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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved into numerous lineages with unique spike mutations and caused multiple epidemics domestically and globally. Although COVID-19 vaccines are available, new variants with the capacity for immune evasion continue to emerge. To understand and characterize the evolution of circulating SARS-CoV-2 variants in the U.S., the Centers for Disease Control and Prevention (CDC) initiated the National SARS-CoV-2 Strain Surveillance (NS3) program and has received thousands of SARS-CoV-2 clinical specimens from across the nation as part of a genotype to phenotype characterization process. Focus reduction neutralization with various antisera was used to antigenically characterize 143 SARS-CoV-2 Delta, Mu and Omicron subvariants from selected clinical specimens received between May 2021 and February 2023, representing a total of 59 unique spike protein sequences. BA.4/5 subvariants BU.1, BQ.1.1, CR.1.1, CQ.2 and BA.4/5 + D420N + K444T; BA.2.75 subvariants BM.4.1.1, BA.2.75.2, CV.1; and recombinant Omicron variants XBF, XBB.1, XBB.1.5 showed the greatest escape from neutralizing antibodies when analyzed against post third-dose original monovalent vaccinee sera. Post fourth-dose bivalent vaccinee sera provided better protection against those subvariants, but substantial reductions in neutralization titers were still observed, especially among BA.4/5 subvariants with both an N-terminal domain (NTD) deletion and receptor binding domain (RBD) substitutions K444M + N460K and recombinant Omicron variants. This analysis demonstrated a framework for long-term systematic genotype to antigenic characterization of circulating and emerging SARS-CoV-2 variants in the U.S., which is critical to assessing their potential impact on the effectiveness of current vaccines and antigen recommendations for future updates. [ABSTRACT FROM AUTHOR]

ABSTRACTInfluenza D virus (IDV) plays an important role in the bovine respiratory disease (BRD) complex. Its potential for the zoonotic transmission is of particular concern. In China, IDV has previously been identified in agricultural animals by molecular surveys with no live virus isolates reported. In this study, live IDVs were successfully isolated from cattle in China, which prompted us to further investigate the national prevalence, antigenic property, and infection biology of the virus. IDV RNA was detected in 11.1% (51/460) of cattle throughout the country in 2022–2023. Moreover, we conducted the first IDV serosurveillance in China, revealing a high seroprevalence (91.4%, 393/430) of IDV in cattle during the 2022–2023 winter season. Notably, all the 16 provinces from which cattle originated possessed seropositive animals, and 3 of them displayed the 100% IDV-seropositivity rate. In contrast, a very low seroprevalence of IDV was observed in pigs (3%, 3/100) and goats (1%, 1/100) during the same period of investigation. Furthermore, besides D/Yama2019 lineage-like IDVs, we discovered the D/660 lineage-like IDV in Chinese cattle, which has not been detected to date in Asia. Finally, the Chinese IDVs replicated robustly in diverse cell lines but less efficiently in the swine cell line. Considering the nationwide distribution, high seroprevalence, and appreciably genetic diversity, further studies are required to fully evaluate the risk of Chinese IDVs for both animal and human health in China, which can be evidently facilitated by IDV isolates reported in this study.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved into numerous lineages with unique spike mutations and caused multiple epidemics domestically and globally. Although COVID-19 vaccines are available, new variants with the capacity for immune evasion continue to emerge. To understand and characterize the evolution of circulating SARS-CoV-2 variants in the U.S., the Centers for Disease Control and Prevention (CDC) initiated the National SARS-CoV-2 Strain Surveillance (NS3) program and has received thousands of SARS-CoV-2 clinical specimens from across the nation as part of a genotype to phenotype characterization process. Focus reduction neutralization with various antisera was used to antigenically characterize 143 SARS-CoV-2 Delta, Mu and Omicron subvariants from selected clinical specimens received between May 2021 and February 2023, representing a total of 59 unique spike protein sequences. BA.4/5 subvariants BU.1, BQ.1.1, CR.1.1, CQ.2 and BA.4/5 + D420N + K444T; BA.2.75 subvariants BM.4.1.1, BA.2.75.2, CV.1; and recombinant Omicron variants XBF, XBB.1, XBB.1.5 showed the greatest escape from neutralizing antibodies when analyzed against post third-dose original monovalent vaccinee sera. Post fourth-dose bivalent vaccinee sera provided better protection against those subvariants, but substantial reductions in neutralization titers were still observed, especially among BA.4/5 subvariants with both an N-terminal domain (NTD) deletion and receptor binding domain (RBD) substitutions K444M + N460K and recombinant Omicron variants. This analysis demonstrated a framework for long-term systematic genotype to antigenic characterization of circulating and emerging SARS-CoV-2 variants in the U.S., which is critical to assessing their potential impact on the effectiveness of current vaccines and antigen recommendations for future updates.

