Virus-infected Animals Research Articles

Highly multiplex volumetric imaging Ce3D-IBEX reveals commensal-induced immune clustering along sensory nerves in the lung

Abstract The airway mucosa is a primary site of the immune response to inhaled antigens. However, how immune homeostasis is maintained in this organ during continuous exposure to airborne particles and microbial antigens is not known. Previous studies have shown that the spatial organization of immune components in relationship to each other and to non-hematopoietic components in tissues is critical to their proper function. To interrogate such relationships in the lung, 3D information is required. However, current methods for fluorescence 3D imaging do not provide the marker depth afforded by high-content 2D imaging. To overcome this constraint, we created Ce3D-IBEX, which enables the visualization of 30+ markers in an optically cleared 3D sample. Using Ce3D-IBEX, we have identified a previously uncharacterized immune cluster in the lung of naïve SPF mice that is enriched with T cells and dendritic cells (DCs) and is localized in close proximity to vagal sensory nerves. Agonistic denervation of the sensory nerves reduced the number of clusters, and that cluster formation was disrupted in germ-free mice as well as OT-I and OT-II TCR transgenic mice. These findings indicate that the clustering requires sensory nerve-derived signals and TCR-mediated recognition of bacterial antigens. Functional studies in virus-infected animals suggest that the T cells in these clusters behave like tissue resident memory T cells, providing acute antigen-independent effector activity.

Gastrointestinal microbiota and metabolites possibly contribute to distinct pathogenicity of SARS-CoV-2 proto or its variants in rhesus monkeys

ABSTRACT Gastrointestinal (GI) infection is evidenced with involvement in COVID-19 pathogenesis caused by SARS-CoV-2. However, the correlation between GI microbiota and the distinct pathogenicity of SARS-CoV-2 Proto and its emerging variants remains unclear. In this study, we aimed to determine if GI microbiota impacted COVID-19 pathogenesis and if the effect varied between SARS-CoV-2 Proto and its variants. We performed an integrative analysis of histopathology, microbiomics, and transcriptomics on the GI tract fragments from rhesus monkeys infected with SARS-CoV-2 proto or its variants. Based on the degree of pathological damage and microbiota profile in the GI tract, five of SARS-CoV-2 strains were classified into two distinct clusters, namely, the clusters of Alpha, Beta and Delta (ABD), and Proto and Omicron (PO). Notably, the abundance of potentially pathogenic microorganisms increased in ABD but not in the PO-infected rhesus monkeys. Specifically, the high abundance of UCG-002, UCG-005, and Treponema in ABD virus-infected animals positively correlated with interleukin, integrins, and antiviral genes. Overall, this study revealed that infection-induced alteration of GI microbiota and metabolites could increase the systemic burdens of inflammation or pathological injury in infected animals, especially in those infected with ABD viruses. Distinct GI microbiota and metabolite profiles may be responsible for the differential pathological phenotypes of PO and ABD virus-infected animals. These findings improve our understanding the roles of the GI microbiota in SARS-CoV-2 infection and provide important information for the precise prevention, control, and treatment of COVID-19.

Removing bovine leukemia virus–infected animals with high proviral load leads to lower within-herd prevalence and new case reduction

Bovine leukosis is prevalent in the North American dairy industry and its effect on animal health and production is widely documented. However, not all bovine leukemia virus (BLV) infected animals transmit the virus equally. Animals with high BLV proviral loads (HPL) are associated with higher transmission risks and therefore, their removal may reduce transmission and eventually within-herd prevalence. We aimed to evaluate the impact of selectively removing HPL cows on the within-herd BLV prevalence and incidence rate of BLV infection in 10 dairy herds. Annual blood and/or milk samples were collected from adult cows over 3 years. Positivity with BLV were determined by ELISA tests and proviral loads in blood of BLV-positive animals were estimated with BLV SS1 quantitative PCR assays. Herd managers were encouraged to consider the proviral load when making culling decisions and implement BLV control practices. High proviral load cows had the highest relative risk of removal indicating the farmers prioritized HPL cows for culling. The within-herd BLV prevalence decreased significantly in 4 herds whereas BLV incidence rate decreased in 9 herds. Over the 3 years, the proviral load demonstrated a relatively stable level, suggesting a single proviral load test in an adult cow may suffice to make culling decisions.

