Emerging Novel Reassortant Influenza A(H5N6) Viruses in Poultry and Humans, China, 2021.

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.

Antigenic characterization of influenza and SARS-CoV-2 viruses.

Antigenic characterization of emerging and re-emerging viruses is necessary for the prevention of and response to outbreaks, evaluation of infection mechanisms, understanding of virus evolution, and selection of strains for vaccine development. Primary analytic methods, including enzyme-linked immunosorbent/lectin assays, hemagglutination inhibition, neuraminidase inhibition, micro-neutralization assays, and antigenic cartography, have been widely used in the field of influenza research. These techniques have been improved upon over time for increased analytical capacity, and some have been mobilized for the rapid characterization of the SARS-CoV-2 virus as well as its variants, facilitating the development of highly effective vaccines within 1 year of the initially reported outbreak. While great strides have been made for evaluating the antigenic properties of these viruses, multiple challenges prevent efficient vaccine strain selection and accurate assessment. For influenza, these barriers include the requirement for a large virus quantity to perform the assays, more than what can typically be provided by the clinical samples alone, cell- or egg-adapted mutations that can cause antigenic mismatch between the vaccine strain and circulating viruses, and up to a 6-month duration of vaccine development after vaccine strain selection, which allows viruses to continue evolving with potential for antigenic drift and, thus, antigenic mismatch between the vaccine strain and the emerging epidemic strain. SARS-CoV-2 characterization has faced similar challenges with the additional barrier of the need for facilities with high biosafety levels due to its infectious nature. In this study, we review the primary analytic methods used for antigenic characterization of influenza and SARS-CoV-2 and discuss the barriers of these methods and current developments for addressing these challenges.

Molecular and antigenic characterization of bovine herpesvirus type 1 (BoHV-1) strains from cattle with diverse clinical cases in Turkey.

The present study reports the molecular and antigenic characterization of 13 bovine herpesvirus type 1 (BoHV-1) field viruses obtained from cattle with different clinical cases in Turkey between 1992 and 2017. We selected glycoprotein C (gC) of BoHV-1 as a target to detect and/or verify presence of the virus in suspect materials followed by virus isolation (VI) in MDBK cells. In seven out of 13 BoHV-1 positive samples, cytophatic effects (CPEs) were observed in MDBK cell cultures, although only four virus samples reached a sufficient titer to use in phylogenetic assay, restriction endonuclease analysis (REA), and virus neutralization test (VNT). According to the results of sequence analysis of the 13 BoHV-1 positive samples, nine BoHV-1 field viruses were determined as BoHV-1.1 and four as BoHV-1.2. Using REA, we demonstrated that two of our isolated viruses could be categorized as BoHV-1.1 while the other two isolates were BoHV-1.2 subtypes. Differences between the BoHV-1.1 and BoHV-1.2 isolates were also detected in the VNT results by assaying 125 suspected serum samples after testing with isolated (KY748023, KY748022, KY748020, and KY748021) and reference viruses (BoHV-1 Cooper and BoHV-5 Texas 89). These results are indicating the need to correctly identify BoHV-1 field isolates to better understand the epidemiology and pathogenesis of infection. In addition, it would be useful to identify the subtypes circulating in the specific geographical area while determining vaccination preferences.

Influenza vaccine effectiveness in adults 65 years and older, Denmark, 2015/16 - a rapid epidemiological and virological assessment.

In Denmark, both influenza A(H1N1)pdm09 and influenza B co-circulated in the 2015/16 season. We estimated the vaccine effectiveness (VE) of the trivalent influenza vaccine in patients 65 years and older using the test-negative case-control design. The adjusted VE against influenza A(H1N1)pdm09 was 35.0% (95% confidence interval (CI): 11.1-52.4) and against influenza B 4.1% (95% CI: -22.0 to 24.7). The majority of influenza A(H1N1)pdm09 circulating in 2015/16 belonged to the new genetic subgroup subclade 6B.1.

Mutations acquired during cell culture isolation may affect antigenic characterisation of influenza A(H3N2) clade 3C.2a viruses.

As elsewhere, few (< 15%) sentinel influenza A(H3N2) clade 3C.2a viruses that dominated in Canada during the 2014/15 season could be antigenically characterised by haemagglutination inhibition (HI) assay. Clade 3C.2a viruses that could be HI-characterised had acquired genetic mutations during in vitro cell culture isolation that modified the potential glycosylation motif found in original patient specimens and the consensus sequence of circulating viruses at amino acid positions 158-160 of the haemagglutinin protein. Caution is warranted in extrapolating antigenic relatedness based on limited HI findings for clade 3C.2a viruses that continue to circulate globally.

Influenza D virus infection in China, 2022-2023.

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.

