in the maintenance and transmission of infectious animal and human diseases in Flanders, we conducted a serologic screening in 12 hunting areas. MATERIALS AND METHODS: Roe deer sera collected between 2008 and 2013 (n=190) were examined for antibodies against 13 infectious agents, using indirect enzyme-linked immunosorbent assay, virus neutralisation, immunofluorescence, or microagglutination test, depending on the agent. RESULTS AND DISCUSSION: High numbers of seropositives were found for Anaplasma phagocytophilum (45.8%), Toxoplasma gondii (43.2%) and Schmallenberg virus (27.9%), the latter with a distinct temporal distribution pattern following the outbreak in domestic ruminants. Lower antibody prevalence was found for Chlamydia abortus (6.7%), tick-borne encephalitis virus (5.1%), Neospora caninum (4.8%), and Mycobacterium avium subsp paratuberculosis (4.1%). The lowest prevalences were found for Leptospira (1.7%), bovine viral diarrhoea virus 1 (1.3%), and Coxiella burnetii (1.2%). No antibodies were found against Brucella sp., bovine herpesvirus 1, and bluetongue virus. A significant difference in seroprevalence between ages (higher in adults >1 year) was found for N. caninum. Four doubtful reacting sera accounted for a significant difference in seroprevalence between sexes for C. abortus (higher in females). CONCLUSIONS: Despite the more intensive landscape use in Flanders, the results are consistent with other European studies. Apart from maintaining C. abortus and MAP, roe deer do not seem to play an important role in the epidemiology of the examined zoonotic and domestic animal pathogens. Nevertheless, their meaning as sentinels should not be neglected in the absence of other wild cervid species.

Autochthonous tick-borne encephalitis virus-seropositive cattle in Belgium: a risk-based targeted serological survey.

The risk of tick-borne encephalitis virus (TBEV) introduction into Belgium remains high, and the presence of infected wildlife in Belgium is suspected. Domestic animals can serve as excellent sentinels for TBEV surveillance to install an early warning surveillance component for this emerging zoonotic disease of public health importance. In a targeted, risk-based and cross-sectional sampling design, serological screening was performed on Belgian cattle (n=650), selected from the 2010 Belgian national cattle surveillance serum bank. All samples were subjected to a gold standard TBEV seroneutralization test (SNT), based on the rapid fluorescent focus inhibition test (RFFIT) protocol. Seventeen bovines were seropositive (titer >1/15) and six had borderline results (1/10 < titer < 1/15). The accuracy of the RFFIT-SNT was confirmed in a mouse inoculation test. The overall bovine TBEV seroprevalence in the targeted area was estimated between 2.61% and 4.29%. This confirms for the first time the presence of infected foci in Belgium. Further surveillance in cattle, other sentinels, ticks, and humans at risk is recommended to further determine the location and size of endemic foci and the risk for public health.

Vaccination of mice using the West Nile virus E-protein in a DNA prime-protein boost strategy stimulates cell-mediated immunity and protects mice against a lethal challenge.

West Nile virus (WNV) is a mosquito-borne flavivirus that is endemic in Africa, the Middle East, Europe and the United States. There is currently no antiviral treatment or human vaccine available to treat or prevent WNV infection. DNA plasmid-based vaccines represent a new approach for controlling infectious diseases. In rodents, DNA vaccines have been shown to induce B cell and cytotoxic T cell responses and protect against a wide range of infections. In this study, we formulated a plasmid DNA vector expressing the ectodomain of the E-protein of WNV into nanoparticles by using linear polyethyleneimine (lPEI) covalently bound to mannose and examined the potential of this vaccine to protect against lethal WNV infection in mice. Mice were immunized twice (prime--boost regime) with the WNV DNA vaccine formulated with lPEI-mannose using different administration routes (intramuscular, intradermal and topical). In parallel a heterologous boost with purified recombinant WNV envelope (E) protein was evaluated. While no significant E-protein specific humoral response was generated after DNA immunization, protein boosting of DNA-primed mice resulted in a marked increase in total neutralizing antibody titer. In addition, E-specific IL-4 T-cell immune responses were detected by ELISPOT after protein boost and CD8(+) specific IFN-γ expression was observed by flow cytometry. Challenge experiments using the heterologous immunization regime revealed protective immunity to homologous and virulent WNV infection.

