Emergence of equine-like G3 strains as the dominant rotavirus among children under five with diarrhea in Sabah, Malaysia during 2018-2019.

Rotavirus infection is a dilemma for developing countries, including Malaysia. Although commercial rotavirus vaccines are available, these are not included in Malaysia's national immunization program. A scarcity of data about rotavirus genotype distribution could be partially to blame for this policy decision, because there are no data for rotavirus genotype distribution in Malaysia over the past 20 years. From January 2018 to March 2019, we conducted a study to elucidate the rotavirus burden and genotype distribution in the Kota Kinabalu and Kunak districts of the state of Sabah. Stool specimens were collected from children under 5 years of age, and rotavirus antigen in these samples was detected using commercially available kit. Electropherotypes were determined by polyacrylamide gel electrophoresis of genomic RNA. G and P genotypes were determined by RT-PCR using type specific primers. The nucleotide sequence of the amplicons was determined by Sanger sequencing and phylogenetic analysis was performed by neighbor-joining method. Rotavirus was identified in 43 (15.1%) children with watery diarrhea. The male:female ratio (1.9:1) of the rotavirus-infected children clearly showed that it affected predominantly boys, and children 12-23 months of age. The genotypes identified were G3P[8] (74% n = 31), followed by G1P[8] (14% n = 6), G12P[6](7% n = 3), G8P[8](3% n = 1), and GxP[8] (3% n = 1). The predominant rotavirus circulating among the children was the equine-like G3P[8] (59.5% n = 25) with a short electropherotype. Eleven electropherotypes were identified among 34 strains, indicating substantial diversity among the circulating strains. The circulating genotypes were also phylogenetically diverse and related to strains from several different countries. The antigenic epitopes present on VP7 and VP4 of Sabahan G3 and equine-like G3 differed considerably from that of the RotaTeq vaccine strain. Our results also indicate that considerable genetic exchange is occurring in Sabahan strains. Sabah is home to a number of different ethnic groups, some of which culturally are in close contact with animals, which might contribute to the evolution of diverse rotavirus strains. Sabah is also a popular tourist destination, and a large number of tourists from different countries possibly contributes to the diversity of circulating rotavirus genotypes. Considering all these factors which are contributing rotavirus genotype diversity, continuous surveillance of rotavirus strains is of utmost importance to monitor the pre- and post-vaccination efficacy of rotavirus vaccines in Sabah.

Identification of Novel Candidate CD8+ T Cell Epitopes of the SARS-CoV2 with Homology to Other Seasonal Coronaviruses.

Cross-reactive T cell immunity to seasonal coronaviruses (HCoVs) may lead to immunopathology or protection during SARS-CoV2 infection. To understand the influence of cross-reactive T cell responses, we used IEDB (Immune epitope database) and NetMHCpan (ver. 4.1) to identify candidate CD8+ T cell epitopes, restricted through HLA-A and B alleles. Conservation analysis was carried out for these epitopes with HCoVs, OC43, HKU1, and NL63. 12/18 the candidate CD8+ T cell epitopes (binding score of ≥0.90), which had a high degree of homology (>75%) with the other three HCoVs were within the NSP12 and NSP13 proteins. They were predicted to be restricted through HLA-A*2402, HLA-A*201, HLA-A*206, and HLA-B alleles B*3501. Thirty-one candidate CD8+ T cell epitopes that were specific to SARS-CoV2 virus (<25% homology with other HCoVs) were predominantly identified within the structural proteins (spike, envelop, membrane, and nucleocapsid) and the NSP1, NSP2, and NSP3. They were predominantly restricted through HLA-B*3501 (6/31), HLA-B*4001 (6/31), HLA-B*4403 (7/31), and HLA-A*2402 (8/31). It would be crucial to understand T cell responses that associate with protection, and the differences in the functionality and phenotype of epitope specific T cell responses, presented through different HLA alleles common in different geographical groups, to understand disease pathogenesis.

Different Cross-Reactivities of IgM Responses in Dengue, Zika and Tick-Borne Encephalitis Virus Infections.

