Antiviral alternatives against important members of the subfamily Parvovirinae: a review.

Parvoviruses are responsible for multiple diseases, and there is a critical need for effective antiviral therapies. Specific antiviral treatments for parvovirus infections are currently lacking, and the available options are mostly supportive and symptomatic. In recent years, significant research efforts have been directed toward understanding the molecular mechanisms of parvovirus replication and identifying potential targets for antiviral interventions. This review highlights the structure, pathogenesis, and treatment options for major viruses of the subfamily Parvovirinae, such as parvovirus B19 (B19V), canine parvovirus type 2 (CPV-2), and porcine parvovirus (PPV) and also describes different approaches in the development of antiviral alternatives against parvovirus, including drug repurposing, serendipity, and computational tools (molecular docking and artificial intelligence) in drug discovery. These advances greatly increase the likelihood of discoveries that will lead to potent antiviral strategies against different parvovirus infections.

Viral Fitness and Antigenic Determinants of Porcine Parvovirus at the Amino Acid Level of the Capsid Protein.

Since 2001, strains of porcine parvovirus (PPV), designated 27a-like strains, were observed in Europe, suggesting a predominance of these viruses over older strains. The reasons for the obvious evolutionary advantage are unknown. Here, a series of mutants containing amino acid replacements found in the predominant field strains were generated in a PPV-NADL2 background, and their impact on replication efficiency and antibody binding activity was determined. Some amino acid substitutions observed in the 27a-like strains significantly increased viral fitness and decreased neutralization activity of serum samples raised against commercial vaccines and old virus strains (e.g., NADL2). These mutant viruses and a monoclonal antibody raised against a classical PPV strain defined a 27a-specific neutralizing epitope around amino acid 228 of the capsid protein VP2. Based on the analysis of the mutant viruses, it is hypothesized that the predominant factor for the global spread of the PPV-27a strain substitutions is an increased viral fitness of the 27a-like viruses, possibly supported by partial immune selection. This is reminiscent to the evolution of canine parvovirus and worldwide replacement of the original virus by the so-called new antigenic types. IMPORTANCE Porcine parvovirus is one of the most important causes of reproductive failure in swine. Recently, despite the continuous use of vaccines, "new" strains emerged, leading to the hypothesis that the emergence of new amino acid substitutions could be a viral adaptation to the immune response against the commercial vaccines. Our results indicate the amino acid substitutions observed in the 27a-like strains can modify viral fitness and antigenicity. However, an absolute immune escape was not evident.

Canine parvovirus type 2 vaccines in Brazil: Viral load in commercial vaccine vials and phylogenetic analysis of the vaccine viruses.

Canine parvovirus type 2 (CPV-2) is the etiological agent of a highly contagious and frequently fatal disease in dogs. Live attenuated vaccines (LAV) are recommended to prevent and control this disease. Commercial vaccines are typically produced with CPV-2 strains adapted to cell culture and usually non-pathogenic. The present study aimed to determine the viral load of CPV-2 vaccines commercially available in Brazil and to characterize the vaccine virus by DNA analysis of its capsid gene. The results demonstrated that all vaccine strains presented high homology of the VP2 gene and they were all closely related to the original CPV-2 strains. However, vaccine strains presented several differences in comparison with field strains currently circulating in Brazil. Seventy-one vials contained viral loads ranging from 7.4E3 to 4.9E10 DNA copies/ml. Nine vials did not contain any detectable CPV-2 DNA. In conclusion, there are genetic and antigenic differences among CPV-2 vaccines and field strains. Additionally, some vaccines have been commercialized with low titers of CPV-2. It is important to improve the quality of the vaccines to prevent or reduce the spread of CPV-2 in Brazil.

