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Zoonotic influenza infections continue to threaten human health. Ongoing surveillance and risk assessment of animal viruses are needed for pandemic preparedness, and population immunity is an important component of risk assessment. We determined age-stratified hemagglutinin inhibition seroprevalence against 5 swine influenza viruses circulating in Hong Kong and Guangzhou in China. Using hemagglutinin inhibition seroprevalence and titers, we modeled the effect of population immunity on the basic reproduction number (R 0 ) if each virus were to become transmissible among humans. Among 353 individual serum samples, we reported low seroprevalence for triple-reassortant H1N2 and Eurasian avian-like H1N1 influenza viruses, which would reduce R 0 by only 18%-20%. The smallest R 0 needed to cause a pandemic was 1.22-1.24, meaning existing population immunity would be insufficient to block the spread of these H1N1 or H1N2 variants. For human-origin H3N2, existing population immunity could suppress R 0 by 47%, thus reducing pandemic risk.

The segmented genome of influenza A virus has conferred significant evolutionary advantages to this virus through genetic reassortment, a mechanism that facilitates the rapid expansion of viral genetic diversity upon influenza co-infections. Therefore, co-infection of genetically diverse avian influenza viruses in poultry may pose a significant public health risk in generating novel reassortants with increased zoonotic potential. This study investigated the reassortment patterns of a Pearl River Delta-lineage avian influenza A(H7N9) virus and four genetically divergent avian influenza A(H9N2) viruses upon co-infection in embryonated chicken eggs and chickens. To characterize "within-host" and "between-host" genetic diversity, we further monitored the viral genotypes that were subsequently transmitted to contact chickens in serial transmission experiments. We observed that co-infection with A(H7N9) and A(H9N2) viruses may lead to the emergence of novel reassortant viruses in ovo and in chickens, albeit with different reassortment patterns. Novel reassortants detected in donor chickens co-infected with different combinations of the same A(H7N9) virus and different A(H9N2) viruses showed distinct onward transmission potential to contact chickens. Sequential transmission of novel reassortant viruses was only observed in one out of four co-infection combinations. Our results demonstrated different patterns by which influenza viruses may acquire genetic diversity through co-infection in ovo , in vivo , and under sequential transmission conditions.

Orbiviruses are arboviruses with 10 double-stranded linear RNA segments, and some have been identified as pathogens of dramatic epizootics in both wild and domestic ruminants. Tibet orbivirus (TIBOV) is a new orbivirus isolated from hematophagous insects in recent decades, and, currently, most of the strains have been isolated from insects in PR China, except for two from Japan. In this study, we isolated a novel reassortment TIBOV strain, YN15-283-01, from Culicoides spp. To identify and understand more characteristics of YN15-283-01, electrophoresis profiles of the viral genome, electron microscopic observations, plaque assays, growth curves in various cell lines, and bioinformatic analysis were conducted. The results indicated that YN15-283-01 replicated efficiently in mosquito cells, rodent cells and several primate cells. Furthermore, the maximum likelihood phylogenetic trees and simplot analysis of the 10 segments indicated that YN15-283-01 is a natural reassortment isolate that had emerged mainly from XZ0906 and SX-2017a.

Reassortant Eurasian avian-like (EA) H1N1 virus, possessing 2009 pandemic (pdm/09) and triple-reassortant (TR)-derived internal genes, namely G4 genotype, has replaced the G1 genotype EA H1N1 virus (all the genes were of EA origin) and become predominant in swine populations in China. Understanding the pathogenicity of G4 viruses in pigs is of great importance for disease control. Here, we conducted comprehensive analyses of replication and pathogenicity of G4 and G1 EA H1N1 viruses in pigs. G4 virus exhibited enhanced replication, increased duration of virus shedding, and caused more severe respiratory lesions in pigs compared with G1 virus. G4 virus, with viral ribonucleoprotein (vRNP) complex genes of pdm/09 origin, exhibited higher levels of nuclear accumulation and higher polymerase activity, which is essential for improved replication of G4 virus. These findings indicate that G4 virus poses a great threat to both swine industry and public health, and control measures should be urgently implemented., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Reassortment of the Rotavirus A (RVA) 11-segment dsRNA genome may generate new genome constellations that allow RVA to expand its host range or evade immune responses. Reassortment may also produce phylogenetic incongruities and weakly linked evolutionary histories across the 11 segments, obscuring reassortment-specific epistasis and changes in substitution rates. To determine the co-segregation patterns of RVA segments, we generated time-scaled phylogenetic trees for each of the 11 segments of 789 complete RVA genomes isolated from mammalian hosts and compared the segments' geodesic distances. We found that segments 4 (VP4) and 9 (VP7) occupied significantly different tree spaces from each other and from the rest of the genome. By contrast, segments 10 and 11 (NSP4 and NSP5/6) occupied nearly indistinguishable tree spaces, suggesting strong co-segregation. Host-species barriers appeared to vary by segment, with segment 9 (VP7) presenting the weakest association with host species. Bayesian Skyride plots were generated for each segment to compare relative genetic diversity among segments over time. All segments showed a dramatic decrease in diversity around 2007 coinciding with the introduction of RVA vaccines. To assess selection pressures, codon adaptation indices and relative codon deoptimization indices were calculated with respect to different host genomes. Codon usage varied by segment with segment 11 (NSP5) exhibiting significantly higher adaptation to host genomes. Furthermore, RVA codon usage patterns appeared optimized for expression in humans and birds relative to the other hosts examined, suggesting that translational efficiency is not a barrier in RVA zoonosis.