Development of a real-time RT-PCR system applicable for rapid and pen-side diagnosis of foot-and-mouth disease using a portable device, PicoGene® PCR1100

Foot-and-mouth disease (FMD) is a highly contagious viral vesicular disease, causing devastating losses to the livestock industry. A diagnostic method that enables quick decisions is required to control the disease, especially in FMD-free countries. Although conventional real-time reverse transcription polymerase chain reaction (RT-PCR) is a highly sensitive method widely used for the diagnosis of FMD, a time lag caused by the transport of samples to a laboratory may allow the spread of FMD. Here, we evaluated a real-time RT-PCR system using a portable PicoGene PCR1100 device for FMD diagnosis. This system could detect the synthetic FMD viral RNA within 20 min with high sensitivity compared to a conventional real-time RT-PCR. Furthermore, the Lysis Buffer S for crude nucleic extraction improved the viral RNA detection of this system in a homogenate of vesicular epithelium samples collected from FMD virus-infected animals. Furthermore, this system could detect the viral RNA in crude extracts prepared using the Lysis Buffer S from the vesicular epithelium samples homogenized using a Finger Masher tube, which allows easy homogenization without any equipment, with a high correlation compared to the standard method. Thus, the PicoGene device system can be utilized for the rapid and pen-side diagnosis of FMD.

Chronological expression and distribution of African swine fever virus p30 and p72 proteins in experimentally infected pigs

African swine fever virus (ASFV), the causative agent of contagious hemorrhagic disease in domestic pigs and wild boars, has temporally regulated gene expression kinetics. The p30 and p72 major structural proteins are involved in viral entry each with different expression kinetics, but neither of their chronological expressions and distribution have been identified in virus-infected animals. Here, we found that both transcription and translation levels of p30 were significantly higher than those of p72 in target organs during the earlier infection-phase. Lymphocyte apoptosis/necrosis and angiectasia were observed as signs of early infection with acute African swine fever. These results show that the chronologically differential expression of ASFV structural proteins tends to be prominent in infected animals, and the p30 protein could play a role in the indication of acute lesions during early infection compared to the late-expressed p72 protein. In conclusion, we propose to consider the chronological expression dynamics of ASFV structural proteins in infected animals to understand virus pathogenesis and antigen targeting for vaccine development.

Determination of Intestinal Viral Loads and Distribution of Bovine Viral Diarrhea Virus, Classical Swine Fever Virus, and Peste Des Petits Ruminants Virus: A Pilot Study.

The aim of this pilot study was to determine viral loads and distribution over the total length, at short distances, and in the separate layers of the intestine of virus-infected animals for future inactivation studies. Two calves, two pigs, and two goats were infected with bovine viral diarrhoea virus (BVDV), classical swine fever virus (CSFV), and peste des petits ruminants virus (PPRV), respectively. Homogenously distributed maximum BVDV viral loads were detected in the ileum of both calves, with a mean titer of 6.0 log10 TCID50-eq/g. The viral loads in colon and caecum were not distributed homogenously. In one pig, evenly distributed CSFV mean viral loads of 4.5 and 4.2 log10 TCID50-eq/g were found in the small and large intestines, respectively. Mucosa, submucosa, and muscular layer/serosa showed mean viral loads of 5.3, 3.4, and 4.0 log10 TCID50-eq/g, respectively. Homogenous distribution of PPRV was shown in the ileum of both goats, with a mean viral load of 4.6 log10 TCID50-eq/g. Mean mucosa, submucosa, and muscular layer/serosa viral loads were 3.5, 2.8, and 1.7 log10 TCID50-eq/g, respectively. This pilot study provides essential data for setting up inactivation experiments with intestines derived from experimentally infected animals, in which the level and the homogeneous distribution of intestinal viral loads are required.

