Defining the antibody cross-reactome directed against the influenza virus surface glycoproteins.

Infection with influenza virus induces antibodies to the viral surface glycoproteins hemagglutinin and neuraminidase, and these responses can be broadly protective. To assess the breadth and magnitude of antibody responses, we sequentially infected mice, guinea pigs and ferrets with divergent H1N1 or H3N2 subtypes of influenza virus. We measured antibody responses by ELISA of an extensive panel of recombinant glycoproteins representing the viral diversity in nature. Guinea pigs developed high titers of broadly cross-reactive antibodies; mice and ferrets exhibited narrower humoral responses. Then, we compared antibody responses after infection of humans with influenza virus H1N1 or H3N2 and found markedly broad responses and cogent evidence for 'original antigenic sin'. This work will inform the design of universal vaccines against influenza virus and can guide pandemic-preparedness efforts directed against emerging influenza viruses.

Extraction of the CDRH3 sequence of the mouse antibody repertoire selected upon influenza virus infection by subtraction of the background antibody repertoire.

Historically, antibody reactivity to pathogens and vaccine antigens has been evaluated using serological measurements of antigen-specific antibodies. However, it is difficult to evaluate all antibodies that contribute to various functions in a single assay, such as the measurement of the neutralizing antibody titer. Bulk antibody repertoire analysis using next-generation sequencing is a comprehensive method for analyzing the overall antibody response; however, it is unreliable for estimating antigen-specific antibodies due to individual variation. To address this issue, we propose a method to subtract the background signal from the repertoire of data of interest. In this study, we analyzed changes in antibody diversity and inferred the heavy-chain complementarity-determining region 3 (CDRH3) sequences of antibody clones that were selected upon influenza virus infection in a mouse model using bulk repertoire analysis. A decrease in the diversity of the antibody repertoire was observed upon viral infection, along with an increase in neutralizing antibody titers. Using kernel density estimation of sequences in a high-dimensional sequence space with background signal subtraction, we identified several clusters of CDRH3 sequences induced upon influenza virus infection. Most of these repertoires were detected more frequently in infected mice than in uninfected control mice, suggesting that infection-specific antibody sequences can be extracted using this method. Such an accurate extraction of antigen- or infection-specific repertoire information will be a useful tool for vaccine evaluation in the future. IMPORTANCE: As specific interactions between antigens and cell-surface antibodies trigger the proliferation of B-cell clones, the frequency of each antibody sequence in the samples reflects the size of each clonal population. Nevertheless, it is extremely difficult to extract antigen-specific antibody sequences from the comprehensive bulk antibody sequences obtained from blood samples due to repertoire bias influenced by exposure to dietary antigens and other infectious agents. This issue can be addressed by subtracting the background noise from the post-immunization or post-infection repertoire data. In the present study, we propose a method to quantify repertoire data from comprehensive repertoire data. This method allowed subtraction of the background repertoire, resulting in more accurate extraction of expanded antibody repertoires upon influenza virus infection. This accurate extraction of antigen- or infection-specific repertoire information is a useful tool for vaccine evaluation.

Relative instantaneous reproduction number of Omicron SARS-CoV-2 variant with respect to the Delta variant in Denmark.

The Omicron variant of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become widespread across the world in a flashing manner. As of December 7, 2021, a total of 758 Omicron cases were confirmed in Denmark. Using the nucleotide sequences of the Delta and Omicron variants registered from Denmark in the GISAID database, we found that the effective (instantaneous) reproduction number of Omicron is 3.19 (95% confidence interval [CI]: 2.82-3.61) times greater than that of Delta under the same epidemiological conditions. The proportion of Omicron infections among all SARS-CoV-2 infections in Denmark was expected to exceed 95% on December 28, 2021, with a 95% CI from December 25 to December 31, 2021. Given that the Delta variant or variants less transmissible than Delta are dominant in most countries, the rapid increase in Omicron in the virus population may be observed as soon as the Omicron is introduced. Preparing proactive control measures is vital, assuming the substantial advantage of the transmission by Omicron.

Predicted dominance of variant Delta of SARS-CoV-2 before Tokyo Olympic Games, Japan, July 2021.

