Chimeric proteins of Mycoplasma hyopneumoniae as vaccine and preclinical model for immunological evaluation.

Mycoplasma hyopneumoniae (M. hyopneumoniae) is a primary agent of porcine enzootic pneumonia, a disease that causes significant economic losses to pig farming worldwide. Commercial vaccines induce partial protection, evidencing the need for a new vaccine against M. hyopneumoniae. In our work, three chimeric proteins were constructed, composed of potentially immunogenic domains from M. hyopneumoniae proteins. We designed three chimeric proteins (Q1, Q2, and Q3) based on bioinformatics analysis that identified five potential proteins with immunogenic potential (MHP418, MHP372, MHP199, P97, and MHP0461). The chimeric proteins were inoculated in the murine model to evaluate the immune response. The mice vaccinated with the chimeras presented IgG and IgG1 against proteins of M. hyopneumoniae. There was induction of IgG in mice immunized with Q3 starting from 30 days post-vaccination, and groups Q1 and Q2 showed induction at 45 days. Mice of the group immunized with Q3 showed the production of IgA. In addition, the mice inoculated with chimeric proteins showed a proinflammatory cytokine response; Q1 demonstrated higher levels of TNF, IL-6, IL2, and IL-17. In contrast, animals immunized with Q2 showed an increase in the concentrations of TNF, IL-6, and IL-4, whereas those immunized with Q3 exhibited an increase in the concentrations of TNF, IL-6, IL-10, and IL-4. The results of the present study indicate that these three chimeric proteins can be used in future vaccine trials with swine because of the promising antigenicity.

A genetic and virulence characterization of Brazilian strains of Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae (M. hyopneumoniae) is considered the primary causative agent of porcine enzootic pneumonia (EP), a chronic contagious respiratory disease that causes economic losses. Obtaining new pathogenic isolates and studying the genome and virulence factors are necessary. This study performed a complete sequencing analysis of two Brazilian strains, UFV01 and UFV02, aiming to characterize the isolates in terms of the virulence factors and sequence type. The complete genome analysis revealed the main virulence genes (mhp385, mhp271, MHP_RS03455, p102, p97, p216, MHP_RS00555, mhp107) and ST-123, the presence of three toxin-related genes (tlyC, PLDc_2 and hcnC), and some genetic groups specific to these two isolates. Subsequently, the pathogenicity of the isolates was evaluated via an experimental infection conducted in a swine model. The study was divided into three groups, namely a negative control group (n = 4) and two test groups (n = 8), totaling 20 animals. They were challenged at 35 days of age with 107 CCU (Color Changing Units) M. hyopneumoniae via the intratracheal route. The UFV01 group showed earlier and higher seroconversion (IgG) (100%), while only 50% of the UFV02 group seroconverted. The same trend was observed when analyzing the presence of IgA in the bronchoalveolar lavage fluid (BALF) at 35 days post-infection (dpi). The UFV01 group had a mean macroscopic lesion score of 11.75% at 35 dpi, while UFV02 had 3.125%. Microscopic lesions were more severe in the UFV01 group. Based on laryngeal swab samples evaluated by qPCR, and the detection began at 14 days. The UFV01 group showed 75% positivity at 14 dpi. The UFV02 group also started excreting at 14 dpi, with a positivity rate of 37.5%. The results indicate that the UFV01 isolate exhibits higher virulence than UFV02. These findings may aid in developing new vaccines and diagnostic kits and establishing experimental models for testing.

Reduced HIV/AIDS diagnosis rates and increased AIDS mortality due to late diagnosis in Brazil during the COVID-19 pandemic.

