Incursion of Highly Pathogenic Avian Influenza A(H5N1) Clade 2.3.4.4b Virus, Brazil, 2023.

We report 4 highly pathogenic avian influenza A(H5N1) clade 2.3.4.4.b viruses in samples collected during June 2023 from Royal terns and Cabot's terns in Brazil. Phylodynamic analysis revealed viral movement from Peru to Brazil, indicating a concerning spread of this clade along the Atlantic Americas migratory bird flyway.

Mortality in sea lions is associated with the introduction of the H5N1 clade 2.3.4.4b virus in Brazil October 2023: whole genome sequencing and phylogenetic analysis.

Clade 2.3.4.4b highly pathogenic avian influenza (HPAI) H5N1 virus was detected in the South American sea lions found dead in Santa Catarina, Brazil, in October 2023. Whole genome sequencing and comparative phylogenetic analysis were conducted to investigate the origin, genetic diversity, and zoonotic potentials of the H5N1 viruses. The H5N1 viruses belonged to the genotype B3.2 of clade 2.3.4.4b H5N1 virus, which was identified in North America and disseminated to South America. They have acquired new amino acid substitutions related to mammalian host affinity. Our study provides insights into the genetic landscape of HPAI H5N1 viruses in Brazil, highlighting the continuous evolutionary processes contributing to their possible adaptation to mammalian hosts.

SARS-CoV-2 Spillback to Wild Coatis in Sylvatic-Urban Hotspot, Brazil.

We tested coatis (Nasua nasua) living in an urban park near a densely populated area of Brazil and found natural SARS-CoV-2 Zeta variant infections by using quantitative reverse transcription PCR, genomic sequencing, and serologic surveillance. We recommend a One Health strategy to improve surveillance of and response to COVID-19.

First detection of Psittacid alphaherpesvirus 5 and coinfection with beak and feather disease virus in naturally infected captive ringneck parakeets (Psittacula krameri) in Brazil.

This study describes a case report in captive rose-ringed parakeets (Psittacula krameri) that developed clinical signs and eventually died after introducing new birds without quarantine. Bronchopneumonia and airsacculitis with syncytial cells associated with intranuclear inclusion bodies were found. Herpesvirus was detected in lungs and liver by PCR, and a nearly complete genome sequence of a Psittacid alphaherpesvirus 5 was obtained from the lung of a bird. Metagenomic analysis also identified beak and feather disease virus in the same samples. The study also highlights the importance of quarantine for avoiding the introduction of new diseases in captive aviaries.

In situ cytokine gene expression in early stage of virulent Newcastle disease in chickens.

Selected lymphoid and reproductive tissues were examined from groups of 3-week-old chickens and 62-week-old hens that were inoculated choanally and conjunctivally with 106 EID50 of a virulent Newcastle disease virus (NDV) isolate from the California 2018-2020 outbreak, and euthanized at 1, 2, and 3 days postinfection. In the 3-week-old chickens, immunohistochemistry for NDV and for T and B cell lymphocytes, as well as in situ hybridization for IL-1ß, IL-6, IFN-γ, and TNF-α revealed extensive expression of IL-1ß and IL-6 in lymphoid tissues, often coinciding with NDV antigen. IFN-γ was only expressed infrequently in the same lymphoid tissues, and TNF-α was rarely expressed. T-cell populations initially expanded but by day 3 their numbers were below control levels. B cells underwent a similar expansion but remained elevated in some tissues, notably spleen, cecal tonsils, and cloacal bursa. Cytokine expression in the 62-week-old hens was overall lower than in the 3-week-old birds, and there was more prolonged infiltration of both T and B cells in the older birds. The strong pro-inflammatory cytokine response in young chickens is proposed as the reason for more severe disease.

Comparative application of IS711-based polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) for canine brucellosis diagnosis.