BLV early diagnosis - key to successful farm recovery

The aim of the research is to use the early diagnosis of cattle leukemia virus-infected animals using serological and molecular genetic methods, as well as to determine the significance of PCR in detecting BLV infected calves. The studies were carried out in the leukemia laboratory of the department for monitoring and forecasting of infectious diseases of the Ural Federal Agrarian Research Center, at the department of infectious and invasive pathology of the Belgorod State Agrarian University named after V.Ya. Gorin, as well as in 20 livestock agricultural enterprises of the Tyumen, Chelyabinsk, Kurgan, Belgorod regions and the Republic of Bashkortostan. The studies were carried out in the framework of the 160th direction of the FNI Program of State Academies of Sciences for 20132020 on the topic «Development of theoretical foundations for the creation and implementation of a monitoring, diagnostic, treatment and wellness program for the protection of animals from epizootically significant infectious diseases» The system of anti-leukemic health measures using complex serological and hematological testing helps to reduce the number of RID+ animals and hematologically sick individuals for a 6-month period by 32.0% and 6.28%, respectively. The use of the polymerase chain reaction method contributes to the detection of an additional 18 to 39% of calves from 15 days of age, infected with the virus of bovine leukemia, in dysfunctional leukemia livestock enterprises. In a PCR study of cows, 29% to 54% of individuals infected with BLV can be detected. This indicates the need for consistent use of serological studies and the importance of molecular genetic tests for the detection of BLV infected calves.

An Experimental Model of Neurodegenerative Disease Based on Porcine Hemagglutinating Encephalomyelitis Virus-Related Lysosomal Abnormalities.

Lysosomes are involved in pathogenesis of a variety of neurodegenerative diseases and play a large role in neurodegenerative disorders caused by virus infection. However, whether virus-infected cells or animals can be used as experimental models of neurodegeneration in humans based on virus-related lysosomal dysfunction remain unclear. Porcine hemagglutinating encephalomyelitis virus displays neurotropism in mice, and neural cells are its targets for viral progression. PHEV infection was confirmed to be a risk factor for neurodegenerative diseases in the present. The findings demonstrated for the first time that PHEV infection can lead to lysosome disorders and showed that the specific mechanism of lysosome dysfunction is related to PGRN expression deficiency and indicated similar pathogenesis compared with human neurodegenerative diseases upon PHEV infection. Trehalose can also increase progranulin expression and rescue abnormalities in lysosomal structure in PHEV-infected cells. In conclusion, these results suggest that PHEV probably serve as a disease model for studying the pathogenic mechanisms and prevention of other degenerative diseases.Electronic supplementary materialThe online version of this article (10.1007/s12035-020-02105-y) contains supplementary material, which is available to authorized users.

Inactivating Three Interferon Antagonists Attenuates Pathogenesis of an Enteric Coronavirus.

