Detection of Parvovirus B19 genome in human heart tissue samples.

OBJECTIVE: Identifying viral genomes in human heart tissues is critical for disease diagnosis and assessment of cardiovascular damage. Human heart tissue samples obtained during a biopsy procedure are routinely used to test for the presence of viruses, as guided by clinical manifestations and prognosis. Furthermore, heart tissue samples obtained post-mortem or during a cardiac transplant procedure serve as a valuable research tool, as they allow for an in-depth assessment of cardiac pathology that can aid in our understanding of molecular pathways associated with disease. Because viral nucleic acid constitutes only a small portion of each sample's genetic material, appropriate methods are necessary for positive viral genome identification. RESULTS: Snap-frozen heart tissue samples obtained either post-mortem or during a cardiac transplant procedure were used to develop conditions for detection of Parvovirus B19. Briefly, total DNA was isolated from the heart tissue under varying conditions. A PCR-based assay with Parvovirus B19 specific primers was implemented to detect the presence of the viral genome, followed by Sanger Sequencing. The mechanical disruption of the heart tissue, as well as the cardiac tissue processing methods, had a significant effect on the DNA quality and the ability to detect the Parvovirus B19 genome.

Prevalence of Borrelia, Neoehrlichia mikurensis and Babesia in ticks collected from vegetation in eastern Poland.

In Poland, tick-borne diseases constitute the majority of diseases related to exposure to biological agents with a predominance of Lyme borreliosis; therefore, research on ticks as a reservoir of various pathogens remains crucial in the epidemiology of human diseases after tick bites. This study aimed to identify the occurrence of Borrelia burgdorferi sensu lato, Borrelia miyamotoi, Neoehrlichia mikurensis, and Babesia spp. in ticks collected from vegetation in eastern Poland. Additionally, the prevalence of co-infections in the adult Ixodes ricinus ticks was determined. Among I. ricinus ticks the predominantly detected pathogen was B. burgdorferi s.l. (23%) with B. burgdorferi sensu stricto as the most frequently identified species, followed by B. garinii. In 2013, the double or triple infections of B. burgdorferi s.s., B. afzelii, and B. garinii species did not exceed 9% in adult ticks, whereas in 2016, the prevalence of mixed infections reached 29%. The prevalence of N. mikurensis and B. miyamotoi in I. ricinus was determined at the same level of 2.8%. Four Babesia species were identified in the examined I. ricinus population: B. microti (1.5%), B. venatorum (1.2%), B. divergens (0.2%), and B. capreoli (0.1%). Co-infections were detected in 10.1% of all infected ticks with the highest prevalence of co-infections with B. burgdorferi s.l. and Babesia species. The changes in the prevalence and the distribution of particular pathogens within tick populations indicate the need for monitoring the current situation related to tick-borne pathogens from the aspect of risk to human health.

Deoptimization of FMDV P1 Region Results in Robust Serotype-Independent Viral Attenuation.

Foot-and-mouth disease (FMD), caused by the FMD virus (FMDV), is a highly contagious disease of cloven-hoofed livestock that can have severe economic impacts. Control and prevention strategies, including the development of improved vaccines, are urgently needed to effectively control FMD outbreaks in endemic settings. Previously, we employed two distinct strategies (codon pair bias deoptimization (CPD) and codon bias deoptimization (CD)) to deoptimize various regions of the FMDV serotype A subtype A12 genome, which resulted in the development of an attenuated virus in vitro and in vivo, inducing varying levels of humoral responses. In the current study, we examined the versatility of the system by using CPD applied to the P1 capsid coding region of FMDV serotype A subtype, A24, and another serotype, Asia1. Viruses carrying recoded P1 (A24-P1Deopt or Asia1-P1Deopt) exhibited different degrees of attenuation (i.e., delayed viral growth kinetics and replication) in cultured cells. Studies in vivo using a mouse model of FMD demonstrated that inoculation with the A24-P1Deopt and Asia1-P1Deopt strains elicited a strong humoral immune response capable of offering protection against challenge with homologous wildtype (WT) viruses. However, different results were obtained in pigs. While clear attenuation was detected for both the A24-P1Deopt and Asia1-P1Deopt strains, only a limited induction of adaptive immunity and protection against challenge was detected, depending on the inoculated dose and serotype deoptimized. Our work demonstrates that while CPD of the P1 coding region attenuates viral strains of multiple FMDV serotypes/subtypes, a thorough assessment of virulence and induction of adaptive immunity in the natural host is required in each case in order to finely adjust the degree of deoptimization required for attenuation without affecting the induction of protective adaptive immune responses.

