Individual Gene Segments Research Articles

Natural recombination of the torque teno canis virus within the ORF1, -2, and -3 genes and the untranslated region

The torque teno canis virus (TTCaV) was first reported in 2001 and it shares similarities with the known Torque teno virus (TTV) in terms of genomic organization and putative transcriptional features. It is a single-stranded DNA virus characterized by high rates of recombination and nucleotide substitution, like RNA viruses. Studies reported recombination events in torque teno virus; however, there is limited reporting of TTCaV reorganization events. This study screened fecal samples from domestic dogs in Henan Province. There was a positivity rate of 16.5% (19/115) for TTCaV. Four nearly complete TTCaV genomes, namely Canine/HeNan/4, 5, 6, and 13/2019, were obtained from the 19 positive fecal samples, whose genome sequence was obtained using gap-filling PCR. Sequence analysis revealed two unique amino acid mutation sites in the TTCaV strains, K278Q (compared with the first isolate Cf-TTV10 in Japan) and V/L268I (compared with the TTCaV strain from southern China). Subsequently, 17 near full-length TTCaV genome sequences were subjected to phylogenetic and recombination detection program analyzes. We obtained evidence supporting recombination events in the Chinese TTCaV strains. These findings suggest that mutation and recombination occurred in the three individual gene segments (ORF1, ORF2, ORF3) and the untranslated region, an area of major recombination in the Chinese TTCaV strain GX265 genome. Interestingly, the TTCaV strain (Canine/HeNan/6/2019) was a major parent involved in the genetic recombination of the GX265 strain. This study provides insights into the genetic variability and evolution of TTCaV.

Cencurut virus: A novel Orthonairovirus from Asian house shrews (Suncus murinus) in Singapore

Orthonairovirus is a genus of viruses in the family Nairoviridae, order Bunyavirales, with a segmented circular RNA genome. They typically infect birds and mammals and are primarily transmitted by ectoparasites such as ticks. Four of nine Orthonairovirus genogroups can infect humans, with Crimean-Congo hemorrhagic fever virus infections displaying case fatality rates up to 40%. Here, we discover and describe a novel Orthonairovirus as Cencurut virus (CENV). CENV was detected in 34 of 37 Asian house shrews (Suncus murinus) sampled in Singapore and in a nymphal Amblyomma helvolum tick collected from an infected shrew. Pairwise comparison of CENV S, M, and L segments had 95.0 to 100% nucleotide and 97.5 to 100% amino acid homology within CENV genomes, suggesting a diverse viral population. Phylogenetic analysis of the individual gene segments showed that CENV is related to Erve, Lamgora, Lamusara, and Thiafora viruses, with only 49.0 to 58.2% nucleotide and 41.7 to 61.1% amino acid homology, which has previously been detected in other shrew species from France, Gabon, and Senegal respectively. The high detection frequency suggests that CENV is endemic among S. murinus populations in Singapore. The discovery of CENV, from a virus family with known zoonotic potential, underlines the importance of surveillance of synanthropic small mammals that are widely distributed across Southeast Asia.

Bluetongue Disease in Small Ruminants in Palestine: A retrospective Study Between the period of 2005-2019

Small ruminant’s livestock industry provides a major source of livelihood for many people worldwide, particularly the rural poor in developing countries. Bluetongue (BT) is a reportable infectious non-contagious disease of the small ruminant that has considerable socioeconomic importance in livestock in Palestine. The objective of the current study was to investigate the epidemiological analysis of BT within small ruminants in Palestine over 14 years 2005 – 2019. The retrospective numbers of BT outbreaks, cases, and deaths from the World Organization for Animal Health were analyzed. The study indicated that BT is enzootic in Palestine, reported started from 2009. A total of 273 outbreaks include 1628 cases resulted in 351 deaths were reported. A total of 273 outbreaks, 1628 cases, and 351 deaths were recorded in small ruminants. The incidence rate average was 5.5%, and the average mortality rate was 1.6%. The case fatality rate ranged from 0.0- 39.29 % with an average of 20.2%. No vaccination practice was applied in Palestine. Temporal analysis obtained that BT is more endemic in the low-temperature season between September and December, with a higher peak in November, displaying `overwintering` of BTV in Palestine. At least five BTV serotypes BTV (2, 4, 5, 6 and 8) were continuously present in Palestine. Multiple serotypes were recorded in the same outbreak period increase the risk for re-assortment of individual BTV gene segments. Efficient data recording, enhance owner awareness, use of the vaccine and a systematic BT monitoring program are required to control and eradicate the disease.