Coronaviruses (CoVs) have repeatedly emerged from wildlife hosts and infected humans and livestock animals to cause epidemics with significant morbidity and mortality. CoVs infect various organs, including respiratory and enteric systems, as exemplified by newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The constellation of viral factors that contribute to developing enteric disease remains elusive. Here, we investigated CoV interferon antagonists for their contribution to enteric pathogenesis. Using an infectious clone of an enteric CoV, porcine epidemic diarrhea virus (icPEDV), we generated viruses with inactive versions of interferon antagonist nonstructural protein 1 (nsp1), nsp15, and nsp16 individually or combined into one virus designated icPEDV-mut4. Interferon-responsive PK1 cells were infected with these viruses and produced higher levels of interferon responses than were seen with wild-type icPEDV infection. icPEDV-mut4 elicited robust interferon responses and was severely impaired for replication in PK1 cells. To evaluate viral pathogenesis, piglets were infected with either icPEDV or icPEDV-mut4. While the icPEDV-infected piglets exhibited clinical disease, the icPEDV-mut4-infected piglets showed no clinical symptoms and exhibited normal intestinal pathology at day 2 postinfection. icPEDV-mut4 replicated in the intestinal tract, as revealed by detection of viral RNA in fecal swabs, with sequence analysis documenting genetic stability of the input strain. Importantly, icPEDV-mut4 infection elicited IgG and neutralizing antibody responses to PEDV. These results identify nsp1, nsp15, and nsp16 as virulence factors that contribute to the development of PEDV-induced diarrhea in swine. Inactivation of these CoV interferon antagonists is a rational approach for generating candidate vaccines to prevent disease and spread of enteric CoVs, including SARS-CoV-2.IMPORTANCE Emerging coronaviruses, including SARS-CoV-2 and porcine CoVs, can infect enterocytes, cause diarrhea, and be shed in the feces. New approaches are needed to understand enteric pathogenesis and to develop vaccines and therapeutics to prevent the spread of these viruses. Here, we exploited a reverse genetic system for an enteric CoV, porcine epidemic diarrhea virus (PEDV), and outline an approach of genetically inactivating highly conserved viral factors known to limit the host innate immune response to infection. Our report reveals that generating PEDV with inactive versions of three viral interferon antagonists, nonstructural proteins 1, 15, and 16, results in a highly attenuated virus that does not cause diarrhea in animals and elicits a neutralizing antibody response in virus-infected animals. This strategy may be useful for generating live attenuated vaccine candidates that prevent disease and fecal spread of enteric CoVs, including SARS-CoV-2.

Non-coding Natural Antisense RNA: Mechanisms of Action in the Regulation of Target Gene Expression and Its Clinical Implications

Recent advances in high-throughput technologies have revealed that 75% of the human genome is transcribed to RNA, whereas only 3% of transcripts are translated into proteins. Consequently, many long non-coding RNAs (lncRNAs) have been identified, which has improved our understanding of the complexity of biological processes. LncRNAs comprise multiple classes of RNA transcripts that regulate the transcription, stability and translation of protein-coding genes in a genome. Natural antisense transcripts (NATs) form one such class, and the GENCODE v30 catalog contains 16193 lncRNA loci, of which 5611 are antisense loci. This review outlines our emerging understanding of lncRNAs, with a particular focus on how lncRNAs regulate gene expression using interferon-α1 (IFN-α1) mRNA and its antisense partner IFN-α1 antisense (as)RNA as an example. We have identified and characterized the asRNA that determines post-transcriptional IFN-α1 mRNA levels. IFN-α1 asRNA stabilizes IFN-α1 mRNA by cytoplasmic sense-antisense duplex formation, which may enhance the accessibility of an RNA stabilizer protein or decrease the affinity of an RNA decay factor for the RNA. IFN-α1 asRNA can also act as competing molecules in the competing endogenous (ce)RNA network with other members of the IFNA multigene family mRNAs/asRNAs, and other cellular mRNA transcripts. Furthermore, antisense oligoribonucleotides representing functional domains of IFN-α1 asRNA inhibit influenza virus proliferation in the respiratory tract of virus-infected animals. Thus, these findings support, at least in part, the rationale that dissecting the activity of NAT on gene expression regulation promises to reveal previously unanticipated biology, with potential to provide new therapeutic approaches to diseases.

Inclusion of the Viral Pentamer Complex in a Vaccine Design Greatly Improves Protection against Congenital Cytomegalovirus in the Guinea Pig Model.