Persistent viral infections and their role in heart disease.

Viral infections are the culprit of many diseases, including inflammation of the heart muscle, known as myocarditis. Acute myocarditis cases have been described in scientific literature, and viruses, such as parvovirus B19, coxsackievirus B3, or more recently, SARS-CoV-2, were the direct cause of cardiac inflammation. If not treated, myocarditis could progress to dilated cardiomyopathy, which permanently impairs the heart and limits a person's lifespan. Accumulated evidence suggests that certain viruses may persist in cardiac tissue after the initial infection, which could open up the door to reactivation under favorable conditions. Whether this chronic infection contributes to, or initiates, cardiac damage over time, remains a pressing issue in the field of virus-induced heart pathology, and it is directly tied to patients' treatment. Previously, large case studies found that a few viruses: parvovirus B19, coxsackievirus, adenovirus, human herpesvirus 6, cytomegalovirus and Epstein-Barr virus, are most commonly found in human endomyocardial biopsy samples derived from patients experiencing cardiac inflammation, or dilated cardiomyopathy. SARS-CoV-2 infection has also been shown to have cardiovascular consequences. This review examines the role of viral persistence in cardiac inflammation and heart disease, and discusses its implications for patients' outcomes.

Residues within the Foot-and-Mouth Disease Virus 3Dpol Nuclear Localization Signal Affect Polymerase Fidelity.

Many RNA viruses encode a proof-reading deficient, low-fidelity RNA-dependent polymerase (RdRp), which generates genetically diverse populations that can adapt to changing environments and thwart antiviral therapies. 3Dpol, the RdRp of the foot-and-mouth disease virus (FMDV), is responsible for replication of viral genomes. The 3Dpol N terminus encodes a nuclear localization signal (NLS) sequence,MRKTKLAPT, important for import of the protein to host nucleus. Previous studies showed that substitutions at residues 18 and 20 of the NLS are defective in proper incorporation of nucleotides and RNA binding. Here, we use a systematic alanine scanning mutagenesis approach to understand the role of individual residues of the NLS in nuclear localization and nucleotide incorporation activities of 3Dpol We identify two residues of 3Dpol NLS, T19 and L21, that are important for the maintenance of enzyme fidelity. The 3Dpol NLS alanine substitutions of T19 and L21 results in aberrant incorporation of nucleoside analogs, conferring a low fidelity phenotype of the enzyme. A molecular dynamics simulation of RNA- and mutagen (RTP)-bound 3Dpol revealed that the T19 residue participates in a hydrogen bond network, including D165 in motif F and R416 at the C terminus of the FMDV 3Dpol and RNA template-primer. Based on these findings and previous studies, we conclude that at least the first six residues of theMRKTKLAPT sequence motif play a vital role in the maintenance of faithful RNA synthesis activity (fidelity) of FMDV 3Dpol, suggesting that the role of the NLS motif in similar viral polymerases needs to be revisited.IMPORTANCE In this study, we employed genetic and molecular dynamics approaches to analyze the role of individual amino acids of the FMDV 3Dpol nuclear localization signal (NLS). The NLS residues were mutated to alanine using a type A full-genome cDNA clone, and the virus progeny was analyzed for defects in growth and in competition with the parental virus. We identified two mutants in 3Dpol, T19A and L21A, that exhibited high rate of mutation, were sensitive to nucleotide analogs, and displayed reduced replicative fitness compared to the parental virus. Using molecular dynamics simulation, we demonstrated that residues T19 and L21 played a role in the structural configuration of the interaction network at the 3Dpol palm subdomain. Cumulatively, our data suggest that the T19 and L21 3Dpol amino acids are important for maintaining the fidelity of the FMDV polymerase and ensuring faithful replication of the FMDV genome.

