Computed Tomographic and Histopathologic Studies of Lung Function Immediately Post Natum in Canine Neonates.

BACKGROUND: The lung tissue in newborn canine neonates is still in a morphologically and functionally immature, canalicular-saccular stage. True alveoli are only formed postnatally. The aim of this study was to analyze the spatial and temporal development of the ventilation of the lung tissue in vital canine neonates during the first 24 h post natum (p.n.). METHODS: Forty pups (birth weight Ø 424 g ± 80.1 g) from three litters of large dog breeds (>20 kg live weight) were included in the studies. Thirty-three pups (29 vital, 2 vitally depressed, 2 stillborn neonates) originated from controlled, uncomplicated births (n = 3); moreover, six stillborn pups as well as one prematurely deceased pup were birthed by other dams with delivery complications. Computed tomography (CT) was used in 39 neonates, and histopathologic tissue classification techniques (HALO) were used in 11 neonates (eight stillborn and three neonates died early post natum, respectively) to quantify the degree of aerated neonatal lung tissue. RESULTS: It was shown that, in vital born pups, within the first 10 min p.n., the degree of ventilation reached mean values of -530 (±114) Hounsfield units (HU) in the dorsal and -453.3 (±133) HU in the ventral lung area. This is about 75-80% of the final values obtained after 24 h p.n. for dorsal -648.0 (±89.9) HU and ventral quadrants -624.7 (±76.8) HU. The dorsal lung areas were always significantly better ventilated than the ventral regions (p = 0.0013). CT as well as histopathology are suitable to clearly distinguish the nonventilated lungs of stillborns from neonates that were initially alive after surviving neonatal respiratory distress syndrome but who died prematurely (p = 0.0398). CONCLUSION: The results of this study are clinically relevant since the lung tissue of canine neonates presents an aeration profile as early as 10 min after birth and continues progressively, with a special regard to the dorsal lung areas. This is the basis for resuscitation measures that should be performed, preferably with the pup in the abdomen-chest position.

Phosphatidylinositolmannoside vaccination induces lipid-specific Th1-responses and partially protects guinea pigs from Mycobacterium tuberculosis challenge.

The concept of donor-unrestricted T cells (DURTs) comprises a heterogeneity of lymphoid cells that respond to an abundance of unconventional epitopes in a non-MHC-restricted manner. Vaccinologists strive to harness this so far underexplored branch of the immune system for new vaccines against tuberculosis. A particular division of DURTs are T cells that recognize their cognate lipid antigen in the context of CD1-molecules. Mycobacteria are characterized by a particular lipid-rich cell wall. Several of these lipids have been shown to be presented to T cells via CD1b-molecules. Guinea pigs functionally express CD1b and are hence an appropriate small animal model to study the role of CD1b-restricted, lipid-specific immune responses. In the current study, guinea pigs were vaccinated with BCG or highly-purified, liposome-formulated phosphatidylinositol-hexa-mannoside (PIM6) to assess the effect of CD1-restricted DURTs on the course of infection after virulent Mycobacterium tuberculosis (Mtb) challenge. Robust PIM6-specific T cell-responses were observed both after BCG- and PIM6-vaccination. The cellular response was significantly reduced in the presence of monoclonal, CD1b-blocking antibodies, indicating that a predominant part of this reactivity was CD1b-restricted. When animals were challenged with Mtb, BCG- and PIM6-vaccinated animals showed significantly reduced pathology, smaller necrotic granulomas in lymph node and spleen and reduced bacterial loads. While BCG conferred an almost sterile protection in this setting, compared to control animals' lesions were reduced roughly by two thirds in PIM6-vaccinated. Comprehensive histological and transcriptional analyses in the draining lymph node revealed that protected animals showed reduced transcription-levels of inflammatory cyto- and chemokines and higher levels of CD1b-expression on professional antigen cells compared to controls. Although BCG as a comparator induced by far stronger effects, our observations in the guinea pig model suggest that CD1b-restricted, PIM6-reactive DURTs contribute to immune-mediated containment of virulent Mtb.

Field survey and molecular characterization of apicomplexan parasites in small mammals from military camps in Afghanistan.