Dissemination and evolution of SARS-CoV-2 in the early pandemic phase in South America.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic spread rapidly and this scenario is concerning in South America, mainly in Brazil with more than seven million cases of infection. Three major pandemic lineages/clades could be identified along with SARS-CoV-2 dissemination (G, GR, and GH) in the Americas. These clades differ according to their genomic characteristics, virulence, and spreading times. The present study describes the main clades and the respective temporal spreading analyses based on SARS-CoV-2 whole-genome sequences (WGS) from South America, obtained in the early pandemic phase (from March 1 to May 31 in 2020). SARS-CoV-2 WGSs with available information from country and year of sampling were obtained from different countries and the main clades were identified and analyzed independently with a Bayesian approach. The results demonstrated the prevalence of clades GR (n = 842; 54.6%), G (n = 529; 34.3%), and GH (n = 171; 11.1%). The frequencies of the clades were significantly different between South American countries. Clade G was the most prevalent in Ecuador, Suriname, and Uruguay, clade GR in Argentina, Brazil, and Peru, and clade GH in Colombia. The phylodynamic analysis indicated that all these main lineages increased viral spreading from February to early March and after an evolutionary stationary phase was observed. The decrease observed in the virus dissemination was directly associated to the reduction of social movement after March. In conclusion, these data demonstrated the current predominance of clades G, GR, and GH in South America because of the early dissemination of them in the first pandemic phase in South America.

Porcine Parvovirus.

Porcine parvovirus (PPV) is considered the main cause of reproductive disorders in pigs, which are summarized under the acronym SMEDI (stillbirth, mummification, embryonic death, and infertility). In this review the biology of the virus and its structure, pathogenic potential and strain variation, as well as the disease induced by the virus, are described. Known aspects of pathogenesis, diagnosis and prevention, particularly by vaccination, are summarized. Furthermore, in recent years 'new' parvoviruses (PPV2 to 7) have been described in pigs. They have been detected in pigs from various parts of the world and their association with clinical signs or disease will be discussed.

Dynamics of vanishing of maternally derived antibodies of Ungulate protoparvovirus 1 suggests an optimal age for gilts vaccination.

The prevention of Ungulate protoparvovirus 1 (UPV1) infection and consequently the reproductive losses is based on vaccination of all pigs intended for breeding. As maternally derived antibodies (MDA) can interfere with the development of immunity following vaccination, it is important to know the duration of anti-UPV1 MDA to determine the optimal age for the best vaccination efficacy. To elucidate the association between dam and piglet antibody levels against UPV1 and to estimate the decrease rate of MDA, sera and colostrum of 127 gilts (before the first vaccination against UPV1; 15 days after the second vaccine dose; at farrowing; and during the second pregnancy) and sera of 276 piglets (prior to initial colostrum intake; at 7, 21, 57, 87, and 128 days-old) were tested by ELISA. Most gilts (85.8%) had anti-UPV1 antibodies before vaccination and after vaccination all were positive. At 7 days old, the majority of the piglets had anti-UPV1 antibodies, but around 57 days old, only 35.3% were positive and at 87 days old, all were negative. The estimated average half-life of MDA was 29.8 (28.8-30.9) days. A strong correlation was determined between piglet serum at 7 days old with gilt serum at farrowing time (r = 0.77, n = 248, P < 0.001) and with piglet serum at 7 days old with colostrum (r = 0.73, n = 248, P < 0.001). The MDA decreased earlier and the antibody half-life was a little longer than previously reported. Based on these findings, UPV1 vaccination can be performed earlier than usual.

An inactivated whole-virus porcine parvovirus vaccine protects pigs against disease but does not prevent virus shedding even after homologous virus challenge.

Inactivated whole-virus vaccines against porcine parvovirus (PPV) can prevent disease but not infection and virus shedding after heterologous virus challenge. Here, we showed that the same is true for a homologous challenge. Pregnant sows were vaccinated with an experimental inactivated vaccine based on PPV strain 27a. They were challenged on day 40 of gestation with the virulent porcine parvovirus PPV-27a from which the vaccine was prepared (homologous challenge). On day 90 of gestation, the fetuses from vaccinated sows were protected against disease, while the fetuses of the non-vaccinated sows (control group) exhibited signs of parvovirus disease. All gilts, whether vaccinated or not vaccinated, showed a boost of PPV-specific antibodies indicative of virus infection and replication. Low DNA copy numbers, but not infectious virus, could be demonstrated in nasal or rectal swabs of immunized sows, but high copy numbers of challenge virus DNA as well as infectious virus could both be demonstrated in non-vaccinated sows.