Avian infectious bronchitis is a serious and highly contagious disease that is caused by the infectious bronchitis virus (IBV). From January 2021 to June 2022, 1008 chicken tissue samples were collected from various regions of southern China, and 15 strains of the IBV were isolated. Phylogenetic analysis revealed that the strains mainly comprised the QX type, belonging to the same genotype as the currently prevalent LX4 type, and identified four recombination events in the S1 gene, among which lineages GI-13 and GI-19 were most frequently involved in recombination. Further study of seven selected isolates revealed that they caused respiratory symptoms, including coughing, sneezing, nasal discharge, and tracheal sounds, accompanied by depression. Inoculation of chicken embryos with the seven isolates resulted in symptoms such as curling, weakness, and bleeding. Immunization of specific pathogen-free (SPF) chickens with inactivated isolates produced high antibody levels that neutralized the corresponding strains; however, antibodies produced by vaccine strains were not effective in neutralizing the isolates. No unambiguous association was found between IBV genotypes and serotypes. In summary, a new trend in IBV prevalence has emerged in southern China, and currently available vaccines do not provide protection against the prevalent IBV strains in this region, facilitating the continued spread of IBV. [ABSTRACT FROM AUTHOR]

Canine parvovirus (CPV) is a pathogen causing hemorrhagic enteritis in puppies and mainly transmitting via feco-oral route. In this study, stool samples were collected from a total of 35 animals suspected of CPV. The samples were examined by Conventional PCR, Nested PCR and Differential PCR tests. 20 out of 35 dogs (57.1%) were detected positive by conventional PCR, 31 (88.6%) by nested PCR and 30 (85.7%) by differential PCR. CPV 2a was stated as the most common antigenic type, male animals and 0–3-month-olds had a high rate of becoming sick and vaccinated animals might also catch the disease, rarely. Accordingly, it is recommended to focus on studies providing molecular epidemiology surveillance in order to detect the existing subtypes and develop reliable diagnosis and vaccination methods.

A novel highly pathogenic avian influenza A(H5N6) clade 2.3.4.4b virus was isolated from a poultry market in China that a person with a confirmed case had visited. Most genes of the avian and human H5N6 isolates were closely related. The virus also exhibited distinct antigenicity to the Re-11 vaccine strain.

Avian infectious bronchitis is a serious and highly contagious disease that is caused by the infectious bronchitis virus (IBV). From January 2021 to June 2022, 1008 chicken tissue samples were collected from various regions of southern China, and 15 strains of the IBV were isolated. Phylogenetic analysis revealed that the strains mainly comprised the QX type, belonging to the same genotype as the currently prevalent LX4 type, and identified four recombination events in the S1 gene, among which lineages GI-13 and GI-19 were most frequently involved in recombination. Further study of seven selected isolates revealed that they caused respiratory symptoms, including coughing, sneezing, nasal discharge, and tracheal sounds, accompanied by depression. Inoculation of chicken embryos with the seven isolates resulted in symptoms such as curling, weakness, and bleeding. Immunization of specific pathogen-free (SPF) chickens with inactivated isolates produced high antibody levels that neutralized the corresponding strains; however, antibodies produced by vaccine strains were not effective in neutralizing the isolates. No unambiguous association was found between IBV genotypes and serotypes. In summary, a new trend in IBV prevalence has emerged in southern China, and currently available vaccines do not provide protection against the prevalent IBV strains in this region, facilitating the continued spread of IBV.

Influenza D virus (IDV) plays an important role in the bovine respiratory disease (BRD) complex. Its potential for the zoonotic transmission is of particular concern. In China, IDV has previously been identified in agricultural animals by molecular surveys with no live virus isolates reported. In this study, live IDVs were successfully isolated from cattle in China, which prompted us to further investigate the national prevalence, antigenic property, and infection biology of the virus. IDV RNA was detected in 11.1% (51/460) of cattle throughout the country in 2022-2023. Moreover, we conducted the first IDV serosurveillance in China, revealing a high seroprevalence (91.4%, 393/430) of IDV in cattle during the 2022-2023 winter season. Notably, all the 16 provinces from which cattle originated possessed seropositive animals, and 3 of them displayed the 100% IDV-seropositivity rate. In contrast, a very low seroprevalence of IDV was observed in pigs (3%, 3/100) and goats (1%, 1/100) during the same period of investigation. Furthermore, besides D/Yama2019 lineage-like IDVs, we discovered the D/660 lineage-like IDV in Chinese cattle, which has not been detected to date in Asia. Finally, the Chinese IDVs replicated robustly in diverse cell lines but less efficiently in the swine cell line. Considering the nationwide distribution, high seroprevalence, and appreciably genetic diversity, further studies are required to fully evaluate the risk of Chinese IDVs for both animal and human health in China, which can be evidently facilitated by IDV isolates reported in this study.