The COVID-19 pandemic has severely affected global health, leading to the suspension of numerous routine healthcare services and posing challenges in efforts to control other diseases, such as HIV/AIDS. This study aimed to assess the impact of the COVID-19 pandemic on HIV/AIDS diagnoses and mortality rates in Brazil during 2020 and 2021. The percentage change was calculated to determine whether there was an increase or decrease in HIV/AIDS diagnoses and mortality, considering the average numbers from the last 5 years. Additionally, a Joinpoint regression model and an interrupted time series analysis were applied to assess time trends before and after the onset of the pandemic. Lastly, choropleth maps were prepared. We observed a reduction of 22.4% (2020) and 9.8% (2021) in the diagnosis of HIV/AIDS in Brazil. Conversely, there was a significant increase in the percentage change of late diagnosis of AIDS deaths in 2020 (6.9%) and 2021 (13.9%), with some states showing an increase of over 87%. Decreasing time trends in the diagnosis of HIV/AIDS were identified before the pandemic in Brazil, especially in the Southeast and South regions, and then time trends stabilized after including the pandemic years. Along with the dissemination of COVID-19, there was a reduction in the diagnosis of HIV/AIDS and an increase in late diagnosis AIDS deaths, signaling a serious impact of the pandemic on HIV/AIDS control strategies in Brazil. Therefore, we highlight the need for continuous efforts to control both diseases, that is, maintaining regular health services even in crisis situations.

Synergism between molecules derived from Garcinia brasiliensis and antimicrobial drugs on field isolates of Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae (M. hyopneumoniae) is one of the smallest free-living bacteria found in nature; it has an extremely small genome and lacks a cell wall. It is the main etiological agent of porcine enzootic pneumonia (EP), a chronic respiratory disease with worldwide distribution that causes significant losses in swine production. Due to the great economic impact caused by EP, new strategies for treating and controlling this agent are researched. The objective of this study was to verify the anti-M. hyopneumoniae activity of compounds derived from Garcinia brasiliensis and the synergism with the main antimicrobials used in the treatment of EP; this is the first study assessing the synergism between bioactive molecules and antimicrobial compounds in vitro against isolates of M. hyopneumoniae. The minimum inhibitory concentrations (MICs) of the antimicrobials tiamulin, valnemulin, and enrofloxacin, as well as the bioactive compounds guttiferone-A (Gut-A), 7-epiculsone (7-Epic), copper 7-epiculsone (7-Epic-Cu), and benzophenone, were determined. Subsequently, the interactions of antibiotics with the compounds were evaluated using the checkerboard method. Three field M. hyopneumoniae isolates were used, and the J strain was used as a control. The MIC values of the antimicrobials compared to the field isolates were equal to and lower than those of the reference strain J. Among the compounds used, 7-Epic-Cu showed the lowest MIC value. Synergistic association was observed for Gut-A with tiamulin and valnemulin, whereas 7-Epic and 7-Epic-Cu showed synergistic action with enrofloxacin. No synergistic effect was observed for benzophenone. Despite being an initial study, the results suggest that these combinations hold promise for the treatment of infections caused by M. hyopneumoniae.

High-Risk Regions of African Swine Fever Infection in Mozambique.

African swine fever (ASF) is a transboundary infectious disease that can infect wild and domestic swine and requires enhanced surveillance between countries. In Mozambique, ASF has been reported across the country, spreading between provinces, mainly through the movement of pigs and their by-products. Subsequently, pigs from bordering countries were at risk of exposure. This study evaluated the spatiotemporal distribution and temporal trends of ASF in swine in Mozambique between 2000 and 2020. During this period, 28,624 cases of ASF were reported across three regions of the country. In total, the northern, central, and southern regions presented 64.9, 17.8, and 17.3% of the total cases, respectively. When analyzing the incidence risk (IR) of ASF per 100,000 pigs, the Cabo Delgado province had the highest IR (17,301.1), followed by the Maputo province (8868.6). In the space-time analysis, three clusters were formed in each region: (i) Cluster A involved the provinces of Cabo Delgado and Nampula (north), (ii) Cluster B involved the province of Maputo and the city of Maputo (south), and (iii) Cluster C consisted of the provinces of Manica and Sofala (central) in 2006. However, when analyzing the temporal trend in the provinces, most were found to be decreasing, except for Sofala, Inhambane, and Maputo, which had a stationary trend. To the best of our knowledge, this is the first study to evaluate the spatial distribution of ASF in Mozambique. These findings will contribute to increasing official ASF control programs by identifying high-risk areas and raising awareness of the importance of controlling the borders between provinces and countries to prevent their spread to other regions of the world.