Impact of Individual Viral Gene Segments from Influenza A/H5N8 Virus on the Protective Efficacy of Inactivated Subtype-Specific Influenza Vaccine

Since its emergence in 2014, the highly pathogenic avian influenza H5N8 virus has continuously and rapidly spread worldwide in the poultry sector resulting in huge economic losses. A typical inactivated H5N8 vaccine is prepared using the six internal genes from A/PR8/1934 (H1N1) and the two major antigenic proteins (HA and NA) from the circulating H5N8 strain with the HA modified to a low pathogenic form (PR8HA/NA-H5N8). The contribution of the other internal proteins from H5N8, either individually or in combination, to the overall protective efficacy of PR8-based H5N8 vaccine has not been investigated. Using reverse genetics, a set of PR8-based vaccines expressing the individual proteins from an H5N8 strain were rescued and compared to the parent PR8 and low pathogenic H5N8 strains and the commonly used PR8HA/NA-H5N8. Except for the PR8-based vaccine strains expressing the HA of H5N8, none of the rescued combinations could efficiently elicit virus-neutralizing antibodies. Compared to PR8, the non-HA viral proteins provided some protection to infected chickens six days post infection. We assume that this late protection was related to cell-based immunity rather than antibody-mediated immunity. This may explain the slight advantage of using full low pathogenic H5N8 instead of PR8HA/NA-H5N8 to improve protection by both the innate and the humoral arms of the immune system.

Genomic Profiling: The Strengths and Limitations of Chloroplast Genome-Based Plant Variety Authentication.

Genomic profiling is a suitable tool for variety authentication and has applications in both operational quality and regulatory raw material control. It can be used to differentiate species or varieties and to identify admixtures as well as field contaminants. To establish a molecular profile, reliable and very accurate sequence data are required. As a result of the influence of the pollinator plant, nuclear genome-based authentication is in most cases not suitable for a direct application on the fruit. Sequences must be used that come exclusively from the localized mother plant. Parts of the fruit of maternal origin, e.g., components derived from the blossom, are suitable as a basis for this. Alternatively, DNA from cell organelles that are maternally inherited, such as mitochondria or chloroplasts, can be used. The latter will be discussed in this review in closer detail. Although individual gene segments on the chloroplast genome are already used for species differentiation in barcoding studies on plants, little is known about the usefulness of the entire chloroplast genome for intraspecies differentiation in general and for differentiation between modern varieties in particular. Results from the literature as well as from our own work suggest that chloroplast genome sequences are indeed very well-suited for the differentiation of old varieties. On the other hand, they are less or not suitable for the genetic differentiation of modern cultivars, because they are often too closely related.

Diversity and Phylogenetic Relationships of Full Genome Sequences of Cassava Brown Streak Viruses in Kenya

Cassava brown streak disease is caused by cassava brown streak virus (CBSV) and Uganda cassava brown streak virus (UCBSV). Many of the CBSV and UCBSV diversity studies utilize partial coat protein sequences due to the unavailability of representative full genome sequences. Hence, there is little information on the diversity of cassava brown streak viruses in the rest of the genomes of the two species that are present in the farmers’ fields. The aim of this study was to determine Kenyan full CBSV and UCBSV genomes, and their sequence diversity and phylogenetic relationships within various genome and genome segments. Twenty four CBSVs positive samples tested by RT PCR from major cassava producing regions in Kenya were sequenced using Illumina MiSeq. Quality assessment of the output reads was done using the CLC Genomics 5.5.1 software programs. Genome assembly was done by de novo and reference guided assembly. Nucleotide sequence similarity of CBSV and UCBSV was determined. Phylogenetic relationships between CBSV and UCBSV were determined by performing the neighbour-joining analysis using MEGA 6.0 software. Six CBSV and 9 UCBSV genomes were generated from this study. The coat protein of the CBSV sequences had nucleotide sequence similarity of 92-100% while P1 and P3 gene had 75-100% and 76-100%, respectively. The coat protein of the UCBSV sequences had nucleotide sequence similarity of 91-99%. P1 and P3 gene had 83-100% and 86-99%, respectively. The phylogenetic analysis of full genomes revealed two distinct clusters one for UCBSV and another cluster for CBSV. Individual gene segments phylogenetic tree resembled that of the whole genome by clustering the nucleotide sequences into two clusters, one belonging to UCBSV and the other CBSV. The study revealed an average genome nucleotide diversity of 21.5% and 15.8% in CBSV and UCBSV respectively. A vast genetic diversity observed in CBSV and UCBSV in this study portends a lot of challenges in developing molecular diagnostic techniques as well as control strategies against the viruses.