Effect of a DIVA vaccine with and without in-feed use of coated calcium-butyrate on transmission of Salmonella Typhimurium in pigs.

BACKGROUND: For satisfactory Salmonella control, good biosecurity along the pork production chain is crucial, although additional control measures on-farm need to be considered. This study evaluated the effect of two potential control measures against the spread of Salmonella Typhimurium via a transmission experiment with 56 piglets (3-15 weeks of age): two groups were orally vaccinated with 107 - 108 Colony Forming Units (CFU)/2 mL of a new attenuated Salmonella Typhimurium vaccine 'Salmoporc-∆rfaJ' with DIVA capacities (Differentiation between Infected and Vaccinated Animals) (n = 2x16); the feed of one group was additionally supplemented with coated calcium-butyrate salt. Two weeks post vaccination, four pigs per group were orally challenged with 107 CFU/2 mL of a Salmonella Typhimurium strain 112910a. Both groups were compared with a positive (challenged/untreated; n = 16) and negative (unchallenged/untreated; n = 8) control group. Until six weeks post challenge, blood, individual faecal and finally tissue samples were examined. Adjusted transmission ratios 'Ra' were estimated, based on the challenge strain isolation from faecal and/or tissue samples. RESULTS: In both intervention groups, Ra values were lower compared to the positive control group, although these differences were not significant. In the combination group DIVA vaccine + coated butyrate, less non-challenged contact animals excreted Salmonella and less tissue samples were found Salmonella-positive in all pigs, when compared to the positive control group (P < 0.01). Seroconversion was detected in none of the vaccinated animals before challenge, when using a commercial lipopolysaccharide (LPS) ELISA targeting only Salmonella O-antigens, deleted in this vaccine. This was in contrast with an in-house whole-cell ELISA testing for various Salmonella antigens, in which Salmonella-specific antibodies were found pre-challenge in the serum of the vaccinated pigs. CONCLUSIONS: Both interventions showed a limited, non-significant reduction of Salmonella transmission between piglets. They may have applications towards Salmonella control and surveillance. Firstly, the number of Salmonella excreting contact pigs was significantly lower in the group where vaccination was combined with coated calcium-butyrate salt in the feed; secondly, the new vaccine confirmed its DIVA capacity. Therefore, these interventions merit further research with larger sample sizes, to optimize their use for Salmonella programmes.

Coupling of drug containing liposomes to microbubbles improves ultrasound triggered drug delivery in mice.

Local extravasation and triggered drug delivery by use of ultrasound and microbubbles is a promising strategy to target drugs to their sites of action. In the past we have developed drug loaded microbubbles by coupling drug containing liposomes to the surface of microbubbles. Until now the advantages of this drug loading strategy have only been demonstrated in vitro. Therefore, in this paper, microbubbles with indocyanine green (ICG) containing liposomes at their surface or a mixture of ICG-liposomes and microbubbles was injected intravenously in mice. Immediately after injection the left hind leg was exposed to 1 MHz ultrasound and the ICG deposition was monitored 1, 4 and 7 days post-treatment by in vivo fluorescence imaging. In mice that received the ICG-liposome loaded microbubbles the local ICG deposition was, at each time point, about 2-fold higher than in mice that received ICG-liposomes mixed with microbubbles. We also showed that the perforations in the blood vessels allow the passage of ICG-liposomes up to 5h after microbubble and ultrasound treatment. An increase in tissue temperature to 41°C was observed in all ultrasound treated mice. However, ultrasound tissue heating was excluded to cause the local ICG deposition. We concluded that coupling of drug containing liposomes to microbubbles may increase ultrasound mediated drug delivery in vivo.

Laboratory activities involving transmissible spongiform encephalopathy causing agents: risk assessment and biosafety recommendations in Belgium.

