Potential outbreak by herpesvirus in equines: detection, clinical, and genetic analysis of equid gammaherpesvirus 2 (EHV-2).

BACKGROUND: Equid herpesvirus (EHV) commonly affects horses causing neurologic and respiratory symptoms beside spontaneous abortions, meaning huge economic losses for equine industry worldwide. In foals, the virus can facilitate secondary infections by Rhodococcus equi, important in morbidity and mortality in equines. A total of five genotypes of EHV were previously described in Brazil including EHV-1, EHV-2, EHV-3, EHV-4, and EHV-5. EHV-2 genotype had only been previously described in Brazil in asymptomatic animals. We report the investigation of the dead of 11 foals in Middle-west region of Brazil showing respiratory and neurological symptoms, as well as several abortions in mares from the same farm. METHODS: Clinical and laboratory exams were performed in this case study. Lung, whole blood, serum, and plasma samples were analyzed by necroscopic and histopathologic techniques followed by molecular assays (conventional and qPCR and Sanger sequencing). RESULTS AND CONCLUSION: Laboratory exams revealed neutrophilia leukocytosis. Necroscopic and histopathologic findings were suppurative bronchopneumonia and ulcerative enteritis. Molecular assays point to the absence of the bacteria Rhodococcus equi and other viruses (including other EHV). The presence of EHV-2 DNA was confirmed by sequencing in serum sample from one foal. This is the first confirmed outbreak of EHV-2 causing disease in Brazilian horses with confirmed presence of the virus, and which highlight the important role of EHV-2 in equine respiratory disease and spontaneous abortions in equid in Brazil.

One-year surveillance of SARS-CoV-2 in wastewater from vulnerable urban communities in metropolitan São Paulo, Brazil.

The current COVID-19 pandemic has emphasized the vulnerability of communities living in the urban outskirts and informal settlements. The lack of reliable COVID-19 case data highlights the importance and application of wastewater-based epidemiology. This study aimed to monitor the COVID-19 trends in four vulnerable urban communities (slums and low-income neighborhoods) in metropolitan São Paulo by assessing the SARS-CoV-2 RNA viral load in wastewater. We analyzed 160 samples from May 2020 to June 2021 with weekly or fortnightly samplings. The samples were ultracentrifuged with glycine elution and quantified by N1/N2 SARS-CoV-2 RT-qPCR. The results of positivity were 100% (Paraisópolis, Heliópolis and Cidade Tiradentes) and 76.9% (Vila Brasilândia). The new case numbers of COVID-19, counted from the onset of symptoms, positively correlated with SARS-CoV-2 N1 viral loads from the two largest communities (p<0.001). SARS-CoV-2 infectivity was tested in Vero E6 cells after concentration with the two techniques, ultrafiltration (Centricon® Plus-70 10 kDa) and sucrose cushion ultracentrifugation, but none of the evaluated samples presented positive results. Next-generation sequencing (NGS) analysis from samples collected in March and August 2021 revealed the presence of the clade 20 J (lineage P.1) belonging to the most prevalent circulating variant in the country. Our results showed that wastewater surveillance data can be used as complementary indicators to monitor the dynamics and temporal trends of COVID-19. The infectivity test results strengthened the evidence of low risk of infection associated with SARS-CoV-2 in wastewater.

Persistence and intra-host genetic evolution of Zika virus infection in symptomatic adults: a special view in the male reproductive system

We followed the presence of Zika virus (ZIKV) in four healthy adults (two men and two women), for periods ranging from 78 to 298 days post symptom onset. The patients were evaluated regarding the presence of the virus in different body fluids (blood, saliva, urine and semen), development of immune responses (including antibodies, cytokines and chemokines), and virus genetic variation within samples collected from semen and urine during the infection course. The analysis was focused primarily on the two male patients who shed the virus for up to 158 days after the initial symptoms. ZIKV particles were detected in the spermatozoa cytoplasm and flagella, in immature sperm cells and could also be isolated from semen in cell culture, confirming that the virus is able to preserve integrity and infectivity during replication in the male reproductive system (MRS). Despite the damage caused by ZIKV infection within the MRS, our data showed that ZIKV infection did not result in infertility at least in one of the male patients. This patient was able to conceive a child after the infection. We also detected alterations in the male genital cytokine milieu, which could play an important role in the replication and transmission of the virus which could considerably increase the risk of ZIKV sexual spread. In addition, full genome ZIKV sequences were obtained from several samples (mainly semen), which allowed us to monitor the evolution of the virus within a patient during the infection course. We observed genetic changes over time in consensus sequences and lower frequency intra-host single nucleotide variants (iSNV), that suggested independent compartmentalization of ZIKV populations in the reproductive and urinary systems. Altogether, the present observations confirm the risks associated with the long-term replication and shedding of ZIKV in the MRS and help to elucidate patterns of intra-host genetic evolution during long term replication of the virus.