Potyviridae is the largest family of plant RNA viruses. Their genomes are expressed through long polyproteins that are usually headed by the leader endopeptidase P1. This protein can be classified as type A or type B based on host proteolytic requirements and RNA silencing suppression (RSS) capacity. The main Potyviridae genus is Potyvirus , and a group of potyviruses infecting sweet potato presents an enlarged P1 protein with a polymerase slippage motif that produces an extra product termed P1N-PISPO. These two proteins display some RSS activity and are expressed followed by HCPro, which appears to be the main RNA silencing suppressor in these viruses. Here, we studied the behavior of the P1 protein of Sweet potato feathery mottle virus (SPFMV) using a viral system based on a canonical potyvirus, Plum pox virus (PPV), and discovered that this protein is able to replace both PPV P1 and HCPro. We also found that P1N-PISPO, produced after polymerase slippage, provides extra RNA silencing suppression capacity to SPFMV P1 in this viral context. In addition, the results showed that presence of two type A P1 proteins was detrimental for viral viability. The ample recombination spectrum that we found in the recovered viruses supports the strong adaptation capacity of P1 proteins and signals the N-terminal part of SPFMV P1 as essential for RSS activity. Further analyses provided data to add extra layers to the evolutionary history of sweet potato-infecting potyvirids. IMPORTANCE Plant viruses represent a major challenge for agriculture worldwide and Potyviridae , being the largest family of plant RNA viruses, is one of the primary players. P1, the leader endopeptidase, is a multifunctional protein that contributes to the successful spread of these viruses over a wide host range. Understanding how P1 proteins work, their dynamic interplay during viral infection, and their evolutionary path is critical for the development of strategic tools to fight the multiple diseases these viruses cause. We focused our efforts on the P1 protein of Sweet potato feathery mottle virus , which is coresponsible for the most devastating disease in sweet potato. The significance of our research is in understanding the capacity of this protein to perform several independent functions, using this knowledge to learn more about P1 proteins in general and the potyvirids infecting this host.

In recent years in China, clade 2.3.4.4 H5N6 plus clade 2.3.2.1 H5N1 subtype highly pathogenic avian influenza (HPAI) viruses have gradually become endemic in poultry, and their co-circulation could inevitably facilitate the gene reassortment between each other. During our routine surveillance in live poultry markets (LPMs) in eastern China in 2017-2018, a novel reassortant H5N6 strain with the HA gene derived from clade 2.3.2.1 was isolated from the cloacal swabs of apparently healthy ducks. Phylogenetic tracing analysis indicated that another two clade 2.3.2.1 H5N1 strains with divergent lineages of PB1 gene and one clade 2.3.4.4 H5N6 isolate of the dominant genotype sharing spatio-temporal proximity were intimately involved in the generation of this rarely reported clade 2.3.2.1 H5N6 reassortant. Distinct with the other three HPAI H5 viruses showing moderate virulence in mice, the H5N1 strain of the homologous internal gene constellation against the clade 2.3.2.1 H5N6 reassortant was highly pathogenic, which might probably attribute to the H3 subtype-derived PB1 gene. However, as compared to the clade 2.3.4.4 H5N6 ancestor, the clade 2.3.2.1 H5N6 reassortant displayed a broader tissue distribution and higher viral titres in mice, which could likely facilitate the viral maintenance and spread in nature. Therefore, our results highlight that continuous epidemiological survey of H5 subtype HPAI viruses in LPMs needs to be strengthened to prevent the potential poultry or even public health threat of the novel reassortants from endemic viruses., (© 2020 Blackwell Verlag GmbH.)

The acid sphingomyelinase/ceramide system has been shown to be important for cellular infection with at least some viruses, for instance, rhinovirus or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Functional inhibition of the acid sphingomyelinase using tricyclic antidepressants prevented infection of epithelial cells, for instance with SARS-CoV-2. The structure of ambroxol, that is, trans-4-[(2,4-dibromanilin-6-yl)-methyamino]-cyclohexanol, a mucolytic drug applied by inhalation, suggests that the drug might inhibit the acid sphingomyelinase and thereby infection with SARS-CoV-2. To test this, we used vesicular stomatitis virus pseudoviral particles presenting SARS-CoV-2 spike protein on their surface (pp-VSV-SARS-CoV-2 spike), a bona fide system for mimicking SARS-CoV-2 entry into cells. Viral uptake and formation of ceramide localization were determined by fluorescence microscopy, activity of the acid sphingomyelinase by consumption of [ 14 C]sphingomyelin and ceramide was quantified by a kinase method. We found that entry of pp-VSV-SARS-CoV-2 spike required activation of acid sphingomyelinase and release of ceramide, events that were all prevented by pretreatment with ambroxol. We also obtained nasal epithelial cells from human volunteers prior to and after inhalation of ambroxol. Inhalation of ambroxol reduced acid sphingomyelinase activity in nasal epithelial cells and prevented pp-VSV-SARS-CoV-2 spike-induced acid sphingomyelinase activation, ceramide release, and entry of pp-VSV-SARS-CoV-2 spike ex vivo. The addition of purified acid sphingomyelinase or C16 ceramide restored entry of pp-VSV-SARS-CoV-2 spike into ambroxol-treated epithelial cells. We propose that ambroxol might be suitable for clinical studies to prevent coronavirus disease 2019., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Natural killer (NK) cells are powerful immune effectors, modulating their anti-tumor function through a balance activating and inhibitor ligands on their cell surface. Though still emerging, cancer immunotherapies utilizing NK cells are proving promising as a modality for the treatment of a number of solid tumors, including glioblastoma (GBM) and other gliomas, but are often limited due to complex immunosuppression associated with the GBM tumor microenvironment which includes overexpression of inhibitory receptors on GBM cells. CD155, or poliovirus receptor (PVR), has recently emerged as a pro-tumorigenic antigen, overexpressed on GBM and contributing to increased GBM migration and aggressiveness. CD155 has also been established as an immunomodulatory receptor, able to both activate NK cells through interactions with CD226 (DNAM-1) and CD96 and inhibit them through interaction with TIGIT. However, NK cell TIGIT expression has been shown to be upregulated in cancer, establishing CD155 as a predominantly inhibitory receptor within the context of GBM and other solid tumors, and rendering it of interest as a potential target for antigen-specific NK cell-based immunotherapy. This review will explore the function of CD155 within GBM as it relates to tumor migration and NK cell immunoregulation, as well as pre-clinical and clinical targeting of CD155/TIGIT and the potential that this pathway holds for the development of emerging NK cell-based immunotherapies.