A vaccine against congenital cytomegalovirus (cCMV) is a high priority. The guinea pig is a small-animal model for cCMV. A disabled infectious single-cycle (DISC) viral vaccine strain based on a guinea pig cytomegalovirus (GPCMV) capsid mutant was evaluated. A previous version of this vaccine did not express the gH/gL-based pentamer complex (PC) and failed to fully protect against cCMV. The PC is necessary for GPCMV epithelial cell/trophoblast tropism and congenital infection and is a potentially important neutralizing antigen. Here, we show that a second-generation PC-positive (PC+) DISC (DISCII) vaccine induces neutralizing antibodies to the PC and other glycoproteins and a cell-mediated response to pp65 (GP83). Additionally, a CRISPR/Cas9 strategy identified guinea pig platelet-derived growth factor receptor alpha (PDGFRA) to be the receptor for PC-independent infection of fibroblast cells. Importantly, PDGFRA was absent in epithelial and trophoblast cells, which were dependent upon the viral PC for infection. Virus neutralization by DISCII antibodies on epithelial and trophoblast cells was similar to that in sera from wild-type virus-infected animals and dependent in part on PC-specific antibodies. In contrast, sera from PC-negative virus-infected animals poorly neutralized virus on non-fibroblast cells. DISCII-vaccinated animals were protected against congenital infection, in contrast to a nonvaccinated group. The target organs of pups in the vaccine group were negative for wild-type virus, unlike those of pups in the control group, with GPCMV transmission being approximately 80%. Overall, the DISCII vaccine had 97% efficacy against cCMV. The complete protection provided by this PC+ DISC vaccine makes the possibility of the use of this approach against human cCMV attractive.IMPORTANCE Cytomegalovirus (CMV) is a leading cause of congenital disease in newborns, and an effective vaccine remains an elusive goal. The guinea pig is the only small-animal model for cCMV. Guinea pig cytomegalovirus (GPCMV) encodes a glycoprotein pentamer complex (PC) for entry into non-fibroblast cells, including placental trophoblasts, to enable cCMV. As with human cytomegalovirus (HCMV), GPCMV uses a specific cell receptor (PDGFRA) for fibroblast entry, but other receptors are required for non-fibroblast cells. A disabled infectious single-cycle (DISC) GPCMV vaccine strain induced an antibody immune response to the viral pentamer to enhance virus neutralization on non-fibroblast cells, and vaccinated animals were fully protected against cCMV. Inclusion of the PC as part of a vaccine design dramatically improved vaccine efficacy, and this finding underlines the importance of the immune response to the PC in contributing toward protection against cCMV. This vaccine represents an important milestone in the development of a vaccine against cCMV.

Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse.

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral load. The purpose of the procedure is to quantify viral titers in peripheral and CNS areas of the mouse at different time points post infection or under different experimental conditions to identify virologic and immunological factors that regulate Zika virus infection. The organ isolation procedures demonstrated allow for both focus forming assay quantification and quantitative PCR assessment of viral titers. The rapid organ isolation techniques are designed for the preservation of virus titer. Viral titer quantification by focus forming assay allows for the rapid throughput assessment of Zika virus. The benefit of the focus forming assay is the assessment of infectious virus, the limitation of this assay is the potential for organ toxicity reducing the limit of detection. Viral titer assessment is combined with quantitative PCR, and using a recombinant RNA copy control viral genome copy number within the organ is assessed with low limit of detection. Overall these techniques provide an accurate rapid high throughput method for the analysis of Zika viral titers in the periphery and CNS of Zika virus infected animals and can be applied to the assessment of viral titers in the organs of animals infected with most pathogens, including Dengue virus.

Involvement of Interferon Regulatory Factor 7 in Nicotine's Suppression of Antiviral Immune Responses.