Determinants of TCRα-β chain pairing in generating TCR diversity

Abstract Advances in next-generation sequencing have allowed insights into the enormous diversity of the human T cell receptor (TCR). Earlier studies have suggested that ~108 unique TRB genes exist at the periphery. In contrast, the factors that determine the pairing of TCRα and TCRβ chains have remained elusive due to the lack of high-throughput single-cell sequencing technology. We have developed a high-throughput single-cell sequencing technology that enables identification of >3×104 paired TRA:TRB sequences per sample and have conducted sequencing of naïve and memory CD4+ and of total T cells from 15 healthy individuals, including six pairs of monozygotic twins. We found that the frequencies of TRAV:TRBV pairing are essentially determined by the frequencies of the individual gene segments, suggesting the absence of structural constraints in the pairing of the α and β chains. Twins are more similar in the frequencies of TRAV and TRBV combinations than are unrelated individuals, but this difference was explained by a genetic influence on AV and BV gene segment frequencies. Twins and unrelated individuals were compared for the sharing of identical TCR sequences. TRA sequence sharing among twins was comparable to that among unrelated individuals, but TRB sharing was significantly higher in twins. Sharing of the entire TCR was regularly found in twins but not detected for naïve CD4+ T cells from unrelated individuals. Results were similar for naïve and memory T cells, with the genetic influence more prominent in memory T cells, suggesting the additional influence of peripheral selection. In support of this interpretation, the sharing of identical TCR sequences in unrelated individuals correlated with the number of shared MHC class II alleles.

VDJ Gene Usage among B-Cell Receptors in ABO-incompatible Kidney Transplantation Determined by RNA-seq Transcriptomic Analysis

Background Studies on B-cell subtypes and V(D)J gene usage of B-cell receptors in kidney transplants are scarce. This study aimed to investigate V(D)J gene segment usage in ABO-incompatible (ABOi) kidney transplant (KT) patients compared to that in ABO-compatible (ABOc) KT patients. Methods We selected 16 ABOi KT patients with accommodation (ABOiA), 6 ABOc stable KT patients (ABOcS), and 6 ABOi KT patients with biopsy-proven acute antibody-mediated rejection (ABOiR) at day 10, whose graft tissue samples had been stored in the biorepository between 2010 and 2014. Complete transcriptomes of graft tissues were sequenced and analyzed through RNA sequencing (RNA-seq). The international ImMunoGeneTics information system (IMGT®) was used for in-depth comparison of V(D)J gene segment usage. Results The mean age of the 28 KT recipients was 43.3 ± 12.8 years, and 53.6% were male. By family, IGHV3, IGHJ4, IGLV2, and IGLJ3 gene segments were most frequently used in all groups, and their usage was not statistically different among the three patient groups. While IGKV3 was most frequently used in both the ABOiA and ABOiR groups, IGKV1 was most commonly used in the ABOcS group. In addition, while IGKJ1 was most commonly used in the ABOiA and ABOcS groups, IGKJ4 was most frequently used in the ABOiR group. According to individual gene segments, IGHV4-34 and IGHV4-30-2 were more commonly used in the ABOiR group than in the ABOiA group, and IGHV6-1 was more commonly used in the ABOcS group than in the ABOiR group. IGLV7-43 was more commonly used in the ABOcS group than in the ABOi group. However, technical variability, small sample size, and potential confounding effects of Rituximab or HLA mismatching are limitations of our study. Conclusions Our findings suggest that RNA-seq transcriptomic analyses can provide information on the V(D)J gene usage of B-cell receptors and the mechanisms of accommodation and immune reaction in ABOi KT. Astellas Pharma Korea, Inc. The Bio & Medical Technology Development Program of the NRF funded by the Korean government, MSIP (grant NRF-2015M3A9D3051413).