Influenza vaccines historically have been multivalent, whole virus inactivated products. The first bivalent, intranasal, live attenuated influenza vaccine (LAIV; Ingelvac Provenza), with H1N1 and H3N2 subtypes, has been approved for use in swine. We investigated the LAIV hemagglutinin ( HA ) sequences in diagnostic cases submitted to the Iowa State University Veterinary Diagnostic Laboratory and potential vaccine virus reassortment with endemic influenza A virus (IAV) in swine. From January 3 to October 11, 2018, IAV HA sequences demonstrating 99.5-99.9% nucleotide homology to the H1 HA or 99.4-100% nucleotide homology to the H3 HA parental strains in the LAIV were detected in 58 of 1,116 (5.2%) porcine respiratory cases (H1 HA A/swine/Minnesota/37866/1999[H1N1; MN99]; H3 HA A/swine/Texas/4199-2/1998[H3N2; TX98]). Nine cases had co-detection of HA genes from LAIV and wild-type IAV in the same specimen. Thirty-five cases had associated epidemiologic information that indicated they were submitted from 11 states representing 31 individual sites and 17 production systems in the United States. Whole genome sequences from 11 cases and another subset of 2 plaque-purified IAV were included in our study. Ten whole genome sequences, including 1 plaque-purified IAV, contained at least one internal gene from endemic IAV detected within the past 3 y. Phylogenetic analysis of whole genome sequences indicated that reassortment occurred between vaccine virus and endemic field strains circulating in U.S. swine. Our data highlight the need and importance of continued IAV surveillance to detect emerging IAV with LAIV genes in the swine population.

The constant crosstalk between the large avian reservoir of influenza A viruses (IAV) and its mammalian hosts drives viral evolution and facilitates their host switching. Direct adaptation of an avian strain to human or reassortment between avian-origin gene segments with that of human strains are the two mechanisms for the emergence of pandemic viruses. While it was suggested that the 1918 pandemic virus is of avian origin, reassortment of 1918 human isolates and avian influenza viruses led to the generation of 1957 and 1968 pandemic viruses. Interestingly, the avian PB1 segment, which encodes the catalytic subunit of IAV polymerase, is present in the 1957 and 1968 pandemic viruses. The biological consequence and molecular basis of such gene exchange remain less well understood. Using the 2009 pandemic H1N1 virus as a model, whose polymerase contains a human-origin PB1 subunit, we demonstrate that the acquisition of an avian PB1 markedly enhances viral RNA synthesis. This enhancement is also effective in the absence of PB2 adaptive mutations, which are key determinants of host switching. Mechanistically, the avian-origin PB1 does not appear to affect polymerase assembly but imparts the reassorted pandemic polymerase-augmented viral primary transcription and replication. Moreover, compared to the parental pandemic polymerase, the reassorted polymerase displays comparable complementary RNA (cRNA)-stabilizing activity but is specifically enhanced in progeny viral RNA (vRNA) synthesis from cRNA in a trans-activating manner. Overall, our results provide the first insight into the mechanism via which avian-origin PB1 enhances viral RNA synthesis of the 2009 pandemic virus polymerase.

Neurotropic viral transsynaptic tracing is an increasingly powerful technique for dissecting the structure and function of neural circuits. Herpes simplex virus type 1 strain H129 has been widely used as an anterograde tracer. However, HSV tracers still have several shortcomings, including high toxicity, low sensitivity and non-specific retrograde labeling. Here, we aimed to construct high-brightness HSV anterograde tracers by increasing the expression of exogenous genes carried by H129 viruses. Using a Trojan horse-like strategy, a HSV/AAV (adeno-associated virus) chimaera termed H8 was generated to enhance the expression of a fluorescent marker. In vitro and in vivo assays showed that the exogenous gene was efficiently replicated and amplified by the synergism of the HSV vector and introduced AAV replication system. H8 reporting fluorescence was brighter than that of currently available H129 tracers, and H8 could be used for fast and effective anterograde tracing without additional immunostaining. These results indicated that foreign gene expression in HSV tracers could be enhanced by integrating HSV with AAV replication system. This approach may be useful as a general enhanced expression strategy for HSV-based tracing tools or gene delivery vectors.

Elucidating the genetic basis of influenza A viruses (IAVs) is important to understand which mutations will determine the virulence and the host range of mammals. Here, seasonal H3N2 influenza was adapted in mice by serial passage and four mutants, each carrying amino acid substitutions related to mouse adaptation in either the PB2, HA, NP, or NA protein, were generated. To confirm the contribution of each gene to enhanced pathogenicity and mouse adaptation, mice were inoculated with the respective variants, and virulence, replication, histopathology, and infectivity were examined. The virus harboring HA mutations displayed increased infection efficiency and replication competence, resulting in higher mortality in mice relative to those infected with wild-type virus. By contrast, the NP D34N mutation caused rapid and widespread infection in multiple organs without presenting virulent symptoms. Additionally, the PB2 F323L mutation presented delayed but elevated replication competence in the respiratory tract, whereas the S331R mutation in NA showed no considerable effects on mouse adaptation. These results suggested that mouse-adapted changes in HA are major factors in increased pathogenicity and that mutations in NP and PB2 also contribute to cross-species adaptability. Our findings offer a better understanding of the molecular basis for IAV pathogenicity and adaptation in a new host., Competing Interests: The authors have declared that no competing interests exist.