Using numbers of SARS-CoV-2 variants detected in Japan as at 13 June 2021, relative instantaneous reproduction numbers (RRI) of the R.1, Alpha, and Delta variants with respect to other strains circulating in Japan were estimated at 1.25, 1.44, and 1.95. Depending on the assumed serial interval distributions, RRI varies from 1.20-1.32 for R.1, 1.34-1.58 for Alpha, and 1.70-2.30 for Delta. The frequency of Delta is expected to take over Alpha in Japan before 23 July 2021.

Upregulated expression of the antioxidant sestrin 2 identified by transcriptomic analysis of Japanese encephalitis virus-infected SH-SY5Y neuroblastoma cells.

Japanese encephalitis virus (JEV) exerts a profound burden of viral encephalitis. We have investigated the differentially expressed transcripts in the neuronal transcriptome during JEV infection by RNA sequencing (RNA-Seq) of virus-infected SH-SY5Y human neuroblastoma cells. Gene ontology analysis revealed significant enrichment from two main pathways: endoplasmic reticulum (ER)-nucleus signaling (P value: 5.75E-18; false discovery rate [FDR] 3.11E-15) and the ER unfolded protein response (P value: 7.58E-18; FDR 3.11E-15). qPCR validation showed significant upregulation and differential expression (P < 0.01) of ER stress-signaling transcripts (SESN2, TRIB3, DDIT3, DDIT4, XBP1, and ATF4) at 24 h post-infection for both low (LN) and high (HN) neurovirulence JEV strains. Immunoblot analysis following JEV infection of SH-SY5Y cells showed an increase in levels of SESN2 protein following JEV infection. Similarly, Zika virus (MR766) infection of SH-SY5Y showed a titer-dependent increase in ER stress-signaling transcripts; however, this was absent or diminished for DDIT4 and ATF4, respectively, suggestive of differences in the induction of stress-response transcripts between flaviviruses. Interestingly, SLC7A11 and SLC3A2 mRNA were also both deregulated in JEV-infected SH-SY5Y cells and encode the two constituent subunits of the plasma membrane xCT amino acid antiporter that relieves oxidative stress by export of glutamate and import of cystine. Infection of SH-SY5Y and HEK293T cells by the JEV HN strain Sw/Mie/40/2004 lead to significant upregulation of the SLC7A11 mRNA to levels comparable to DDIT3. Our findings suggest upregulation of antioxidants including SESN2 and, also, the xCT antiporter occurs to counteract the oxidative stress elicited by JEV infection.

Discovery of African bat polyomaviruses and infrequent recombination in the large T antigen in the Polyomaviridae.

Bat species represent natural reservoirs for a number of high-consequence human pathogens. The present study investigated the diversity of polyomaviruses (PyVs) in Zambian insectivorous and fruit bat species. We describe the complete genomes from four newly proposed African bat PyV species employing the recently recommended criteria provided by the Polyomaviridae Study Group of the International Committee on Taxonomy of Viruses. A comprehensive phylogenetic and recombination analysis was performed to determine genetic relationships and the distribution of recombination events in PyV from mammalian and avian species. The novel species of PyV from Zambian bats segregated with members of the genera Alphapolyomavirus and Betapolyomavirus, forming monophyletic clades with bat and non-human primate PyVs. Miniopterus schreibersii polyomavirus 1 and 2 segregated in a clade with South American bat PyV species, Old World monkey and chimpanzee PyVs and Human polyomavirus 13 (New Jersey PyV). Interestingly, the newly described Egyptian fruit bat PyV, tentatively named Rousettus aegyptiacus polyomavirus 1, had the highest nucleotide sequence identity to species of PyV from Indonesian fruit bats, and Rhinolophus hildebrandtii polyomavirus 1 was most closely related to New World monkey PyVs. The distribution of recombination events in PyV genomes was non-random: recombination boundaries existed in the intergene region between VP1 and LTAg and also at the 3' end of VP2/3 in the structural genes, whereas infrequent recombination was present within the LTAg gene. These findings indicate that recombination within the LTAg gene has been negatively selected against during polyomaviral evolution and support the recent proposal for taxonomic classification based on LTAg to define novel PyV species.