Immunomodulatory activity of trifluoromethyl arylamides derived from the SRPK inhibitor SRPIN340 and their potential use as vaccine adjuvant.

The serine/arginine-rich protein kinases (SRPK) specifically phosphorylate their substrates at RS-rich dipeptides, which are abundantly found in SR splicing factors. SRPK are classically known for their ability to affect the splicing and expression of gene isoforms commonly implicated in cancer and diseases associated with infectious processes. Non-splicing functions have also been attributed to SRPK, which highlight their functional plasticity and relevance as therapeutic targets for pharmacological intervention. In this sense, different SRPK inhibitors have been developed, such as the well-known SRPIN340 and its derivatives, with anticancer and antiviral activities. Here we evaluated the potential immunomodulatory activity of SRPIN340 and three trifluoromethyl arylamide derivatives. In in vitro analysis with RAW 264.7 macrophages and primary splenocytes, all the compounds modulated the expression of immune response mediators and antigen-presentation molecules related to a tendency for M2 macrophage polarization. Immunization experiments were carried out in mice to evaluate their potential as vaccine immunostimulants. When administrated alone, the compounds altered the expression of immune factors at the injection site and did not produce macroscopic or microscopic local reactions. In addition, when prepared as an adjuvant with inactivated EHV-1 antigens, all the compounds increased the anti-EHV-1 neutralizing antibody titers, a change that is consistent with an increased Th2 response. These findings demonstrate that SRPIN340 and its derivatives exhibit a noticeable capacity to modulate innate and adaptative immune cells, disclosing their potential to be used as vaccine adjuvants or in immunotherapies.

Two Years into the COVID-19 Pandemic: Lessons Learned.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible and virulent human-infecting coronavirus that emerged in late December 2019 in Wuhan, China, causing a respiratory disease called coronavirus disease 2019 (COVID-19), which has massively impacted global public health and caused widespread disruption to daily life. The crisis caused by COVID-19 has mobilized scientists and public health authorities across the world to rapidly improve our knowledge about this devastating disease, shedding light on its management and control, and spawned the development of new countermeasures. Here we provide an overview of the state of the art of knowledge gained in the last 2 years about the virus and COVID-19, including its origin and natural reservoir hosts, viral etiology, epidemiology, modes of transmission, clinical manifestations, pathophysiology, diagnosis, treatment, prevention, emerging variants, and vaccines, highlighting important differences from previously known highly pathogenic coronaviruses. We also discuss selected key discoveries from each topic and underline the gaps of knowledge for future investigations.

The SRPK inhibitor N-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl) isonicotinamide (SRPIN340) increases the immune response against metastatic melanoma in mice.

Cancers have a strong relationship with immune cells in their microenvironment, which significantly influences tumor proliferation and progression. Thus, pharmacological strategies that stimulate the immune system to combat tumor cells are promising for better therapeutic efficacy. Deregulated expression of the splicing regulatory serine arginine protein kinases (mostly SRPK1 and SRPK2) has been found in different cancer types, leading to the expression of isoforms related to tumor growth and metastasis. The microenvironment of melanoma exhibits a strong presence of immune cells, which significantly influences tumor progression, and around 50% of cutaneous melanoma patients benefit from targeted immunotherapy. Here, we analyzed human malignant melanoma single-cell gene expression data and observed that SRPK1/2 overexpression correlates with immune system pathway alterations. In further analysis, we observed an increased presence of immune cells in biopsies from mice bearing metastatic melanoma treated with SRPIN340, a well-known SRPK1/2 pharmacological inhibitor. Local treatments increased the expression of proinflammatory cytokines at the tumor lesions and the activity of the spleen, accompanied by reduced pulmonary metastasis foci, edema formation, and alveolar congestion. In in vitro assays, SRPIN340 also potentiated immunological susceptibility, by increasing the expression of the antigen presenting MHCI and MHCII molecules and by increasing the ability of B16F10 cells to attract splenic cells in transwell assays. Taken together, these results reveal that the antimetastatic effect of SRPIN340 can also involve an increased immune response, which suggests additional functional clues for SRPKs in tumor biology.