This report describes the detection of a triple reassortant swine influenza A virus of H1 av N2 subtype. It evolved from an avian-like swine H1 av N1 that first acquired the N2 segment from a seasonal H3N2, then the M segment from a 2009 pandemic H1N1, in two reassortments estimated to have occurred 10 years apart. This study illustrates how recurrent influenza infections increase the co-infection risk and facilitate evolutionary jumps by successive gene exchanges. It recalls the importance of appropriate biosecurity measures inside holdings to limit virus persistence and interspecies transmissions, which both contribute to the emergence of new potentially zoonotic viruses.

Cyprinid herpesvirus 2 (CyHV-2) is the causative pathogen of herpesviral haematopoietic necrosis disease, which has caused huge economic losses to aquaculture industry in China. In this study, nine truncated CyHV-2 membrane glycoproteins (ORF25, ORF25C, ORF25D, ORF30, ORF124, ORF131, ORF136, ORF142A, ORF146) and a GFP reporter protein were respectively expressed using baculovirus surface displaying system. Western blot showed that the proteins were successfully packaged in the recombinant virus particles. In baculovirus transduced gibel carp kidney cells, the target proteins were expressed and displayed on the fish cell surface. Healthy gibel carp were immunized by immersion with the recombinant baculoviruses and the fish treated with phosphate-buffered saline (PBS) were served as mock group. The expression of interleukin-11 (IL-11), interferon α (IFNα) and a complement component gene C3 were significantly up-regulated in most experimental groups, and interferon γ (IFNγ) expression in some groups were also induced after immunization. Subsequently, the immunized gibel carp were challenged by intraperitoneal injection of CyHV-2 virus. All the immunized groups exhibited reduced mortality after CyHV-2 challenge. In the groups immunized with baculoviruses displaying and expressing ORF25, ORF25C and ORF146, the relative percentage survival values reached 83.3%, 87.5% and 70.8%, respectively. Our data suggested that baculovirus-displayed ORF25, ORF25C and ORF146 could be potential vaccine candidates for the prevention of CyHV-2 infection in gibel carp., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Potato virus Y (PVY) exists as several strains with distinct symptomology and tuber yield effects in different potato varieties. Recently, new recombinant strains have proliferated and dominated local populations around the world. In this study, PVY O , PVY N:O , PVY N-Wi , and PVY NTN strains were tracked across Canada from 2014 to 2017, showing rapid evolution of populations away from the traditionally dominant PVY O to recombinants PVY N-Wi (western Canada) and PVY NTN (eastern Canada). Simultaneously, 30 potato varieties were inoculated with PVY O , PVY N:O , and PVY NTN in controlled greenhouse experiments. Foliar symptoms of primary (mechanical inoculation mimicking aphid infection) and secondary (tuber seedborne) infection were cataloged, and tuber yield measured. On average, and generally similar in primary and secondary infection, symptom expression and yield reduction were most severe with PVY O , followed by PVY N:O and PVY NTN . Strong mosaic symptoms were most commonly expressed with PVY O infection, and only seen with PVY N:O or PVY NTN in 15 and 3 varieties, respectively. Across variety-strain combinations, yield reduction was correlated with symptom severity, most strongly in PVY O -infected plants (e.g., AC Chaleur, Beljade, Envol, Norland, and Pacific Russet), and four varieties exhibited tuber necrotic ringspot disease with PVY NTN (AC Chaleur, Envol, Pacific Russet, and Yukon Gold).

Avian influenza virus H9N2 has been endemic in birds in the Middle East, in particular in Egypt with multiple cases of human infections since 1998. Despite concerns about the pandemic threat posed by H9N2, little is known about the biological properties of H9N2 in this epicentre of infection. Here, we investigated the evolutionary dynamics of H9N2 in the Middle East and identified phylogeny-associated PB2 mutations that acted cooperatively to increase H9N2 replication/transcription in human cells. The accumulation of PB2 mutations also correlated with an increase in H9N2 virus growth in the upper and lower airways of mice and in virulence. These mutations clustered on a solvent-exposed region in the PB2-627 domain in proximity to potential interfaces with host factors. These PB2 mutations have been found at high prevalence during evolution of H9N2 in the field, indicating that they have provided a selective advantage for viral adaptation to infect poultry. Therefore, continuous prevalence of H9N2 virus in the Middle East has generated a far more fit or optimized replication phenotype, leading to an expanded viral host range, including to mammals, which may pose public health risks beyond the current outbreaks., Competing Interests: The authors have declared that no competing interests exist.