Canine brucellosis is caused by Brucella canis, a gram negative and facultative intracellular bacterium that is commonly associated with reproductive failures in dogs. The accurate diagnosis of the infection relies on the use of serological tests associated with blood culturing to guarantee sensitivity. The polymerase chain reaction (PCR) can replace the culturing procedure for the direct diagnosis of the infection because of its speed, high specificity and sensitivity values; however, it depends on some laboratory infrastructure to be conducted. The loop-mediated isothermal amplification (LAMP) may be an alternative method for DNA amplification in a shorter period, using simpler equipment, and with a lower cost. This study evaluated the potential of molecular tools based on PCR and LAMP using primers targeting the insertion sequence IS711 for Brucella detection in three groups of dogs (infected, non-infected and suspected of brucellosis), which were determined according to the results of blood culturing and clinical examination. The performance of the three diagnostic tests was also determined using McNemar test and Kappa coefficient. The proportion of positive samples detected by blood culturing, PCR and LAMP was respectively 31.57% (18/57), 33.34% (19/57), and 14.03% (8/57). The agreement between blood culturing and PCR was almost perfect, while the agreement of PCR and blood culturing compared to LAMP was fair. The diagnostic sensitivity of PCR and LAMP was respectively 100% (18/18) and 44.44% (8/18), while the diagnostic specificity of both tests was 100% (21/21). LAMP performance was not satisfactory for canine brucellosis diagnosis because of the low diagnostic sensitivity of the test. The IS711 based PCR, otherwise, showed high values of sensitivity and specificity, which makes it a good alternative for use for the rapid diagnosis of canine brucellosis.

Detection of Leishmania infantum DNA in conjunctival swabs of cats by quantitative real-time PCR.

Although some studies have investigated the potential role of cats as a reservoir for Leishmania, their role in the epidemiology of visceral leishmaniasis (VL) is still poorly understood. Molecular diagnostic techniques are an important tool in VL diagnosis, and PCR shows high sensitivity and specificity for Leishmania spp. detection. Quantitative real-time PCR (qPCR) is a method that permits quantitative analysis of a large number of samples, resulting in more sensitive, accurate, and reproducible measurements of specific DNA present in the sample. This study compared real-time PCR (qPCR) and conventional PCR (cPCR) for detection of Leishmania spp. in blood and conjunctival swab (CS) samples of healthy cats from a non-endemic area in the state of São Paulo, Brazil. Of all CS samples, 1.85% (2/108) were positive for Leishmania spp. by both cPCR as qPCR (kappa index = 1), indicating excellent agreement between the two methods. The DNA from the two CS-cPCR- and CS-qPCR-positive samples was further tested with a PCR test amplifying the Leishmania spp. discriminative rRNA internal transcribed spacer 1 (ITS 1), of which one sample generated a 300-350-bp DNA fragment whose size varies according to the Leishmania species. Following sequencing, the fragment showed 100% similarity to a GenBank L. infantum sequence obtained from a cat in Italy. In conclusion, the association of qPCR and CS proved to be effective for detection of Leishmania in cats. Conjunctival swab samples were shown to be a practical and better alternative to blood samples and may be useful in the diagnosis and studies of feline leishmaniasis.

Differential Viral Fitness Between H1N1 and H3N8 Avian Influenza Viruses Isolated from Mallards (Anas platyrhynchos).

Homosubtypic and heterosubtypic immunity in mallards (Anas platyrhynchos) play an important role in the avian influenza virus (AIV) diversity. The mechanisms of AIV replication among wild birds and the role of immunity in AIV diversity have thus not been completely clarified. During the monitoring of AI circulation among wild waterfowl in 2007-2008, two viruses (H3N8 and H1N1) were isolated from ducks caught in a funnel trap located in La Hulpe wetland in Belgium. H3N8 viruses were revealed to be more prevalent in the mallard population than was H1N1, which might suggest a better adaptation to this species. In order to investigate this hypothesis, we characterized both isolated viruses biologically by experimental inoculation. Virus excretion and humoral response induced by both isolated viruses were evaluated in mallards after a first infection followed by a homo- or heterosubtypic reinfection under controlled experimental conditions. The H1N1 virus had a delayed peak of excretion of 4 days compared to the H3N8, but the virus shedding was more limited, earlier, and shorter after each reinfection. Moreover, the H3N8 virus could spread to all ducks after homo- or heterosubtypic reinfections and during a longer period. Although the humoral response induced by both viruses after infection and reinfection could be detected efficiently by competitive ELISA, only a minimal H1 antibody response and almost no H3-specific antibodies could be detected by the HI test. Our results suggest that the H3N8 isolate replicates better in mallards under experimental controlled conditions.

Isolation and molecular characterization of Brazilian turkey reovirus from immunosuppressed young poults.

In this study, we investigated turkey reovirus (TReoV) in tissue samples from young birds, aged 15 days. RT-PCR for TReoV detected 3.3 % positive samples and TReoV was successfully isolated in Vero cells. Histological analysis of positive bursa of Fabricius (BF) revealed atrophied follicles and lymphocyte depletion. The number of CD8+, CD4+ and IgM+ cells was lower in infected BF. Phylogenetic analysis based on S3 gene showed that the Brazilian TReoV isolates clustered in a single group with 98-100 % similarity to TReoV strains circulating in the United States. This is the first indication that TReoV infection may be a contributing factor to immunosuppression in young birds.

