Protein-Coding Region Derived Small RNA in Exosomes from Influenza A Virus-Infected Cells.

Exosomes may function as multifactorial mediators of cell-to-cell communication, playing crucial roles in both physiological and pathological processes. Exosomes released from virus-infected cells may contain RNA and proteins facilitating infection spread. The purpose of our study was to analyze how the small RNA content of exosomes is affected by infection with the influenza A virus (IAV). Exosomes were isolated by ultracentrifugation after hemadsorption of virions and their small RNA content was identified using high-throughput sequencing. As compared to mock-infected controls, 856 RNA transcripts were significantly differentially expressed in exosomes from IAV-infected cells, including fragments of 458 protein-coding (pcRNA), 336 small, 28 long intergenic non-coding RNA transcripts, and 33 pseudogene transcripts. Upregulated pcRNA species corresponded mainly to proteins associated with translation and antiviral response, and the most upregulated among them were RSAD2, CCDC141 and IFIT2. Downregulated pcRNA species corresponded to proteins associated with the cell cycle and DNA packaging. Analysis of differentially expressed pseudogenes showed that in most cases, an increase in the transcription level of pseudogenes was correlated with an increase in their parental genes. Although the role of exosome RNA in IAV infection remains undefined, the biological processes identified based on the corresponding proteins may indicate the roles of some of its parts in IAV replication.

Chorioallantoic membranes of embryonated chicken eggs as an alternative system for isolation of equine influenza virus.

BACKGROUND: Influenza virus isolation in embryonated chicken eggs (ECEs) is not applicable for rapid diagnosis, however it allows the recovery and propagation of the viable virus. A low number of infectious virus particles in the swabs, poor quality of samples or individual strain properties can lead to difficulties during the virus isolation process. We propose to utilize chorioallantoic membranes (CAM) of ECEs with the assistance of real-time RT PCR to facilitate equine influenza virus isolation. METHODS: Real-time RT PCR was used to detect influenza virus genetic material in amniotic/allantoic fluids (AF) and CAM of ECEs. Haemagglutination assay was used for AF. We used highly diluted virus as a substitute of clinical specimen for ECEs inoculation. RESULTS: Our study demonstrated that real-time RT PCR testing of CAM homogenates was more useful than testing of AF for EIV detection in ECEs. Positive results from CAM allowed to select the embryos from those with haemagglutination assay (HA) - and real-time RT PCR-negative AF for further passages. Using homogenates of CAM for subsequent passages, we finally obtained HA-positive AF, which confirmed virus replication. CONCLUSION: We postulate that real-time RT PCR testing of CAM homogenates and their subsequent passages may facilitate the isolation of equine influenza viruses.

Mass spectrometry identification of granins and other proteins secreted by neuroblastoma cells.

We used mass spectrometry-based protein identification to determine the presence of granins and other proteins in the mouse neuroblastoma secretome. We detected polypeptides derived from four members of the granin family: chromogranin A, chromogranin B, secretogranin III, and VGF. Many of them are derived from previously described biologically active regions; however, for VGF and CgB, we detected peptides not related to known bioactivities. Along with granins, we identified 115 other proteins secreted by mouse neuroblastoma cells, belonging to different functional categories. Fifty-six out of 119 detected proteins possess the signal fragments required for translocation into endoplasmic reticulum. Sequences of remaining 63 proteins were analyzed using SecretomeP algorithm to determine probability of nonclassical secretion. Identified proteins are involved in the regulation of cell cycle, proliferation, apoptosis, angiogenesis, proteolysis, and cell adhesion.

Tick-Borne Encephalitis-Review of the Current Status.

The tick-borne encephalitis virus (TBEV) is the arboviral etiological agent of tick-borne encephalitis (TBE), considered to be one of the most important tick-borne viral diseases in Europe and Asia. In recent years, an increase in the incidence of TBE as well as an increasing geographical range of the disease have been noted. Despite the COVID-19 pandemic and the imposition of restrictions that it necessitated, the incidence of TBE is rising in more than half of the European countries analyzed in recent studies. The virus is transmitted between ticks, animals, and humans. It seems that ticks and small mammals play a role in maintaining TBEV in nature. The disease can also affect dogs, horses, cattle, and small ruminants. Humans are incidental hosts, infected through the bite of an infected tick or by the alimentary route, through the consumption of unpasteurized milk or milk products from TBEV-infected animals. TBEV infections in humans may be asymptomatic, but the symptoms can range from mild flu-like to severe neurological. In Europe, cases of TBE are reported every year. While there is currently no effective treatment for TBE, immunization and protection against tick bites are critical in preventing this disease.