Flaviviruses circulate worldwide and cause a number of medically relevant human diseases, such as dengue, Zika, yellow fever, and tick-borne encephalitis (TBE). Serology plays an important role in the diagnosis of flavivirus infections, but can be impeded by antigenic cross-reactivities among flaviviruses. Therefore, serological diagnosis of a recent infection can be insufficiently specific, especially in areas where flaviviruses co-circulate and/or vaccination coverage against certain flaviviruses is high. In this study, we developed a new IgM assay format, which is well suited for the specific diagnosis of TBE, Zika and dengue virus infections. In the case of TBE and Zika, the IgM response proved to be highly specific for the infecting virus. In contrast, primary dengue virus infections induced substantial amounts of cross-reactive IgM antibodies, which is most likely explained by structural peculiarities of dengue virus particles. Despite the presence of cross-reactive IgM, the standardized nature and the quantitative read-out of the assay even allowed the serotype-specific diagnosis of recent dengue virus infections in most instances.

High prevalence of DS-1-like rotavirus infection in Thai adults between 2016 and 2019.

Rotavirus infection is the most common cause of viral diarrhea in infants and young children but uncommon and usually asymptomatic in adults. In the winter of 2017-2018, a large-scale outbreak of rotavirus in both children and adults was reported in Thailand. The current study focused on the prevalence, genotyping, and molecular characterization of rotavirus infections in Thai adults from July 2016 to December 2019. In 2,598 stool samples collected from adult residents of Bangkok (aged #x2265; 15 years) with acute gastroenteritis, rotavirus was detected via real-time RT-PCR analysis of the VP6 gene. G, P and I genotypes were determined by direct sequencing of VP7, VP4, and VP6 genes, respectively. Our results showed 8.7% (226/2,598) of stool samples were positive for rotavirus. The incidence of rotavirus was high during the winter season of 2017-2018 (17.7%) compared to another studied periods (4.5% between July 2016- October 2017 and 2.8% between March 2018- December 2019). Nucleotide sequencing of VP7 and VP4 revealed G3P[8] as the predominant strain (33.2%,75/226), followed by G9P[8] (17.3%,39/226), and G2P[4] (15.0%,34/226). Uncommon G and P combinations were additionally detected at low frequencies. VP6 sequencing was conducted to discriminate I genotype between the Wa and DS-1 genogroup. The unusual DS-1-like G3P[8] strain was most prevalent amomg rotavirus strains detected in this study (29.6%, 67/226), and the corresponding VP7 sequences showed high nucleotide identity with unusual DS-1-like globally circulating strains. Our study demonstrates that rotavirus outbreaks in adults are attributable not only to high prevalence of RV infection but also the unusual DS-like genogroup. The collective findings reinforce the importance of investigating rotavirus diagnosis in adults suffering from acute gastroenteritis and taking appropriate preventive measures.

Antigenic analysis of genetic variants of Canine distemper virus.

Canine distemper virus (CDV) is an RNA virus of the genus Morbillivirus within the family Paramyxoviridae. CDV produces multi-systemic disease in dogs and other terrestrial carnivores. With the development of modified live vaccines in the 1950s and 1960s, the disease, with a few exceptions, has been successfully controlled. However, recently the cases of CDV in vaccinated dogs have been increasing throughout the world, including the United States. There are many reasons that can lead to vaccine failure, including antigenic differences between the vaccine strains and the currently circulating wild-type strains. Currently, there are at least three genetically different CDV lineages circulating in the US. Therefore, in this study, we evaluated various wild-type CDV and vaccine isolates to determine if the genetic differences observed among various strains result in significant antigenic differences based on changes to the neutralizing epitopes. The results of a cross-neutralization assay revealed that there are antigenic differences among the tested CDV wild-type isolates as well as between the tested isolates and the vaccine strains currently used in the US. Therefore, these results suggest the need to develop an updated CDV vaccine.

Antigenicity characterization of four representative natural reassortment IBDVs isolated from commercial three-yellow chickens from Southern China reveals different subtypes co-prevalent in the field.