Swine play an important role in the ecology of influenza A viruses (IAVs), acting as mixing vessels. Swine (sw) IAVs of H1N1 (including H1N1pdm09), H3N2, and H1N2 subtypes are enzootic in pigs globally, with different geographic distributions. This study investigated the genetic diversity of swIAVs detected during passive surveillance of pig farms in Northern Italy between 2017 and 2020. A total of 672 samples, IAV-positive according to RT-PCR, were subtyped by multiplex RT-PCR. A selection of strains was fully sequenced. High genotypic diversity was detected among the H1N1 and H1N2 strains, while the H3N2 strains showed a stable genetic pattern. The hemagglutinin of the H1Nx swIAVs belonged to HA-1A, HA-1B, and HA-1C lineages. Increasing variability was found in HA-1C strains with the circulation of HA-1C.2, HA-1C.2.1 and HA-1C.2.2 sublineages. Amino acid deletions in the HA-1C receptor binding site were observed and antigenic drift was confirmed. HA-1B strains were mostly represented by the Δ146-147 Italian lineage HA-1B.1.2.2, in combination with the 1990s human-derived NA gene. One antigenic variant cluster in HA-1A strains was identified in 2020. SwIAV circulation in pigs must be monitored continuously since the IAVs’ evolution could generate strains with zoonotic potential.

Abstract Here we report studies of the antigenic relationship of West Nile virus (WNV) and Usutu virus (USUV), two zoonotic flaviviruses from Italy, together with a Japanese encephalitis virus (JEV) strain and compared them with their genetic relationship using the immunodominant viral E protein. Thirty-nine isolates and reference strains were inactivated and used to immunize rabbits to produce hyper immune sera. Serum samples were tested by neutralization against all isolates and results visualized by generating antigenic map. Strains of WNV, USUV, and JEV grouped in separate clusters on the antigenic map. JEV was closer antigenically to USUV (mean of 3.5 Antigenic Unit, AU, equivalent to a 2-fold change in antibody titer) than to WNV strains (mean of 6 AU). A linear regression model predicted, on average, one unit of antigenic change, equivalent to a 2-fold change in antibody titer, for every 22 amino acid substitutions in the E protein ectodomain. Overall, antigenic map was demonstrated to be robust and consistent with phylogeny of the E protein. Indeed, the map provided a reliable means of visualizing and quantifying the relationship between these flaviviruses. Further antigenic analyses employing representative strains of extant serocomplexes are currently underway. This will provide a more in deep knowledge of antigenic relationships between flaviviruses. Highlights • Antigenic relationship of WNV, USUV and JEV is reported. • They grouped in separate clusters on the antigenic map. • JEV was closer antigenically to USUV than to WNV. • One unit of antigenic change for every 22 amino acid substitutions was shown in the E protein ectodomain. [ABSTRACT FROM AUTHOR]

Low pathogenic avian influenza (LPAI) H9N2 viruses have established endemic status in Egyptian poultry populations since 2012. Recently, four cases of human H9N2 virus infections in Egypt demonstrated the zoonotic potential of these viruses. Egyptian H9N2 viruses obtained from 2011 to 2014 phylogenetically grouped into three clusters (1−3) within subclade B of the G1 lineage. Antigenically, a close clustering of the Egyptian H9N2 viruses with other recent G1-B like H9N2 strains and a significant antigenic distance from viruses outside the G1-B lineage was evident. Recent Egyptian LPAIV H9N2 showed a tendency to increased binding with erythrocytes expressing α 2,6-linked sialic acid which correlated with the Q226L amino acid substitution at the receptor binding unit of the hemagglutinin (Q234L, H9 numbering). Sequence analyses of the N2 neuraminidase (NA) revealed substitutions in the NA hemadsorption site similar to the N2 of prepandemic H3N2/1968, but no distinct antigenic or functional characteristics of the H9N2 NA associated with increased zoonotic potential could be identified. [ABSTRACT FROM AUTHOR]

Background In Denmark, influenza A virus of the subtype H3N2 has been dominating the 2016/17 season, as in most countries of the Northern Hemisphere. Objectives This study was conducted as part of the Danish seasonal influenza surveillance programme to genetically characterize circulating H3N2 viruses and determine the seasonal vaccine effectiveness (VE) overall in the Danish population and further on the virus cluster level. Study design Influenza virus positive samples submitted for the national surveillance programme were genetically characterized by sequencing. VE estimates against influenza A and the circulating virus clusters were determined in patients above 65 years using the test-negative case–control design. Results The genetic characterization revealed several genetically drifted viruses, which could be divided into four main clusters by the defining amino acid substitutions: 3C.2a/N121K/S144K, 3C.2a/T131K/R142K, 3C.2a1, and 3C.2a1/N121K. Some of the drifted viruses appeared to be more prominent in vaccinated or non-vaccinated individuals, respectively. Overall the adjusted VE was 7.4% (95% confidence interval (CI): −6.0–19.2) among inpatients and 19.3% (95% CI: −5.7–38.4) among outpatients, respectively. VE for the four main virus clusters was; cluster 3C.2a1: 38.8% (95% CI: −29.8–71.1), cluster 3C.2a/N121K/S144K: 9.2% (95% CI: −63.0–49.4), cluster 3C.2a/T131K/R142K: 19.0% (95% CI: −85.3–64.6), and cluster 3C.2a1/N121K: −12.2% (95%CI: −129.7–45.2). Conclusions Several genetically drifted H3N2 viruses have been circulating in Denmark in the 2016-17 influenza season. An overall low VE was estimated and VE for the four main virus cluster indicate different VEs between the circulating drifted H3N2 viruses. [ABSTRACT FROM AUTHOR]

