Dengue virus 3 genotype I shows natural changes in heparan sulphate binding sites, cell interactions, and neurovirulence in a mouse model.

Dengue virus (DENV) is the most prevalent pathogen of the Flaviviridae family. Due to the considerable increase in DENV incidence and spread, symptoms such as CNS involvement have increased. Heparan sulphate (HS) was the first molecule identified as an adhesion factor for DENV in mammalian cells. Viral phenotypes with different HS interactions are associated with various clinical symptoms, including neurological alterations. Here, using in silico analyses, in vitro studies, and the in vivo mouse model, we characterized two natural circulating DENV3 genotype I (GI) lineage 1 (L1) in Brazil-DENV3 MG-20 (from Minas Gerais) and DENV3 PV_BR (from Rondônia) that present divergent neurovirulent profiles and sensitivity to sulphated molecules. We identified substitutions at the viral envelope (E) in positions 62 and 123 as likely responsible for the differences in neurovirulence. The E62K and E123Q substitutions in DENV3 MG-20 and DENV3 PV_BR, respectively, greatly influenced in silico electrostatic density and heparin docking results. In vivo, mice inoculated with DENV3 MG-20 died, but not those infected with DENV3 PV_BR. The clinical symptoms, such as paralysis of the lower limbs and meningoencephalitis, and histopathology, also differed between the inoculated groups. In vitro heparin and heparinases assays further demonstrated the biological impact of these substitutions. Other characteristics that have been previously associated with alterations in cell tropism and neurovirulence, such as changes in the size of lysis plaques and differences in cytopathic effects in glioblastoma cells, were also observed.

Active Circulation of Madariaga Virus, a Member of the Eastern Equine Encephalitis Virus Complex, in Northeast Brazil.

Madariaga virus (MADV) is a member of the eastern equine encephalitis virus (EEEV) complex that circulates in Central and South America. It is a zoonotic, mosquito-borne pathogen, belonging to the family Togaviridae. Disturbances in the natural transmission cycle of this virus result in outbreaks in equines and humans, leading to high case fatality in the former and acute febrile illness or neurological disease in the latter. Although a considerable amount of knowledge exists on the eco-epidemiology of North American EEEV strains, little is known about MADV. In Brazil, the most recent isolations of MADV occurred in 2009 in the States of Paraíba and Ceará, northeast Brazil. Because of that, health authorities have recommended vaccination of animals in these regions. However, in 2019 an equine encephalitis outbreak was reported in a municipality in Ceará. Here, we present the isolation of MADV from two horses that died in this outbreak. The full-length genome of these viruses was sequenced, and phylogenetic analyses performed. Pathological findings from postmortem examination are also discussed. We conclude that MADV is actively circulating in northeast Brazil despite vaccination programs, and call attention to this arbovirus that likely represents an emerging pathogen in Latin America.

Fluorescent quantum dots-zika virus hybrid nanoconjugates for biolabeling, bioimaging, and tracking host-cell interactions.

The earliest possible diagnosis and understanding of the infection mechanisms play a crucial role in the outcome of fighting viral diseases. Thus, we designed and developed for the first time, novel bioconjugates made of Ag-In-S@ZnS (ZAIS) fluorescent quantum dots coupled with ZIKA virus via covalent amide bond with carboxymethylcellulose (CMC) biopolymer for labeling and bioimaging the virus-host cell interactions mechanisms through confocal laser scanning microscopy. This work offers relevant insights regarding the profile of the ZIKA virus-nanoparticle conjugates interactions with VERO cells, which can be applied as a nanoplatform to elucidate the infection mechanisms caused by this viral disease.

Short communication: Parapoxvirus and Orthopoxvirus coinfection in milk of naturally infected cows.

Several studies have shown the occurrence of poxvirus infections associated with exanthematic lesions in cattle from many Brazilian states. Coinfection between viruses belonging to 2 genera, Orthopoxvirus (OPXV) and Parapoxvirus (PPV), was already identified from the lesions of affected cows and humans. The DNA and infectious viral particles of Vaccinia virus, an OPXV, have been detected in milk of naturally and experimentally infected cows. However, to date no reports have described the detection of Pseudocowpox virus, a PPV, in milk. Thus, we investigated the presence of PPV and OPXV in milk samples obtained from dairy cows from a Brazilian region with exanthematic disease outbreaks. From 2011 to 2014, 6 dairy farms with exanthematic disease outbreaks involving dairy cows, calves, and humans were visited. Twelve crusts of cows' teat lesions and 60 milk samples were collected. The crusts and milk samples were analyzed by PCR to detect OPXV or PPV DNA. According to the analyzed crusts, we detected PPV infection in 4 of the 6 visited farms, from which we investigated the PPV contamination in milk. From the 40 milk samples tested, PPV DNA was detected in 12 samples. Of these milk samples, 8 were positive for both PPV and OPXV. This is the first report of PPV DNA detection in milk samples from affected cows, indicating that the virus may be present in milk and potentially contaminating dairy products associated or not with OPXV. In addition to the lesions caused by direct contact, the presence of 2 or more poxvirus species in milk showed that the effect of zoonotic exanthematic diseases on public health and animal husbandry is relevant and cannot be overlooked.

Short communication: Survival of Vaccinia virus in inoculated cheeses during 60-day ripening.