Comparison of single 1-day-old chick vaccination using a Newcastle disease virus vector with a prime/boost vaccination scheme against a highly pathogenic avian influenza H5N1 challenge.

Avian influenza (AI) vaccines should be used as part of a whole comprehensive AI control programme. Vectored vaccines based on Newcastle disease virus (NDV) are very promising, but are so far licensed in only a few countries. In the present study, the immunogenicity and protection against a highly pathogenic H5N1 influenza challenge were evaluated after vaccination with an enterotropic NDV vector expressing an H5 haemagglutinin (rNDV-H5) in 1-day-old specific pathogen free chickens inoculated once, twice or once followed by a heterologous boost with an inactivated H5N9 vaccine (iH5N9). The heterologous prime/boost rNDV-H5/iH5N9 combination afforded the best level of protection against the H5N1 challenge performed at 6 weeks of age. Two rNDV-H5 administrations conferred a good level of protection after challenge, although only a cellular H5-specific response could be detected. Interestingly, a single administration of rNDV-H5 gave the same level of protection as the double administration but without any detectable H5-specific immune response. In contrast to AI immunity, a high humoral, mucosal and cellular NDV-specific immunity could be detected up to 6 weeks post vaccination after using the three different vaccination schedules. NDV-specific mucosal and cellular immune responses were slightly higher after double rNDV-H5 vaccination when compared with single inoculation. Finally, the heterologous prime/boost rNDV-H5/iH5N9 combination induced a broader detectable immunity including systemic, mucosal and cellular AI and NDV-specific responses.

Whole genome sequencing of low pathogenicity avian influenza virus (H6N2) detected from a Brazilian teal (Amazonnetta brasiliensis) in Brazil, 2023.

The whole genome sequence of a low pathogenicity avian influenza virus (H6N2) was sequenced from a Brazilian teal (Amazonetta brasiliensis) in Brazil, 2023. Phylogenetic analysis of the whole genome revealed a distinct genome pertaining to South American LPAIV from 2014 to 2016, indicating extensive circulation among South American wild birds.

33rd Brazilian Society for Virology (SBV) 2022 Annual Meeting.

Each year, the Brazilian Society for Virology promotes a national meeting during the second semester of the year. In October 2022, the 33rd meeting took place at Arraial da Ajuda, Porto Seguro, Bahia, in-person:.this was the first in-person meeting since 2019, as the 2020 and 2021 events occurred online due to the issues imposed by COVID-19. It was a great pleasure for the whole audience to return to an in-person event, which certainly improved the interactions between the attendees in all ways. As usual, the meeting involved massive participation of undergraduate, graduate, and postdoc students, and several noteworthy international researchers were present. During five afternoons and evenings, attendees could discuss and learn about the most recent data presented by distinguished scientists from Brazil and other countries. In addition, young virology researchers from all levels could present their latest results as oral presentations and posters. The meeting covered all virology areas, with conferences and roundtables about human, veterinary, fundamental, environmental, invertebrate, and plant virology. The costs associated with attending the in-person event caused a slight reduction in the number of attendees compared to the two online events. However, even with this issue, the attendance was impressive. The meeting successfully achieved its most important goals: inspiring young and senior scientists and discussing high-quality, up-to-date virology research.

Genetic diversity of adenovirus in neotropical bats from Brazil.

Bats can harbor a diversity of viruses, such as adenovirus. Ten different species of bat adenoviruses (BtAdV A to J) have been previous described worlwide. In Brazil, BtAdV was described in three species of phyllostomid species: Artibeus lituratus, Desmodus rotundus, and Sturnira lilium. There are around 180 bat species in Brazil, with 67% inhabiting the Atlantic Forest, with few information about the circulation of BtAdV in this biome. We aimed to describe the molecular detection and the phylogenetic characterization and suggest a classification of BtAdVs circulating in bats from the Brazilian Atlantic Forest. We collected 382 oral and rectal swabs from 208 bats between 2014-2015 and 2020-2021 from São Paulo, Pernambuco, and Santa Catarina Brazilian states. The adenovirus detection was done by a nested PCR targeting the DNA polymerase gene, and all positive samples were sequenced by the Sanger method. The phylogenetic analyses were based on the amino acid sequences using the MEGA 7 and BEAST software. We obtained 16 positive animals (detection rate 7.7%) belonging to seven bat species: Artibeus lituratus, Carollia perspicillata, Sturnira lilium, Molossus molossus, and the first record of Phyllostomus discolor, Eptesicus diminutus, and Myotis riparius. The phylogenetic analysis based on partial amino acid sequences showed that all obtained AdV sequences belong to the Mastadenovirus genus. We observed a high genetic diversity of BtAdV and identified eleven potential BtAdV species circulating in Brazil (BtAdV K to U). Our results contribute to the epidemiological surveillance of adenovirus, increasing the knowledge about the viral diversity and the distribution of AdV in bats from the Atlantic Forest.