HIV-1 does not persistently infect macaques due in part to restriction by several macaque host factors. This has been partially circumvented by generating chimeric SIV/HIV-1 viruses (SHIVs) that encode SIV antagonist of known restriction factors. However, most SHIVs replicate poorly in macaques unless they are further adapted in culture and/or macaques (adapted SHIVs). Therefore, development of SHIVs encoding HIV-1 sequences derived directly from infected humans without adaptation (unadapted SHIVs) has been challenging. In contrast to the adapted SHIVs, the unadapted SHIVs have lower replication kinetics in macaque lymphocytes and are sensitive to type-1 interferon (IFN). The HIV-1 Envelope (Env) in the chimeric virus determines both the reduced replication and the IFN-sensitivity differences. There is limited information on macaque restriction factors that specifically limit replication of the more biologically relevant, unadapted SHIV variants. In order to identify the IFN-induced host factor(s) that could contribute to the inhibition of SHIVs in macaque lymphocytes, we measured IFN-induced gene expression in immortalized pig-tailed macaque (Ptm) lymphocytes using RNA-Seq. We found 147 genes that were significantly upregulated upon IFN treatment in Ptm lymphocytes and 31/147 were identified as genes that encode transmembrane helices and thus are likely present in membranes where interaction with viral Env is plausible. Within this group of upregulated genes with putative membrane-localized proteins, we identified several interferon-induced transmembrane protein (IFITM) genes, including several previously uncharacterized Ptm IFITM3-related genes. An evolutionary genomic analysis of these genes suggests the genes are IFITM3 duplications not found in humans that are both within the IFITM locus and also dispersed elsewhere in the Ptm genome. We observed that Ptm IFITMs are generally packaged at higher levels in unadapted SHIVs when compared to adapted SHIVs. CRISPR/Cas9-mediated knockout of Ptm IFITMs showed that depletion of IFITMs partially rescues the IFN sensitivity of unadapted SHIV. Moreover, we found that the depletion of IFITMs also increased replication of unadapted SHIV in the absence of IFN treatment, suggesting that Ptm IFITMs are likely important host factors that limit replication of unadapted SHIVs. In conclusion, this study shows that Ptm IFITMs selectively restrict replication of unadapted SHIVs. These findings suggest that restriction factors including IFITMs vary in their potency against different SHIV variants and may play a role in selecting for viruses that adapt to species-specific restriction factors., Competing Interests: The authors have declared that no competing interests exist.

Influenza A virus (IAV) has a segmented genome, which ( i ) allows for exchange of gene segments in coinfected cells, termed reassortment, and ( ii ) necessitates a selective packaging mechanism to ensure incorporation of a complete set of segments into virus particles. Packaging signals serve as segment identifiers and enable segment-specific packaging. We have previously shown that packaging signals limit reassortment between heterologous IAV strains in a segment-dependent manner. Here, we evaluated the extent to which packaging signals prevent reassortment events that would raise concern for pandemic emergence. Specifically, we tested the compatibility of hemagglutinin (HA) packaging signals from H5N8 and H7N9 avian IAVs with a human seasonal H3N2 IAV. By evaluating reassortment outcomes, we demonstrate that HA segments carrying H5 or H7 packaging signals are significantly disfavored for incorporation into a human H3N2 virus in both cell culture and a guinea pig model. However, incorporation of the heterologous HAs was not excluded fully, and variants with heterologous HA packaging signals were detected at low levels in vivo, including in naïve contact animals. This work indicates that the likelihood of reassortment between human seasonal IAV and avian IAV is reduced by divergence in the RNA packaging signals of the HA segment. These findings offer important insight into the molecular mechanisms governing IAV emergence and inform efforts to estimate the risks posed by H7N9 and H5N8 subtype avian IAVs., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Two novel influenza A virus-like genomes were detected in fruit bats in Central and South America. However, the biological properties of these bat-derived influenza viruses (BatIVs) are still largely unknown since infectious viral particles have never been isolated from the infected host species. In this study, a reverse genetics approach was used to generate infectious BatIV particles entirely from plasmids encoding full-length sequences in eight gene segments. We inoculated BatIV particles into various cell cultures including bat-derived cell lines and found that BatIVs infected particular bat-derived cells efficiently but not the other cell lines tested. Reassortant viruses between the two BatIVs were also successfully generated and their replication in the susceptible bat cell lines was confirmed. These findings suggest a limited host range and reassortment potential of BatIVs in nature, providing fundamental information for understanding of the ecology of BatIVs.

Betanodavirus reassortant strains (RGNNV/SJNNV) isolated from Senegalese sole harbour an SJNNV capsid featuring several changes with respect to the SJNNV-type strain, sharing three hallmark substitutions. Here, we have employed recombinant strains harbouring mutations in these positions (r20 and r20 + 247 + 270) and have demonstrated that the three substitutions affect different steps of the viral replication process. Adsorption ability and efficiency of viral attachment were only affected by substitutions in the C-terminal side of the capsid. However, the concurrent mutation in the N-terminal side seems to slightly decrease these properties, suggesting that this region could also be involved in viral binding. Differences in the intracellular and extracellular production of the mutant strains suggest that both the C-terminal and N-terminal regions of the capsid protein may be involved in the particle budding. Furthermore, viral replication in sole brain tissue of the mutant strains, and especially double- and triple-mutant strains, is clearly delayed with respect to the wt strain. These data support previous findings indicating that the C-terminal side plays a role in virulence because of a slower spread in the fish host brain and suggest that the concurrent participation of the N-terminal side is also important for viral replication in vivo., (© 2018 John Wiley & Sons Ltd.)

Envelope glycoprotein E2 of Classical Swine Fever Virus (CSFV) is involved in several critical virus functions. To analyze the role of E2 in virus replication, a series of recombinant CSFVs harboring chimeric forms of E2 CSFV and Bovine viral diarrhea virus (BVDV) were created and tested for their ability to infect swine or bovine cell lines. Substitution of native CSFV E2 by BVDV E2 abrogates virus replication in both cell lines. Substitution of individual domains in CSFV Brescia E2 by the homologous from BVDV produces chimeras that efficiently replicate in SK6 cells with the exception of a chimera harboring BVDV E2 residues 93-168. Further mapping revealed a critical area in E2 required for CSFV replication in SK6 cells between protein residues 136-156. This is the first report categorically defining a discrete portion of E2 as essential to pestivirus infection in susceptible cells., (Published by Elsevier Inc.)