Identification of the same polyomavirus species in different African horseshoe bat species is indicative of short-range host-switching events.

Polyomaviruses (PyVs) are considered to be highly host-specific in different mammalian species, with no well-supported evidence for host-switching events. We examined the species diversity and host specificity of PyVs in horseshoe bats (Rhinolophus spp.), a broadly distributed and highly speciose mammalian genus. We annotated six PyV genomes, comprising four new PyV species, based on pairwise identity within the large T antigen (LTAg) coding region. Phylogenetic comparisons revealed two instances of highly related PyV species, one in each of the Alphapolyomavirus and Betapolyomavirus genera, present in different horseshoe bat host species (Rhinolophus blasii and R. simulator), suggestive of short-range host-switching events. The two pairs of Rhinolophus PyVs in different horseshoe bat host species were 99.9 and 88.8 % identical with each other over their respective LTAg coding sequences and thus constitute the same virus species. To corroborate the species identification of the bat hosts, we analysed mitochondrial cytb and a large nuclear intron dataset derived from six independent and neutrally evolving loci for bat taxa of interest. Bayesian estimates of the ages of the most recent common ancestors suggested that the near-identical and more distantly related PyV species diverged approximately 9.1E4 (5E3-2.8E5) and 9.9E6 (4E6-18E6) years before the present, respectively, in contrast to the divergence times of the bat host species: 12.4E6 (10.4E6-15.4E6). Our findings provide evidence that short-range host-switching of PyVs is possible in horseshoe bats, suggesting that PyV transmission between closely related mammalian species can occur.

Isolation of a simian immunodeficiency virus from a malbrouck (Chlorocebus cynosuros).

To investigate the diversity of simian immunodeficiency virus (SIV) among nonhuman primates (NHPs) in Zambia, next-generation sequencing was performed to determine the complete genome sequence of a novel SIV recovered by co-culturing African green monkey (AGM) peripheral blood lymphocytes with human CD4+ T-cell lines. We report the first described SIV (SIVagmMAL-ZMB) from a malbrouck (Chlorocebus cynosuros). SIVagmMAL-ZMB was detected by real-time PCR analysis of splenic RNA in 3.2% (3/94) of AGMs and was undetectable in baboons (0/105). SIVagmMAL-ZMB possessed <80% nucleotide sequence identity to known SIV isolates and was located basally to vervet monkey SIV strains in all phylogenies.

Multi-reassortant G3P[3] group A rotavirus in a horseshoe bat in Zambia.

Group A rotavirus is a major cause of diarrhoea in humans, especially in young children. Bats also harbour group A rotaviruses, but the genetic backgrounds of bat rotavirus strains are usually distinct from those of human rotavirus strains. We identified a new strain of group A rotavirus in the intestinal contents of a horseshoe bat in Zambia. Whole genome sequencing revealed that the identified virus, named RVA/Bat-wt/ZMB/LUS12-14/2012/G3P[3], possessed the genotype constellation G3-P[3]-I3-R2-C2-M3-A9-N2-T3-E2-H3. Several genome segments of LUS12-14 were highly similar to those of group A rotaviruses identified from humans, cows and antelopes, indicating interspecies transmission of rotaviruses between bats and other mammals with possible multiple genomic reassortment events.

Seroepidemiological Prevalence of Multiple Species of Filoviruses in Fruit Bats (Eidolon helvum) Migrating in Africa.

Fruit bats are suspected to be a natural reservoir of filoviruses, including Ebola and Marburg viruses. Using an enzyme-linked immunosorbent assay based on the viral glycoprotein antigens, we detected filovirus-specific immunoglobulin G antibodies in 71 of 748 serum samples collected from migratory fruit bats (Eidolon helvum) in Zambia during 2006-2013. Although antibodies to African filoviruses (eg, Zaire ebolavirus) were most prevalent, some serum samples showed distinct specificity for Reston ebolavirus, which that has thus far been found only in Asia. Interestingly, the transition of filovirus species causing outbreaks in Central and West Africa during 2005-2014 seemed to be synchronized with the change of the serologically dominant virus species in these bats. These data suggest the introduction of multiple species of filoviruses in the migratory bat population and point to the need for continued surveillance of filovirus infection of wild animals in sub-Saharan Africa, including hitherto nonendemic countries.