Since the appearance in 1986 of epidemic of bovine spongiform encephalopathy (BSE), a new form of neurological disease in cattle which also affected human beings, many diagnostic and research activities have been performed to develop detection and therapeutic tools. A lot of progress was made in better identifying, understanding and controlling the spread of the disease by appropriate monitoring and control programs in European countries. This paper reviews the recent knowledge on pathogenesis, transmission and persistence outside the host of prion, the causative agent of transmissible spongiform encephalopathies (TSE) in mammals with a particular focus on risk (re)assessment and management of biosafety measures to be implemented in diagnostic and research laboratories in Belgium. Also, in response to the need of an increasing number of European diagnostic laboratories stopping TSE diagnosis due to a decreasing number of TSE cases reported in the last years, decontamination procedures and a protocol for decommissioning TSE diagnostic laboratories is proposed.

Cholesterol granuloma associated with otitis media and leptomeningitis in a cat due to a Streptococcus canis infection.

Cholesterol granuloma in the middle ear is a pathological condition often associated with otitis media in humans. Cholesterol granulomas in cats are rarely described. To our knowledge, this is the first report of middle ear cholesterol granuloma in a cat, associated with otitis media and leptomeningitis due to a Streptococcus canis septicemia.

Granulome à cholestérol associé à une otite moyenne et à une leptoméningite chez un chat causé par une infection parStreptococcus canis. Un granulome à cholestérol dans l'oreille moyenne est une affection pathologique souvent associée à l'otite moyenne chez les humains. Les granulomes à cholestérol chez les chats sont rarement décrits. À notre connaissance, il s'agit du premier rapport d'un granulome à cholestérol de l'oreille moyenne chez un chat, associé à l'otite moyenne et à la letpoméningite, causé par une septicémie à Streptococcus canis.(Traduit par Isabelle Vallières).

An essential role for γ-herpesvirus latency-associated nuclear antigen homolog in an acute lymphoproliferative disease of cattle.

Wildebeests carry asymptomatically alcelaphine herpesvirus 1 (AlHV-1), a γ-herpesvirus inducing malignant catarrhal fever (MCF) to several ruminant species (including cattle). This acute and lethal lymphoproliferative disease occurs after a prolonged asymptomatic incubation period after transmission. Our recent findings with the rabbit model indicated that AlHV-1 infection is not productive during MCF. Here, we investigated whether latency establishment could explain this apparent absence of productive infection and sought to determine its role in MCF pathogenesis. First, whole-genome cellular and viral gene expression analyses were performed in lymph nodes of MCF-developing calves. Whereas a severe disruption in cellular genes was observed, only 10% of the entire AlHV-1 genome was expressed, contrasting with the 45% observed during productive infection in vitro. In vivo, the expressed viral genes included the latency-associated nuclear antigen homolog ORF73 but none of the regions known to be essential for productive infection. Next, genomic conformation analyses revealed that AlHV-1 was essentially episomal, further suggesting that MCF might be the consequence of a latent infection rather than abortive lytic infection. This hypothesis was further supported by the high frequencies of infected CD8(+) T cells during MCF using immunodetection of ORF73 protein and single-cell RT-PCR approaches. Finally, the role of latency-associated ORF73 was addressed. A lack of ORF73 did not impair initial virus replication in vivo, but it rendered AlHV-1 unable to induce MCF and persist in vivo and conferred protection against a lethal challenge with a WT virus. Together, these findings suggest that a latent infection is essential for MCF induction.

M2e-displaying virus-like particles with associated RNA promote T helper 1 type adaptive immunity against influenza A.

The ectodomain of influenza A matrix protein 2 (M2e) is a candidate for a universal influenza A vaccine. We used recombinant Hepatitis B core antigen to produce virus-like particles presenting M2e (M2e-VLPs). We produced the VLPs with and without entrapped nucleic acids and compared their immunogenicity and protective efficacy. Immunization of BALB/c mice with M2e-VLPs containing nucleic acids induced a stronger, Th1-biased antibody response compared to particles lacking nucleic acids. The former also induced a stronger M2e-specific CD4(+) T cell response, as determined by ELISPOT. Mice vaccinated with alum-adjuvanted M2e-VLPs containing the nucleic acid-binding domain were better protected against influenza A virus challenge than mice vaccinated with similar particles lacking this domain, as deduced from the loss in body weight following challenge with X47 (H3N2) or PR/8 virus. Challenge of mice that had been immunized with M2e-VLPs with or without nucleic acids displayed significantly lower mortality, morbidity and lung virus titers than control-immunized groups. We conclude that nucleic acids present in M2e-VLPs correlate with improved immune protection.