Small mammals are an important reservoir for causative agents of numerous infectious diseases, including zoonotic and vector-borne diseases. The occurrence of these pathogens represents a regional but permanent threat for humans and animals in general and might especially weaken military personnel and companion animals in abroad missions. In our study, small mammals collected in military camps in Afghanistan (Feyzabad, Mazar-e Sharif, and Kunduz) were investigated for the presence of apicomplexans using histopathology and molecular methods. For this purpose, well-established and newly developed real-time PCR assays were applied. A high prevalence was detected not only in house mice (Mus musculus), but also in shrews (Crocidura cf. suaveolens) and grey dwarf hamsters (Cricetulus migratorius). The molecular characterization based on the 18S rRNA gene revealed a close relationship to a cluster of Hepatozoon sp. detected in voles of the genus Microtus. Hepatozoon canis DNA was detected in one house mouse as well as in two Rhipicephalus ticks from a dog puppy. In addition, around 5% of the house mice were found to be infected with far related adeleorinids showing the highest sequence identity of 91.5% to Klossiella equi, the only published Klossiella sequence at present. For their better phylogenetic characterization, we conducted metagenomics by sequencing of two selected samples. The resulting 18S rRNA gene sequences have a length of about 2400 base pairs including an insertion of about 500 base pairs and are 100% identical to each other. Histopathology together with organ tropism and detection rates verified this sequence as of Klossiella muris. In conclusion, we documented naturally occurring protozoan stages and the additional taxonomic characterization of a well-known commensal in mice by applying a combination of different approaches. The study is of medical, social, and biological importance for ensuring human and animal health in military camps and also stresses the required awareness for the potential risk of zoonoses.

Tropism of Puumala orthohantavirus and Endoparasite Coinfection in the Bank Vole Reservoir.

In Europe, most cases of human hantavirus disease are caused by Puumala orthohantavirus (PUUV) transmitted by bank voles (Clethrionomys glareolus, syn. Myodes glareolus), in which PUUV causes inconspicuous infection. Little is known about tropism and endoparasite coinfections in PUUV-infected reservoir and spillover-infected rodents. Here, we characterized PUUV tropism, pathological changes and endoparasite coinfections. The voles and some non-reservoir rodents were examined histologically, immunohistochemically, by in situ hybridization, indirect IgG enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction. PUUV RNA and anti-PUUV antibodies were detected simultaneously in a large proportion of the bank voles, indicating persistent infection. Although PUUV RNA was not detected in non-reservoir rodents, the detection of PUUV-reactive antibodies suggests virus contact. No specific gross and histological findings were detected in the infected bank voles. A broad organ tropism of PUUV was observed: kidney and stomach were most frequently infected. Remarkably, PUUV was detected in cells lacking the typical secretory capacity, which may contribute to the maintenance of virus persistence. PUUV-infected wild bank voles were found to be frequently coinfected with Hepatozoon spp. and Sarcocystis (Frenkelia) spp., possibly causing immune modulation that may influence susceptibility to PUUV infection or vice versa. The results are a prerequisite for a deeper understanding of virus-host interactions in natural hantavirus reservoirs.

Pharmacokinetics and Efficacy of the Benzothiazinone BTZ-043 against Tuberculous Mycobacteria inside Granulomas in the Guinea Pig Model.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is the world's leading cause of mortality from a single bacterial pathogen. With increasing frequency, emergence of drug-resistant mycobacteria leads to failures of standard TB treatment regimens. Therefore, new anti-TB drugs are urgently required. BTZ-043 belongs to a novel class of nitrobenzothiazinones, which inhibit mycobacterial cell wall formation by covalent binding of an essential cysteine in the catalytic pocket of decaprenylphosphoryl-ß-d-ribose oxidase (DprE1). Thus, the compound blocks the formation of decaprenylphosphoryl-ß-d-arabinose, a precursor for the synthesis of arabinans. An excellent in vitro efficacy against M. tuberculosis has been demonstrated. Guinea pigs are an important small-animal model to study anti-TB drugs, as they are naturally susceptible to M. tuberculosis and develop human-like granulomas after infection. In the current study, dose-finding experiments were conducted to establish the appropriate oral dose of BTZ-043 for the guinea pig. Subsequently, it could be shown that the active compound was present at high concentrations in Mycobacterium bovis BCG-induced granulomas. To evaluate its therapeutic effect, guinea pigs were subcutaneously infected with virulent M. tuberculosis and treated with BTZ-043 for 4 weeks. BTZ-043-treated guinea pigs had reduced and less necrotic granulomas than vehicle-treated controls. In comparison to the vehicle controls a highly significant reduction of the bacterial burden was observed after BTZ-043 treatment at the site of infection and in the draining lymph node and spleen. Together, these findings indicate that BTZ-043 holds great promise as a new antimycobacterial drug.