The hemagglutination inhibition (HI) assay has been used for the antigenic characterization of influenza viruses for decades. However, the majority of recent seasonal influenza A viruses of the H3N2 subtype has lost the capacity to agglutinate erythrocytes of various species. The hemagglutination (HA) activity of other A(H3N2) strains is generally sensitive to the action of the neuraminidase inhibitor oseltamivir, which indicates that the neuraminidase and not the hemagglutinin is responsible for the HA activity. These findings complicate the antigenic characterization and selection of A(H3N2) vaccine strains, calling for alternative antigenic characterization assays. Here we describe the development and use of the ViroSpot microneutralization (MN) assay as a reliable and robust alternative for the HI assay. Serum neutralization of influenza A(H3N2) reference virus strains and epidemic isolates was determined by automated readout of immunostained cell monolayers, in a format designed to minimize the influence of infectious virus doses on serum neutralization titers. Neutralization of infection was largely independent from rates of viral replication and cell-to-cell transmission, facilitating the comparison of different virus isolates. Other advantages of the ViroSpot MN assay include its relative insensitivity to variation in test dose of infectious virus, automated capture and analyses of residual infection patterns, and compatibility with standardized large scale analyses. Using this assay, a number of epidemic influenza A(H3N2) strains that failed to agglutinate erythrocytes, were readily characterized antigenically. [ABSTRACT FROM AUTHOR]

The present research investigated the presence of T. evansi antibodies in animals from the subregion of Nhecolandia, in the Pantanal Sul-mato-grossense, by means of an enzyme linked immunosorbent assay (ELISA) and indirect immunofluorescence antibody test (IFAT), and the pattern of polypeptide recognition by sera from experimentally and naturally infected hosts using Western blotting. Serum samples were obtained from bovines (n = 102), horses (n = 98), and dogs (n = 55), and from 32 free-ranging coatis (Nasua nasua). None of the bovines were found positive, while sera from 16 dogs (29%) and 23 horses (23.4%) were positive by ELISA. Sera from 8 coatis (25%) were found positive using IFAT. Western blotting revealed major polypeptides of T. evansi with molecular weight ranging from 74 to 38 kDa. The polypeptides of 66, 48-46, and 38 kDa were identified by sera from experimentally infected bovines, donkeys, dogs, and coatis. The 48-46 and 38 kDa bands were mainly recognized in chronic phase of infection. The antigen with apparent molecular weight of 66 kDa, revealed by antibodies from all experimental animals, was also recognized in sera of horses and dogs from the Pantanal. The 48-46 kDa polypeptide was identified by antibodies from all naturally infected animals and must be further evaluated for use in specific diagnosis of T. evansi infection.O trabalho de pesquisa investigou a presença de anticorpos anti- T. evansi em animais da sub-região da Nhecolândia, no Pantanal sul-mato-grossense, pelo ensaio imunoenzimático (ELISA) e a reação de imunofluorescência indireta (RIFI). O reconhecimento de polipeptídeos de T. evansi foi realizado pela técnica de "Western blotting", utilizando soros de animais experimentalmente e naturalmente infectados. As amostras de soro foram obtidas de bovinos (n = 102), cavalos (n = 98) e cães (n = 55) e de 32 quatis de vida livre (Nasua nasua) do pantanal mato-grossense. Todos os soros dos bovinos foram negativos, enquanto soros de 16 cães (29%) e 23 cavalos (23,4%) foram positivos pelo ELISA. Soros de oito quatis (25%) foram positivos pela RIFI. Pelo "Western-blotting" foi possível revelar polipeptídeos de T. evansi, com peso molecular variando de 74 a 38 kDa. Os polipeptídeos de 66, 48-46 e 38 kDa foram identificados por soros de bovinos, cavalos, cães e quatis experimentalmente infectados. As bandas de 48-46 e 38 kDa foram reconhecidas principalmente na fase crônica da infecção. O antígeno com peso molecular aparente de 66 kDa, revelado por anticorpos de todos os animais experimentais, também foi reconhecido por soros de cavalos e cães do Pantanal. O polipeptídeo de 48-46 kDa foi identificado por anticorpos de todos os animais naturalmente infectados, devendo ser avaliado para o diagnóstico específico da infecção pelo T. evansi.