Potential sources of infection with selected zoonotic agents in the veterinary work environment - pilot studies.

INTRODUCTION AND OBJECTIVE: The problem of occupational biohazards is very important, especially in the field of agriculture and in human and veterinary medicine. The aim of the study was to determine the potential sources of infection in veterinary professionals with selected zoonotic agents, including: Toxoplasma gondii, Giardia duodenalis, Leptospira spp., Cryptosporidium spp. and Coxiella burnetii. MATERIAL AND METHODS: A total of 50 air samples from barns, piggeries and veterinary surgeries were examined for the presence of Leptospira spp. and C. burnetii DNA. Serum samples of 86 pigs and 80 cows were tested for the presence of antibodies to Leptospira spp. and to phase I and II C. burnetii antigens. Serum of 70 cats were tested for the presence of antibodies to T. gondii and 65 samples of cat faeces for the presence of T. gondii oocysts. The presence of G. duodenalis and Cryptosporidium spp. were examined in 50 of dog faeces and 50 of bovine faeces samples. RESULTS: DNA of Leptospira spp. was detected in 2 air samples from the piggeries (4%). C. burnetii DNA was not found in any sample. Anti-Leptospira spp. antibodies were detected in 51 (59.3%) of examined pigs. Neither anti-Leptospira spp. nor anti-C. burnetii antibodies were found among samples of bovine serum. Anti-T. gondii antibodies was found in 52 cat serum samples (74.3%). Among samples of cat faeces, no T. gondii oocysts were detected. In one sample of cattle stool (2%), G. duodenalis was detected and in another (2%) - Cryptosporidium spp. G. duodenalis was detected in 7 samples (14%) and Cryptosporidium spp. in 2 samples (2%) of dog faeces. CONCLUSIONS: The results of this study demonstrate the potential risk of infection with Leptospira spp. in veterinarians working with pigs. Veterinarians could be also be at risk of infection with T. gondii and G. duodenalis.

Use of Synonymous Deoptimization to Derive Modified Live Attenuated Strains of Foot and Mouth Disease Virus.

Foot-and-mouth disease (FMD) is one of the most economically important viral diseases that can affect livestock. In the last 70 years, use of an inactivated whole antigen vaccine has contributed to the eradication of disease from many developed nations. However, recent outbreaks in Europe and Eastern Asia demonstrated that infection can spread as wildfire causing economic and social devastation. Therefore, it is essential to develop new control strategies that could confer early protection and rapidly stop disease spread. Live attenuated vaccines (LAV) are one of the best choices to obtain a strong early and long-lasting protection against viral diseases. In proof of concept studies, we previously demonstrated that "synonymous codon deoptimization" could be applied to the P1 capsid coding region of the viral genome to derive attenuated FMDV serotype A12 strains. Here, we demonstrate that a similar approach can be extended to the highly conserved non-structural P2 and P3 coding regions, providing a backbone for multiple serotype FMDV LAV development. Engineered codon deoptimized P2, P3 or P2, and P3 combined regions were included into the A24Cruzeiro infectious clone optimized for vaccine production, resulting in viable progeny that exhibited different degrees of attenuation in cell culture, in mice, and in the natural host (swine). Derived strains were thoroughly characterized in vitro and in vivo. Our work demonstrates that overall, the entire FMDV genome tolerates codon deoptimization, highlighting the potential of using this technology to derive novel improved LAV candidates.

The Roles of Picornavirus Untranslated Regions in Infection and Innate Immunity.

Viral genomes have evolved to maximize their potential of overcoming host defense mechanisms and to induce a variety of disease syndromes. Structurally, a genome of a virus consists of coding and noncoding regions, and both have been shown to contribute to initiation and progression of disease. Accumulated work in picornaviruses has stressed out the importance of the noncoding RNAs, or untranslated 5'- and 3'-regions (UTRs), in both replication and translation of viral genomes. Unsurprisingly, defects in these processes have been reported to cause viral attenuation and affect viral pathogenicity. However, substantial evidence suggests that these untranslated RNAs may influence the outcome of the host innate immune response. This review discusses the involvement of 5'- and 3'-terminus UTRs in induction and regulation of host immunity and its consequences for viral life cycle and virulence.