Persistence and intra-host genetic evolution of Zika virus infection in symptomatic adults: a special view in the male reproductive system

We followed the presence of Zika virus (ZIKV) in four healthy adults (two men and two women), for periods ranging from 78 to 298 days post symptom onset. The patients were evaluated regarding the presence of the virus in different body fluids (blood, saliva, urine and semen), development of immune responses (including antibodies, cytokines and chemokines), and virus genetic variation within samples collected from semen and urine during the infection course. The analysis was focused primarily on the two male patients who shed the virus for up to 158 days after the initial symptoms. ZIKV particles were detected in the spermatozoa cytoplasm and flagella, in immature sperm cells and could also be isolated from semen in cell culture, confirming that the virus is able to preserve integrity and infectivity during replication in the male reproductive system (MRS). Despite the damage caused by ZIKV infection within the MRS, our data showed that ZIKV infection did not result in infertility at least in one of the male patients. This patient was able to conceive a child after the infection. We also detected alterations in the male genital cytokine milieu, which could play an important role in the replication and transmission of the virus which could considerably increase the risk of ZIKV sexual spread. In addition, full genome ZIKV sequences were obtained from several samples (mainly semen), which allowed us to monitor the evolution of the virus within a patient during the infection course. We observed genetic changes over time in consensus sequences and lower frequency intra-host single nucleotide variants (iSNV), that suggested independent compartmentalization of ZIKV populations in the reproductive and urinary systems. Altogether, the present observations confirm the risks associated with the long-term replication and shedding of ZIKV in the MRS and help to elucidate patterns of intra-host genetic evolution during long term replication of the virus.

Adjuvant-Mediated Epitope Specificity and Enhanced Neutralizing Activity of Antibodies Targeting Dengue Virus Envelope Protein.

The heat-labile toxins (LT) produced by enterotoxigenic Escherichia coli display adjuvant effects to coadministered antigens, leading to enhanced production of serum antibodies. Despite extensive knowledge of the adjuvant properties of LT derivatives, including in vitro-generated non-toxic mutant forms, little is known about the capacity of these adjuvants to modulate the epitope specificity of antibodies directed against antigens. This study characterizes the role of LT and its non-toxic B subunit (LTB) in the modulation of antibody responses to a coadministered antigen, the dengue virus (DENV) envelope glycoprotein domain III (EDIII), which binds to surface receptors and mediates virus entry into host cells. In contrast to non-adjuvanted or alum-adjuvanted formulations, antibodies induced in mice immunized with LT or LTB showed enhanced virus-neutralization effects that were not ascribed to a subclass shift or antigen affinity. Nonetheless, immunosignature analyses revealed that purified LT-adjuvanted EDIII-specific antibodies display distinct epitope-binding patterns with regard to antibodies raised in mice immunized with EDIII or the alum-adjuvanted vaccine. Notably, the analyses led to the identification of a specific EDIII epitope located in the EF to FG loop, which is involved in the entry of DENV into eukaryotic cells. The present results demonstrate that LT and LTB modulate the epitope specificity of antibodies generated after immunization with coadministered antigens that, in the case of EDIII, was associated with the induction of neutralizing antibody responses. These results open perspectives for the more rational development of vaccines with enhanced protective effects against DENV infections.