VDJ Gene Usage of B Cell Receptors in Peripheral Blood of ABO-incompatible Kidney Transplantation Patients

IntroductionB cell subtypes and immunoglobulin variable (V), diversity (D), joining (J) gene segment usage of B cell receptors in ABO-incompatible (ABOi) kidney transplantation (KT) in comparison to ABO-compatible KT have not been studied. The aims of this study were to analyze the VDJ gene segment usages of B cell receptors in peripheral blood of ABOi KT recipients. MethodsEighteen ABOi KT patients with accommodation (ABOiA), 10 ABO-compatible stable KT patients (ABOcS), and 10 ABOi KT patients with biopsy-proven acute antibody-mediated rejection (ABOiR) at day 10 after transplantation were selected. Complete transcriptomes of their peripheral blood samples were sequenced and analyzed through RNA sequencing. ResultsBy family, immunoglobulin heavy chain variable 3 (IGHV3), immunoglobulin light kappa chain variable 1 (IGKV1), immunoglobulin light lambda chain variable 2 (IGLV2), and immunoglobulin light lambda chain joining 3 (IGLJ3) gene segments were most frequently used in all groups, and their usage was not statistically different among the three groups except for IGHV3 and IGKV1. IGKV1 was more frequently used in the ABOiA group than in the ABOcS group. According to individual gene segments, IGHV3-7, IGHV3-15, IGHV4-59, IGKV3-11, IGLV1-44, IGLV2-14, IGLV4-69, and IGLV7-46 were more frequently used in the ABOcS group than other groups, and IGKV3-7 was more frequently used in the ABOiR group than other groups. IGLV5-52 and IGLV7-43 were more frequently used in the ABOiA group than in ABOcS group. ConclusionsOur findings suggest that RNA sequencing transcriptomic analyses of peripheral blood can provide information on the VDJ gene usage of B cell receptors and the mechanisms of accommodation and immune reaction in ABOi KT.

Phylogenetic Analysis and Characterization of a Sporadic Isolate of Equine Influenza A H3N8 from an Unvaccinated Horse in 2015.

Equine influenza, caused by the H3N8 subtype, is a highly contagious respiratory disease affecting equid populations worldwide and has led to serious epidemics and transboundary pandemics. This study describes the phylogenetic characterization and replication kinetics of recently-isolated H3N8 virus from a nasal swab obtained from a sporadic case of natural infection in an unvaccinated horse from Montana, USA. The nasal swab tested positive for equine influenza by Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR). Further, the whole genome sequencing of the virus confirmed that it was the H3N8 subtype and was designated as A/equine/Montana/9564-1/2015 (H3N8). A BLASTn search revealed that the polymerase basic protein 1 (PB1), polymerase acidic (PA), hemagglutinin (HA), nucleoprotein (NP), and matrix (M) segments of this H3N8 isolate shared the highest percentage identity to A/equine/Tennessee/29A/2014 (H3N8) and the polymerase basic protein 2 (PB2), neuraminidase (NA), and non-structural protein (NS) segments to A/equine/Malaysia/M201/2015 (H3N8). Phylogenetic characterization of individual gene segments, using currently available H3N8 viral genomes, of both equine and canine origin, further established that A/equine/Montana/9564-1/2015 belonged to the Florida Clade 1 viruses. Interestingly, replication kinetics of this H3N8 virus, using airway derived primary cells from multiple species, such as equine, swine, bovine, and human lung epithelial cells, demonstrated appreciable titers, when compared to Madin–Darby canine kidney epithelial cells. These findings indicate the broad host spectrum of this virus isolate and suggest the potential for cross-species transmissibility.

Characterization of the naive murine antibody repertoire using unamplified high-throughput sequencing.

Antibody specificity and diversity are generated through the enzymatic splicing of genomic gene segments within each B cell. Antibodies are heterodimers of heavy- and light-chains encoded on separate loci. We studied the antibody repertoire from pooled, splenic tissue of unimmunized, adult female C57BL/6J mice, using high-throughput sequencing (HTS) without amplification of antibody transcripts. We recovered over 90,000 heavy-chain and over 135,000 light-chain immunoglobulin sequences. Individual V-, D-, and J-gene segment usage was uniform among the three mouse pools, particularly in highly abundant gene segments, with low frequency V-gene segments not being detected in all pools. Despite the similar usage of individual gene segments, the repertoire of individual B-cell CDR3 amino acid sequences in each mouse pool was highly varied, affirming the combinatorial diversity in the B-cell pool that has been previously demonstrated. There also was some skewing in the V-gene segments that were detected depending on chromosomal location. This study presents a unique, non-primer biased glimpse of the conventionally housed, unimmunized antibody repertoire of the C57BL6/J mouse.