Streptococcus suis: a re-emerging pathogen associated with occupational exposure to pigs or pork products. Part II - Pathogenesis.

Streptococcus suis is a re-emerging zoonotic pathogen that may cause severe disease, mostly meningitis, in pigs and in humans having occupational contact with pigs and pork, such as farmers, slaughterhose workers and butchers. The first stage of the pathogenic process, similar in pigs and humans, is adherence to and colonisation of mucosal and/or epithelial surface(s) of the host. The second stage is invasion into deeper tissue and extracellular translocation of bacterium in the bloodstream, either free in circulation or attached to the surface of monocytes. If S. suis present in blood fails to cause fatal septicaemia, it is able to progress into the third stage comprising penetration into host's organs, mostly by crossing the blood-brain barrier and/or blood-cerebrospinal fluid barrier to gain access to the central nervous system (CNS) and cause meningitis. The fourth stage is inflammation that plays a key role in the pathogen esis of both systemic and CNS infections caused by S. suis. The pathogen may induce the overproduction of pro-inflammatory cytokines that cause septic shock and/or the recruitment and activation of different leukocyte populations, causing acute inflammation of the CNS. Streptococcus suis can also evoke - through activation of microglial cells, astrocytes and possibly other cell types - a fulminant inflammatory reaction of the brain which leads to intracranial complications, including brain oedema, increased intracranial pressure, cerebrovascular insults, and deafness, as a result of cochlear sepsis. In all stages of the pathogenic process, S. suis interacts with many types of immunocompetent host's cells, such as polymorphonuclear leukocytes, mononuclear macrophages, lymphocytes, dendritic cells and microglia, using a range of versatile virulence factors for evasion of the innate and adaptive immune defence of the host, and for overcoming environmental stress. It is estimated that S. suis produces more than 100 different virulence factors that could be classified into 4 groups: surface components or secreted elements, enzymes, transcription factors or regulatory systems and transporter factors or secretion systems. A major virulence factor is capsular polysaccharide (CPS) that protects bacteria from phagocytosis. However, it hampers adhesion to and invasion of host's cells, release of inflammatory cytokines and formation of the resistant biofilm which, in many cases, is vital for the persistence of bacteria. It has been demonstrated that the arising by mutation unencapsulated S. suis clones, which are more successful in penetration to and propagation within the host's cells, may coexist in the organism of a single host together with those that are encapsulated. Both 'complementary' clones assist each other in the successful colonization of host's tissues and persistence therein. S. suis has an open pan-genome characterized by a frequent gene transfer and a large diversity. Of the genetic determinants of S. suis pathogenicity, the most important are pathogenicity islands (PAI), in particular, a novel DNA segment of 89 kb length with evident pathogenic traits that has been designated as 89K PAI. It has been estimated that more than one-third of the S. suis virulence factors is associated with this PAI. It has been proved that the virulent S. suis strains possess smaller genomes, compared to avirulent ones, but more genes associated with virulence. Overall, the evolution of the species most probably aims towards increased pathogenicity, and hence the most significant task of the current research is an elaboration of a vaccine, efficient both for humans and pigs.

Study on Toxoplasma Gondii, Leptospira Spp., Coxiella Burnetii, and Echinococcus Granulosus Infection in Veterinarians from Poland.