Distinct Lineages of Bufavirus in Wild Shrews and Nonhuman Primates.

Viral metagenomic analysis identified a new parvovirus genome in the intestinal contents of wild shrews in Zambia. Related viruses were detected in spleen tissues from wild shrews and nonhuman primates. Phylogenetic analyses showed that these viruses are related to human bufaviruses, highlighting the presence and genetic diversity of bufaviruses in wildlife.

Metagenomic analysis of the shrew enteric virome reveals novel viruses related to human stool-associated viruses.

Shrews are small insectivorous mammals that are distributed worldwide. Similar to rodents, shrews live on the ground and are commonly found near human residences. In this study, we investigated the enteric virome of wild shrews in the genus Crocidura using a sequence-independent viral metagenomics approach. A large portion of the shrew enteric virome was composed of insect viruses, whilst novel viruses including cyclovirus, picornavirus and picorna-like virus were also identified. Several cycloviruses, including variants of human cycloviruses detected in cerebrospinal fluid and stools, were detected in wild shrews at a high prevalence rate. The identified picornavirus was distantly related to human parechovirus, inferring the presence of a new genus in this family. The identified picorna-like viruses were characterized as different species of calhevirus 1, which was discovered previously in human stools. Complete or nearly complete genome sequences of these novel viruses were determined in this study and then were subjected to further genetic characterization. Our study provides an initial view of the diversity and distinctiveness of the shrew enteric virome and highlights unique novel viruses related to human stool-associated viruses.

Efficient isolation of Swine influenza viruses by age-targeted specimen collection.

The control of swine influenza virus (SIV) infection is paramount for increasing the productivity of pig farming and minimizing the threat of pandemic outbreaks. Thus, SIV surveillance should be conducted by region and on a regular basis. Here, we established a microneutralization assay specific for SIV seroprevalence surveillance by using reporter gene-expressing recombinant influenza viruses. Growth-based SIV seroprevalence revealed that most sows and piglets were positive for neutralizing antibodies against influenza viruses. In contrast, the 90-day-old growing pigs exhibited limited neutralizing activity in their sera, suggesting that this particular age of population is most susceptible to SIV infection and thus is an ideal age group for SIV isolation. From nasal swab specimens of healthy pigs in this age population, we were able to isolate SIVs at a higher incidence (5.3%) than those of previous reports. Nucleotide sequencing and phylogenetic analysis of the hemagglutinin (HA) genes revealed that the isolated SIVs have circulated and evolved in pigs but not have been recently introduced from humans, implying that a large number of SIV lineages may remain "undiscovered" in the global porcine populations. We propose that the 90-day-old growing pig-targeted nasal swab collection presented in this study facilitates global SIV surveillance and contributes to the detection and control of SIV infection.

Tobacco calmodulin-like protein provides secondary defense by binding to and directing degradation of virus RNA silencing suppressors.

RNA silencing (RNAi) induced by virus-derived double-stranded RNA (dsRNA), which is in a sense regarded as a pathogen-associated molecular pattern (PAMP) of viruses, is a general plant defense mechanism. To counteract this defense, plant viruses express RNA silencing suppressors (RSSs), many of which bind to dsRNA and attenuate RNAi. We showed that the tobacco calmodulin-like protein, rgs-CaM, counterattacked viral RSSs by binding to their dsRNA-binding domains and sequestering them from inhibiting RNAi. Autophagy-like protein degradation seemed to operate to degrade RSSs with the sacrifice of rgs-CaM. These RSSs could thus be regarded as secondary viral PAMPs. This study uncovered a unique defense system in which an rgs-CaM-mediated countermeasure against viral RSSs enhanced host antiviral RNAi in tobacco.

ELM: enhanced lowest common ancestor based method for detecting a pathogenic virus from a large sequence dataset.