Bovine vaccinia is a neglected zoonosis caused by Vaccinia virus (VACV) and has a major economic and public health effect in Brazil. Previous studies showed infectious VACV particles in milk from either experimentally or naturally infected cows and in fresh cheeses prepared with experimentally contaminated milk. Ripening is a process that leads to major changes in the physical and chemical characteristics of cheese, reducing contamination by spoilage, pathogenic microorganisms, or both. However, it is not known if VACV infectious particles persist after the ripening process. To investigate this issue, viral infectivity at different ripening times was studied in cheeses manufactured with milk experimentally contaminated with VACV strain Guarani P2 (GP2). Cheeses were analyzed at 1, 7, 14, 21, 45, and 60 d of ripening at 25°C. Viral DNA was quantified by real-time PCR, and VACV isolation and titration were performed in Vero cells. The whole experiment was repeated 4 times. Analysis of the mean viral DNA quantification and infectivity indicated a reduction of approximately 2 logs along the ripening process; however, infectious viral particles (1.7 × 102 pfu/mL) could still be recovered at d 60 of ripening. These findings indicate that the ripening process reduces VACV infectivity, but it was not able to inactivate completely the viral particles after 60 d.

Clinical, hematological and biochemical parameters of dairy cows experimentally infected with Vaccinia virus.

Vaccinia virus (VACV) is the etiological agent of bovine vaccinia (BV), an important zoonosis that affects dairy cattle. There are many aspects of the disease that remain unknown, and aiming to answer some of these questions, the clinical, hematological, and biochemical parameters of VACV experimentally infected cows were evaluated. In the first part of the study, lactating cows were infected with VACV-GP2 strain. In the second part, animals previously infected with VACV-GP2 were divided into two treatment groups: Group 1, immunosuppressed cows; and Group 2, re-infected cows. In this study, BV could be experimentally reproduced, with similar lesions as observed in natural infections. Moreover, a short incubation period and local lymphadenopathy were also observed. VACV could be detected by PCR and isolated from scabs taken from teat lesions of all inoculated and re-inoculated animals. Lymphocytosis and neutrophilia were observed in all animals from the first part of the experiment, and lymphopenia and relative neutrophilia were observed in the immunosuppressed animals. Detection of viral DNA in oral mucosa lesions suggests that viral reactivation might occur in immunosuppressed animals. Moreover, clinical disease with teat lesions may occur in previously VACV-infected cows under the experimental conditions of the present study.

Bovine vaccinia, a systemic infection: evidence of fecal shedding, viremia and detection in lymphoid organs.

Bovine vaccinia (BV) is a zoonosis caused by Vaccinia virus (VACV) that affects dairy cattle and milkers, causing economic losses and impacting animal and human health. Based on the clinical presentation, BV appears to be a localized disease, with lesions restricted to the skin of affected individuals. However, there are no studies on the pathogenesis of the disease in cows to determine if there is a systemic spread of the virus and if there are different ways of VACV shedding. The objective of this work was to study if there is a systemic spread of VACV in experimentally infected cows and to study the kinetics of VACV circulation in the blood and shedding in the feces of these animals. To this end, eight crossbred lactating cows were used. Three teats of each cow were inoculated with the GP2V strain of VACV. All animals were monitored daily, and blood and fecal samples were collected for 67 days post-infection (dpi). After this period, four of these previously infected cows were immunosuppressed using dexamethasone. Viral DNA was continuously detected and quantified in the blood and feces of these animals in an intermittent way, even after the resolution of the lesions. At slaughter, tissues were collected, and viral DNA was detected and quantified in the mesenteric and retromammary lymph nodes, ileum, spleen and liver. The detection of VACV DNA in the feces for a longer period (67 dpi) and in the lymphatic organs provides new evidence about VACV elimination and suggests that BV could be a systemic infection with a chronic course and viral shedding through the feces.

Validation of an immunoperoxidase monolayer assay for total anti-Vaccinia virus antibody titration.

Vaccinia virus (VACV) has been associated with zoonotic exanthemic outbreaks affecting bovids and human beings, with significant public health and economic impacts. Rapid and reliable diagnostic methods are needed to detect and epidemiologically monitor antibodies to VACV. The current study describes the development of an immunoperoxidase monolayer assay (IPMA) for detection of total VACV antibodies in bovine serum. The assay was validated by comparison with a plaque reduction neutralization test (PRNT). Kappa index of agreement, diagnostic sensitivity, specificity, and accuracy of the IPMA were -1.008, 100%, 96%, and 98%, respectively, when compared with PRNT on 148 field bovine sera. Repeatability tests on 32 field-positive serum samples revealed that intraclass coefficient correlation was 0.86. In experimentally infected cattle, VACV antibodies were detectable by IPMA 4 days postinfection, which was more than 2 weeks earlier than with the PRNT, indicating that IPMA could be a more sensitive test than the latter. In 4 naturally VACV-diseased cows monitored for 13 months, IPMA could detect VACV antibodies up to 13 months, a longer time than PRNT. The IPMA is simpler to produce and perform when compared with PRNT and is time saving and suitable for large-scale surveys of VACV infection in bovine.

Reactivation of porcine cytomegalovirus through allogeneic stimulation.

Reactivation of latent porcine cytomegalovirus after coculture of peripheral blood mononuclear cells (PBMCs) from pigs with different genetic backgrounds was investigated. Nine of 10 allogeneic coculture pairs were PCR (DNA) positive, whereas 7 coculture pairs had porcine cytomegalovirus (PCMV) RNA, an indication of virus replication. The cell subpopulations harboring PCMV were monocytes and CD8+ T cells.