INTRODUCTION: Exposure to zoonotic factors in veterinary practice is closely related to the nature of the work. The main aim of the study was to determine the risk of selected zoonotic infections among the occupational group of veterinarians in Poland. MATERIAL AND METHODS: Blood samples of 373 veterinarians (162 males and 211 females) from 12 provinces of Poland were collected by the venipuncture of a forearm for serological tests. Commercial immunoenzymatic tests (ELISA) were used for detection of specific IgG antibodies to Echinococcus granulosus, IgM and IgG to Leptospira spp., and IgM, IgA, and I and II phase IgG to Coxiella burnetii. Enzyme-linked fluorescence assays (ELFA) were used to detect IgM and IgG antibodies to Toxoplasma gondii. RESULTS: Positive results were found in 209 (56.0%) veterinarians for at least one of the examined diseases. The overall proportion of participants found to have specific Toxoplasma gondii antibodies in the IgM and/or IgG assays amounted to 44.5%. The presence of Coxiella burnetii antibodies was found in 16 (4.3%) subjects, while Leptospira spp. antibodies were detected in 63 (16.9%) veterinarians. Among the 373 veterinarians examined, no Echinococcus granulosus antibodies were found. CONCLUSION: Results of the study seem to indicate a slightly elevated risk of Toxoplasma gondii infection and a moderate risk of infection with Leptospira spp. and Coxiella burnetii in veterinarians.

Streptococcus suis: a re-emerging pathogen associated with occupational exposure to pigs or pork products. Part I - Epidemiology.

Streptococcus suis (ex Elliot 1966, Kilpper-Bälz & Schleifer 1987) is a facultatively anaerobic Gram-positive ovoid or coccal bacterium surrounded by a polysaccharide capsule. Based on the antigenic diversity of the capsule, S. suis strains are classified serologically into 35 serotypes. Streptococcus suis is a commensal of pigs, commonly colonizing their tonsils and nasal cavities, mostly in weaning piglets between 4-10 weeks of age. This species occurs also in cattle and other mammals, in birds and in humans. Some strains, mostly those belonging to serotype 2, are also pathogenic for pigs, as well as for other animals and humans. Meningitis is the primary disease syndrome caused by S. suis, both in pigs and in humans. It is estimated that meningitis accounted for 68.0% of all cases of human disease reported until the end of 2012, followed by septicaemia (including life-threatening condition described as 'streptococcal toxic shock-like syndrome' - STSLS), arthritis, endocarditis, and endophthalmitis. Hearing loss and/or ves tibular dysfunction are the most common sequelae after recovery from meningitis caused by S. suis, occurring in more than 50% of patients. In the last two decades, the number of reported human cases due to S. suis has dramatically increased, mostly due to epidemics recorded in China in 1998 and 2005, and the fulminant increase in morbidity in the countries of south-eastern Asia, mostly Vietnam and Thailand. Out of 1,642 cases of S. suis infections identified between 2002-2013 worldwide in humans, 90.2% occurred in Asia, 8.5% in Europe and 1.3% in other parts of the globe. The human disease has mostly a zoonotic and occupational origin and occurs in pig breeders, abattoir workers, butchers and workers of meat processing facilities, veterinarians and meat inspectors. Bacteria are transmitted to workers by close contact with pigs or pig products, usually through contamination of minor cuts or abrasions on skin of hands and/or arms, or by pig bite. A different epidemiologic situation occurs in the Southeast Asian countries where most people become infected by habitual consumption of raw or undercooked pork, blood and offal products in the form of traditional dishes. Prevention of S. suis infections in pigs includes vaccination, improvement in pig-raising conditions, disinfection and/or fumigation of animal houses, and isolation of sick animals at the outbreak of disease. Prevention of human infections comprises: protection of skin from pig bite or injury with sharp tools by people occupationally exposed to pigs and pig products, prompt disinfection and dressing of wounds and abrasions at work, protection of the respiratory tract by wearing appropriate masks or repirators, consulting a doctor in the case of febrile illness after exposure to pigs or pork meat, avoidance of occupations associated with exposure to pigs and pork by immunocompomised people, avoidance of consumption of raw pork or pig blood, adequate cooking of pork, and health education.

Foot-and-mouth disease virus 5'-terminal S fragment is required for replication and modulation of the innate immune response in host cells.

The S fragment of the FMDV 5' UTR is predicted to fold into a long stem-loop structure and it has been implicated in virus-host protein interactions. In this study, we report the minimal S fragment sequence required for virus viability and show a direct correlation between the extent of the S fragment deletion mutations and attenuated phenotypes. Furthermore, we provide novel insight into the role of the S fragment in modulating the host innate immune response. Importantly, in an FMDV mouse model system, all animals survive the inoculation with the live A24 FMDV-S4 mutant, containing a 164 nucleotide deletion in the upper S fragment loop, at a dose 1000 higher than the one causing lethality by parental A24 FMDV, indicating that the A24 FMDV-S4 virus is highly attenuated in vivo. Additionally, mice exposed to high doses of live A24 FMDV-S4 virus are fully protected when challenged with parental A24 FMDV virus.