BACKGROUND: Emerging viral diseases, most of which are caused by the transmission of viruses from animals to humans, pose a threat to public health. Discovering pathogenic viruses through surveillance is the key to preparedness for this potential threat. Next generation sequencing (NGS) helps us to identify viruses without the design of a specific PCR primer. The major task in NGS data analysis is taxonomic identification for vast numbers of sequences. However, taxonomic identification via a BLAST search against all the known sequences is a computational bottleneck. DESCRIPTION: Here we propose an enhanced lowest-common-ancestor based method (ELM) to effectively identify viruses from massive sequence data. To reduce the computational cost, ELM uses a customized database composed only of viral sequences for the BLAST search. At the same time, ELM adopts a novel criterion to suppress the rise in false positive assignments caused by the small database. As a result, identification by ELM is more than 1,000 times faster than the conventional methods without loss of accuracy. CONCLUSIONS: We anticipate that ELM will contribute to direct diagnosis of viral infections. The web server and the customized viral database are freely available at http://bioinformatics.czc.hokudai.ac.jp/ELM/.

Molecular mechanisms underlying oseltamivir resistance mediated by an I117V substitution in the neuraminidase of subtype H5N1 avian influenza A viruses.

BACKGROUND: The neuraminidase (NA) inhibitor oseltamivir is widely used to treat patients infected with influenza viruses. An Ile-to-Val change at position 117 in influenza A virus subtype H5N1 NA (NA-I117V) confers a reduction in susceptibility to oseltamivir carboxylate. However, the in vivo relevance and molecular basis of the decreased sensitivity mediated by this mutation are poorly understood. METHODS: We created single-point-mutant viruses with 3 genetically different backgrounds (ie, 1 belonging to clade 1 and 2 belonging to clade 2.3.4) and evaluated the effects of the I117V mutation on oseltamivir susceptibility in vitro, in vivo, and in silico. RESULTS: The NA-I117V mutation conferred a slight reduction in susceptibility to oseltamivir in vitro (1.3- to 6.3-fold changes), although it did not substantially compromise NA enzymatic activity. Mice infected with I117V virus exhibited reduced susceptibility to oseltamivir and decreased survival in 2 of 3 virus pairs tested. Molecular dynamics simulations revealed that I117V caused the loss of hydrogen bonds between an arginine at position 118 and the carboxyl group of oseltamivir, leading to a lower binding affinity for oseltamivir. CONCLUSIONS: Our findings provide new insight into the mechanism of NA-I117V-mediated oseltamivir resistance in highly pathogenic H5N1 avian influenza viruses.

Spatiotemporal dynamics and epidemiological impact of SARS-CoV-2 XBB lineage dissemination in Brazil in 2023.

The SARS-CoV-2 XBB is a group of highly immune-evasive lineages of the Omicron variant of concern that emerged by recombining BA.2-descendent lineages and spread worldwide during 2023. In this study, we combine SARS-CoV-2 genomic data (n = 11,065 sequences) with epidemiological data of severe acute respiratory infection (SARI) cases collected in Brazil between October 2022 and July 2023 to reconstruct the space-time dynamics and epidemiologic impact of XBB dissemination in the country. Our analyses revealed that the introduction and local emergence of lineages carrying convergent mutations within the Spike protein, especially F486P, F456L, and L455F, propelled the spread of XBB* lineages in Brazil. The average relative instantaneous reproduction numbers of XBB* + F486P, XBB* + F486P + F456L, and XBB* + F486P + F456L + L455F lineages in Brazil were estimated to be 1.24, 1.33, and 1.48 higher than that of other co-circulating lineages (mainly BQ.1*/BE*), respectively. Despite such a growth advantage, the dissemination of these XBB* lineages had a reduced impact on Brazil's epidemiological scenario concerning previous Omicron subvariants. The peak number of SARI cases from SARS-CoV-2 during the XBB wave was approximately 90%, 80%, and 70% lower than that observed during the previous BA.1*, BA.5*, and BQ.1* waves, respectively. These findings revealed the emergence of multiple XBB lineages with progressively increasing growth advantage, yet with relatively limited epidemiological impact in Brazil throughout 2023. The XBB* + F486P + F456L + L455F lineages stand out for their heightened transmissibility, warranting close monitoring in the months ahead. IMPORTANCE: Brazil was one the most affected countries by the SARS-CoV-2 pandemic, with more than 700,000 deaths by mid-2023. This study reconstructs the dissemination of the virus in the country in the first half of 2023, a period characterized by the dissemination of descendants of XBB.1, a recombinant of Omicron BA.2 lineages evolved in late 2022. The analysis supports that XBB dissemination was marked by the continuous emergence of indigenous lineages bearing similar mutations in key sites of their Spike protein, a process followed by continuous increments in transmissibility, and without repercussions in the incidence of severe cases. Thus, the results suggest that the epidemiological impact of the spread of a SARS-CoV-2 variant is influenced by an intricate interplay of factors that extend beyond the virus's transmissibility alone. The study also underlines the need for SARS-CoV-2 genomic surveillance that allows the monitoring of its ever-shifting composition.