VDJ gene usage among B-cell receptors in ABO-incompatible kidney transplantation determined by RNA-seq Transcriptomic analysis

BackgroundStudies on B-cell subtypes and V(D)J gene usage of B-cell receptors in kidney transplants are scarce. This study aimed to investigate V(D)J gene segment usage in ABO-incompatible (ABOi) kidney transplant (KT) patients compared to that in ABO-compatible (ABOc) KT patients.MethodsWe selected 16 ABOi KT patients with accommodation (ABOiA), 6 ABOc stable KT patients (ABOcS), and 6 ABOi KT patients with biopsy-proven acute antibody-mediated rejection (ABOiR) at day 10, whose graft tissue samples had been stored in the biorepository between 2010 and 2014. Complete transcriptomes of graft tissues were sequenced and analyzed through RNA sequencing (RNA-seq). The international ImMunoGeneTics information system (IMGT®) was used for in-depth comparison of V(D)J gene segment usage.ResultsThe mean age of the 28 KT recipients was 43.3 ± 12.8 years, and 53.6% were male. By family, IGHV3, IGHJ4, IGLV2, and IGLJ3 gene segments were most frequently used in all groups, and their usage was not statistically different among the three patient groups. While IGKV3 was most frequently used in both the ABOiA and ABOiR groups, IGKV1 was most commonly used in the ABOcS group. In addition, while IGKJ1 was most commonly used in the ABOiA and ABOcS groups, IGKJ4 was most frequently used in the ABOiR group. According to individual gene segments, IGHV4–34 and IGHV4–30-2 were more commonly used in the ABOiR group than in the ABOiA group, and IGHV6–1 was more commonly used in the ABOcS group than in the ABOiR group. IGLV7–43 was more commonly used in the ABOcS group than in the ABOi group. However, technical variability, small sample size, and potential confounding effects of Rituximab or HLA mismatching are limitations of our study.ConclusionsOur findings suggest that RNA-seq transcriptomic analyses can provide information on the V(D)J gene usage of B-cell receptors and the mechanisms of accommodation and immune reaction in ABOi KT.

Shifting Clade Distribution, Reassortment, and Emergence of New Subtypes of Highly Pathogenic Avian Influenza A(H5) Viruses Collected from Vietnamese Poultry from 2012 to 2015.

Whole-genome sequences of representative highly pathogenic avian influenza A(H5) viruses from Vietnam were generated, comprising samples from poultry outbreaks and active market surveillance collected from January 2012 to August 2015. Six hemagglutinin gene clades were characterized. Clade 1.1.2 was predominant in southern Mekong provinces throughout 2012 and 2013 but gradually disappeared and was not detected after April 2014. Clade 2.3.2.1c viruses spread rapidly during 2012 and were detected in the south and center of the country. A number of clade 1.1.2 and 2.3.2.1c interclade reassortant viruses were detected with different combinations of internal genes derived from 2.3.2.1a and 2.3.2.1b viruses, indicating extensive cocirculation. Although reassortment generated genetic diversity at the genotype level, there was relatively little genetic drift within the individual gene segments, suggesting genetic stasis over recent years. Antigenically, clade 1.1.2, 2.3.2.1a, 2.3.2.1b, and 2.3.2.1c viruses remained related to earlier viruses and WHO-recommended prepandemic vaccine strains representing these clades. Clade 7.2 viruses, although detected in only low numbers, were the exception, as indicated by introduction of a genetically and antigenically diverse strain in 2013. Clade 2.3.4.4 viruses (H5N1 and H5N6) were likely introduced in April 2014 and appeared to gain dominance across northern and central regions. Antigenic analyses of clade 2.3.4.4 viruses compared to existing clade 2.3.4 candidate vaccine viruses (CVV) indicated the need for an updated vaccine virus. A/Sichuan/26221/2014 (H5N6) virus was developed, and ferret antisera generated against this virus were demonstrated to inhibit some but not all clade 2.3.4.4 viruses, suggesting consideration of alternative clade 2.3.4.4 CVVs.IMPORTANCE Highly pathogenic avian influenza (HPAI) A(H5) viruses have circulated continuously in Vietnam since 2003, resulting in hundreds of poultry outbreaks and sporadic human infections. Despite a significant reduction in the number of human infections in recent years, poultry outbreaks continue to occur and the virus continues to diversify. Vaccination of poultry has been used as a means to control the spread and impact of the virus, but due to the diversity and changing distribution of antigenically distinct viruses, the utility of vaccines in the face of mismatched circulating strains remains questionable. This study assessed the putative amino acid changes in viruses leading to antigenic variability, underscoring the complexity of vaccine selection for both veterinary and public health purposes. Given the overlapping geographic distributions of multiple, antigenically distinct clades of HPAI A(H5) viruses in Vietnam, the vaccine efficacy of bivalent poultry vaccine formulations should be tested in the future.