Influenza D in Domestic and Wild Animals.

Influenza D virus (IDV) infections have been observed in animals worldwide, confirmed through both serological and molecular tests, as well as virus isolation. IDV possesses unique properties that distinguish it from other influenza viruses, primarily attributed to the hemagglutinin-esterase fusion (HEF) surface glycoprotein, which determines the virus' tropism and wide host range. Cattle are postulated to be the reservoir of IDV, and the virus is identified as one of the causative agents of bovine respiratory disease (BRD) syndrome. Animals associated with humans and susceptible to IDV infection include camels, pigs, small ruminants, and horses. Notably, high seroprevalence towards IDV, apart from cattle, is also observed in camels, potentially constituting a reservoir of the virus. Among wild and captive animals, IDV infections have been confirmed in feral pigs, wild boars, deer, hedgehogs, giraffes, wildebeests, kangaroos, wallabies, and llamas. The transmission potential and host range of IDV may contribute to future viral differentiation. It has been confirmed that influenza D may pose a threat to humans as a zoonosis, with seroprevalence noted in people with professional contact with cattle.

Vector-Borne Viral Diseases as a Current Threat for Human and Animal Health-One Health Perspective.

Over the last decades, an increase in the emergence or re-emergence of arthropod-borne viruses has been observed in many regions. Viruses such as dengue, yellow fever, or zika are a threat for millions of people on different continents. On the other hand, some arboviruses are still described as endemic, however, they could become more important in the near future. Additionally, there is a group of arboviruses that, although important for animal breeding, are not a direct threat for human health. Those include, e.g., Schmallenberg, bluetongue, or African swine fever viruses. This review focuses on arboviruses and their major vectors: mosquitoes, ticks, biting midges, and sandflies. We discuss the current knowledge on arbovirus transmission, ecology, and methods of prevention. As arboviruses are a challenge to both human and animal health, successful prevention and control are therefore only possible through a One Health perspective.

Occurrence of Equine Foamy Virus Infection in Horses from Poland.

Equine foamy virus (EFVeca) is a foamy virus of non-primate origin and among the least-studied members of this retroviral subfamily. By sequence comparison, EFVeca shows the highest similarity to bovine foamy virus. In contrast to simian, bovine or feline foamy viruses, knowledge about the epidemiology of EFVeca is still limited. Since preliminary studies suggested EFVeca infections among horses in Poland, we aimed to expand the diagnostics of EFVeca infections by developing specific diagnostic tools and apply them to investigate its prevalence. An ELISA test based on recombinant EFVeca Gag protein was developed for serological investigation, while semi-nested PCR for the detection of EFVeca DNA was established. 248 DNA and serum samples from purebred horses, livestock and saddle horses, Hucul horses and semi-feral Polish primitive horses were analyzed in this study. ELISA was standardized, and cut off value, sensitivity and specificity of the test were calculated using Receiver Operating Characteristic and Bayesian estimation. Based on the calculated cut off, 135 horses were seropositive to EFVeca Gag protein, while EFVeca proviral DNA was detected in 85 animals. The rate of infected individuals varied among the horse groups studied; this is the first report confirming the existence of EFVeca infections in horses from Poland using virus-specific tools.

Rift Valley Fever - a Growing Threat To Humans and Animals.

Rift Valley fever (RVF) is a zoonotic, vector-borne infectious disease of ruminants and camels transmitted mainly by the Aedes and Culex mosquito species. Contact with the blood or organs of infected animals may infect humans. Its etiological factor is the Rift Valley fever virus (RVFV) of the Phlebovirus genus and Bunyaviridae family. Sheep and goats are most susceptible to infection and newborns and young individuals endure the most severe disease course. High abortion rates and infant mortality are typical for RVF; its clinical signs are high fever, lymphadenitis, nasal and ocular secretions and vomiting. Conventional diagnosis is done by the detection of specific IgM or IgG antibodies and RVFV nucleic acids and by virus isolation. Inactivated and live-attenuated vaccines obtained from virulent RVFV isolates are available for livestock. RVF is endemic in sub-Saharan Africa and the Arabian Peninsula, but in the last two decades, it was also reported in other African regions. Seropositive animals were detected in Turkey, Tunisia and Libya. The wide distribution of competent vectors in non-endemic areas coupled with global climate change threaten to spread RVF transboundarily. The EFSA considers the movement of infected animals and vectors to be other plausible pathways of RVF introduction into Europe. A very low risk both of introduction of the virus through an infected animal or vector and of establishment of the virus, and a moderate risk of its transmission through these means was estimated for Poland. The risk of these specific modes of disease introduction into Europe is rated as very low, but surveillance and response capabilities and cooperation with the proximal endemic regions are recommended.