The antigenic relationships between the natural reassortment field strains of infectious bursal disease virus (IBDV), and between the field strains and the vaccine strains are poorly understood. In the present study, the antigenicity of four representative natural reassortment IBDV isolates designated JS7, GD10111, NN1005 and NN1172 from southern China during the years 2005-2011 and their antigenic relationship with the most commonly used vaccine strain B87 were investigated in vivo. For this purpose, cross-challenge studies were performed on 28-day-old birds, which were 2 weeks post-vaccination by oil-emulsion vaccines (OEVs) prepared from the four field viruses and B87, respectively. The protection related values (PRV) were evaluated based on the protection rate measured by clinical signs and mortality, bursa/body weight (B/BW) ratio and the viral load in the bursal samples at 3 and 7 days post challenge. As a result, the PRV showed that the isolates NN1172 and GD10111 belonged to the same antigenic subtype, while the isolates NN1005 and JS7 belong to another subtype. The vaccine strain B87 was grouped with the isolates NN1005 and JS7 but actually belongs to another small subgroup and provided only 60-80% protection against the challenge of the four field strains. The results demonstrated that different antigenic subtypes co-existed among the field natural reassortment IBDV strains and the commonly used vaccine strain B87 was antigenically different from the prevalent IBDVs in southern China.

Is It Possible to Develop Cancer Vaccines to Neoantigens, What Are the Major Challenges, and How Can These Be Overcome? Neoantigens: Nothing New in Spite of the Name.

The term "neoantigen," as applied to molecules newly expressed on tumor cells, has a long history. The groundbreaking discovery of a cancer causing virus in chickens by Rous over 100 years ago, followed by discoveries of other tumor-causing viruses in animals, suggested a viral etiology of human cancers. The search for other oncogenic viruses in the 1960s and 1970s resulted in the discoveries of Epstein-Barr virus (EBV), hepatitis B virus (HBV), and human papilloma virus (HPV), and continues until the present time. Contemporaneously, the budding field of immunology was posing the question can the immune system of animals or humans recognize a tumor that develops from one's own tissues and what types of antigens would distinguish the tumor from normal cells. Molecules encoded by oncogenic viruses provided the most logical candidates and evidence was quickly gathered for both humoral and cellular recognition of viral antigens, referred to as neoantigens. Often, however, serologic responses to virus-bearing tumors revealed neoantigens unrelated to viral proteins and expressed on multiple tumor types, foreshadowing later findings of multiple changes in other genes in tumor cells creating nonviral neoantigens.

Comparative analysis of the immunogenicity of monovalent and multivalent rotavirus immunogens.

The strategies for developing rotavirus (RV) vaccines have always been controversial. At present, both the monovalent RV vaccine and the multivalent RV vaccine have displayed excellent safety and efficacy against RV infection and shown cross-reactive immunity, which laid the question whether the multivalent RV vaccine could be replaced by the monovalent RV vaccine. In this study, we focused on comparing the immunogenicity (serum neutralization activity and protection against homotypic and heterotypic RVs' challenge) of individual standard RV strains (monovalent RV immunogens) and different combinations of them (multivalent RV immunogens). In result, RV immunogens showed general immunogenicity and heterotypic reaction but the multivalent RV immunogens exhibited greater serum neutralization activity and stronger heterotypic reaction than the monovalent RV immunogens (P<0.05). As to the protection, the multivalent RV immunogens also revealed more rapid and stronger protection against homotypic and heterotypic RVs' challenge than the monovalent RV immunogens. The results demonstrated that both the monovalent and multivalent RV immunogens exhibited high immunogenicity, but the monovalent RV immunogens could not provide enough neutralization antibodies to protect MA104 cells against the infection with heterotypic RV strains and timely protection against homotypic and heterotypic RVs, so the multivalent RV vaccine could not be replaced by the monovalent RV vaccine.

Rapid Detection of Bovine Respiratory Syncytial Virus in Poland Using a Human Patient-Side Diagnostic Assay.