Several studies conducted all over the world have reported that the influenza virus is associated with great morbidity and mortality rates. In this study, we analyzed the incidence of the influenza virus between 2000 and 2003 in Curitiba. We studied 1621 samples obtained from outpatients and hospitalized patients of both sexes and all ages. The study was conducted at the local primary care health units (outpatients) and at the tertiary care unit (hospitalized) of the General Hospital of the Federal University in the state of Paraná, Brazil. Nasopharyngeal aspirates and, eventually, bronchoalveolar lavage were assayed for the presence of viral antigens, either by indirect immunofluorescence or cell culture. Of the samples studied, 135 (8.3%) were positive for influenza virus, and of those, 103 (76.3%) were positive for type A and 32 (23.7%) for type B. Additionally, positive samples were analyzed by reverse transcription followed by polymerase chain reaction and subtypes H1 and H3 were identified from this group. A high incidence of positive samples was observed mainly in the months with lower temperatures. Furthermore, outpatients showed a higher incidence of influenza viruses than hospitalized patients.

Introduction: In Brazil, various isolates of rabies virus (RABV) show antigenic profiles distinct from those established by the reduced panel of eight monoclonal antibodies (MAbs) determined by the Centers for Disease Control and Prevention (CDC), utilized for the antigenic characterization of RABV in the Americas. The objective of this study was to produce MAbs from RABV isolates from insectivorous bats with an antigenic profile incompatible with the pre-established one. Methodology: An isolate of RABV from the species Eptesicus furinalis that showed an antigenic profile incompatible with the panel utilized was selected. Hybridomas were produced utilizing the popliteal lymph nodes of mice immunized with ribonucleoproteins purified from the isolate. Results: Two MAbs-producing clones were obtained, BR/IP1-3A7 and BR/IP2-4E10. Fifty-seven isolates of RABV from different species of animals and different regions of Brazil were analyzed utilizing the MAbs obtained. In the analysis of 23 RABV isolates from nonhematophagous bats, the MAbs cross-reacted with ten isolates, of which four were of the species Nyctinomops laticaudatus, one of the species Eptesicus furinalis, and five of the genus Artibeus. Of the nine isolates of non-hematophagous isolates that displayed an incompatible profile analyzed, characteristic of insectivorous bats, BR/IP1-3A7 reacted with five (55.55%) and BR/IP2-4E10 with four (44.44%). Conclusions: The MAbs obtained were able to recognize epitopes common between the three genera, Artibeus, Eptesicus, and Nyctinomops, thereby allowing the antigenic characterization of RABV isolates in Brazil. [ABSTRACT FROM AUTHOR]

Antigenic characterization of Anaplasma marginale isolates, by identifying conserved and variable epitopes of major surface proteins (MSP), is an important tool for vaccine development against this rickettsia. The B cell epitopes of A. marginale isolates from three microregions of the State of Pernambuco and one from the State of Mato Grosso do Sul, Brazil, were characterized by indirect fluorescent antibody technique (IFAT) and Western blot (WB) with 15 monoclonal antibodies (MAbs). The epitope recognized by MAb ANA22B1 (MSP-1a) was conserved by IFAT and WB (73-81 kDa). MSP-2 epitopes recognized by MAbs ANAO58A2 and ANAO70A2 were conserved by IFAT, while ANAO50A2 and ANA66A2 epitopes were polymorphic; in the WB, the MAbs ANAO50A2 and ANAO70A2 identified bands of 45 kDa only in the Pernambuco-Mata isolate. None of the isolates reacted with MAb ANAR75C2 (MSP-3). The MSP-4 epitope recognized by MAb ANAR76A1 was conserved by IFAT, as well as the MSP-5 epitope recognized by MAb ANAF16C1 by IFAT and WB (16 kDa). The MAbs ANAR17A6, ANAR83B3, ANAR94C1, ANAO24D5 and ANAR19A6 identified conserved epitopes by IFAT. MSP-1, MSP-2 and MSP-4, which previously showed partial protection in experimental trials, are also potential immunogens to be employed in Brazil, due to the B cell epitope conservation.

HoBi-like pestiviruses have been sporadically reported from naturally infected cattle in South America, Asia and Europe. While the closely related bovine viral diarrhoea virus 1 (BVDV-1) and BVDV-2 have been reported from cattle in India, the prevalence and diversity of HoBi-like viruses have not yet been studied. Here we report the genetic diversity and molecular characteristics of HoBi-like viruses, through systematic surveillance in cattle ( n = 1049) from 21 dairy farms across India during 2012–2013. On the basis of real-time RT-PCR, virus isolation and nucleotide sequencing results, of the 20 pestivirus positive cattle, HoBi-like viruses were identified in 19 cattle from four farms in three states and BVDV-1b in one cattle. Phylogenetic analysis of 5′-UTR and N pro region identified the circulation of two lineages of HoBi-like viruses in India, that were distinct to those circulating globally, highlighting the independent evolution of at least three lineages of HoBi-like viruses globally. Antigenic differences were also evident between the two Indian lineages. In addition to revealing that HoBi-like virus may be more widespread in Indian cattle than previously reported, this study shows greater genetic divergence of HoBi-like viruses indicating a need for continued pestivirus surveillance in cattle. [ABSTRACT FROM AUTHOR]