Antiviral activity of stachyflin on influenza A viruses of different hemagglutinin subtypes.

BACKGROUND: The hemagglutinin (HA) of influenza viruses is a possible target for antiviral drugs because of its key roles in the initiation of infection. Although it was found that a natural compound, Stachyflin, inhibited the growth of H1 and H2 but not H3 influenza viruses in MDCK cells, inhibitory activity of the compound has not been assessed against H4-H16 influenza viruses and the precise mechanism of inhibition has not been clarified. METHODS: Inhibitory activity of Stachyflin against H4-H16 influenza viruses, as well as H1-H3 viruses was examined in MDCK cells. To identify factors responsible for the susceptibility of the viruses to this compound, Stachyflin-resistant viruses were selected in MDCK cells and used for computer docking simulation. RESULTS: It was found that in addition to antiviral activity of Stachyflin against influenza viruses of H1 and H2 subtypes, it inhibited replication of viruses of H5 and H6 subtypes, as well as A(H1N1)pdm09 virus in MDCK cells. Stachyflin also inhibited the virus growth in the lungs of mice infected with A/WSN/1933 (H1N1) and A/chicken/Ibaraki/1/2005 (H5N2). Substitution of amino acid residues was found on the HA2 subunit of Stachyflin-resistant viruses. Docking simulation indicated that D37, K51, T107, and K121 are responsible for construction of the cavity for the binding of the compound. In addition, 3-dimensional structure of the cavity of the HA of Stachyflin-susceptible virus strains was different from that of insusceptible virus strains. CONCLUSION: Antiviral activity of Stachyflin was found against A(H1N1)pdm09, H5, and H6 viruses, and identified a potential binding pocket for Stachyflin on the HA. The present results should provide us with useful information for the development of HA inhibitors with more effective and broader spectrum.

Link prediction on bipartite networks using matrix factorization with negative sample selection.

We propose a new method for bipartite link prediction using matrix factorization with negative sample selection. Bipartite link prediction is a problem that aims to predict the missing links or relations in a bipartite network. One of the most popular solutions to the problem is via matrix factorization (MF), which performs well but requires reliable information on both absent and present network links as training samples. This, however, is sometimes unavailable since there is no ground truth for absent links. To solve the problem, we propose a technique called negative sample selection, which selects reliable negative training samples using formal concept analysis (FCA) of a given bipartite network in advance of the preceding MF process. We conduct experiments on two hypothetical application scenarios to prove that our joint method outperforms the raw MF-based link prediction method as well as all other previously-proposed unsupervised link prediction methods.

Comparative epidemic expansion of SARS-CoV-2 variants Delta and Omicron in the Brazilian State of Amazonas.

The SARS-CoV-2 variants of concern (VOCs) Delta and Omicron spread globally during mid and late 2021, respectively. In this study, we compare the dissemination dynamics of these VOCs in the Amazonas state, one of Brazil's most heavily affected regions. We sequenced the virus genome from 4128 patients collected in Amazonas between July 1st, 2021, and January 31st, 2022, and investigated the viral dynamics using a phylodynamic approach. The VOCs Delta and Omicron BA.1 displayed similar patterns of phylogeographic spread but different epidemic dynamics. The replacement of Gamma by Delta was gradual and occurred without an upsurge of COVID-19 cases, while the rise of Omicron BA.1 was extremely fast and fueled a sharp increase in cases. Thus, the dissemination dynamics and population-level impact of new SARS-CoV-2 variants introduced in the Amazonian population after mid-2021, a setting with high levels of acquired immunity, greatly vary according to their viral phenotype.