Toxoplasma gondii and Alternaria sp.: An Original Association in an Immunosuppressed Dog with Persistent Skin Lesions.

Dogs and cats may suffer from a variety of diseases, mainly immune mediated, that require the administration of immunosuppressive drugs. Such therapies can cause adverse effects either by the toxicity of the drugs or as a consequence of immune suppression and associated opportunistic infections. Here we present an, yet unknown, association of Toxoplasma gondii and Alternaria fungus, within cutaneous lesions in a dog under long-term immunosuppressive therapy. The diagnosis of such infections is laborious and not obvious at first glance, since the clinical signs of cutaneous toxoplasmosis, neosporosis or alternariosis are not specific. A further laboratory confirmation is needed. Therefore, we currently recommend that dogs and cats should undergo serologic testing for toxoplasmosis or neosporosis prior to immunosuppressive therapy and a regular dermatological evaluation during the immunosuppressive therapy.

Small Mammals as Reservoir for Zoonotic Agents in Afghanistan.

INTRODUCTION: Rodents and other small mammals can serve as reservoirs for a large number of zoonotic pathogens. A higher risk of infection with rodent-borne pathogens exists for humans with direct contact to rodents and/or their excretions, e.g., soldiers in operation areas. To date, little is known about endemic human pathogenic disease agents that are naturally associated with small mammals in Afghanistan. The aim of this study was to screen abundant rodents and insectivores collected from 2009 to 2012 in four field camps of the German Federal Armed Forces (Bundeswehr) in Northern Afghanistan for the presence of different pathogens. MATERIALS AND METHODS: Isolated nucleic acids from ear pinna were screened by real-time PCR for spotted fever group (SFG) rickettsiae and from liver samples for Francisella spp., Coxiella burnetii, Brucella spp., Yersinia pestis, and poxvirus. Chest cavity lavage (CCL) samples were tested for antibodies against SFG and typhus group (TG) rickettsiae, as well as against flaviviruses using an indirect immunofluorescence assay. RESULTS: Rickettsial DNA was detected in 7/750 (1%) ear pinna samples with one being identified as Rickettsia conorii. Antibodies against SFG rickettsiae were detected in 15.3% (n = 67/439) of the small mammals; positive samples were only from house mice (Mus musculus). Antibodies against TG rickettsiae were found in 8.2% (n = 36/439) of the samples, with 35 from house mice and one from gray dwarf hamster (Cricetulus migratorius). Flavivirus-reactive antibodies were detected in 2.3% (n = 10/439) of the investigated CCL samples; again positive samples were exclusively identified in house mice. All 199 investigated liver-derived DNA preparations were negative in the Francisella spp., C. burnetii, Brucella spp., Y. pestis, and poxvirus-specific PCRs. CONCLUSIONS: Further investigations will have to prove the potential value of rodents in army camps as sentinel animals.

Clinical, neuropathological, and immunological short- and long-term feature of a mouse model mimicking human herpes virus encephalitis.

Herpes simplex encephalitis (HSE) is one of the most serious diseases of the nervous system in humans. However, its pathogenesis is still only poorly understood. Although several mouse models of predominantly herpes simplex virus 1 (HSV-1) infections mimic different crucial aspects of HSE, central questions remain unanswered. They comprise the specific temporofrontal tropism, viral spread within the central nervous system (CNS), as well as potential molecular and immunological barriers that drive virus into latency while only rarely resulting in severe HSE. We have recently proposed an alternative mouse model by using a pseudorabies virus (PrV) mutant that more faithfully represents the striking features of human HSE: temporofrontal meningoencephalitis with few severely, but generally only moderately to subclinically affected mice as well as characteristic behavioral abnormalities. Here, we characterized this animal model using 6- to 8-week-old female CD-1 mice in more detail. Long-term investigation over 6 months consistently revealed a biphasic course of infection accompanied by recurring clinical signs including behavioral alterations and mainly mild meningoencephalitis restricted to the temporal and frontal lobes. By histopathological and immunological analyses, we followed the kinetics and spatial distribution of inflammatory lesions as well as the underlying cytokine expression in the CNS over 21 days within the acute phase of infection. Affecting the temporal lobes, the inflammatory infiltrate was composed of lymphocytes and macrophages showing a predominantly lymphocytic shift 15 days after infection. A strong increase was observed in cytokines CXCL10, CCL2, CCL5, and CXCL1 recruiting inflammatory cells to the CNS. Unlike the majority of infected mice, strongly affected animals demonstrated extensive temporal lobe edema, which is typically present in severe human HSE cases. In summary, these results support the validity of our animal model for in-depth investigation of HSE pathogenesis.