Attenuation of Foot-and-Mouth Disease Virus by Engineered Viral Polymerase Fidelity.

Foot-and-mouth disease virus (FMDV) RNA-dependent RNA polymerase (RdRp) (3Dpol) catalyzes viral RNA synthesis. Its characteristic low fidelity and absence of proofreading activity allow FMDV to rapidly mutate and adapt to dynamic environments. In this study, we used the structure of FMDV 3Dpol in combination with previously reported results from similar picornaviral polymerases to design point mutations that would alter replication fidelity. In particular, we targeted Trp237 within conserved polymerase motif A because of the low reversion potential inherent in the single UGG codon. Using biochemical and genetic tools, we show that the replacement of tryptophan 237 with phenylalanine imparts higher fidelity, but replacements with isoleucine and leucine resulted in lower-fidelity phenotypes. Viruses containing these W237 substitutions show in vitro growth kinetics and plaque morphologies similar to those of the wild-type (WT) A24 Cruzeiro strain in BHK cells, and both high- and low-fidelity variants retained fitness during coinfection with the wild-type virus. The higher-fidelity W237F (W237FHF) mutant virus was more resistant to the mutagenic nucleoside analogs ribavirin and 5-fluorouracil than the WT virus, whereas the lower-fidelity W237I (W237ILF) and W237LLF mutant viruses exhibited lower ribavirin resistance. Interestingly, the variant viruses showed heterogeneous and slightly delayed growth kinetics in primary porcine kidney cells, and they were significantly attenuated in mouse infection experiments. These data demonstrate, for a single virus, that either increased or decreased RdRp fidelity attenuates virus growth in animals, which is a desirable feature for the development of safer and genetically more stable vaccine candidates.IMPORTANCE Foot-and-mouth disease (FMD) is the most devastating disease affecting livestock worldwide. Here, using structural and biochemical analyses, we have identified FMDV 3Dpol mutations that affect polymerase fidelity. Recombinant FMDVs containing substitutions at 3Dpol tryptophan residue 237 were genetically stable and displayed plaque phenotypes and growth kinetics similar to those of the wild-type virus in cell culture. We further demonstrate that viruses harboring either a W237FHF substitution or W237ILF and W237LLF mutations were highly attenuated in animals. Our study shows that obtaining 3Dpol fidelity variants by protein engineering based on polymerase structure and function could be exploited for the development of attenuated FMDV vaccine candidates that are safer and more stable than strains obtained by selective pressure via mutagenic nucleotides or adaptation approaches.

Prevalence of infections and co-infections with 6 pathogens in Dermacentor reticulatus ticks collected in eastern Poland.