In silico designing of multi-epitope vaccine against canine parvovirus using reverse vaccinology.

Canine parvovirus (CPV-2) is a highly contagious virus affecting dogs worldwide, posing a significant threat. The VP2 protein stands out as the predominant and highly immunogenic structural component of CPV-2. Soon after its emergence, CPV-2 was replaced by variants known as CPV-2a, 2b and 2c, marked by changes in amino acid residue 426 of VP2. Additional amino acid alterations have been identified within VP2, with certain modifications serving as signatures of emerging variants. In Brazil, CPV-2 outbreaks persist with diverse VP2 profiles. Vaccination is the main preventive measure against the virus. However, the emergence of substitutions presents challenges to conventional vaccine methods. Commercial vaccines are formulated with strains that usually do not match those currently circulating in the field. To address this, the study aimed to investigate CPV-2 variants in Brazil, predict epitopes, and design an in silico vaccine tailored to local variants employing reverse vaccinology. The methodology involved data collection, genetic sequence analysis, and amino acid comparison between field strains and vaccines, followed by the prediction of B and T cell epitope regions. The predicted epitopes were evaluated for antigenicity, allergenicity and toxicity. The final vaccine construct consisted of selected epitopes linked to an adjuvant and optimized for expression in Escherichia coli. Structural predictions confirmed the stability and antigenicity of the vaccine, while molecular docking demonstrated interaction with the canine toll-like receptor 4. Molecular dynamics simulations indicated a stable complex formation. In silico immune simulations demonstrated a progressive immune response post-vaccination, including increased antibody production and T-helper cell activity. The multi-epitope vaccine design targeted prevalent CPV-2 variants in Brazil and potentially other regions globally. However, experimental validation is essential to confirm our in silico findings.

Canine parvovirus type 2 (CPV-2) serological and molecular patterns in dogs with viral gastroenteritis from southern Brazil.

Canine Parvovirus type 2 (CPV-2) is a highly contagious virus that can cause severe systemic disease with gastroenteric symptoms in dogs, particularly in young puppies. Originating from the feline parvovirus in the late 1970s, it swiftly propagated globally, instigating a pandemic in dogs. Despite vaccination advancements, CPV-2 remains a substantial challenge for veterinary professionals and pet owners. This study aimed to contribute knowledge about the current situation of CPV-2 among dogs in southern Brazil. In this study, the sera of 125 dogs (mostly with gastroenteritis symptoms) were screened for antibodies against CPV-2 and their faeces for the virus itself. The results showed that 40% (50/125) of dogs were infected with CPV-2. Most animals (65.5%) had previously been exposed to CPV-2 (with serotitres equal or above 1:40), and only 37.6% had protective antibody titres equal or above 1:80. The findings have also demonstrated that vaccination against CPV-2 significantly reduced the risk of infection, with positive cases decreasing from 56.9% (unvaccinated) to 2.0% (fully vaccinated). Furthermore, the prevalence of CPV-2 decreased as dogs aged, with younger dogs and those with an incomplete or non-existent vaccination history at the highest risk of infection. In conclusion, this study provides valuable insight into the prevalence and risk factors associated with CPV-2 infection in dogs in southern Brazil, thereby providing valuable knowledge for the improvement of veterinary care and pet health.

Epidemiological Insights into Feline Leukemia Virus Infections in an Urban Cat (Felis catus) Population from Brazil.