A double deletion prevents replication of the pestivirus bovine viral diarrhea virus in the placenta of pregnant heifers.

In contrast to wild type bovine viral diarhea virus (BVDV) specific double deletion mutants are not able to establish persistent infection upon infection of a pregnant heifer. Our data shows that this finding results from a defect in transfer of the virus from the mother animal to the fetus. Pregnant heifers were inoculated with such a double deletion mutant or the parental wild type virus and slaughtered pairwise on days 6, 9, 10 and 13 post infection. Viral RNA was detected via qRT-PCR and RNAscope analyses in maternal tissues for both viruses from day 6 p.i. on. However, the double deletion mutant was not detected in placenta and was only found in samples from animals infected with the wild type virus. Similarly, high levels of wild type viral RNA were present in fetal tissues whereas the genome of the double deletion mutant was not detected supporting the hypothesis of a specific inhibition of mutant virus replication in the placenta. We compared the induction of gene expression upon infection of placenta derived cell lines with wild type and mutant virus via gene array analysis. Genes important for the innate immune response were strongly upregulated by the mutant virus compared to the wild type in caruncle epithelial cells that establish the cell layer on the maternal side at the maternal-fetal interface in the placenta. Also, trophoblasts which can be found on the fetal side of the interface showed significant induction of gene expression upon infection with the mutant virus although with lower complexity. Growth curves recorded in both cell lines revealed a general reduction of virus replication in caruncular epithelial cells compared to the trophoblasts. Compared to the wild type virus this effect was dramtic for the mutant virus that reached only a TCID50 of 1.0 at 72 hours post infection.

Dry-fog decontamination of microbiological safety cabinets after activities with SARS-CoV-2: cycle development and process validation for dry fogging with peroxyacetic acid.

Background: Technical protection measures for laboratory activities involving biological agents include biological safety cabinets (BSC) that may be contaminated. In the case of diagnostic activities with SARS-CoV-2, this may also affect BSC that are operated at protection level 2; therefore, decontamination of all contaminated surfaces of the BSC may be required. In addition to fumigation with hydrogen peroxide (H2O2), dry fogging of H2O2-stabilized peroxyacetic acid (PAA) represents another alternative to fumigation with formalin. However, to prove their efficacy, these alternatives need to be validated for each model of BSC. Methods: The validation study was performed on 4 different BSCs of Class II A2 using the "Mini Dry Fog" system. Results: An aerosol concentration of 0.03% PAA and 0.15% H2O2 during a 30 min exposure was sufficient to inactivate SARS-CoV-2. Effective concentrations of 1.0% PAA and 5% H2O2 were required to decontaminate the custom-prepared biological indicators loaded with spores of G. stearothermophilus and deployed at 9 different positions in the BSC. Commercial spore carriers were easier to inactivate by a factor of 4, which corresponded to a reduction of 106 in all localizations. Conclusions: Dry fogging with PAA is an inexpensive, robust, and highly effective decontamination method for BSCs for enveloped viruses such as SARS-CoV-2. The good material compatibility, lack of a requirement for neutralization, low pH - which increases the range of efficacy compared to H2O2 fumigation - the significantly shorter processing time, and the lower costs argue in favor of this method.

After nasopharyngeal infection, foot-and-mouth disease virus serotype A RNA is shed in bovine milk without associated mastitis.

Foot-and-mouth disease (FMD) is a highly contagious aphthoviral infection of cloven-hoofed animals, inducing vesiculopustular stomatitis, pododermatitis, and thelitis. Vesicular fluid represents a major pathway of virus excretion, but bovine milk is another important source of virus shedding. We describe here the time course of FMD virus (FMDV) excretion in the milk and characterize associated lesions in the mammary gland. Three dairy cows were infected by nasopharyngeal instillation of FMDV and monitored over 12 d. Autopsy was performed at the end of the study, and specimens were collected for histopathology, IHC, and RT-qPCR. All 3 cows developed fever, drooling, vesiculopustular stomatitis, interdigital dermatitis, and thelitis. FMDV RNA was detectable in whole milk until the end of the trial, but only transiently in saliva, nasal secretions, and blood serum. Although histology confirmed vesiculopustular lesions in the oral and epidermal specimens, the mammary glands did not have unequivocal evidence of FMDV-induced inflammation. FMDV antigen was detectable in skin and oral mucosa, but not in the mammary gland, and FMDV RNA was detectable in 9 of 29 samples of squamous epithelia but only in 1 of 12 samples of mammary gland.