Bovine respiratory syncytial virus (BRSV) plays a significant role in the etiopathogenesis of the respiratory syndrome in young cattle during their first year of life. Development of rapid and accurate BRSV diagnostic tools would aid in the appropriate control of this important pathogen. The objective of this study was to characterize infections induced by BRSV by means of rapid patient-side immunomigration assays used for diagnosis of human respiratory syncytial virus (hRSV) in humans. Nasal and tracheal swabs were obtained from healthy calves of various beef and dairy breeds - Holstein-Friesian, Simmental, Charolais, Belgian Blue and Limousin, between the ages of 5 and 12 months, from 26 farms. BRSV was identified using two rapid immunomigration assays, TruRSV® and Clearview® RSV, and compared with RT-PCR as a reference technique. BRSV was found in 73.1% of all the herds tested. High agreement with RT-PCR was obtained for TruRSV® (κ = 0.824), while in the case of the Clearview® RSV test, agreement with PCR was moderate (κ = 0.420). The results demonstrate that rapid patient-side immunomigration assays designed to detect hRSV can be used to accurately detect BRSV in field samples collected from cattle.

[THE SELECTION OF ANTIGENS FOR DETECTING OF IMMUNOGLOBULIN G TO CYTOMEGALOVIRUS ON THE BASIS OF FOSFAN TECHNOLOGY].

The results of selection of composition of antigens to cytomegalovirus in the structure of multiplex test on the basis of FOSFAN™ technique are presented. In the process of detection of immunoglobulin G (IgG) to this virus the best indicators of sensitivity were registered with mosaic antigen containing immunodominant sequences of proteins pp150, gB, pp28 and pp52; reliably lower indicators of sensitivity was registered with phosphoprotein pp150; the lowest indicators of sensitivity were registered with proteins gB and pp65. The specificity made up from 93.5% to 96.8% independently of type of antigen. The mosaic antigen ensured the best ratio between sensitivity and specificity of immunoassay and is considered as the main component of immunochip for detecting IgG to cytomegalovirus.

Evidence of VP7 and VP4 intra-lineage diversification in G4P[8] Italian human rotaviruses.

Intragenotypic heterogeneity of co-circulating rotaviruses is remarkable. Sequence and phylogenetic analyses of the rotavirus VP7 and VP4 genes were performed on selected human G4P[8] strains identified in Parma, Northern Italy, during 2004-2005 and 2008-2012. All the strains clustered into lineages Ic (VP7) and P[8]-III (VP4) in different subclusters with a nucleotide sequence variation up to 4 %. VP7 and VP4 amino acid sequences of the Italian rotaviruses showed multiple changes with the corresponding reference strains as well as with vaccine viruses in the neutralizing epitopes. There is concern that the progressive intra-lineage diversification in the VP7 and VP4 through the accumulation of point mutations and amino acid differences between vaccine strains and currently circulating rotaviruses could generate, over the years, vaccine-resistant variants.

Antigenic characterization of H3N2 influenza A viruses from Ohio agricultural fairs.

The demonstrated link between the emergence of H3N2 variant (H3N2v) influenza A viruses (IAVs) and swine exposure at agricultural fairs has raised concerns about the human health risk posed by IAV-infected swine. Understanding the antigenic profiles of IAVs circulating in pigs at agricultural fairs is critical to developing effective prevention and control strategies. Here, 68 H3N2 IAV isolates recovered from pigs at Ohio fairs (2009 to 2011) were antigenically characterized. These isolates were compared with other H3 IAVs recovered from commercial swine, wild birds, and canines, along with human seasonal and variant H3N2 IAVs. Antigenic cartography demonstrated that H3N2 IAV isolates from Ohio fairs could be divided into two antigenic groups: (i) the 2009 fair isolates and (ii) the 2010 and 2011 fair isolates. These same two antigenic clusters have also been observed in commercial swine populations in recent years. Human H3N2v isolates from 2010 and 2011 are antigenically clustered with swine-origin IAVs from the same time period. The isolates recovered from pigs at fairs did not cross-react with ferret antisera produced against the human seasonal H3N2 IAVs circulating during the past decade, raising the question of the degree of immunity that the human population has to swine-origin H3N2 IAVs. Our results demonstrate that H3N2 IAVs infecting pigs at fairs and H3N2v isolates were antigenically similar to the IAVs circulating in commercial swine, demonstrating that exhibition swine can function as a bridge between commercial swine and the human population.