Whole genome characterization of human influenza A(H1N1)pdm09 viruses isolated from Kenya during the 2009 pandemic

An influenza pandemic caused by a novel influenza virus A(H1N1)pdm09 spread worldwide in 2009 and is estimated to have caused between 151,700 and 575,400 deaths globally. While whole genome data on new virus enables a deeper insight in the pathogenesis, epidemiology, and drug sensitivities of the circulating viruses, there are relatively limited complete genetic sequences available for this virus from African countries. We describe herein the full genome analysis of influenza A(H1N1)pdm09 viruses isolated in Kenya between June 2009 and August 2010. A total of 40 influenza A(H1N1)pdm09 viruses isolated during the pandemic were selected. The segments from each isolate were amplified and directly sequenced. The resulting sequences of individual gene segments were concatenated and used for subsequent analysis. These were used to infer phylogenetic relationships and also to reconstruct the time of most recent ancestor, time of introduction into the country, rates of substitution and to estimate a time-resolved phylogeny. The Kenyan complete genome sequences clustered with globally distributed clade 2 and clade 7 sequences but local clade 2 viruses did not circulate beyond the introductory foci while clade 7 viruses disseminated country wide. The time of the most recent common ancestor was estimated between April and June 2009, and distinct clusters circulated during the pandemic. The complete genome had an estimated rate of nucleotide substitution of 4.9×10−3 substitutions/site/year and greater diversity in surface expressed proteins was observed. We show that two clades of influenza A(H1N1)pdm09 virus were introduced into Kenya from the UK and the pandemic was sustained as a result of importations. Several closely related but distinct clusters co-circulated locally during the peak pandemic phase but only one cluster dominated in the late phase of the pandemic suggesting that it possessed greater adaptability.

Dynamic Control of Long-Range Genomic Interactions at the Immunoglobulin κ Light-Chain Locus.

The Igκ locus, which is spread over 3Mb of genomic DNA and contains >100 variable (V) genes, serves as an important model system to study long-range chromatin interactions. Here, we will discuss how in developing B cells in the bone marrow the accessibility of individual Vκ segments is controlled by many lineage-specific and ubiquitously expressed transcription factors that act on various cis-regulatory elements, including promoters, enhancers, and insulators. This dynamic control furthermore involves changes in subnuclear localization, histone modification, DNA demethylation, and three-dimensional locus compaction. In pro-B cells, the Igκ locus adopts a poised conformation as full contraction has been achieved and many key transcription factors already occupy the locus. Subsequently, the combined activation of pre-B cell antigen receptor signaling pathways and attenuation of IL-7R signaling in small resting pre-B cells dramatically modifies the transcription factor landscape, supporting the induction of monoallelic Igκ gene rearrangements. Hereby, the intronic and 3' Igκ enhancer elements coordinately focus their activities in the Vκ region toward frequently used Vκ genes. Recent work has drawn attention to the intriguing role of the CTCF-associated regulatory elements Cer and Sis, which are located in the Vκ-Jκ intervening region and control Igκ locus contraction and Vκ repertoire diversity. This involves CTCF-mediated locus insulation, restricting enhancer activity to the Vκ region and suppressing the preferential recombination to proximal Vκ genes. A picture emerges in which the dynamic control of long-range genomic interactions ensures correct timing of Igκ locus recombination and provides appropriate opportunities for individual Vκ gene segments to engage in Vκ-Jκ rearrangement.

Influenza A virus nucleoprotein selectively decreases neuraminidase gene-segment packaging while enhancing viral fitness and transmissibility.

The influenza A virus (IAV) genome is divided into eight distinct RNA segments believed to be copackaged into virions with nearly perfect efficiency. Here, we describe a mutation in IAV nucleoprotein (NP) that enhances replication and transmission in guinea pigs while selectively reducing neuraminidase (NA) gene segment packaging into virions. We show that incomplete IAV particles lacking gene segments contribute to the propagation of the viral population through multiplicity reactivation under conditions of widespread coinfection, which we demonstrate commonly occurs in the upper respiratory tract of guinea pigs. NP also dramatically altered the functional balance of the viral glycoproteins on particles by selectively decreasing NA expression. Our findings reveal novel functions for NP in selective control of IAV gene packaging and balancing glycoprotein expression and suggest a role for incomplete gene packaging during host adaptation and transmission.