Nicotine, the active ingredient in tobacco smoke, suppresses antiviral responses. Interferon regulatory factors (IRFs) regulate transcription of type I interferons (IFNs) and IFN-stimulated genes (ISGs) in this response. IRF7 is a key member of the IRF family. Expression of Irf7 is elevated in the brains of virus-infected animals, including human immunodeficiency virus-1 transgenic (HIV-1Tg) rats. We hypothesized that IRF7 affects nicotine's modulation of antiviral responses. Using CRISPR/Cas9 system, IRF7-mutant cell lines were created from human embryonic kidney 293FT cells in which 16 nicotinic acetylcholine receptors (nAChRs) were detected. Decreased expression of IRF7 was confirmed at both the mRNA and protein levels, as was IRF7-regulated cell growth in two IRF7-mutant cell lines, designated IRF7-Δ7 and IRF7-Δ11. In IRF7-Δ7 cells, expression of two nAChR genes, CHRNA3 and CHRNA9, changed modestly. After stimulation with polyinosinic-polycytidylic acid (poly I:C) (0.25μg/ml) for 4h to mimic viral infection, 293FT wild-type (WT) and IRF7-Δ7 cells were treated with 0, 1, or 100μM nicotine for 24h, which increased IFN-β expression in both types of cells but elevation was higher in WT cells (p < 0.001). Expression was significantly suppressed in WT cells (p < 0.001) but not in IRF7-Δ7 cells by 24-h nicotine exposure. Poly I:C stimulation increased mRNA expression of retinoic-acid-inducible protein I (RIG-I), melanoma-differentiation-associated gene 5 (MDA5), IFN-stimulated gene factor 3 (ISG3) complex, and IFN-stimulated genes (IRF7, ISG15, IFIT1, OAS1); nicotine attenuated mRNA expression only in WT cells. Overall, IRF7 is critical to nicotine's effect on the antiviral immune response. Graphical Abstract Involvement of IRF7 in nicotine's suppression of poly I:C-induced antiviral immune responses. PAMPs, such as a synthetic viral analogue of dsRNA poly I:C attack cells, will be recognized by PRRs, and the host innate immunity against viral infection will be activated. PRRs signaling trigger phosphorylation of IRF7 and IRF3 to induce their translocation to the nucleus and result in the production of type I IFNs. Then IFNs bind to IFNAR to activate the transcription factor ISGF3, a complex consisting of STAT1, STAT2, and IRF9. Further, it induces the expression of ISGs, including IFIT1, OAS1, IRF7, ISG15, etc. Nicotine suppresses the immune responses stimulated by poly I:C. In the IRF7-mutant cells, nicotine's suppressive effects on poly I:C-stimulated immune responses were restrained.

Serological evidence of Rift Valley fever virus infection in slaughtered ruminants in Nigeria

ABSTRACT The risk of exposure of slaughterhouse workers to Rift Valley fever (RVF) virus-infected animals in Nigeria was assessed by determining the prevalence of anti-RVF IgM in cattle, goats and sheep slaughtered in a major abattoir in Ibadan, Nigeria. Blood samples were collected from 290 animals in Bodija Municipal abattoir, Ibadan, Nigeria in January and February 2017 and analyzed for the presence of RVF virus using IgM Enzyme-Linked Immunosorbent Assay (ELISA) and Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) for detection of the virus RNA. Descriptive statistics was used to analyze data. Overall, an IgM prevalence of 0.7% (2/290) was found among the blood samples of the animals, suggesting recent exposure to the virus. Antibody was detected in the sera from a cow and one goat. RVF virus RNA was not detected in the 2 IgM positive blood samples. There was no statistically significant relationship between RVF IgM infection and some variables of the animals, including age, sex and breed (p ≥ 0.05). Results of this study indicate active RVF virus transmission in domestic livestock in Nigeria. The study emphasizes the need to embark on monitoring of human and animal populations to prevent outbreak of the virus in the country.

IFN-Alpha1 antisense RNA represses human influenza A virus growth in a guinea pig system.

We reported a natural antisense (AS) long non-coding RNA as an important modulator of interferon-Alpha1 (IFNA1) mRNA levels. We showed that IFN-Alpha1 AS promotes IFNA1 mRNA stability by transient duplex formation and inhibition of miR-1270-induced mRNA decay. Here, we performed a proof-of-concept experiment to verify that the AS-mRNA regulatory axis exerts in vivo control of innate immunity. We established a model system for influenza virus infection using guinea pig, which encodes a functional MX1 gene for the type I IFN pathway. This system allowed us to investigate the effects of antisense oligoribonucleotides representing functional domains of guinea pig IFN-Alpha1 AS on gpIFNA1 mRNA levels and, consequently, on viral proliferation in the respiratory tract of influenza virus-infected animals. We demonstrated that pulmonary-administered asORNs inhibited the proliferation of the virus in the animals by modulating IFNA1 mRNA levels. These results indicate that, in light of the proposed actions, asORNs may modulate the level of IFNA1 mRNA in vivo, indicating that IFN-Alpha1 AS plays a pivotal role in determining the outcome of type I IFN responses.