Analysis of Single Nucleotide Variants (SNVs) Induced by Passages of Equine Influenza Virus H3N8 in Embryonated Chicken Eggs.

Vaccination is an effective method for the prevention of influenza virus infection. Many manufacturers use embryonated chicken eggs (ECE) for the propagation of vaccine strains. However, the adaptation of viral strains during subsequent passages can lead to additional virus evolution and lower effectiveness of the resulting vaccines. In our study, we analyzed the distribution of single nucleotide variants (SNVs) of equine influenza virus (EIV) during passaging in ECE. Viral RNA from passage 0 (nasal swabs), passage 2 and 5 was sequenced using next generation technology. In total, 50 SNVs with an occurrence frequency above 2% were observed, 29 of which resulted in amino acid changes. The highest variability was found in passage 2, with the most variable segment being IV encoding hemagglutinin (HA). Three variants, HA (W222G), PB2 (A377E) and PA (R531K), had clearly increased frequency with the subsequent passages, becoming dominant. None of the five nonsynonymous HA variants directly affected the major antigenic sites; however, S227P was previously reported to influence the antigenicity of EIV. Our results suggest that although host-specific adaptation was observed in low passages of EIV in ECE, it should not pose a significant risk to influenza vaccine efficacy.

Proteomic analysis of the secretome of equine herpesvirus-1 infected rabbit kidney cells.

Herpesviruses are the main cause of abortions and respiratory or neurological disorders in horses. Various disease patterns are suspected to be associated with the A2254G point mutation in the DNA polymerase sequence (ORF30) of the herpesvirus genome, although the importance of this link is still under debate. Based on a label-free quantitative proteomic analysis, the differences in the secretion of some host proteins between rabbit kidney cells infected with A2254 and cells of the same line infected with G2254 equine herpesvirus 1 (EHV-1) strains were identified. In both groups, downregulation of proteins involved in insulin growth factor and extracellular matrix pathway regulation was observed. Among 12 proteins with increased secretion, 8 were regulated only in G2254 EHV-1 infection. Those were endoplasmic reticulum chaperones with calcium binding properties, related to unfolded protein response and mitochondria. It was presumed that the secretion of proteins such as calreticulin, Hspa5 or endoplasmin may contribute to the pathogenesis of EHV-1 infection.

Serologic investigation of influenza A virus infection in dogs in Poland.

The 2 predominant circulating subtypes of influenza A virus in the dog population, equine-origin H3N8 and avian-origin H3N2, constitute a potential zoonotic risk. We determined the prevalence of influenza A antibodies in 496 dogs in Poland and found 2.21% of sera positive by commercial ELISA. Hemagglutination inhibition (HI) assays indicated 7.25% of sera positive using equine H3N8, swine H3N2, and pandemic H1N1 antigens, with the most frequently detected immune response being to H3N2. Considering interspecies transfer, reassortment ability, and close contact between dogs and humans, infections of dogs with influenza A virus should be monitored.

Biomarkers of HIV-1 associated dementia: proteomic investigation of sera.

BACKGROUND: New, more sensitive and specific biomarkers are needed to support other means of clinical diagnosis of neurodegenerative disorders. Proteomics technology is widely used in discovering new biomarkers. There are several difficulties with in-depth analysis of human plasma/serum, including that there is no one proteomic platform that can offer complete identification of differences in proteomic profiles. Another set of problems is associated with heterogeneity of human samples in addition intrinsic variability associated with every step of proteomic investigation. Validation is the very last step of proteomic investigation and it is very often difficult to validate potential biomarker with desired sensitivity and specificity. Even though it may be possible to validate a differentially expressed protein, it may not necessarily prove to be a valid diagnostic biomarker. RESULTS: In the current study we report results of proteomic analysis of sera from HIV-infected individuals with or without cognitive impairment. Application of SELDI-TOF analysis followed by weak cation exchange chromatography and 1-dimensional electrophoresis led to discovery of gelsolin and prealbumin as differentially expressed proteins which were not detected in this cohort of samples when previously investigated by 2-dimensional electrophoresis with Difference Gel Electrophoresis technology. CONCLUSION: Validation using western-blot analysis led us to conclude that relative change of the levels of these proteins in one patient during a timeframe might be more informative, sensitive and specific than application of average level estimated based on an even larger cohort of patients.

Multidimensional protein fractionation using ProteomeLab PF 2D for profiling amyotrophic lateral sclerosis immunity: A preliminary report.