In vivo reassortment of influenza viruses.

The genetic material of influenza A virus consists of eight negative-sense RNA segments. Under suitable conditions, the segmented structure of the viral genome allows an exchange of the individual gene segments between different strains, causing formation of new reassorted viruses. For reassortment to occur, co-infection with two or more influenza virus strains is necessary. The reassortment is an important evolutionary mechanism which can result in antigenic shifts that modify host range, pathology, and transmission of the influenza A viruses. In this process, the influenza virus strain with epidemic and/or pandemic potential can be created. Cases of this kind were in 1957 (Asian flu), 1968 (Hong Kong flu) and recently in 2009 (Mexico). Viruses containing genes of avian, swine, and/or human origin are widespread around the world, for example the triple reassortant H1N1 virus causing the 2009 influenza pandemic in 2009 that has become a seasonal virus. The aim of the study is to present the mechanism of reassortment and the results of experimental co-infection with different influenza viruses.

The temperature-sensitive and attenuation phenotypes conferred by mutations in the influenza virus PB2, PB1, and NP genes are influenced by the species of origin of the PB2 gene in reassortant viruses derived from influenza A/California/07/2009 and A/WSN/33 viruses.

Live attenuated influenza vaccines in the United States are derived from a human virus that is temperature sensitive (ts), characterized by restricted (≥ 100-fold) replication at 39 °C. The ts genetic signature (ts sig) has been mapped to 5 loci in 3 genes: PB1 (391 E, 581 G, and 661 T), PB2 (265 S), and NP (34 G). However, when transferred into avian and swine influenza viruses, only partial ts and attenuation phenotypes occur. To investigate the reason for this, we introduced the ts sig into the human origin virus A/WSN/33 (WSN), the avian-origin virus A/Vietnam/1203/04 (VN04), and the swine origin triple-reassortant 2009 pandemic H1N1 virus A/California/07/2009 (CA07), which contains gene segments from human, avian, and swine viruses. The VN04(ts sig) and CA07(ts sig) viruses replicated efficiently in Madin-Darby canine kidney (MDCK) cells at 39 °C, but the replication of WSN(ts sig) was restricted ≥ 100-fold compared to that at 33 °C. Reassortant CA07(ts sig) viruses were generated with individual polymerase gene segments from WSN, and vice versa. Only ts sig viruses with a PB2 gene segment derived from WSN were restricted in replication ≥ 100-fold at 39 °C. In ferrets, the CA07(ts sig) virus replicated in the upper and lower respiratory tract, but the replication of a reassortant CA07(ts sig) virus with a WSN PB2 gene was severely restricted in the lungs. Taken together, these data suggest that the origin of the PB2 gene segment influences the ts phenotype in vitro and attenuation in vivo. This could have implications for the design of novel live vaccines against animal origin influenza viruses. Live attenuated influenza vaccines (LAIVs) on temperature-sensitive (ts) backbones derived from animal origin influenza viruses are being sought for use in the poultry and swine industries and to protect people against animal origin influenza. However, inserting the ts genetic signature from a licensed LAIV backbone fails to fully attenuate these viruses. Our data indicate this is associated with the presence of a PB2 gene segment derived from an avian influenza virus. We show that a reassortant 2009 pandemic H1N1 virus with the ts signature from a licensed LAIV donor virus is ts in vitro and attenuated in vivo when the PB2 gene is derived from a human origin virus but not from an avian virus. Our study provides information that could benefit the rational design of alternative LAIV backbones against animal origin influenza viruses.

Genomic characterization of H14 subtype Influenza A viruses in new world waterfowl and experimental infectivity in mallards (Anas platyrhynchos).