Trends in genotype frequency resulting from breeding for resistance to classical scrapie in Belgium (2006 ˜ 2011).

In sheep, susceptibility to scrapie is mainly determined by codons 136, 154, and 171 of the PRNP gene. Five haplotypes are usually present (ARR, ARQ, ARH, AHQ, and VRQ). The ARR haplotype confers the greatest resistance to classical scrapie while VRQ renders animals most susceptible. In 2004, the European Union implemented a breeding program that promotes selection of the ARR haplotype while reducing the incidence of VRQ. From 2006 to 2011 in Belgium, frequency for the ARR/ARR genotypes increased from 38.3% to 63.8% (n = 6,437), the ARQ haplotype diminished from 21.1% to 12.9%, and the VRQ haplotype decreased from 2.0% to 1.7%. The status of codon 141, a determinant for atypical scrapie, was also evaluated. Out of 27 different breeds (n = 5,163), nine were abundant. The ARR/ARR frequency increased in eight of these nine major breeds. The selection program has had a major impact on the ARR haplotype frequency in Belgium. However, the occurrence of atypical scrapie represents a critical point for this program that warrants the continuous monitoring of scrapie. Additionally, genotype frequencies among the breeds varied greatly. Texel, a breed that is common in Belgium, can still be selected for due to its average ARR frequency.

Infectivity of rabies virus-exposed macrophages.

Rabies virus distributes widely in infected mice, including lymphoid tissues and spleen macrophages. The infection characteristics in murine macrophages and the infectivity of virus-exposed macrophages were examined upon inoculation in mice. In vitro, Mf4/4 spleen macrophages supported mild virus production (10(4)-fold less than neuroblastoma), with formation of typical virions. Bone marrow-derived macrophages (BMM) were most efficient to capture virus, but new virus production was not detected. Virus-induced cell death was significantly stronger in BMM, which might have eliminated BMM with productive infection. Still, viral RNA remained detectable in the remaining BMM for at least 4 weeks. Injection of in vitro-infected Mf4/4 in the nose or brain proved efficient to propagate infection in mice, even when cells were pre-incubated with neutralizing antibodies. Surprisingly, injection of ex-vivo-infected BMM in the brain also led to lethal infection in 8 out of 12 mice. Injection of infected Mf4/4 in the muscle mostly favoured a protective antibody response. Despite that macrophages are less fit to support virus production, they can still act as a source of infectious virus upon transfer in mice. This may be relevant for screening donor organs/cells, for which RT-PCR should be preferred over the traditional antigen or virus isolation assays.

Spatiotemporal dynamics of Puumala hantavirus in suburban reservoir rodent populations.

The transmission of pathogens to susceptible hosts is dependent on the vector population dynamics. In Europe, bank voles (Myodes glareolus) carry Puumala hantavirus, which causes nephropathia epidemica (NE) in humans. Fluctuations in bank vole populations and epidemics in humans are correlated but the main factors influencing this relationship remain unclear. In Belgium, more NE cases are reported in spring than in autumn. There is also a higher incidence of human infections during years of large vole populations. This study aimed to better understand the link between virus prevalence in the vector, vole demography, habitat quality, and human infections. Three rodent populations in different habitats bordering Brussels city, Belgium, were studied for two years. The seroprevalence in voles was influenced first by season (higher in spring), then by vole density, vole weight (a proxy for age), and capture site but not by year or sex. Moreover, voles with large maximal distance between two captures had a high probability for Puumala seropositivity. Additionally, the local vole density showed similar temporal variations as the number of NE cases in Belgium. These results showed that, while season was the main factor influencing vole seroprevalence, it was not sufficient to explain human risks. Indeed, vole density and weight, as well as the local habitat, were essential to understanding the interactions in these host-pathogen dynamics. This can, in turn, be of importance for assessing the human risks.