Feline leukemia virus (FeLV) is a retrovirus distributed worldwide in domestic cats and with different outcomes (progressive, regressive, abortive, focal). The present study reports an epidemiological survey of FeLV frequency and the evaluation of some risk factors and the two main disease outcomes (progressive and regressive) in an urban cat population from Brazil. A total of 366 cats with sociodemographic information and p27 FeLV antigen test performed were included in the study. FeLV DNA (provirus) in the blood samples of all cats was detected via real-time polymerase chain reaction (qPCR). Plasma samples from 109 FeLV-positive and FeLV-negative cats were also submitted to reverse transcription (RT-qPCR) to determine the FeLV viral load. The results demonstrated that 112 (30.6%) cats were positive through the p27 antigen and/or qPCR. A risk factor analysis demonstrated that cats without vaccination against FeLV (OR 9.9, p < 0.001), clinically ill (OR 2.9, p < 0.001), with outdoors access (OR 2.7, p < 0.001), and exhibiting apathetic behavior (OR 3.1, p < 0.001) were more likely to be infected with FeLV. FeLV-infected cats were also more likely to present with anemia (OR 13, p < 0.001) and lymphoma (OR 13.7, p = 0.001). A comparative analysis of the different detection methods in a subset of 109 animals confirmed FeLV infection in 58 cats, including 38 (65.5%) with progressive, 16 (27.6%) with regressive, and 4 (6.9%) with probably focal outcome diseases. In conclusion, this study demonstrates a high prevalence of FeLV in this urban cat population from Brazil and highlights the need to establish more effective prevention strategies (such as viral testing, vaccination programs, specific care for FeLV-positive cats) to reduce diseases associated with this virus in Brazil.

Population dynamics and in vitro antibody pressure of porcine parvovirus indicate a decrease in variability.

To estimate the impact of porcine parvovirus (PPV) vaccines on the emergence of new phenotypes, the population dynamic history of the virus was calculated using the Bayesian Markov chain Monte Carlo method with a Bayesian skyline coalescent model. Additionally, an in vitro model was performed with consecutive passages of the 'Challenge' strain (a virulent field strain) and NADL2 strain (a vaccine strain) in a PK-15 cell line supplemented with polyclonal antibodies raised against the vaccine strain. A decrease in genetic diversity was observed in the presence of antibodies in vitro or after vaccination (as estimated by the in silico model). We hypothesized that the antibodies induced a selective pressure that may reduce the incidence of neutral selection, which should play a major role in the emergence of new mutations. In this scenario, vaccine failures and non-vaccinated populations (e.g. wild boars) may have an important impact in the emergence of new phenotypes.

Analysis of porcine parvoviruses in tonsils and hearts from healthy pigs reveals high prevalence and genetic diversity in Germany.

In the present study, tonsils and hearts from 100 pigs were collected in a German slaughterhouse in 2010 and tested for porcine parvoviruses (PPV, PPV2, PPV3 and PPV4). PPV was observed in 60 of 100 hearts and 61 of 100 tonsils, and PPV2 was observed in 55 of 100 hearts and 78 of 100 tonsils. PPV3 and PPV4 were found in 20 and 7, respectively, of the 100 tonsils tested, but not in the heart samples. Positive samples of PPV, PPV2 and PPV3 were analyzed by nucleotide sequencing, and phylogenetic analysis revealed at least two distinct lineages for each virus in the German samples. The high detection rate of PPV, PPV2 and PPV3 in healthy animals and their genetic diversity highlights the importance of continuous monitoring of these viruses and their zoonotic potential.

Antimicrobial activity of essential oils against Staphylococcus aureus and Staphylococcus chromogenes isolated from bovine mastitis.