Occurrence of co-infections with various pathogens in ixodid ticks creates a risk of increased severity of tick-borne diseases in humans and animals exposed to bite of the ticks carrying multiple pathogens. Accordingly, co-infections in ticks were subject of numerous analyses, but almost exclusively with regard to Ixodes ricinus complex whereas potential tick vectors belonging to other genera were much less studied. Taking into consideration the role of Dermacentor reticulatus in the transmission of various pathogens, we carried out for the first time the comprehensive statistical analysis of co-infections occurring in this tick species. An attempt was made to determine the significance of the associations between 6 different pathogens occurring in D. reticulatus (Tick-borne encephalitis virus = TBEV, Anaplasma phagocytophilum, Rickettsia raoultii, Borrelia burgdorferi s. l., Babesia spp., Toxoplasma gondii), using 2 statistical methods: determination of Odds Ratios (ORs) and the Fisher's exact test. 634 questing Dermacentor reticulatus ticks (370 females and 264 males) were collected in 2011- 2013 by flagging the lower vegetation in 3 localities in the area of Leczynsko-Wlodawskie Lakeland, situated in the Lublin region of eastern Poland. The presence of individual pathogens was detected by PCR. Ticks were infected most often with Rickettsia raoultii (43.8%), less with TBEV (8.5%), and much less with Babesia spp., Toxoplasma gondii, Borrelia burgdorferi s.l., and Anaplasma phagocytophilum (2.5%, 2.1%, 1.6% and 1.1%, respectively). The locality-dependent variability proved to be significant for TBEV (c2=11.063; P=0.004) and Toxoplasma gondii (c2=11.298; P=0.0035), but not for other pathogens. Two hundred seventy (42.6%) of the examined ticks were infected only with a single pathogen, and 54 (8.5%) showed the presence of dual co-infections, each with 2 pathogens. The most common were dual infections with participation of Rickettsia raoultii (7.41%); next, those with participation of the TBEV (5.21%), Toxoplasma gondii (1.58%), Borrelia burgdorferi s.l. (1.26%), Anaplasma phagocytophilum (0.95%), and Babesia spp. (0.63%). On the total number of 15 possible associations, in 9 cases co-infections occurred whereas in 6 cases they were not detected. The most noteworthy were positive co-infections with the participation of TBEV, which proved to be weakly significant (0.05

Interaction between FMDV Lpro and transcription factor ADNP is required for optimal viral replication.

The foot-and-mouth disease virus (FMDV) leader protease (Lpro) inhibits host translation and transcription affecting the expression of several factors involved in innate immunity. In this study, we have identified the host transcription factor ADNP (activity dependent neuroprotective protein) as an Lpro interacting protein by mass spectrometry. We show that Lpro can bind to ADNP in vitro and in cell culture. RNAi of ADNP negatively affected virus replication and higher levels of interferon (IFN) and IFN-stimulated gene expression were detected. Importantly, infection with FMDV wild type but not with a virus lacking Lpro (leaderless), induced recruitment of ADNP to IFN-α promoter sites early during infection. Furthermore, we found that Lpro and ADNP are in a protein complex with the ubiquitous chromatin remodeling factor Brg-1. Our results uncover a novel role of FMDV Lpro in targeting ADNP and modulation of its transcription repressive function to decrease the expression of IFN and ISGs.

Presence of Pathogenic Rickettsiae and Protozoan in Samples of Raw Milk from Cows, Goats, and Sheep.

OBJECTIVE: The aim of the present work was to determine the presence of various rickettsiae and protozoan in raw milk and the assessment the potential, milk-borne route in the spread of selected zoonotic pathogens. MATERIALS: A total of 119 raw milk samples collected randomly from 63 cows, 29 goats, and 27 sheep bred on 34 farms situated on eight communities in eastern Poland were examined by polymerase chain reaction (PCR) method for the presence of pathogenic rickettsiae (Coxiella burnetii, Anaplasma phagocytophilum, and Rickettsia spp.) and protozoan (Toxoplasma gondii). RESULTS: The only prevalent pathogen was T. gondii, which was found in 10 samples of cow milk (15.9%), in one sample of goat milk (3.4%), and in one sample of sheep milk (3.7%). One sample of cow milk was positive for C. burnetii; however, the sequence analysis did not confirm any species of Coxiella or Coxiella-like organisms, but showed 100% homology to Psychrobacter alimentarius. None of the examined samples showed the presence of A. phagocytophilum or Rickettsia spp. CONCLUSIONS: The results of this study suggest a potential hazard of milk-borne Toxoplasma infection, mostly by consumption of raw cow milk. The milk-borne spread seems to be limited or nonsignificant in the case of C. burnetii, A. phagocytophilum, and Rickettsia spp. The false-positive sample for Coxiella spp. suggests that some care should be taken in the interpretation of the results obtained by using the PCR method.

Analysis of the interaction between host factor Sam68 and viral elements during foot-and-mouth disease virus infections.