The occurrence of rabies virus antigenic variants in North and Central West regions of Brazil was studied using 61 rabies viruses isolated from different species: 30 from domestic dogs, 20 from cattle, four from horses, two from cats, one from a human and four from unidentified species. The isolates were submitted to antigenic analyses by indirect immunofluorescence with a panel of 12 monoclonal antibodies (Mabs) to lyssavirus antigens. Antigenic analyses revealed consistent differences between isolates whose natural hosts were dogs and those of haematophagous bats, often isolated from cattle. Three out of four isolates from horses and one from a domestic dog showed patterns of reactivity found only in viruses of insectivorous bats, indicating that non-haematophagous bats do play a unique role in the transmission of the virus to other species.

Swine play an important role in the ecology of influenza A viruses (IAVs), acting as mixing vessels. Swine (sw) IAVs of H1N1 (including H1N1pdm09), H3N2, and H1N2 subtypes are enzootic in pigs globally, with different geographic distributions. This study investigated the genetic diversity of swIAVs detected during passive surveillance of pig farms in Northern Italy between 2017 and 2020. A total of 672 samples, IAV-positive according to RT-PCR, were subtyped by multiplex RT-PCR. A selection of strains was fully sequenced. High genotypic diversity was detected among the H1N1 and H1N2 strains, while the H3N2 strains showed a stable genetic pattern. The hemagglutinin of the H1Nx swIAVs belonged to HA-1A, HA-1B, and HA-1C lineages. Increasing variability was found in HA-1C strains with the circulation of HA-1C.2, HA-1C.2.1 and HA-1C.2.2 sublineages. Amino acid deletions in the HA-1C receptor binding site were observed and antigenic drift was confirmed. HA-1B strains were mostly represented by the Δ146-147 Italian lineage HA-1B.1.2.2, in combination with the 1990s human-derived NA gene. One antigenic variant cluster in HA-1A strains was identified in 2020. SwIAV circulation in pigs must be monitored continuously since the IAVs' evolution could generate strains with zoonotic potential. [ABSTRACT FROM AUTHOR]

• Principal Component Analysis identified antigenic clusters and relationships. • Antigenic clusters may be a method for determining antigenic relationships between vaccine strains and field isolates. • Antigenic relationships may exist between BVDV isolates that belong to same or different BVDV species and subgenotype. • This methodology may be a useful a tool to improve development of vaccine. • This methodology provides a tool to evaluate strains that generate broader cross reactivity. Bovine viral diarrhea virus (BVDV) comprises two species, BVDV-1 and BVDV-2. But given the genetic diversity among pestiviruses, at least 22 subgenotypes are described for BVDV-1 and 3-4 for BVDV-2. Genetic characterization is generally accomplished through complete or partial sequencing and phylogeny, but it is not a reliable method to define antigenic relationships. The traditional method for evaluating antigenic relationships between pestivirus isolates is the virus neutralization (VN) assay, but interpretation of the data to define antigenic relatedness can be difficult to discern for BVDV isolates within the same BVDV species. Data from this study utilized a multivariate analysis for visualization of VN results to analyze the antigenic relationships between US vaccine strains and field isolates from Switzerland, Italy, Brazil, and the UK. Polyclonal sera were generated against six BVDV strains currently contained in vaccine formulations, and each serum was used in VNs to measure the titers against seven vaccine strains (including the six homologous strains) and 23 BVDV field isolates. Principal component analysis (PCA) was performed using VN titers, and results were interpreted from PCA clustering within the PCA dendrogram and scatter plot. The results demonstrated clustering patterns among various isolates suggesting antigenic relatedness. As expected, the BVDV-1 and BVDV-2 isolates did not cluster together and had the greatest spatial distribution. Notably, a number of clusters representing antigenically related BVDV-1 subgroups contain isolates of different subgenotypes. The multivariate analysis may be a method to better characterize antigenic relationships among BVDV isolates that belong to the same BVDV species and do not have distinct antigenic differences. This might be an invaluable tool to ameliorate the composition of current vaccines, which might well be important for the success of any BVDV control program that includes vaccination in its scheme. [ABSTRACT FROM AUTHOR]

A novel highly pathogenic avian influenza A(H5N6) clade 2.3.4.4b virus was isolated from a poultry market in China that a person with a confirmed case had visited. Most genes of the avian and human H5N6 isolates were closely related. The virus also exhibited distinct antigenicity to the Re-11 vaccine strain.