Staphylococcus aureus and Staphylococcus chromogenes are pathogens frequently detected in bovine mastitis. Treatment and prevention of this disease have been usually carried on with antimicrobials. However, the emergence of bacterial isolates with antimicrobial resistance has aroused interest in new therapeutic alternatives. Plant essential oils (EOs) have been largely studied as antibacterial treatments. In the present study, EOs from five plants were evaluated for their antibacterial activities against S. aureus and S. chromogenes. Bacterial isolates were obtained in a previous study of clinical cases of bovine mastitis. EOs from lemongrass, eucalyptus, lavender, peppermint, and thyme were obtained by hydrodistillation and their chemical compositions were evaluated by gas chromatography (GC). Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were evaluated for all EOs. The results demonstrated that citral (40.9%), myrcene (24.7%), and geraniol (1.9%) were detected in lemongrass EO; 1,8-cineole (76.9%), α-pinene (8.2%), and ledene (5.1%) in eucalyptus EO; 1,8-cineole (45.2%), camphor (18.2%), and fenchone (14.6%) in lavender EO; L-menthol (38.5%), menthofuran (16.3%), and citronellal (10.6%) in peppermint EO; and thymol (44.2%), p-cymene (24.6%) and 1,8-cineole (9.9%) in thyme EO. More effective antibacterial activities were observed only with the use of lemongrass (MIC and MBC ranging from 0.39 to 3.12 mg/mL and 0.39 to 6.35 mg/mL, respectively) and thyme (MIC and MBC ranging from 0.39 to 1.56 mg/mL and 0.39 to 3.12 mg/mL, respectively). Peppermint, lavender and eucalyptus EOs did not show bactericidal activities. In conclusion, lemongrass and thyme EOs are promising antibacterial alternatives against Staphylococcus species associated with bovine mastitis.

Canine Parvovirus 2 in Free-Living Wild Mammals from Southern Brazil.

Pathogens from domestic canines represent a significant and constant threat to wildlife. This study looked for four common canine pathogens, Babesia vogeli, Ehrlichia canis, Leishmania infantum, and canine parvovirus 2 (CPV-2) in mammals from the Pampa Biome, southern Brazil. Animals killed by vehicular trauma on a road traversing this biome were evaluated over a 1-yr period. Tissues collected from 31 wild mammals and six dogs were further analyzed by specific real-time PCR assays for each pathogen. Babesia vogeli and L. infantum were not detected in any investigated animal. Ehrlichia canis was detected in one dog and CPV-2 in nine animals: four dogs, three white-eared opossums (Didelphis albiventris), one pampas fox (Lycalopex gymnocercus), and one brown rat (Rattus norvegicus). These results demonstrate the occurrence of important carnivore pathogens (E. canis and CPV-2) in domestic dogs and wild mammals from the Pampa Biome in southern Brazil.

Emergence, Dissemination and Antimicrobial Resistance of the Main Poultry-Associated Salmonella Serovars in Brazil.

Salmonella infects poultry, and it is also a human foodborne pathogen. This bacterial genus is classified into several serovars/lineages, some of them showing high antimicrobial resistance (AMR). The ease of Salmonella transmission in farms, slaughterhouses, and eggs industries has made controlling it a real challenge in the poultry-production chains. This review describes the emergence, dissemination, and AMR of the main Salmonella serovars and lineages detected in Brazilian poultry. It is reported that few serovars emerged and have been more widely disseminated in breeders, broilers, and layers in the last 70 years. Salmonella Gallinarum was the first to spread on the farms, remaining as a concerning poultry pathogen. Salmonella Typhimurium and Enteritidis were also largely detected in poultry and foods (eggs, chicken, turkey), being associated with several human foodborne outbreaks. Salmonella Heidelberg and Minnesota have been more widely spread in recent years, resulting in frequent chicken/turkey meat contamination. A few more serovars (Infantis, Newport, Hadar, Senftenberg, Schwarzengrund, and Mbandaka, among others) were also detected, but less frequently and usually in specific poultry-production regions. AMR has been identified in most isolates, highlighting multi-drug resistance in specific poultry lineages from the serovars Typhimurium, Heidelberg, and Minnesota. Epidemiological studies are necessary to trace and control this pathogen in Brazilian commercial poultry production chains.

Molecular phylogenetic assessment of the canine parvovirus 2 worldwide and analysis of the genetic diversity and temporal spreading in Brazil.