Effects of glyphosate residues and different concentrate feed proportions in dairy cow rations on hepatic gene expression, liver histology and biochemical blood parameters.

Glyphosate (GLY) is worldwide one of the most used active substances in non-selective herbicides. Although livestock might be orally exposed via GLY-contaminated feedstuffs, not much is known about possible hepatotoxic effects of GLY. As hepatic xenobiotic and nutrient metabolism are interlinked, toxic effects of GLY residues might be influenced by hepatic nutrient supply. Therefore, a feeding trial with lactating dairy cows was conducted to investigate effects of GLY-contaminated feedstuffs and different concentrate feed proportions (CFP) in the diets as tool for varying nutrient supply to the liver. For this, 61 German Holstein cows (207 ± 49 days in milk; mean ± standard deviation) were either fed a GLY-contaminated total mixed ration (TMR, GLY groups, mean GLY intake 122.7 µg/kg body weight/day) or control TMR (CON groups, mean GLY intake 1.2 µg/kg body weight/day) for 16 weeks. Additionally, both groups were further split into subgroups fed a lower (LC, 30% on dry matter basis) or higher (HC, 60% on dry matter basis) CFP resulting in groups CONHC (n = 16), CONLC (n = 16), GLYHC (n = 15), GLYLC (n = 14). Blood parameters aspartate aminotransferase, γ-glutamyltransferase, glutamate dehydrogenase, cholesterol, triglyceride, total protein, calcium, phosphorus, acetic acid and urea and histopathological evaluation were not influenced by GLY, whereas all mentioned parameters were at least affected by time, CFP or an interactive manner between time and CFP. Total bilirubin blood concentration was significantly influenced by an interaction between GLY and CFP with temporarily elevated concentrations in GLYHC, whereas the biological relevance remained unclear. Gene expression analysis indicated 167 CFP-responsive genes, while seven genes showed altered expression in GLY groups compared to CON groups. Since expression changes of GLY-responsive genes were low and liver-related blood parameters changed either not at all or only slightly, the tested GLY formulation was considered to have no toxic effects on the liver of dairy cows.

Technical and Anatomical Considerations for Reproducible Inactivation of Large Animal Carcasses by Steam Sterilization.

Introduction: The complete inactivation of infectious tissues of large animal carcasses is one of the most challenging tasks in high-containment facilities. Steam sterilization is a method frequently in use to achieve biological inactivation of liquid and solid waste. Objective: This study aims to highlight parameters most effective in creating reproducible cycles for steam sterilization of pig and calf carcasses. Methods: Two pigs or 1 calf were sterilized by running a liquid cycle (n = 3) at 121°C for at least 120 minutes in a pass-through autoclave. To assess the physical and biological parameters, temperature data loggers and biological indicators (BIs) with spores of Geobacillus stearothermophilus (ATCC 7953) were placed at defined positions within animal carcasses. After completion of each cycle, data loggers were analyzed and BIs were incubated for 7 days at 60°C. Results: Initial testing with an undissected pig carcass resulted in suboptimal temperatures at the tissue level with growth on 1 BI. After modifications of the used stainless-steel boxes and by placing the reference probe of the autoclave in the animal carcass, reproducible cycles could be created. A complete inactivation of BIs and a temperature profile of >121°C for at least 20 minutes could be achieved in almost all probed tissues. Conclusion: Only minor modifications in carcass preparation and the used sterilization equipment resulted in effective and reproducible cycles to inactivate large animal carcasses by using a steam autoclave.

Airborne Disinfection by Dry Fogging Efficiently Inactivates Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Mycobacteria, and Bacterial Spores and Shows Limitations of Commercial Spore Carriers.