Abstract: Virological and antigenic characteristics of two Indian Peste des Petits Ruminants (PPR) vaccine viruses namely “PPRV-Sungri/96” and “PPRV-AR/87” were investigated. This investigation included, type of cytopathic effect (CPE) produced, one-step growth curve, antigenic reactivity of viral antigens using a panel of 18 monoclonal antibodies (MAbs) and ability of viruses for neutralization using a monoclonal antibody directed to haemagglutinin (H) protein. Findings surprisingly indicated that the PPRV-AR/87 is a fast growing virus with an entirely different pattern of cytopathic effect in Vero cell system. This virus has a relatively short replication cycle of 21h with no clearly defined eclipse phase. Whereas, PPRV-Sungri/96 has a replication cycle of 72h with distinct eclipse phase. Both viruses showed very good antigenic correlation (r =0.823) based on reactivity with 18 MAbs in an indirect ELISA, indicating that they are closely antigenically related. PPRV-AR/87 showed a poor neutralization index with antihaemaglutinin MAb 4B11 and poor reactivity in sandwich ELISA using an anti-nucleocapsid (N) MAb as compared to PPRV-Sungri/96. The findings suggest that both these vaccine viruses can easily be differentiated based on the pattern of cytopathic effect and degree of neutralization using MAb 4B11. [Copyright &y& Elsevier]

Abstract: The open reading frame 2 (ORF2) of human astrovirus (HAstV) encodes the structural VP26 protein that seems to be the main antigenic viral protein. However, its functional role remains unclear. Bioinformatic predictions revealed that VP29 and VP26 proteins could be involved in virus–cell interaction. In this study, we describe for the first time the cloning and expression in Escherichia coli (E. coli) of a recombinant VP26 (rVP26) protein and a VP26 C-terminal truncated form (VP26ΔC), followed by purification by NTA-Ni2+ agarose affinity chromatography. Protein expression and purification were evaluated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (WB). Then, the purified proteins were evaluated for antigenic properties in enzyme linked immunosorbent assay (ELISA) using a polyclonal antibody (PAb) and a neutralizing monoclonal antibody (nMAb) named PL2, both of them directed to HAstV. The results presented herein indicate that the C-terminal end of the VP26 protein is essential to maintain the neutralizing epitope recognized by nMAb PL2 and that the N-terminus of VP26 protein may contain antigenic lineal-epitopes recognized by PAb. Thus, these recombinant proteins can be ideal tools for further antigenic, biochemical, structural and functional VP26 protein characterization, in order to evaluate its potential role in immunodiagnosis and vaccine studies. [Copyright &y& Elsevier]

A polyclonal antiserum with high titer, great specificity and sensitivity was obtained against the hemagglutinin of Influenza A/Johannesburg/33/94, belonging to subtype A (H3N2). The obtaining process of the antigen or immunogen, the immunization schedule and its administration to Wistar rats as animal model are described. All of these were validated by the hemagglutinin inhibition technique. The antiserum obtained was treated with receptor-destroying enzymes of national production (EDR/IPK) in order to eliminate the unspecific inhibitors of the hemagglutinin inhibition. Validation was carried out in front of a panel including 9 influenza reference strains belonging to subtypes A(H3N2), A(H1N1), and to type B. No unspecific reactions against strains A(H1N1) and of type B were detected. Five autochthonous isolates were satisfactorily typed and subtyped. [ABSTRACT FROM AUTHOR]

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Antigenic characterization of A. marginale isolates has contributed to identifying the presence of common and restricts epitopes of major surface proteins (MSPs). The data may improve vaccine development to protect against A. marginale isolates from different regions. Brazilian A. marginale isolates were characterized antigenically by Western blot with monoclonal antibodies (MAbs) against MSPs and rabbit anti-MSP-4 from Florida strain. Six A. marginale isolates from MS, MG (AUFV1), SP, PR-L1, PR-HV, RS and Florida strain were tested with ANA22B1 to MSP-1a, AMR36A6 to MSP-1b, ANAF19E2 to MSP-2, AMG75C1 and AMG76B2 to MSP-3 and ANAF16C1 to MSP-5. ANA22B1 recognized MSP-1a epitope in all A. marginale isolates, and reacted with polypeptides of different size ranging 46–105 kDa. MSP2 was not detected in MS and SP isolates by ANAF19E2, and only PR-L1 and MG (AUFV1) isolates reacted with MAbs which recognize MSP3 epitope. MSP4 and MSP5 were detected in all A. marginale isolates analyzed. The results revealed conservation of MSP-1a and MSP-5 epitopes among all Brazilian isolates, and showed antigenic variability to MSP-1b, MSP-2 and MSP-3 proteins, agreeing with recent data about the genetic diversity found in the polimorphic multigene family responsible for these proteins. [Copyright &y& Elsevier]

Swine play an important role in the ecology of influenza A viruses (IAVs), acting as mixing vessels. Swine (sw) IAVs of H1N1 (including H1N1pdm09), H3N2, and H1N2 subtypes are enzootic in pigs globally, with different geographic distributions. This study investigated the genetic diversity of swIAVs detected during passive surveillance of pig farms in Northern Italy between 2017 and 2020. A total of 672 samples, IAV-positive according to RT-PCR, were subtyped by multiplex RT-PCR. A selection of strains was fully sequenced. High genotypic diversity was detected among the H1N1 and H1N2 strains, while the H3N2 strains showed a stable genetic pattern. The hemagglutinin of the H1Nx swIAVs belonged to HA-1A, HA-1B, and HA-1C lineages. Increasing variability was found in HA-1C strains with the circulation of HA-1C.2, HA-1C.2.1 and HA-1C.2.2 sublineages. Amino acid deletions in the HA-1C receptor binding site were observed and antigenic drift was confirmed. HA-1B strains were mostly represented by the Δ146-147 Italian lineage HA-1B.1.2.2, in combination with the 1990s human-derived NA gene. One antigenic variant cluster in HA-1A strains was identified in 2020. SwIAV circulation in pigs must be monitored continuously since the IAVs' evolution could generate strains with zoonotic potential.