Evidence for Viral Interference and Cross-reactive Protective Immunity Between Influenza B Virus Lineages.

Two influenza B virus lineages, B/Victoria and B/Yamagata, cocirculate in the human population. While the lineages are serologically distinct, cross-reactive responses to both lineages have been detected. Viral interference describes the situation whereby infection with one virus limits infection and replication of a second virus. We investigated the potential for viral interference between the influenza B virus lineages. Ferrets were infected and then challenged 3, 10, or 28 days later with pairs of influenza B/Victoria and B/Yamagata viruses. Viral interference occurred at challenge intervals of 3 and 10 days and occasionally at 28 days. At the longer interval, shedding of challenge virus was reduced, and this correlated with cross-reactive interferon γ responses from lymph nodes from virus-infected animals. Viruses from both lineages could prevent or significantly limit subsequent infection with a virus from the other lineage. Coinfections were rare, indicating the potential for reassortment between lineages is limited. These data suggest that innate and cross-reactive immunity mediate viral interference and that this may contribute to the dominance of a specific influenza B virus lineage in any given influenza season. Furthermore, infection with one influenza B virus lineage may be beneficial in protecting against subsequent infection with either influenza B virus lineage.

Food as a passible source af H5N1 influenza virus infection in mammals

The recent infections of humans and other mammals with die highly padiogenic H5N1 avian influenza virus have raised questions about the safety of the consumption of poultry products. The aim of this review is to collect and present information regarding the risk of infection in humans with H5N1 virus via the oral route. The presence of virus in edible poultry products and the chances for viral entry via the gastrointestinal (GI) tract, from a pathogenetic point of view, are the main focus points. The transmission of H5N1 from poultry to humans is a rare event which has been associated with very close contact with infected animals. It is generally accepted that the most likely candidate routes for the virus entry are the oropharygeal and/or respiratory tract tissues where virus replication may occur and leading to clinical symptoms. However, the low number of human cases, compared to the high number of humans that have been exposed to H5N1 virus infected animals, shows clearly that a readily accessible portal of entry does not exist. The possibility of virus entry through the GI tract has been proposed, but, so far, no proof that the virus can replicate in the human intestines has been shown. The presence of diarrhoea in several patients and the detection of viral RNA in the intestines and rectal swabs of 3 patients do not allow one to safely conclude that the GI tract can serve as a portal of entry or a target organ. Furthermore, the occurrence of disease has not clearly or definitely been associated with the consumption of poultry meat in any human case. Feline species, on the other hand, have been naturally and experimentally infected after the consumption of infected poultry. However, also in these mammals, it was not established that the intestine is the initial portal of entry of the virus. In conclusion, the possibility that the intestinal tract serves as a portal of entry in mammals remains unlikely, since there is no convincing evidence that the GI tract tissues can support virus replication. It can, however, not be excluded that food containing virus may be a source of infection when passing oropharyngeal tissues, which have been identified as target sites for virus replication.

Influenza-derived peptides cross-react with allergens and provide asthma protection

The hygiene hypothesis is the leading concept to explain the current asthma epidemic, which is built on the observation that a lack of bacterial contact early in life induces allergic TH2 immune responses. Because little is known about the contribution of respiratory tract viruses in this context, we evaluated the effect of prior influenza infection on the development of allergic asthma. Mice were infected with influenza and, once recovered, subjected to an ovalbumin- or house dust mite-induced experimental asthma protocol. Influenza-polarized effector memory T (Tem) cells were transferred adoptively to allergen-sensitized animals before allergen challenge. Acomprehensive in silico analysis assessed homologies between virus- and allergen-derived proteins. Influenza-polarized Tem cells were stimulated exvivo with candidate peptides. Mice were immunized with a pool of virus-derived T-cell epitopes. In 2 murine models we found a long-lasting preventive effect against experimental asthma features. Protection could be attributed about equally to CD4+ and CD8+ Tem cells from influenza-infected mice. An in silico bioinformatic analysis identified 4 influenza- and 3 allergen-derived MHC class I and MHC class II candidate T-cell epitopes with potential antigen-specific cross-reactivity between influenza and allergens. Lymphocytes from influenza-infected mice produced IFN-γ and IL-2 but not IL-5 on stimulation with the aforementioned peptides. Immunization with a mixture of the influenza peptides conferred asthma protection, and peptide-immunized mice transferred protection through CD4+ and CD8+ Tem cells. For the first time, our results illustrate heterologous immunity of virus-infected animals toward allergens. This finding extends the original hygiene hypothesis.