Viroids can be transmitted vertically and/or horizontally by pollen. Tomato planta macho viroid (TPMVd) has a high rate of horizontal transmission by pollen, whereas potato spindle tuber viroid (PSTVd) does not. To specify the domain(s) involved in horizontal transmission, four viroid chimeras were created by exchanging the terminal left (TL) and/or pathogenicity (P) domains between PSTVd and TPMVd. PSTVd-based chimeras containing TPMVd-TL and P, or TPMVd-TL alone, displayed a high rate of horizontal transmission. TPMVd-based chimeras containing PSTVd-TL and P lost infectivity, and those containing PSTVd-TL alone displayed a low rate of horizontal transmission. In addition, the vertical transmission rate was also higher in the mutants containing TPMVd-TL than in the others. These findings indicate that the sequences or structures in the TL and P (although the role is limited) domains are important not only for horizontal but also for vertical transmission by pollen., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Subgroup C Avian Metapneumoviruses (AMPV-C) has two lineages, one mostly in turkeys and one mostly in ducks. To investigate the molecular basis of AMPV-C host tropism, a reverse genetics system for a duck AMPV-C virus was developed. A recombinant copy and a recombinant virus in which the SH protein had been exchanged for that of a turkey AMPV-C were rescued. No change in cytopathogenic effect or replication profile in vitro were observed for either virus compared to the wild type. In SPF Muscovy ducks the wild type and its recombinant copy were equally pathogenic. Exchanging the SH in the recombinant copy produced the same results. In SPF turkeys, neither recombinant virus was pathogenic, although both showed a low level of replication. Thus, from the current model, it appears that AMPV-C SH proteins derived from the different species are compatible and that turkey SH does not affect duck AMPV-C pathogenicity., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The H3N2 influenza viruses became widespread in humans during the 1968 H3N2 pandemic and have been a major cause of influenza epidemics ever since. Different lineages of H3N2 influenza viruses are also commonly found in animals. If a different lineage of H3N2 virus jumps to humans, a human influenza pandemic could occur with devastating consequences. Here, we studied the genetics, receptor-binding properties, and replication and transmission in mammals of 15 H3N2 avian influenza viruses detected in live poultry markets in China. We found that the H3N2 avian influenza viruses are complicated reassortants with distinct replication phenotypes in mice. Five viruses replicated efficiently in mice and bound to both human-type and avian-type receptors. These viruses transmitted efficiently to direct-contact guinea pigs, and three of them also transmitted among guinea pigs and ferrets via respiratory droplets. Moreover, ferret antiserum induced by human H3N2 viruses did not react with any of the H3N2 avian influenza viruses. Our study demonstrates that the H3N2 avian influenza viruses pose a clear threat to human health and emphasizes the need for continued surveillance and evaluation of the H3N2 influenza viruses circulating in nature.

Following attachment to host receptors via σ1, reovirus particles are endocytosed and disassembled to generate infectious subvirion particles (ISVPs). ISVPs undergo conformational changes to form ISVP*, releasing σ1 and membrane-targeting peptides from the viral μ1 protein. ISVP* formation is required for delivery of the viral core into the cytoplasm for replication. We characterized the properties of T3D F /T3D C S1, an S1 gene monoreassortant between two laboratory isolates of prototype reovirus strain T3D: T3D F and T3D C T3D F /T3D C S1 is poorly infectious. This deficiency is a consequence of inefficient encapsidation of S1-encoded σ1 on T3D F /T3D C S1 virions. Additionally, compared to T3D F , T3D F /T3D C S1 undergoes ISVP-to-ISVP* conversion more readily, revealing an unexpected role for σ1 in regulating ISVP* formation. The σ1 protein is held within turrets formed by the λ2 protein. To test if the altered properties of T3D F /T3D C S1 are due to a mismatch between σ1 and λ2 proteins from T3D F and T3D C , properties of T3D F /T3D C L2 and T3D F /T3D C S1L2, which express a T3D C -derived λ2, were compared. The presence of T3D C λ2 allowed more efficient σ1 incorporation, producing particles that exhibit T3D F -like infectivity. Compared to T3D F , T3D F /T3D C L2 prematurely converts to ISVP*, uncovering a role for λ2 in regulating ISVP* formation. Importantly, a virus with matching σ1 and λ2 displayed a more regulated conversion to ISVP* than either T3D F /T3D C S1 or T3D F /T3D C L2. In addition to identifying new regulators of ISVP* formation, our results highlight that protein mismatches produced by reassortment can alter virus assembly and thereby influence subsequent functions of the virus capsid. IMPORTANCE Cells coinfected with viruses that possess a multipartite or segmented genome reassort to produce progeny viruses that contain a combination of gene segments from each parent. Reassortment places new pairs of genes together, generating viruses in which mismatched proteins must function together. To test if such forced pairing of proteins that form the virus shell or capsid alters the function of the particle, we investigated properties of reovirus variants in which the σ1 attachment protein and the λ2 protein that anchors σ1 on the particle are mismatched. Our studies demonstrate that a σ1-λ2 mismatch produces particles with lower levels of encapsidated σ1, consequently decreasing virus attachment and infectivity. The mismatch between σ1 and λ2 also altered the capacity of the viral capsid to undergo conformational changes required for cell entry. These studies reveal new functions of reovirus capsid proteins and illuminate both predictable and novel implications of reassortment., (Copyright © 2018 American Society for Microbiology.)