BACKGROUND: The ProteomeLab PF 2D platform is a relatively new approach to global protein profiling. Herein, it was used for investigation of plasma proteome changes in amyotrophic lateral sclerosis (ALS) patients before and during immunization with glatiramer acetate (GA) in a clinical trial. RESULTS: The experimental design included immunoaffinity depletion of 12 most abundant proteins from plasma samples with the ProteomeLab IgY-12 LC10 column kit as first dimension separation, also referred to as immuno-partitioning. Second and third dimension separations of the enriched proteome were performed on the PF 2D platform utilizing 2D isoelectric focusing and RP-HPLC with the resulting fractions collected for analysis. 1D gel electrophoresis was added as a fourth dimension when sufficient protein was available. Protein identification from collected fractions was performed using nano-LC-MS/MS approach. Analysis of differences in the resulting two-dimensional maps of fractions obtained from the PF 2D and the ability to identify proteins from these fractions allowed sensitivity threshold measurements. Masked proteins in the PF 2D fractions are discussed. CONCLUSION: We offer some insight into the strengths and limitations of this emerging proteomic platform.

Genetic Analysis of the M Gene of Equine Influenza Virus Strains Isolated in Poland, in the Context of the Asian-like Group Formation.

INTRODUCTION: Traditionally, evolutionary analysis of equine influenza virus (EIV) is based on the HA gene. However, the specificity of the influenza virus enables the classification of viral strains into different phylogenetic groups, depending on the gene being analysed. The aim of the study was to analyse phylogenetic paths of EIV based on M gene with reference to the HA gene. MATERIAL AND METHODS: M gene of Polish isolates has been sequenced and analysed along with all M sequences of EIV available in GenBank database. Phylogenetic analysis was performed using BioEdit, ClustalW, and MEGA7 softwares. RESULTS: The clustering of the strains isolated not only from Asia but also from Europe into one common Asian-like group of EIV was observed. Twelve nucleotide substitutions in the M gene of strains from the Asian-like group were crucial for the evolutionary analysis. We also observed homology in the M gene of the Asian-like and H7N7 strains. CONCLUSIONS: M gene specific for the Asian-like group is present in strains recently isolated in Europe and Asia, which were classified previously in the Florida 2 clade based on HA. Therefore, Asian-like group does not seem to be assigned to a specific geographical region. Traces of H7N7 strains in more conservative genes like M of some contemporary EIV strains may indicate the link between the old phylogenetic group and recent H3N8 strains. Analysis of conservative genes may be more useful in tracking the direction of virus evolution than in the genes where the high variability rate may blur the original relationships.

HIV-1 infected monocyte-derived macrophages affect the human brain microvascular endothelial cell proteome: new insights into blood-brain barrier dysfunction for HIV-1-associated dementia.

Blood-brain barrier (BBB) compromise and transendothelial migration of HIV-infected leukocytes into the central nervous system (CNS) underlies the neuropathogenesis of HIV-1 infection. How this occurs is incompletely understood. We used a proteomic platform integrating difference gel electrophoresis and tandem mass spectrometry peptide sequencing to determine the effects that HIV-1-infected macrophages have on human brain microvascular endothelial cell (HBMEC) protein profiles. HIV-1 infected monocyte-derived macrophages (MDM) induced the upregulation of over 200 HBMEC proteins. These included metabolic, voltage-gated ion channels, heat shock, transport, cytoskeletal, regulatory, and calcium binding proteins. Results were validated by Western blot analysis. We conclude that HIV-1-infected MDM affect the HBMEC proteome and, in this way, affect BBB dysfunction and the development of HIV-1 CNS disease.

CSF proteomic fingerprints for HIV-associated cognitive impairment.

Cognitive impairment remains a major complication of advanced human immunodeficiency virus (HIV) infection despite the widespread use of anti-retroviral therapy. Diagnosis is made by exclusion making biomarkers of great potential use. Thus, we used an integrated proteomics platform to assess cerebrospinal fluid protein profiles from 50 HIV-1 seropositive Hispanic women. Nine of 38 proteins identified were unique in those patients with cognitive impairment (CI). These proteins were linked to cell signaling, structural function, and antioxidant activities. This work highlights, in a preliminary manner, the utility of proteomic profiling for biomarker discovery for HIV-1 associated cognitive dysfunction.

NS-gene based phylogenetic analysis of equine influenza viruses isolated in Poland.