Interferon-alpha treatment rapidly clears Hepatitis E virus infection in humanized mice

Antiviral treatment options for chronic Hepatitis E Virus (HEV) infections are limited and immunological determinants of viral persistence remain largely unexplored. We studied the antiviral potency of pegylated interferon-α (pegIFNα) against HEV infections in humanized mice and modelled intrahepatic interferon stimulated gene (ISG) responses. Human gene expression levels in humanized mouse livers were analyzed by qPCR and Nanostring. Human CXCL10 was measured in mouse serum. HEV genotype 3 (gt3) infections were cleared from liver and feces within 8 pegIFNα doses in all mice and relapsed after a single pegIFNα injection in only half of treated animals. Rapid viral clearance by pegIFNα was confirmed in HEV gt1, but not in Hepatitis B Virus infected animals. No ISG induction was observed in untreated HEV gt3 and gt1 infected humanized livers compared to control chimeric mice, irrespective of the human hepatocyte donor, viral isolate or HEV infection duration. Human specific ISG transcript levels in mouse liver increased significantly after pegIFNα treatment and induced high circulating human CXCL10 in mouse serum. In conclusion, HEV gt1 and gt3 infections do not elicit innate intrahepatic immune responses and remain highly sensitive to pegIFNα in immunocompromised humanized mice.

Direct Targeting of Macrophages With Methylglyoxal-Bis-Guanylhydrazone Decreases SIV-Associated Cardiovascular Inflammation and Pathology.

Despite effective combination antiretroviral therapy, HIV-infected individuals develop comorbidities, including cardiovascular disease, where activated macrophages play a key role. To date, few therapies target activated monocytes and macrophages. We evaluated a novel oral form of the polyamine biosynthesis inhibitor methylglyoxal-bis-guanylhydrazone (MGBG) on cardiovascular inflammation, carotid artery intima-media thickness (cIMT), and fibrosis in a simian immunodeficiency virus infection model of AIDS. Eleven simian immunodeficiency virus-infected animals received MGBG (30 mg/kg) once daily and 8 received a placebo control both beginning at 21 days postinfection (dpi). Animals were time sacrificed at 49 days post infection (dpi), when their matched placebo controls developed AIDS (63, 70, 77, 80), or at the study end-point (84 dpi). Aorta, carotid artery, and cardiac tissues were analyzed. Quantitative analyses of macrophage populations and T lymphocytes were done and correlated with cIMT and fibrosis. MGBG treatment resulted in 2.19-fold (CD163), 1.86-fold (CD68), 2.31-fold (CD206), and 2.12-fold (MAC387) decreases in macrophages in carotid arteries and significant 2.07-fold (CD163), 1.61-fold (CD68), 1.95-fold (MAC387), and 1.62-fold (CD206) decreases in macrophages in cardiac tissues. cIMT (1.49-fold) and fibrosis (2.05-fold) also were significantly decreased with MGBG treatment. Numbers of macrophage and the degree of fibrosis in treated animals were similar to uninfected animals. A positive correlation between decreased macrophage in the carotid artery and cIMT, and cardiac macrophages and fibrosis was found. These data demonstrate that directly targeting macrophages with MGBG can reduce cardiovascular inflammation, cIMT, and fibrosis. They suggest that therapies targeting macrophages with HIV could be used in conjunction with combination antiretroviral therapy.