Infectious bursal disease virus (IBDV) is one of the most important immunosuppressive viral agents in poultry production. Prophylactic vaccinations of chicken flocks are the primary tool for disease control. Widely used immunoprophylaxis can, however, provide high pressure which contributes to the genetic diversification of circulating viruses, e.g. through reassortment of genome segments. We report the genetic and phenotypic characterization of a field reassortant IBDV (designated as Bpop/03) that acquired segment A from very virulent IBDV and segment B from classical attenuated D78-like IBDV. Despite the mosaic genetic make-up, the virus caused high mortality (80%) in experimentally infected SPF chickens and induced lesions typical of the acute form of IBD. The in vivo study results are in contrast with the foregoing experimental investigations in which the natural reassortants exhibited an intermediate pathotype, and underline the complex nature of IBDV virulence.

Novel H1N2 and H3N2 swine influenza A viruses (IAVs) were identified in commercial farms in Chile. These viruses contained H1, H3 and N2 sequences, genetically divergent from IAVs described worldwide, associated with pandemic internal genes. Guinea pigs were used as human surrogate to evaluate the infection dynamics of these reassortant viruses, compared with a pandemic H1N1 virus. All viruses replicated and were shed in the upper respiratory tract without prior adaptation although H1N2 viruses showed the highest shedding titers. This could have public health importance, emphasizing the need to carry out further studies to evaluate the zoonotic potential of these viruses.

Genome reassortment in Lassa virus (LASV) has been reported in nature, but phenotypic consequences of this phenomenon are not well described. Here we characterize, both in vitro and in vivo , reassortment between 2 LASV strains: the prototypic 1976 Josiah strain and a more recently isolated 2015 Liberian strain. In vitro analysis showed that although cis- and trans-acting elements of viral RNA synthesis were compatible between strains, reassortants demonstrated different levels of viral replication. These differences were also apparent in vivo , as reassortants varied in pathogenicity in the guinea pig model of LASV infection. The reassortant variant containing the Josiah S segment retained the virulence of the parental Josiah strain, but the reassortant variant containing the S segment of the Liberian isolate was highly attenuated compared to both parental strains. Contrary to observations in reassortants between LASV and other arenavirus species, which suggest that L segment-encoded factors are responsible for virulence, these studies highlight a role for S segment-encoded virulence factors in disease, and also suggest that inefficient interactions between proteins of heterologous strains may limit the prevalence of reassortant LASV variants in nature.

Influenza A viruses cause an annual contagious respiratory disease in humans and are responsible for periodic high-mortality human pandemics. Pandemic influenza A viruses usually result from the reassortment of gene segments between human and avian influenza viruses. These avian influenza virus gene segments need to adapt to humans. Here we focus on the human adaptation of the synonymous codons of the avian influenza virus PB1 gene of the 1968 H3N2 pandemic virus. We generated recombinant H3N2 viruses differing only in codon usage of PB1 mRNA and demonstrated that codon usage of the PB1 mRNA of recent H3N2 virus isolates enhances replication in interferon (IFN)-treated human cells without affecting replication in untreated cells, thereby partially alleviating the interferon-induced antiviral state. High-throughput sequencing of tRNA pools explains the reduced inhibition of replication by interferon: the levels of some tRNAs differ between interferon-treated and untreated human cells, and evolution of the codon usage of H3N2 PB1 mRNA is skewed toward interferon-altered human tRNA pools. Consequently, the avian influenza virus-derived PB1 mRNAs of modern H3N2 viruses have acquired codon usages that better reflect tRNA availabilities in IFN-treated cells. Our results indicate that the change in tRNA availabilities resulting from interferon treatment is a previously unknown aspect of the antiviral action of interferon, which has been partially overcome by human-adapted H3N2 viruses. IMPORTANCE Pandemic influenza A viruses that cause high human mortality usually result from reassortment of gene segments between human and avian influenza viruses. These avian influenza virus gene segments need to adapt to humans. Here we focus on the human adaptation of the avian influenza virus PB1 gene that was incorporated into the 1968 H3N2 pandemic virus. We demonstrate that the coding sequence of the PB1 mRNA of modern H3N2 viruses enhances replication in human cells in which interferon has activated a potent antiviral state. Reduced interferon inhibition results from evolution of PB1 mRNA codons skewed toward the pools of tRNAs in interferon-treated human cells, which, as shown here, differ significantly from the tRNA pools in untreated human cells. Consequently, avian influenza virus-derived PB1 mRNAs of modern H3N2 viruses have acquired codon usages that better reflect tRNA availabilities in IFN-treated cells and are translated more efficiently., (Copyright © 2018 Smith et al.)

Lychnis ringspot virus (LRSV, genus Hordeivirus) was first isolated in 1959, and has been shown to infect several dicot plants in nature. However, due to the lack of infectious cDNA clones, the biological properties and mechanisms underlying LRSV infection are obscure. In this work, we constructed infectious cDNA clones of LRSV and have compiled the complete LRSV genomic (g) RNA sequence. Comparison of nucleotide and amino acid sequences between LRSV and barley stripe mosaic virus (BSMV), the type member of genus Hordeivirus, reveals that despite belonging to the same genus, and replicating in chloroplasts, the viruses are only distantly related. This could be further indicated by the failure of different LRSV/BSMV reassortants to infect N. benthamiana. LRSV infectious cDNA clones provide a useful tool for studies of biological diversity among hordeiviruses, and also may contribute to the understanding of seed transmission in dicot plants., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The fifth epidemic wave of avian influenza A(H7N9) virus in China during 2016-2017 demonstrated a geographic range expansion and caused more human cases than any previous wave. The factors that may explain the recent range expansion and surge in incidence remain unknown. We investigated the effect of anthropogenic, poultry, and wetland variables on all epidemic waves. Poultry predictor variables became much more important in the last 2 epidemic waves than they were previously, supporting the assumption of much wider H7N9 transmission in the chicken reservoir. We show that the future range expansion of H7N9 to northern China may increase the risk of H7N9 epidemic peaks coinciding in time and space with those of seasonal influenza, leading to a higher risk of reassortments than before, although the risk is still low so far.