Airborne disinfection of high-containment facilities before maintenance or between animal studies is crucial. Commercial spore carriers (CSC) coated with 106 spores of Geobacillus stearothermophilus are often used to assess the efficacy of disinfection. We used quantitative carrier testing (QCT) procedures to compare the sensitivity of CSC with that of surrogates for nonenveloped and enveloped viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), mycobacteria, and spores, to an aerosolized mixture of peroxyacetic acid and hydrogen peroxide (aPAA-HP). We then used the QCT methodology to determine relevant process parameters to develop and validate effective disinfection protocols (≥4-log10 reduction) in various large and complex facilities. Our results demonstrate that aPAA-HP is a highly efficient procedure for airborne room disinfection. Relevant process parameters such as temperature and relative humidity can be wirelessly monitored. Furthermore, we found striking differences in inactivation efficacies against some of the tested microorganisms. Overall, we conclude that dry fogging a mixture of aPAA-HP is highly effective against a broad range of microorganisms as well as material compatible with relevant concentrations. Furthermore, CSC are artificial bioindicators with lower resistance and thus should not be used for validating airborne disinfection when microorganisms other than viruses have to be inactivated.IMPORTANCE Airborne disinfection is not only of crucial importance for the safe operation of laboratories and animal rooms where infectious agents are handled but also can be used in public health emergencies such as the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. We show that dry fogging an aerosolized mixture of peroxyacetic acid and hydrogen peroxide (aPAA-HP) is highly microbicidal, efficient, fast, robust, environmentally neutral, and a suitable airborne disinfection method. In addition, the low concentration of dispersed disinfectant, particularly for enveloped viral pathogens such as SARS-CoV-2, entails high material compatibility. For these reasons and due to the relative simplicity of the procedure, it is an ideal disinfection method for hospital wards, ambulances, public conveyances, and indoor community areas. Thus, we conclude that this method is an excellent choice for control of the current SARS-CoV-2 pandemic.

Comparative Pathology of Pseudorabies in Different Naturally and Experimentally Infected Species-A Review.

The pseudorabies virus (PRV) is an alphaherpesvirus and the causative agent of Aujeszky's disease (AD). PRV infects a wide range of animal species including swine as the natural host as well as ruminants, carnivores, rodents and lagomorphs. In these species, except for the pig, PRV infection causes acute, severe disease, characterized by insatiable itching, and is always lethal. Horses, chickens and non-human primates have been shown to be largely resistant to PRV infection, while disease in humans is still controversial. PRV is a pantropic virus, which preferably invades neural tissue, but also infects epithelia of various organs, whereupon multisystemic lesions may result. Although AD is mainly associated with severe pruritus, also known as "mad itch", there are notable differences regarding infection route, clinical signs, viral distribution and lesion patterns in different animal species. In this comprehensive review, we will present clinico-pathologic findings from different species, which have been either shown to be susceptible to PRV infection or have been tested experimentally.

Canine papillomavirus type 16 associated to squamous cell carcinoma in a dog: virological and pathological findings.

Papillomaviruses (PVs) are circular double-stranded DNA virus belonging to Papillomaviridae family. During the infection cycle, PVs translate proteins that can influence cell growth and differentiation, leading to epidermal hyperplasia and papillomas (warts) or malignant neoplasms. Canis familiaris papillomaviruses (CPVs) have been associated with different lesions, such as oral and cutaneous papillomatosis, pigmented plaques, and squamous cell carcinomas (SCCs). Here, we report a clinical case of a mixed bred female dog with pigmented plaques induced by CPV16 (Chipapillomavirus 2) that progressed to in situ and invasive SCCs. Gross and histological findings were characterized, and the lesions were mainly observed in ventral abdominal region and medial face of the limbs. In situ hybridization (ISH) revealed strong nuclear hybridization signals in the neoplastic epithelial cells, as well as in the keratinocytes and koilocytes of the pigmented viral plaques. The full genome of the CPV16 recovered directly from the lesions was characterized, and the phylogenetic relationships were determined. The identification of oncoprotein genes (E5, E6, and E7) by high throughput sequencing (HTS) and their expected domains are suggestive of the malignant transformation by CPV16.

Peste Des Petits Ruminants Virus and Goatpox Virus Co-Infection in Goats.

Infection of small ruminants with peste des petits ruminants virus (PPRV) and goatpox virus (GTPV) are endemic and can have devastating economic consequences in Asia and Africa. Co-infection with these viruses have recently been reported in goats and sheep in Nigeria. In this study, we evaluated samples from the lips of a red Sokoto goat, and describe co-infection of keratinocytes with PPRV and GTPV using histopathology and transmission electron microscopy. Eosinophilic cytoplasmic inclusion bodies were identified histologically, and ultrastructural analysis revealed numerous large cytoplasmic viral factories containing poxvirus particles and varying sizes of smaller cytoplasmic inclusions composed of PPRV nucleocapsids. These histopathological and ultrastructural findings show concurrent infection with the 2 viruses for the first time as well as the detection of PPRV particles in epithelial cells of the mucocutaneous junction of the lip.