Detection of substantial porcine group B rotavirus genetic diversity in the United States, resulting in a modified classification proposal for G genotypes.

Rotavirus (RV) is an important cause of gastrointestinal disease in animals and humans. In this study, we developed an RT-PCR to detect RV group B (RVB) and characterized the VP7 (G) gene segment detected in porcine samples. One hundred seventy three samples were tested for RV group A (RVA), RVB, and C (RVC) by RT-PCR and examined for RV-like lesion using histopathology. A majority (86.4%) of the samples had mixed RV infections and co-infections of RVA/RVB/RVC were detected at a higher rate (24.3%) than previously reported. RVB was identified in 46.8% of the 173 samples. An adapted VP7 classification was developed using previously published (n=57) and newly sequenced (n=68) RVB strains, resulting in 20 G genotypes based on an 80% nucleotide identity cutoff value. Our results revealed a broad genetic diversity of porcine RVB strains, suggesting RVB has been the cause of common/pre-existing, yet undiagnosed, disease in pigs.

Use of rapid human respiratory syncytial virus strip tests for detection of bovine respiratory syncytial virus in experimentally vaccinated calves.

Three different rapid strip tests: TRU RSV, BinaxNOW RSV and RSV Respi-strip were compared with RT-PCR and ELISA BRSV Ag for the ability to detect bovine respiratory syncytial virus (BRSV) in nasal swabs collected from calves experimentally vaccinated with live vaccine Rispoval RS-PI3. The reference strains of BRSV (375 and A51908) were detected by ELISA BRSV Ag whereas the strains of human respiratory syncytial virus (HRSV) and bovine parainfluenza virus type 3 (BPIV-3) were not. All rapid strip tests as well as RT-PCR reacted positively both to HRSV and BRSV reference strains and negatively to BPIV-3. The detection limit for RT-PCR was 39.1 TCID50 (strain 375 of BRSV), whereas for each of the rapid tests it was approximately 156 TCID50 and 312 TCID50 for antigen ELISA. Diagnostic sensitivity in detecting BRSV in nasal swabs for TRU RSV and RSV Respi-strip tests was 33% and 50% for BinaxNOW RSV. Diagnostic specificity of TRU RSV was 100%, whereas for both BinaxNOW and Respi-strip it was 87%. We concluded that TRU RSV could be used as a supportive rapid test for BRSV screening in nasal swabs taken directly on a farm. However, due to the small group of animals used in the experiment, the results should be regarded as preliminary and the study should be repeated on a larger number of animals.

Antigenic and genetic evolution of equine influenza A (H3N8) virus from 1968 to 2007.

Equine influenza virus is a major respiratory pathogen in horses, and outbreaks of disease often lead to substantial disruption to and economic losses for equestrian industries. The hemagglutinin (HA) protein is of key importance in the control of equine influenza because HA is the primary target of the protective immune response and the main component of currently licensed influenza vaccines. However, the influenza virus HA protein changes over time, a process called antigenic drift, and vaccine strains must be updated to remain effective. Antigenic drift is assessed primarily by the hemagglutination inhibition (HI) assay. We have generated HI assay data for equine influenza A (H3N8) viruses isolated between 1968 and 2007 and have used antigenic cartography to quantify antigenic differences among the isolates. The antigenic evolution of equine influenza viruses during this period was clustered: from 1968 to 1988, all isolates formed a single antigenic cluster, which then split into two cocirculating clusters in 1989, and then a third cocirculating cluster appeared in 2003. Viruses from all three clusters were isolated in 2007. In one of the three clusters, we show evidence of antigenic drift away from the vaccine strain over time. We determined that a single amino acid substitution was likely responsible for the antigenic differences among clusters.

Update: influenza activity--United States and worldwide, May 22-September 3, 2011.

During May 22-September 3, 2011, the United States experienced low levels of influenza activity; 2009 influenza A (H1N1), influenza A (H3N2), and influenza B viruses were detected worldwide and identified sporadically in the United States. Typical seasonal patterns of influenza activity occurred in the Southern Hemisphere. This report summarizes influenza activity in the United States and worldwide since the last update.