BACKGROUND: The nuclear protein Src-associated protein of 68 kDa in mitosis (Sam68) is known to bind RNA and be involved in cellular processes triggered in response to environmental stresses, including virus infection. Interestingly, Sam68 is a multi-functional protein implicated in the life cycle of retroviruses and picornaviruses and is also considered a marker of virus-induced stress granules (SGs). Recently, we demonstrated the partial redistribution of Sam68 to the cytoplasm in FMDV infected cells, its interaction with viral protease 3C(pro), and found a significant reduction in viral titers as consequence of Sam68-specific siRNA knockdowns. Despite of that, details of how it benefits FMDV remains to be elucidated. METHODS: Sam68 cytoplasmic localization was examined by immunofluorescent microscopy, counterstaining with antibodies against Sam68, a viral capsid protein and markers of SGs. The relevance of RAAA motifs in the IRES was investigated using electromobility shift assays with Sam68 protein and parental and mutant FMDV RNAs. In addition, full genome WT and mutant or G-luc replicon RNAs were tested following transfection in mammalian cells. The impact of Sam68 depletion to virus protein and RNA synthesis was investigated in a cell-free system. Lastly, through co-immunoprecipitation, structural modeling, and subcellular fractionation, viral protein interactions with Sam68 were explored. RESULTS: FMDV-induced cytoplasmic redistribution of Sam68 resulted in it temporarily co-localizing with SG marker: TIA-1. Mutations that disrupted FMDV IRES RAAA motifs, with putative affinity to Sam68 in domain 3 and 4 cause a reduction on the formation of ribonucleoprotein complexes with this protein and resulted in non-viable progeny viruses and replication-impaired replicons. Furthermore, depletion of Sam68 in cell-free extracts greatly diminished FMDV RNA replication, which was restored by addition of recombinant Sam68. The results here demonstrated that Sam68 specifically co-precipitates with both FMDV 3D(pol) and 3C(pro) consistent with early observations of FMDV 3C(pro)-induced cleavage of Sam68. CONCLUSION: We have found that Sam68 is a specific binding partner for FMDV non-structural proteins 3C(pro) and 3D(pol) and showed that mutations at RAAA motifs in IRES domains 3 and 4 cause a decrease in Sam68 affinity to these RNA elements and rendered the mutant RNA non-viable. Interestingly, in FMDV infected cells re-localized Sam68 was transiently detected along with SG markers in the cytoplasm. These results support the importance of Sam68 as a host factor co-opted by FMDV during infection and demonstrate that Sam68 interact with both, FMDV RNA motifs in the IRES and viral non-structural proteins 3C(pro) and 3D(pol).

Chromatin and pluripotency: the MYSTerious connection.

Although histone acetylation is critical for maintaining embryonic stem cell pluripotency, the molecular machinery involved remains poorly understood. Li et al. (2012) now show that Mof, a MYST family histone acetyltransferase, functions as a coactivator of Nanog-mediated transcription, maintains the expression of pluripotency-associated genes, and primes developmental genes for differentiation.

RNAi promotes heterochromatic silencing through replication-coupled release of RNA Pol II.

Heterochromatin comprises tightly compacted repetitive regions of eukaryotic chromosomes. The inheritance of heterochromatin through mitosis requires RNA interference (RNAi), which guides histone modification during the DNA replication phase of the cell cycle. Here we show that the alternating arrangement of origins of replication and non-coding RNA in pericentromeric heterochromatin results in competition between transcription and replication in Schizosaccharomyces pombe. Co-transcriptional RNAi releases RNA polymerase II (Pol II), allowing completion of DNA replication by the leading strand DNA polymerase, and associated histone modifying enzymes that spread heterochromatin with the replication fork. In the absence of RNAi, stalled forks are repaired by homologous recombination without histone modification.

RNAi, heterochromatin and the cell cycle.

For many decades after its initial characterization, heterochromatin was considered to be transcriptionally inert, but newer work indicates that this highly condensed chromosomal material is transcribed, and rapidly silenced, by an orchestrated sequence of events directed by RNA interference (RNAi). Recent studies shed light on the timely assembly and inheritance of heterochromatin within a short period during the cell cycle, thereby providing an explanation for how 'silent' heterochromatin can be transcribed during the S phase of the cell cycle. Together, these findings suggest a model of RNAi-directed epigenetic inheritance.