Several hot springs in the Rotorua-Taupo regions, North Island, New Zealand, were tested for the presence of extremely thermophilic acidophilic bacteria. In the majority of the springs, ranging in temperature from 43-96°C and in pH from 2.1-6.9, direct microscopic observations revealed the presence of both rod-shaped and spherical bacteria. Isolations were attempted at 70°C and pH 2.0 and 7.0, with either yeast extract for heterotrophic growth, or elemental sulfur as the sole source of energy for autotrophic growth. Eight of the samples produced grwoth at pH 2.0 with either yeast extract or sulfur, but none of the samples grew at pH 7.0. All the isolates obtained, resembled Sulfolobus acidocaldarius, a thermophilic acidophilic bacterium which has previously been reported from various regions in the Northern Hemisphere. Immunofluorescence examination of six of these isolates revealed varying degrees of cross reactions with two already characterized Sulfolobus isolates from the Yellowstone National Park, U.S.A. This paper is the first published record of Sulfolobus from the Southern Hemisphere. [ABSTRACT FROM AUTHOR]

Twenty strains of rabies virus were recovered by tissue culture technique from 32 samples of different animal brains collected between 1984 and 1986 in North Italy in the course of routine rabies diagnosis: they were characterized by using specific Wistar antirabies monoclonal antibodies (Mo-Abs). It was of interest to identify the isolated virus strains to avoid doubts of possible vaccine induced rabies since an experiment with oral wildlife immunization, using live SAD strain vaccine was being carried out for the last two years and is still on-going in different areas of North Italy (provinces: Brescia, Trento, Bolzano). All the isolates were found to be street rabies viruses resembling the fox strains of rabies virus currently present in Europe to date. Different aspects of rabies virus isolation, using tissue culture technique, are discussed. Zusammenfassung 20 Tollwutvirus-Stämme wurden mittels Zellkulturtechnik aus 32 Proben von verschiedenen Tiergehirnen isoliert, die zwischen 1984 und 1986 in Norditalien im Zuge der Routine-Diagnostik gesammelt wurden: sie wurden mit spezifischen Wistar-Antitollwut-monoklonalen Antikörpern charakterisiert. Die Identifizierung dieser Stämme war von Interesse, um Zweifel an einer möglichen, impfbedingten Tollwutvirusinfektion auszuräumen, da seit zwei Jahren ein Versuch mit oraler Immunisierung an Wildtieren mit dem lebenden Impfstamm SAD im Gange ist und weiterhin in verschiedenen Gebieten Norditaliens (Provinzen: Brescia, Trento, Bozen) durchgeführt wird. Alle Isolate waren Tollwutvirus-Feldstämme, die den gegenwärtig in Europa auftretenden Tollwutvirus-Stämmen von Füchsen ähnelten. Verschiedene Aspekte der Tollwutvirus-Isolierung mittels Zellkulturtechniken werden diskutiert. [ABSTRACT FROM AUTHOR]

The hemagglutination inhibition (HI) assay for influenza A virus has been used since the 1940s. The assay may be utilized to detect or quantify antibodies to influenza A viruses and can be used to characterize differences in antigenic reactivity between influenza isolates. In addition, data from HI assays are routinely used for antigenic cartography, influenza virus surveillance, epidemiology, and vaccine-seed strain selection. For antibody quantification, the HI assay is a fast and inexpensive method; other than a source of red blood cells, no expensive or unusual lab equipment is needed, and results can be obtained within a few hours. Historically, the HI assay has also served as a primary method of subtype identification and is still used widely. However, as gene sequencing technology has evolved to be cheaper and faster, it is replacing the HI assay for this purpose.

Antigenic cartography is a powerful method that allows for the calculation of antigenic distances between influenza viruses or sera and their positioning on a map, by quantifying raw data from hemagglutination inhibition assays. As a consequence, the antigenic drift of influenza viruses over time can be visualized in a straightforward manner. Antigenic cartography is not only useful in the research of influenza virus evolution but also in the surveillance of influenza viruses. Most importantly, antigenic cartography plays a very important role in vaccine updating decisions, since by calculating the antigenic distances between a vaccine strain and circulating strains, an informed decision can be made on whether the distances are large enough to warrant a vaccine update or not. Recent improvements in antigenic cartography calculations have significantly improved its accuracy.