Intranasal DNA vaccination induces potent mucosal and systemic immune responses and cross-protective immunity against influenza viruses.

The induction of potent virus-specific immune responses at mucosal surfaces where virus transmission occurs is a major challenge for vaccination strategies. In the case of influenza vaccination, this has been achieved only by intranasal delivery of live-attenuated vaccines that otherwise pose safety problems. Here, we demonstrate that potent mucosal and systemic immune responses, both cellular and humoral, are induced by intranasal immunization using formulated DNA. We show that formulation with the DNA carrier polyethylenimine (PEI) improved by a 1,000-fold the efficiency of gene transfer in the respiratory track following intranasal administration of luciferase-coding DNA. Using PEI formulation, intranasal vaccination with DNA-encoding hemagglutinin (HA) from influenza A H5N1 or (H1N1)2009 viruses induced high levels of HA-specific immunoglobulin A (IgA) antibodies that were detected in bronchoalveolar lavages (BALs) and the serum. No mucosal responses could be detected after parenteral or intranasal immunization with naked-DNA. Furthermore, intranasal DNA vaccination with HA from a given H5N1 virus elicited full protection against the parental strain and partial cross-protection against a distinct highly pathogenic H5N1 strain that could be improved by adding neuraminidase (NA) DNA plasmids. Our observations warrant further investigation of intranasal DNA as an effective vaccination route.

Immune responses and protection against H5N1 highly pathogenic avian influenza virus induced by the Newcastle disease virus H5 vaccine in ducks.

Ducks play an important role in the epidemiology of avian influenza, and there is a need for new avian influenza vaccines that are suitable for mass vaccination in ducks. The immune responses as well as highly pathogenic avian influenza (HPAI) H5N1 protection induced by a Newcastle disease virus (NDV) vector expressing an H5N1 hemagglutinin (rNDV-H5) were investigated in mule ducks, a hybrid between Muscovy (Cairina moschata domesticus) males and Pekin (Anas platyrhynchos domesticus) females. Immunological tools to measure NDV and H5-specific serum antibody, mucosal, and cell-mediated immune (CMI) responses in ducks have been validated after infection with the vector NDV and an H5N1 low pathogenic avian influenza virus. The effect of maternally-derived antibodies (MDAs) to NDV on the humoral and CMI responses after NDV-H5 vaccination was also investigated. Our results showed the rNDV-H5 vaccine elicits satisfactory humoral and cellular responses in 11-day-old ducks correlating with a complete clinical and virological protection against the H5N1 strain. However, vaccination with rNDV-H5 in the presence of NDV MDA induced lower NDV-specific serum antibody, mucosal, and CMI responses than in ducks with no MDA, while interestingly the H5-specific serum antibody and duodenal IgY response were higher in ducks with NDV MDA. To our knowledge, this is the first report of the use of an NDV vector in ducks and of an HPAI H5N1 challenge in mule ducks, which appeared to be as resistant as Pekin ducks.

Long-Term Wastewater Surveillance for SARS-CoV-2: One-Year Study in Brazil.

Wastewater-based epidemiology (WBE) is a tool involving the analysis of wastewater for chemicals and pathogens at the community level. WBE has been shown to be an effective surveillance system for SARS-CoV-2, providing an early-warning-detection system for disease prevalence in the community via the detection of genetic materials in the wastewater. In numerous nation-states, studies have indicated the presence of SARS-CoV-2 in wastewater. Herein, we report the primary time-course monitoring of SARS-CoV-2 RNA in wastewater samples in São José do Rio Preto-SP/Brazil in order to explain the dynamics of the presence of SARS-CoV-2 RNA during one year of the SARS-CoV-2 pandemic and analyze possible relationships with other environmental parameters. We performed RNA quantification of SARS-CoV-2 by RT-qPCR using N1 and N2 targets. The proportion of positive samples for every target resulted in 100% and 96.6% for N1 and N2, respectively. A mean lag of -5 days is observed between the wastewater signal and the new SARS-CoV-2-positive cases reported. A correlation was found between the air and wastewater temperatures and therefore between the SARS-CoV-2 viral titers for N1 and N2 targets. We also observed a correlation between SARS-CoV-2 viral titers and media wastewater flow for the N1 target. In addition, we observed higher viral genome copies within the wastewater samples collected on non-rainy days for the N1 target. Thus, we propose that, based on our results, monitoring raw wastewater may be a broadly applicable strategy that might contribute to resolving the pressing problem of insufficient diagnostic testing; it may represent an inexpensive and early-warning method for future COVID-19 outbreaks, mainly in lower- and middle-income countries.