The phylogenetic analysis of influenza virus is based mainly on the variable hemagglutinin or neuraminidase genes. However, some discrete evolutionary trends might be revealed when more conservative genes are considered. We compared all available in GenBank database full length NS sequences of equine influenza virus including Polish isolates. Four nucleotides at positions A202, A237, T672 and A714 and three amino acids at positions H59, K71 and S216 which are also present in A/eq/Pulawy/2006 and A/eq/Pulawy/2008 may be discriminating for the Florida sublineage. Threonine at position 83 seems to be characteristic for EIV strains of Florida 2 isolated after 2007. There are nine common substitutions in the NS sequences of A/eq/Pulawy/2005, A/eq/Aboyne/1/2005 and A/eq/Lincolnshire/1/2006 in relation to the reference strain A/eq/Miami/63, resulting in four amino acid changes in NS1 protein (I56, E76, K140, E179) and one in NEP (R22). We grouped these strains as "Aboyne-like". Some of the listed changes were also observed in H7N7 strains isolated between 1956 and 1966, in A/eq/Jilin/89 or in pre-divergent H3N8 strains. Two hypotheses regarding the origin of this group were postulated: three independent transfers of avian influenza viruses into the equine population or reassortation between H7N7 and H3N8 EIV. Similarities of the NS sequences of "Aboyne like" viruses to viruses isolated in the fifties or seventies can reflect a phenomenon of "frozen evolution".

Genetic typing of equine influenza virus isolated in Poland in 2005 and 2006.

Two equine influenza virus strains were isolated from horses during the local respiratory disease outbreaks in Poland in 2005 and 2006. The H3 equine influenza viral RNA was amplified directly from the clinical specimens with RT-PCR and HA1 fragments were sequenced. The highest homology of HA1 nucleotide sequences of A/eq/Pulawy/05 with A/eq/Aboyne/1/05 and A/eq/Pulawy/06 with A/eq/Essex/2/05 was found. The phylogenetic analysis based on amino acid sequences of HA1 fragments of 84 equine influenza virus strains isolated in Europe during the period of 1976-2007 was conducted to determine the evolutionary relationship of the two Polish and the other European isolates. The resulting phylogenetic tree clearly clustered A/eq/Pulawy/05 with the strains belonging to the European lineage of the equine influenza virus. On the other hand A/eq/Pulawy/06 was placed in the Florida sub-lineage of the American type strains. The presence of the same amino acids: methionine, asparagine and threonine at the positions 48, 159 and 163 respectively, in both Polish isolates, despite the fact that the strains are grouped in two different lineages may indicate the existence of the common ancestor. It is possible that A/eq/Pulawy/06 evolved locally rather than was introduced.

Sera proteomic biomarker profiling in HIV-1 infected subjects with cognitive impairment.

HIV-1 infection of the brain commonly leads to cognitive impairments (CIs). In its most severe form, HIV-1 associated dementia (HAD) is associated with advanced immune suppression and debilitating loss of memory, behavioral, and motor functions. Despite significant research activities, diagnosis remains one of exclusion. Bioimaging, neuropsychological testing, and viral and immune biomarkers serve to support but not define a diagnosis of HIV-1 associated CI. This is timely and required as brain injury triggered by HIV-1 can be controlled, in part, by antiretroviral medicines. The recent development of proteomics has opened new ways to study viral-host interactions which may provide new insight into treatment and disease monitoring. To this end, we developed a proteomics platform for HIV-1 associated CI biomarker discovery and used it to perform a pilot study for sera-associated HAD proteins. A 2-DE map of a serum proteome was focused on differentially expressed proteins. Differential expression of two proteins was validated by Western blot tests identifying afamin and ceruloplasmin as a potential biomarkers for CI associated with advanced HIV-1 infection.

Cerebrospinal fluid proteomic profiling of HIV-1-infected patients with cognitive impairment.

Advanced HIV-1 infection is commonly associated with progressive immune suppression and the development of cognitive, motor, and behavior disturbances. In its most severe form, it is diagnosed as HIV-1 associated dementia (HAD) and can progress to profound functional disability and death. Despite prodigious efforts to uncover biomarkers of HAD, none can adequately reflect disease onset or progression. Thus, we developed a proteomics platform for HAD biomarker discovery and used it to perform a pilot study on cerebrospinal fluid (CSF) from HIV-1-infected people with or without HAD. A 2-dimensional electrophoresis (2-DE) map of a HAD CSF proteome was focused on differentially expressed proteins. 2-DE difference gel electrophoresis (2-D DIGE) analysis showed >90 differences in protein spots of which 20 proteins were identified. Differential expression of 6 proteins was validated by Western blot tests and included vitamin D binding protein, clusterin, gelsolin, complement C3, procollagen C-endopeptidase enhancer 1, and cystatin C. We posit that these proteins, alone or together, are potential HAD biomarkers.