Identification of extracellular vesicles from J strain and wild isolate of Mycoplasma hyopneumoniae.

Respiratory diseases constitute a major health challenge for the worldwide pork industry. Porcine enzootic pneumonia (PES) is caused by Mycoplasma hyopneumoniae (Mhyo). Mycoplasmas have the ability to produce extracellular vesicles (EVs), which can be useful for pathogenicity studies and as delivery systems for vaccines. The aim of this study was to demonstrate and compare, under laboratory conditions, EVs produced by Mhyo strain J and wild isolate in stressed and non-stressed in vitro conditions. Using differential centrifugation, density gradient ultracentrifugation, and transmission electron microscopy, the ability of Mhyo strains to produce EVs was demonstrated under favorable and unfavorable conditions.

Urea, salts, and Tween 20 influence on adsorption of IgG and Leishmania rNTPDase2 to nitrocellulose.

Lateral flow immunochromatography is a widely used technique for immunological assays. Construction of test and control lines is mostly done by antigen adsorption to nitrocellulose membranes, a process not fully understood. This study aimed to evaluate the influence of urea, salts, and Tween 20, on adsorption. The performance of canine IgG in water and in buffer containing urea and salts (pH 8.3) were compared to observe if the interferents would lead to protein stripping when challenged with increasing concentrations of Tween 20 in the lateral flow buffer. Immobilization of the rLiNTPDase2, an antigen for Canine Leishmaniasis diagnosis, was evaluated and compared to the rLbNTPDase2 by the same method. There were no differences between adsorption coefficients of IgG in water and in buffer, but high salt and urea concentrations seems to stabilize and enhance IgG immobilization. Adsorption performance between canine IgG and rNTPDases had different patterns, but was highly similar between rNTPDases, indicating that protein identity may have an important role. Also, low concentrations of Tween 20 in the flow solution may aid the maintenance of rNTPDase2 on the strips. Our results bring insights about protein adsorption and perspectives about the influence of urea, salts and Tween 20 on this process.

Geographical and temporal spread of equine rabies in Brazil.

In Brazil, the horse is frequently used in cultural activities, sports, and in rural and urban work, implementing the economy in different social classes. Among the diseases in horses with zoonotic potential, rabies has been neglected in the country, increasing the risk of spreading the disease across borders. The present study evaluated the spatiotemporal distribution and temporal trend of rabies in horses in Brazil between 2010 and 2019. During this period, 1290 cases of rabies were detected in horses in Brazil, mainly in the states of São Paulo (21.7%) and Mato Grosso (13.3%). However, Espírito Santo stood out, with an incidence risk (IR) of 139.7 cases of rabies per 100,000 horses. The years 2013 and 2017 had higher peaks of IR for the disease, and the states that contributed to this increase were Mato Grosso, São Paulo, and Espírito Santo. There was no monthly seasonality of the disease among the states. The temporal trend revealed an increase for the northeastern region (Annual Percentage Change [APC]: 8.9%) and for Alagoas State (APC: 26.6%). In the spatiotemporal analysis, three high-risk clusters were formed: (i) cluster A (Relative Risk [RRs]: 6.21), involving only Minas Gerais, between 2017 and 2019; (ii) cluster B (RRs: 6.18), involving only Mato Grosso, between 2011 and 2013; and (iii) cluster C (RRs:4.71), involving the states of Rio de Janeiro and Espírito Santo, between 2010 and 2014. Only the states of Roraima and Amapá had no cases of equine rabies during the study period. Therefore, rabies in horses occurs in all Brazilian regions, with areas at high risk of infection concentrated in the Southeast. However, attention should be directed to the north-eastern and northern states, where notifications were infrequent, with an unknown risk in relation to the spread of rabies to transboundary regions. This is the first study evaluating the interstate distribution of rabies in equine species in regions of Brazil.