A proof-of-principle study to identify suitable vaccine seed candidates to combat introductions of Eurasian lineage H5 and H7 subtype avian influenza viruses.

Vaccination against avian influenza (AI) is now included amongst the prevention and control measures recommended by international animal health organizations to combat the disease in poultry. For optimal control of human influenza infections, the antigenic variability within subtypes requires the annual update of seed strains for inclusion in vaccines. The decisions taken are based on serological cross-reactivity of viral strains measured by haemagglutination inhibition (HI) tests. The reason for this is to ensure that the vaccine contains strains that are related antigenically to the current circulating field strain as field viruses evolve or are substituted by variants of distinct antigenicity. Such an annual approach is not viable economically for the poultry industry. In the current study, we have applied a similar HI-based approach to demonstrate, as proof of principle, that cross-reactive strains can be identified. Applying the same approach used by the World Health Organization to investigate antigenic differences among human influenza viruses, we assessed the serological cross-reactivity of a selection of natural H5 and H7 subtype viruses. Analysing HI data, we have identified strains that are cross-reactive and may have the potential to act as seed viruses for future vaccine development. This study should be considered a starting point for a more informed approach to the selection of seed strains for the development of avian influenza vaccines against field infections caused by viruses of H5 and H7 subtypes.

Use of antigenic cartography in vaccine seed strain selection.

Human influenza A viruses are classic examples of antigenically variable pathogens that have a seemingly endless capacity to evade the host's immune response. The viral hemagglutinin (HA) and neuraminidase (NA) proteins are the main targets of our antibody response to combat infections. HA and NA continuously change to escape from humoral immunity, a process known as antigenic drift. As a result of antigenic drift, the human influenza vaccine is updated frequently. The World Health Organization (WHO) coordinates a global influenza surveillance network that, by the hemagglutination inhibition (HI) assay, routinely characterizes the antigenic properties of circulating strains in order to select new seed viruses for such vaccine updates. To facilitate a quantitative interpretation and easy visualization of HI data, a new computational technique called "antigenic cartography" was developed. Since its development, antigenic cartography has been applied routinely to assist the WHO with influenza surveillance activities. Until recently, antigenic variation was not considered a serious issue with influenza vaccines for poultry. However, because of the diversification of the Asian H5N1 lineage since 1996 into multiple genetic clades and subclades, and because of the long-term use of poultry vaccines against H5 in some parts of the world, this issue needs to be re-addressed. The antigenic properties of panels of avian H5N1 viruses were characterized by HI assay, using mammalian or avian antisera, and analyzed using antigenic cartography methods. These analyses revealed antigenic differences between circulating H5N1 viruses and the H5 viruses used in poultry vaccines. Considerable antigenic variation was also observed within and between H5N1 clades. These observations have important implications for the efficacy and long-term use of poultry vaccines.

Establishment of an H6N2 influenza virus lineage in domestic ducks in southern China.

Multiple reassortment events between different subtypes of endemic avian influenza viruses have increased the genomic diversity of influenza viruses circulating in poultry in southern China. Gene exchange from the natural gene pool to poultry has contributed to this increase in genetic diversity. However, the role of domestic ducks as an interface between the natural gene pool and terrestrial poultry in the influenza virus ecosystem has not been fully characterized. Here we phylogenetically and antigenically analyzed 170 H6 viruses isolated from domestic ducks from 2000 to 2005 in southern China, which contains the largest population of domestic ducks in the world. Three distinct hemagglutinin lineages were identified. Group I contained the majority of isolates with a single internal gene complex and was endemic in domestic ducks in Guangdong from the late 1990s onward. Group II was derived from reassortment events in which the surface genes of group I viruses were replaced with novel H6 and N2 genes. Group III represented H6 viruses that undergo frequent reassortment with multiple virus subtypes from the natural gene pool. Surprisingly, H6 viruses endemic in domestic ducks and terrestrial poultry seldom reassort, but gene exchanges between viruses from domestic ducks and migratory ducks occurred throughout the surveillance period. These findings suggest that domestic ducks in southern China mediate the interaction of viruses between different gene pools and facilitate the generation of novel influenza virus variants circulating in poultry.