Recent repeated isolation of H14 hemagglutinin subtype influenza A viruses (IAVs) in the New World waterfowl provides evidence to suggest that host and/or geographic ranges for viruses of this subtype may be expanding. In this study, we used genomic analyses to gain inference on the origin and evolution of H14 viruses in New World waterfowl and conducted an experimental challenge study in mallards (Anas platyrhynchos) to evaluate pathogenicity, viral replication, and transmissibility of a representative viral strain in a natural host species. Genomic characterization of H14 subtype IAVs isolated from New World waterfowl, including three isolates sequenced specifically for this study, revealed high nucleotide identity among individual gene segments (e.g. ≥95% shared identity among H14 HA gene segments). In contrast, lower shared identity was observed among internal gene segments. Furthermore, multiple neuraminidase subtypes were observed for H14 IAVs isolated in the New World. Gene segments of H14 viruses isolated after 2010 shared ancestral genetic lineages with IAVs isolated from wild birds throughout North America. Thus, genomic characterization provided evidence for viral evolution in New World waterfowl through genetic drift and genetic shift since purported introduction from Eurasia. In the challenge study, no clinical disease or lesions were observed among mallards experimentally inoculated with A/blue-winged teal/Texas/AI13-1028/2013(H14N5) or exposed via contact with infected birds. Titers of viral shedding for mallards challenged with the H14N5 IAV were highest at two days post-inoculation (DPI); however shedding was detected up to nine DPI using cloacal swabs. The distribution of viral antigen among mallards infected with H14N5 IAV was largely restricted to enterocytes lining the villi in the lower intestinal tract and in the epithelium of the bursa of Fabricius. Characterization of the infectivity of A/blue-winged teal/Texas/AI13-1028/2013(H14N5) in mallards provides support for similarities in viral replication and shedding as compared to previously described waterfowl-adapted, low pathogenic IAV strains in ducks.

On The Organization Of Human T Cell Receptor Loci

T cell repertoire in health and disease is complex, comprised of millions of unique T cell clones defined by hetero-dimeric T cell receptors (TCR). The a and b subunits of the TCR are products of genetic loci consisting of variable (V) and joining (J) segments in the case of TCR a (TRA), and V, J and diversity (D) segments in the case of TCR b (TRB). These gene segments are recombined to yield TCR a (VJ) or TCR b (VDJ). Recently we reported that T cell clonal frequency in normal stem cell donors, from the point of view of TCR b gene segment usage rather than being random, has a fractal ordering (Meier et al, BBMT 2013). Fractals are mathematical constructs characterized by self-similarity at different scales of measurement. Thus whether T cell repertoire is examined from the standpoint of DJ, VDJ or VDJ+nucleotide inserted TCR b clones the frequency distribution remains the same when log transformed data are examined yielding a fractal dimension (FD) of ~ 1.5.In order to identify the origin of the observed fractal ordering of TCR b gene segment containing clones, the distribution of gene segments across the TRA and TRB loci was evaluated for self-similarity. Data describing the numeric positions of the initial and final nucleotides of the start and stop codons respectively, was obtained for each gene segment on the two loci was from the NCBI public data base. Resulting gene segment length and intervening space between successive gene segments (in base pairs) were calculated and log-transformed to assess self-similarity by calculating FD using the formula, FD = Log (magnitude)/Log (scaling factor). Length of individual gene segments was considered as scaling factor and the inter-segment space, as the magnitude of the line segment; FD was calculated for each segment. This demonstrated that the V and J gene segments for both TRA and TRB loci were arranged in a fractal, self-similar manner when the spacing between the loci was considered as a function of the size of the gene segment. Despite the apparent variability in the observed sizes of the V and J gene segments and the spacing between them, on a logarithmic scale there was marked uniformity in their size and spacing, with an average FD of 1.4±0.1 and 1.3±0.2 for TRB, V and J segments respectively (Figure). Corresponding values for TRA locus were 1.5±0.1 and 1.7±0.1, for the V and J segments. [Display omitted] During T cell receptor recombination, the J and the D segments in TRA and TRB loci respectively, are recombined with the V segments therefore the distribution of each V segment in the TRA and TRB loci was determined relative to the position of the J and the D segments in the respective loci, by the using the following formula spacing V-J or V-D = INP-D or J segment / INP nthV segment, (INP: initial nucleotide positions of gene segments). This measure demonstrated a strikingly consistent proportionality across both TRA and TRB loci, and when plotted for successive V segments on the TRB locus, the value declined logarithmically, with a slope of approximately 1.6, and similarly an average slope of 1.3 for the TRA locus, consistent with the earlier observed FD for these loci. We conclude that T cell receptor gene segments are organized in an iterating logarithmically scaled manner. This implies that aside from mechanisms such as the 12/23 rule, gene segment distribution across the T cell receptor loci may help determine the order of recombination, and thus contribute to the observed fractal ordering of the T cell repertoire. Disclosures:No relevant conflicts of interest to declare.