Antigenic Characterization of Circulating and Emerging SARS-CoV-2 Variants in the U.S. throughout the Delta to Omicron Waves.

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.

Antigenic Characterization of Infectious Bronchitis Virus in the South China during 2021-2022.

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.

Detection and Characterization of Feline Calicivirus Associated with Paw and Mouth Disease.

Feline calicivirus (FCV) infection in cats can led to several diverse clinical presentations, ranging from mild upper respiratory signs to virulent systemic disease. Herein, we report a paw and mouth disease case in a 7-year-old household cat due to an FCV infection. An asymptomatic cat living in the same household was also infected with FCV. Clinical and pathological investigations were combined with the molecular and phenotypical characterization of the FCV strains. The RNA of the FCV was detected using qualitative and quantitative reverse transcription (RT)-PCR assays, and FCV antigen was confirmed by immunohistochemistry. After the whole genome analysis, the strains detected in the two cats appeared to be genetically diverse from FCVs previously detected in association with paw and mouth disease and with virulent systemic disease. Interestingly, the isolates obtained in this study were resistant to low pH conditions and slightly susceptible to bile salts, but they were susceptible to a trypsin treatment, revealing a phenotype pattern that is different from that which has been observed for respiratory FCVs.

Use of multivariate analysis to evaluate antigenic relationships between US BVDV vaccine strains and non-US genetically divergent isolates.

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.

Genetic Variability among Swine Influenza Viruses in Italy: Data Analysis of the Period 2017-2020.

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.

Antigenic Cartography: Overview and Current Developments.

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.

Hemagglutination Inhibition Assay.

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 relationship among zoonotic flaviviruses from Italy.

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.

Changes in genetically drifted H3N2 influenza A viruses and vaccine effectiveness in adults 65 years and older during the 2016/17 season in Denmark.

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.

ViroSpot microneutralization assay for antigenic characterization of human influenza viruses.

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.

Insights into genetic diversity and biological propensities of potentially zoonotic avian influenza H9N2 viruses circulating in Egypt.

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.

Antigenic typing of canine parvovirus using differential PCR.

Canine parvovirus (CPV) is an enteric pathogen causing hemorrhagic enteritis in pups of 3-6 months of age and is mainly transmitted via feco-oral route. In the present study, a total of 85 animals rectal swabs suspected of CPV were tested using a PCR, nested PCR and a newly designed differential PCR. Using PCR 7 (8.23 %) animals were positive whereas 39 (45.88 %) were positive by using nested PCR and 40 (47.05 %) were positive for either one or more than one antigenic types of CPV using differential PCR. Using differential PCR it was found that CPV-2a and CPV-2b were the most prevailing antigenic types. Also it was found that dogs that were vaccinated too yielded positive CPV indicating a possible presence of additional CPV antigenic types. Thus, the primers used in differential PCR can be used in a single PCR reaction to detect various antigenic types of CPV.

Identification and molecular characterization of novel and divergent HoBi-like pestiviruses from naturally infected cattle in India.

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.

Genetic characterization of seasonal influenza A (H3N2) viruses in Ontario during 2010-2011 influenza season: high prevalence of mutations at antigenic sites.

BACKGROUND: The direct effect of antigenic site mutations in influenza viruses on antigenic drift and vaccine effectiveness is poorly understood. OBJECTIVE: To investigate the genetic and antigenic characteristics of human influenza A (H3N2) viruses circulating in Ontario during the early 2010-2011 winter season. STUDY DESIGN: We sequenced the hemagglutinin (HA) and neuraminidase (NA) genes from 41 A(H3N2) viruses detected in nasopharyngeal specimens. Strain typing was performed by hemagglutination inhibition (HI) assay. Molecular and phylogenetic tree analyses were conducted. RESULTS: HA and NA genes showed high similarity to the 2010-2011 vaccine strain, A/Perth/16/2009 (H3N2)-like virus (97·7-98·5% and 98·7-99·5% amino acid (AA) identity, respectively). Compared to A/Perth/16/2009 strain, HA gene mutations were documented at 28 different AA positions across all five H3 antigenic sites, with a range of 5-11 mutations in individual viruses. Thirty-six (88%) viruses had 8 AA substitutions in common; none of these had reduced HI titer. Among Ontario isolates, 11 antigenic site AAs were positively selected with an increase in glycosylation sites. CONCLUSION: The presence of antigenic site mutations with high frequency among 2010-2011 influenza H3N2 isolates confirms ongoing adaptive H3N2 evolution. These may represent early phylogenetic changes that could cause antigenic drift with further mutations. Clinical relevance of antigenic site mutations not causing drift in HI assays is unknown and requires further investigation. In addition, viral sequencing information will assist with vaccine strain planning and may facilitate early detection of vaccine escape.