An improved animal model for herpesvirus encephalitis in humans.

Herpesviral encephalitis caused by Herpes Simplex Virus 1 (HSV-1) is one of the most devastating diseases in humans. Patients present with fever, mental status changes or seizures and when untreated, sequelae can be fatal. Herpes Simplex Encephalitis (HSE) is characterized by mainly unilateral necrotizing inflammation effacing the frontal and mesiotemporal lobes with rare involvement of the brainstem. HSV-1 is hypothesized to invade the CNS via the trigeminal or olfactory nerve, but viral tropism and the exact route of infection remain unclear. Several mouse models for HSE have been developed, but they mimic natural infection only inadequately. The porcine alphaherpesvirus Pseudorabies virus (PrV) is closely related to HSV-1 and Varicella Zoster Virus (VZV). While pigs can control productive infection, it is lethal in other susceptible animals associated with severe pruritus leading to automutilation. Here, we describe the first mutant PrV establishing productive infection in mice that the animals are able to control. After intranasal inoculation with a PrV mutant lacking tegument protein pUL21 and pUS3 kinase activity (PrV-ΔUL21/US3Δkin), nearly all mice survived despite extensive infection of the central nervous system. Neuroinvasion mainly occurred along the trigeminal pathway. Whereas trigeminal first and second order neurons and autonomic ganglia were positive early after intranasal infection, PrV-specific antigen was mainly detectable in the frontal, mesiotemporal and parietal lobes at later times, accompanied by a long lasting lymphohistiocytic meningoencephalitis. Despite this extensive infection, mice showed only mild to moderate clinical signs, developed alopecic skin lesions, or remained asymptomatic. Interestingly, most mice exhibited abnormalities in behavior and activity levels including slow movements, akinesia and stargazing. In summary, clinical signs, distribution of viral antigen and inflammatory pattern show striking analogies to human encephalitis caused by HSV-1 or VZV not observed in other animal models of disease.

Roles of the Different Isoforms of the Pseudorabies Virus Protein Kinase pUS3 in Nuclear Egress.

Protein kinases homologous to the US3 gene product (pUS3) of herpes simplex virus (HSV) are conserved throughout the alphaherpesviruses but are absent from betaherpesviruses and gammaherpesviruses. pUS3 homologs are multifunctional and are involved in many processes, including modification of the cytoskeleton, inhibition of apoptosis, and immune evasion. pUS3 also plays a role in efficient nuclear egress of alphaherpesvirus nucleocapsids. In the absence of pUS3, primary enveloped virions accumulate in the perinuclear space (PNS) in large invaginations of the inner nuclear membrane (INM), pointing to a modulatory function for pUS3 during deenvelopment. The HSV and pseudorabies virus (PrV) US3 genes are transcribed into two mRNAs encoding two pUS3 isoforms, which have different aminoterminal sequences and abundances. To test whether the two isoforms in PrV serve different functions, we constructed mutant viruses expressing exclusively either the larger minor or the smaller major isoform, a mutant virus with decreased expression of the smaller isoform, or a mutant with impaired kinase function. Respective virus mutants were investigated in several cell lines. Our results show that absence of the larger pUS3 isoform has no detectable effect on viral replication in cell culture, while full expression of the smaller isoform and intact kinase activity is required for efficient nuclear egress. Absence of pUS3 resulted in only minor titer reduction in most cell lines tested but disclosed a more severe defect in Madin-Darby bovine kidney cells. However, accumulations of primary virions in the PNS do not account for the observed titer reduction in PrV.IMPORTANCE A plethora of substrates and functions have been assigned to the alphaherpesviral pUS3 kinase, including a role in nuclear egress. In PrV, two different pUS3 isoforms are expressed, which differ in size, abundance, and intracellular localization. Their respective role in replication is unknown, however. Here, we show that efficient nuclear egress of PrV requires the smaller isoform and intact kinase activity, whereas absence of the larger isoform has no significant effect on viral replication. Thus, there is a clear distinction in function between the two US3 gene products of PrV.