Antigens and their diagnostic performance for Canine Visceral Leishmaniasis: A critical review.

Canine visceral leishmaniasis (CVL) is the most aggressive and lethal form of leishmaniasis manifesting in dogs and represents a major public health concern. Although there are sufficiently sensitive molecular tools for CVL diagnosis, they are not accessible at the main points of disease dissemination, in which context serodiagnosis has been used as an alternative tool on the epidemiological control. As an attempt to develop more accurate immunodiagnostic assays, many antigens have been tested over the years, on different platforms. This review aimed to access studies reporting new antigens that can be applied for CVL serodiagnosis. Articles published from January of 2016 to March of 2021 were retrieved from Google Scholar, Science Direct, and PubMed, using "Canine Visceral Leishmaniasis" and "Serodiagnosis" as keywords. In total, 1527 articles were identified, of which 42 were selected based on exclusion factors. Sensitivity, specificity, sample size, and sample quality data were extracted by manual curation and analyzed. Of the selected articles, 26 contemplated ELISA, which enabled a more thorough comparison and a critical review of these studies. Soluble Leishmania Antigens (SLA) and the A2 protein were used as controls in 53.8 and 46.15 % of these articles, respectively, and were evaluated separately; their frequent use was questioned. Subsequently, articles that evaluated other assay platforms, such as immunochromatography, immunosensors, and others, were also reported and evaluated. Finally, data relative to validation studies of commercial kits were briefly discussed. Our results show that there are several antigens with great potential for the development of accurate diagnostic tools, but further testing is required. The critical analysis also brings insights that can be useful for more assertive diagnostic development of more robust tools for CVL serodiagnosis.

Apoptosis in the late replication phase of Bovine alphaherpesvirus 1 in experimentally infected calves.

Bovine alphaherpesvirus 1 (BoHV-1) is a pathogen causing respiratory and reproductive clinical signs in cattle. Infected animals may develop rhinotracheitis, vulvovaginitis, balanoposthitis, and abortion. Viral latency is generally established in neuronal ganglia simultaneously to a decrease in both genes or genome expression and viral replication. Under stressful conditions, infection is reactivated leading to viral replication and the manifestation of clinical signs. In this study, we evaluated both viral reactivation and apoptosis in trigeminal ganglia cells as BoHV-1 progressed from the latent to the acute phase of infection after dexamethasone administration in experimentally infected calves. To test ganglia cell death as a consequence of BoHV-1 infection, we stained the BoHV-1 samples with TUNEL after the viral shedding by the calves. RT-qPCR of apoptotic genes was also performed, showing the upregulation of the caspase 8 gene in the trigeminal ganglia from cattle experimentally infected with BoHV-1. These results showed the occurrence of apoptosis in ganglion cells of calves infected by BoHV-1.

Porcine circovirus 3 in North and South America: Epidemiology and genetic diversity.

Porcine circovirus 3 (PCV3) is a recently discovered virus that has been detected in the swine population worldwide. PCV3 infection has been associated with several signs, but its pathogenicity is currently uncertain. This review article aimed to analyse the PCV3 strains that circulate in different countries in North and South America. We demonstrated the main regions of polymorphisms in the capsid protein structure. Furthermore, we found that PCV3 has at least six different lineages circulating in the Americas. Additional studies are required to determine the role of PCV3 in different clinical syndromes and its epidemiology in swine herds in North and South American countries.

First complete genome sequence and molecular characterization of Canine morbillivirus isolated in Central Brazil.

The Brazilian regions are still highly endemic areas for Canine morbillivirus [canine distemper virus (CDV)]. However, little is known regarding the genetic variability of the strain circulating in several Brazilian regions. Here, we report the first full-length genome and molecular characterization of CDV isolated from domestic dogs in the Brazilian Center-West region. Sequence alignment and phylogenetic analyses based on deduced amino acid and nucleotide sequences showed that the isolated strain is characterized as the South America-I/Europe genotype. However, it segregates into a CDV subgenotype branch. Interestingly, both H and F proteins have a gain of a potential N-glycosylation sites compared to the Onderstepoort vaccine strain. Therefore, this study provides a reference to further understand the epidemic and molecular characteristics of the CDV in Brazil.