Adjuvant-mediated epitope specificity and enhanced neutralizing activity of antibodies targeting dengue virus envelope protein

The heat-labile toxins (LT) produced by enterotoxigenic Escherichia coli display adjuvant effects to coadministered antigens, leading to enhanced production of serum antibodies. Despite extensive knowledge of the adjuvant properties of LT derivatives, including in vitro-generated non-toxic mutant forms, little is known about the capacity of these adjuvants to modulate the epitope specificity of antibodies directed against antigens. This study characterizes the role of LT and its non-toxic B subunit (LTB) in the modulation of antibody responses to a coadministered antigen, the dengue virus (DENV) envelope glycoprotein domain III (EDIII), which binds to surface receptors and mediates virus entry into host cells. In contrast to non-adjuvanted or alum-adjuvanted formulations, antibodies induced in mice immunized with LT or LTB showed enhanced virus-neutralization effects that were not ascribed to a subclass shift or antigen affinity. Nonetheless, immunosignature analyses revealed that purified LT-adjuvanted EDIII-specific antibodies display distinct epitope-binding patterns with regard to antibodies raised in mice immunized with EDIII or the alum-adjuvanted vaccine. Notably, the analyses led to the identification of a specific EDIII epitope located in the EF to FG loop, which is involved in the entry of DENV into eukaryotic cells. The present results demonstrate that LT and LTB modulate the epitope specificity of antibodies generated after immunization with coadministered antigens that, in the case of EDIII, was associated with the induction of neutralizing antibody responses. These results open perspectives for the more rational development of vaccines with enhanced protective effects against DENV infections.(AU) i

Epitope Sequences in Dengue Virus NS1 Protein Identified by Monoclonal Antibodies.

Dengue nonstructural protein 1 (NS1) is a multi-functional glycoprotein with essential functions both in viral replication and modulation of host innate immune responses. NS1 has been established as a good surrogate marker for infection. In the present study, we generated four anti-NS1 monoclonal antibodies against recombinant NS1 protein from dengue virus serotype 2 (DENV2), which were used to map three NS1 epitopes. The sequence 193AVHADMGYWIESALNDT209 was recognized by monoclonal antibodies 2H5 and 4H1BC, which also cross-reacted with Zika virus (ZIKV) protein. On the other hand, the sequence 25VHTWTEQYKFQPES38 was recognized by mAb 4F6 that did not cross react with ZIKV. Lastly, a previously unidentified DENV2 NS1-specific epitope, represented by the sequence 127ELHNQTFLIDGPETAEC143, is described in the present study after reaction with mAb 4H2, which also did not cross react with ZIKV. The selection and characterization of the epitope, specificity of anti-NS1 mAbs, may contribute to the development of diagnostic tools able to differentiate DENV and ZIKV infections.

Adjuvant-mediated epitope specificity and enhanced neutralizing activity of antibodies targeting dengue virus envelope protein

The heat-labile toxins (LT) produced by enterotoxigenic Escherichia coli display adjuvant effects to coadministered antigens, leading to enhanced production of serum antibodies. Despite extensive knowledge of the adjuvant properties of LT derivatives, including in vitro-generated non-toxic mutant forms, little is known about the capacity of these adjuvants to modulate the epitope specificity of antibodies directed against antigens. This study characterizes the role of LT and its non-toxic B subunit (LTB) in the modulation of antibody responses to a coadministered antigen, the dengue virus (DENV) envelope glycoprotein domain III (EDIII), which binds to surface receptors and mediates virus entry into host cells. In contrast to non-adjuvanted or alum-adjuvanted formulations, antibodies induced in mice immunized with LT or LTB showed enhanced virus-neutralization effects that were not ascribed to a subclass shift or antigen affinity. Nonetheless, immunosignature analyses revealed that purified LT-adjuvanted EDIII-specific antibodies display distinct epitope-binding patterns with regard to antibodies raised in mice immunized with EDIII or the alum-adjuvanted vaccine. Notably, the analyses led to the identification of a specific EDIII epitope located in the EF to FG loop, which is involved in the entry of DENV into eukaryotic cells. The present results demonstrate that LT and LTB modulate the epitope specificity of antibodies generated after immunization with coadministered antigens that, in the case of EDIII, was associated with the induction of neutralizing antibody responses. These results open perspectives for the more rational development of vaccines with enhanced protective effects against DENV infections.