Canine parvovirus type 2 (CPV-2) is a relevant pathogen for dogs and causes a severe disease in carnivore species. CPV-2 reached pandemic proportions after the 1970s with the worldwide dissemination, generating antigenic and genetic variants (CPV-2a, CPV-2b, and CPV-2c) with different pathobiology in comparison with the original type CPV-2. The present study aimed to assess the current global CPV-2 molecular phylogeny and to analyze genetic diversity and temporal spreading of variants from Brazil. A total of 284 CPV-2 whole-genome sequences (WGS) and 684 VP2 complete genes (including 23 obtained in the present study) were compared to analyze phylogenetic relationships. Bayesian coalescent analysis estimated the time to the most recent common ancestor (tMRCA) and the population dynamics of the different CPV-2 lineages in the last decades. The WGS phylogenetic tree demonstrated two main clades disseminated worldwide today. The VP2 gene tree showed a total of four well-defined clades distributed in different geographic regions, including one with CPV-2 sequences exclusive from Brazil. These clades do not have a relationship with the previous classification into CPV-2a, CPV-2b, and CPV-2c, despite some having a predominance of one or more antigenic types. Temporal analysis demonstrated that the main CPV-2 clades evolved within a few years (from the 1980s to 1990s) in North America and they spread worldwide afterwards. Population dynamics analysis demonstrated that CPV-2 presented a major dissemination increase at the end of the 1980s / beginning of the 1990s followed by a period of stability and a second minor increase from 2000 to 2004.

High rate of viral evolution in the capsid protein of porcine parvovirus.

In recent years, it has been shown that some parvoviruses exhibit high substitution rates, close to those of RNA viruses. In order to monitor and determine new mutations in porcine parvovirus (PPV), recent PPV field isolates from Austria, Brazil, Germany and Switzerland were sequenced and analysed. These samples, together with sequences retrieved from GenBank, were included in three datasets, consisting of the complete NS1 and VP1 genes and a partial VP1 gene. For each dataset, the nucleotide substitution rate and the molecular clock were determined. Analysis of the PPV field isolates revealed that a recently described amino acid substitution, S436T, appeared to be common in the VP2 protein in the Austrian, Brazilian and German virus populations. Furthermore, new amino acid substitutions were identified, located mainly in the viral capsid loops. By inferring the evolutionary dynamics of the PPV sequences, nucleotide substitution rates of approximately 10(-5) substitutions per site per year for the non-structural protein gene and 10(-4) substitutions per site per year for the capsid protein gene (for both viral protein datasets) were found. The latter rate is similar to those commonly found in RNA viruses. An association of the phylogenetic tree with the molecular clock analysis revealed that the mutations on which the divergence for both capsid proteins was based occurred in the past 30 years. Based on these findings, it was concluded that PPV variants are continuously evolving and that vaccines, which are based mainly on strains isolated about 30 years ago, should perhaps be updated.

Use of plant extracts and essential oils in the control of bovine mastitis.

Bovine mastitis is the most important disease affecting dairy herds worldwide, causing direct impacts on farms' profitability and food safety issues. The prevention and treatment of this pathology is especially done through antimicrobials, but the increasing antimicrobial resistance of pathogens to this disease may affect the efficiency of conventional drugs. Besides, antimicrobials residues in milk and the environment are a potential threat to human health. Thereby, the use of plant extracts and essential oils may become promising alternatives for the control of bovine mastitis. Antimicrobial properties present in several plants are well described and plant extracts and essential oils are often considered safe to animals, humans and environment. This review summarizes the current problems encountered in the conventional treatment of mastitis, the possibilities of the use of plant extracts and essential oils as alternative agents for the control of these pathogens and the limitations found in the use of these plant derivatives. Finally, the perspectives to the use of plant extracts and essential oils for the treatment of bovine mastitis are presented.

First detection of canine parvovirus type 2c in Brazil.

The presence of canine parvovirus type 2 (CPV-2), 2a and 2b has been described in Brazil, however, the type 2c had not been reported until now. In the current study, seven out of nine samples from dogs with diarrhea were characterized as CPV-2c, indicating that this virus is already circulating in the Brazilian canine population.