Genetic diversity of porcine circovirus 3 strains and the first detection of two different PCV3 strains coinfecting the same host in Minas Gerais, Brazil.

Porcine circovirus 3 (PCV3) is a recently emerged circovirus discovered in 2016 that has drawn the attention of the swine industry worldwide. In this study, we evaluated the genetic diversity of PCV3 strains on pig farms. A total of 261 samples from sows, weaning pigs, growing pigs, and stillborn/mummified fetuses were analyzed by quantitative real-time PCR. The results revealed that at least two main lineages of PCV3 are circulating in Brazil. For the first time, it was possible to detect the presence of two different PCV3 strains in the same host.

Bovine alphaherpesvirus 1 (BHV1) infection in testes and epididymis from bulls from a slaughterhouse.

Bovine alphaherpesvirus 1 (BHV1) is an agent associated with reproductive disease in cattle. Viral pathogenicity is related to disorders such as temporary infertility, embryonic death, and abortions in affected animals. Considering that natural infections in genital organs of males are understudied, this investigation evaluated the presence of BHV1 in both testicular and epididymal tissues obtained from naturally infected bulls by the evaluation of the presence of the BHV1 genome and antigens. Sixty samples of blood and genital organs of 60 bulls that were not vaccinated against BHV1 were assayed. Fragments from testes and head, body, and tail of epididymides were processed and analyzed by nested PCR and immunofluorescence with confocal laser scanning microscopy. Also, the BHV1 gB glycoprotein gene of 14 positive samples was partially sequenced. The percentage of BHV1 presence obtained by the immunolocalization assay corresponded to 95.9% of the testes, 100% of the epidydimal tissue in the head and tail portions, and 98% of the epididymal body. The nested PCR assay detected the viral nucleic acid in 59.2% of the testicular tissue and in 65.3, 75.5, and 83.7% of epididymis head, body, and tail samples, respectively. The partial sequences analyzed presented 100% of identity with other BHV1 strains. Accordingly, BHV1 detection in testes and epididymides of naturally infected bulls suggests that these organs may be sources of viral infection for semen.

Multi-targeted gene silencing strategies inhibit replication of Canine morbillivirus.

BACKGROUND: Canine morbilivirus (canine distemper virus, CDV) is a highly contagious pathogen associated with high morbidity and mortality in susceptible carnivores. Although there are CDV vaccines available, the disease poses a huge threat to dogs and wildlife hosts due to vaccine failures and lack of effective treatment. Thus, the development of therapeutics is an urgent need to achieve rapid outbreak control and reduce mortality in target species. Gene silencing by RNA interference has emerged as a promising therapeutic approach against different human and animal viruses. In this study, plasmid-based short hairpin RNAs (shRNAs) against three different regions in either CDV nucleoprotein (N), or large polymerase (L) genes and recombinant adenovirus-expressing N-specific multi-shRNAs were generated. Viral cytopathic effect, virus titration, plaque-forming unit reduction, and real-time quantitative RT-PCR analysis were used to check the efficiency of constructs against CDV. RESULTS: In CDV-infected VerodogSLAM cells, shRNA-expressing plasmids targeting the N gene markedly inhibited the CDV replication in a dose-dependent manner, with viral genomes and titers being decreased by over 99%. Transfection of plasmid-based shRNAs against the L gene displayed weaker inhibition of viral RNA level and virus yield as compared to CDV N shRNAs. A combination of shRNAs targeting three sites in the N gene considerably reduced CDV RNA and viral titers, but their effect was not synergistic. Recombinant adenovirus-expressing multiple shRNAs against CDV N gene achieved a highly efficient knockdown of CDV N mRNAs and successful inhibition of CDV replication. CONCLUSIONS: We found that this strategy had strong silencing effects on CDV replication in vitro. Our findings indicate that the delivery of shRNAs using plasmid or adenovirus vectors potently inhibits CDV replication and provides a basis for the development of therapeutic strategies for clinical trials.