The 32nd Brazilian Society of Virology (SBV) 2021 Annual Meeting.

The Brazilian Society of Virology has been organizing annual meetings for 32 years now. The 32nd annual meeting, which occurred in 2021, was once again an online meeting in consequence of the issues imposed by COVID-19, even with the vaccination advances. As in the 2020 meeting, the number of attendees was high, with considerable participation by undergraduate, graduate, and postdoc students. Distinguished scientists from different countries offered high-quality conferences, and oral presentation sessions were presented by young scientists showing their newest research results. For almost five hours a day during five days, attendees discussed high-quality science related to all areas of virology. Even with the difficulties imposed by another pandemic year, the 32nd SBV annual meeting achieved its most important goal-to inspire young scientists and discuss high-quality virology research.

Hemorrhagic fever viruses: Pathogenesis, therapeutics, and emerging and re-emerging potential.

Hemorrhagic fever viruses (HFVs) pose a threat to global public health owing to the emergence and re-emergence of highly fatal diseases. Viral hemorrhagic fevers (VHFs) caused by these viruses are mostly characterized by an acute febrile syndrome with coagulation abnormalities and generalized hemorrhage that may lead to life-threatening organ dysfunction. Currently, the events underlying the viral pathogenicity associated with multiple organ dysfunction syndrome still underexplored. In this minireview, we address the current knowledge of the mechanisms underlying VHFs pathogenesis and discuss the available development of preventive and therapeutic options to treat these infections. Furthermore, we discuss the potential of HFVs to cause worldwide emergencies along with factors that favor their spread beyond their original niches.

H6N8 avian influenza virus in Antarctic seabirds demonstrates connectivity between South America and Antarctica.

Wild aquatic birds are the natural reservoirs of avian influenza viruses (AIVs). It is estimated that 100 million seabirds live in the Antarctic Peninsula and adjacent islands, regularly encountering migratory birds that use the islands to nest. Between 2010 and 2013, we collected samples from 865 seabirds in Elephant, King George and Livingston islands, around Antarctica Peninsula: chinstrap penguin (n = 143); gentoo penguin (n = 208); Adelie penguin (n = 46); brown skua (n = 90); Cape petrel (n = 115) and southern giant petrel (n = 263). Serum (n = 673) samples were analysed by competitive ELISA and swabs (n = 614) were tested by one step real-time RT-PCR for avian influenza virus (AIV). Sera from 30 chinstrap penguins, 76 brown skuas and a single Adelie penguin were seropositive for AIV. Thirteen swab samples were AIV positive by RT-PCR, and complete genome sequences of H6N8 AIVs isolated from brown skua and chinstrap penguin in 2011 were obtained. Phylogenetic analyses indicated that all gene segments of the H6N8 viruses were closely related to Argentinian and Chilean AIVs. The prevalence with which we identified evidence for AIVs infection in various Antarctic seabirds suggest viral circulation in Antarctic avifauna and interspecies viral transmission in the sub-Antarctic region.

Impact of SARS-CoV-2 Gamma lineage introduction and COVID-19 vaccination on the epidemiological landscape of a Brazilian city.

Background: The emergence of the Brazilian variant of concern, Gamma lineage (P.1), impacted the epidemiological profile of COVID-19 cases due to its higher transmissibility rate and immune evasion ability. Methods: We sequenced 305 SARS-CoV-2 whole-genomes and performed phylogenetic analyses to identify introduction events and the circulating lineages. Additionally, we use epidemiological data of COVID-19 cases, severe cases, and deaths to measure the impact of vaccination coverage and mortality risk. Results: Here we show that Gamma introduction in São José do Rio Preto, São Paulo, Brazil, was followed by the displacement of seven circulating SARS-CoV-2 variants and a rapid increase in prevalence two months after its first detection in January 2021. Moreover, Gamma variant is associated with increased mortality risk and severity of COVID-19 cases in younger age groups, which corresponds to the unvaccinated population at the time. Conclusions: Our findings highlight the beneficial effects of vaccination indicated by a pronounced reduction of severe cases and deaths in immunized individuals, reinforcing the need for rapid and massive vaccination.