Two subtypes of influenza A virus (IAV), avian-origin canine influenza virus (CIV) H3N2 (CIV-H3N2) and equine-origin CIV H3N8 (CIV-H3N8), are enzootic in the canine population. Dogs have been demonstrated to seroconvert in response to diverse IAVs, and naturally occurring reassortants of CIV-H3N2 and the 2009 H1N1 pandemic virus (pdmH1N1) have been isolated. We conducted a thorough phenotypic evaluation of CIV-H3N2 in order to assess its threat to human health. Using ferret-generated antiserum, we determined that CIV-H3N2 is antigenically distinct from contemporary human H3N2 IAVs, suggesting that there may be minimal herd immunity in humans. We assessed the public health risk of CIV-H3N2 × pandemic H1N1 (pdmH1N1) reassortants by characterizing their in vitro genetic compatibility and in vivo pathogenicity and transmissibility. Using a luciferase minigenome assay, we quantified the polymerase activity of all possible 16 ribonucleoprotein (RNP) complexes (PB2, PB1, PA, NP) between CIV-H3N2 and pdmH1N1, identifying some combinations that were more active than either parental virus complex. Using reverse genetics and fixing the CIV-H3N2 hemagglutinin (HA), we found that 51 of the 127 possible reassortant viruses were viable and able to be rescued. Nineteen of these reassortant viruses had high-growth phenotypes in vitro , and 13 of these replicated in mouse lungs. A single reassortant with the NP and HA gene segments from CIV-H3N2 was selected for characterization in ferrets. The reassortant was efficiently transmitted by contact but not by the airborne route and was pathogenic in ferrets. Our results suggest that CIV-H3N2 reassortants may pose a moderate risk to public health and that the canine host should be monitored for emerging IAVs. IMPORTANCE IAV pandemics are caused by the introduction of novel viruses that are capable of efficient and sustained transmission into a human population with limited herd immunity. Dogs are a a potential mixing vessel for avian and mammalian IAVs and represent a human health concern due to their susceptibility to infection, large global population, and close physical contact with humans. Our results suggest that humans are likely to have limited preexisting immunity to CIV-H3N2 and that CIV-H3N2 × pdmH1N1 reassortants have moderate genetic compatibility and are transmissible by direct contact in ferrets. Our study contributes to the increasing evidence that surveillance of the canine population for IAVs is an important component of pandemic preparedness., (Copyright © 2017 American Society for Microbiology.)

A novel reassortant H5N8 highly pathogenic avian influenza (HPAI) virus was recently identified in Asia, Europe, and North America. The H5N8 HPAI virus has raised serious concerns regarding the potential risk for human infection. However, the molecular changes responsible for allowing mammalian infection in H5N8 HPAI viruses are not clear. The objective of this study was to identify amino acid substitutions that are potentially associated with the adaptation of H5N8 HPAI viruses to mammals. In this study, an avian-origin H5N8 virus was adapted to mice through serial lung-to-lung passage. The virulence of mouse-adapted virus was increased and adaptive mutations, HA (A149V) and PB2 (E627K), were detected after the ninth passage in each series of mice. Reverse genetics were used to generate reassortants of the wild type and mouse-adapted viruses. Substitutions in the HA (A149V) and PB2 (E627K) proteins led to enhanced viral virulence in mice, the viruses displayed expanded tissue tropism, and increased replication kinetics in mammalian cells. Continued surveillance in poultry for amino acid changes that might indicate H5N8 HPAI viruses pose a threat to human health is required., (Copyright © 2017. Published by Elsevier B.V.)

Avian influenza A H5N1 and H9N2 viruses have been extensively circulating in various avian species and frequently infect mammals, including humans. The synchronous circulation of both viruses in Egypt provides an opportunity for possible genetic assortment, posing a probable threat to global public health. To assess the potential risk of the IAV reassortants derived from co-circulation of these two AI subtypes, reverse genetics technology was used to generate a set of IAV reassortants carrying single genetic segments of clade 2.2.1.2 virus A/duck/Egypt/Q4596D/2012 (H5N1), a representative of the most prevalent H5N1 clade in Egypt, in the genetic backbone of A/chicken/Egypt/S4456B/2011 (H9N2), a representative of G1-like H9N2 lineage which is widely circulating in Egypt. Furthermore, the genetic compatibility, growth kinetics and virulence were evaluated in vitro in mammalian systems using the MDCK cell line and avian system using SPF embryonated chicken eggs. Pathogenicity and virus shedding were further tested using SPF chickens. Out of the eight desired H9-reassortants, we could rescue only 5 reassortant viruses, either due to difficulty in cloning (PB1 of H5N1 virus) or genetic incompatibility (NP-H5/H9 and NA-H5/H9). Results revealed higher replication rates for the H9N2 virus having the NS segment of H5N1 virus. The lowest survival rate in both SPF eggs and SPF chickens was associated with the H5N1 parent virus infection, followed by the HA-H5/H9 virus. Our findings also suggest that all other reassortant viruses were of lower pathogenicity than the wild type H5N1 virus.

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to Africa and the Arabian Peninsula. The causative agent, Rift Valley fever phlebovirus (RVFV), belongs to the genus Phlebovirus in the family Phenuiviridae and causes high rates of abortions in ruminants, and hemorrhagic fever, encephalitis, or blindness in humans. Viral maintenance by mosquito vectors has led to sporadic RVF outbreaks in ruminants and humans in endemic countries, and effective vaccination of animals and humans may minimize the impact of this disease. A live-attenuated MP-12 vaccine strain is one of the best characterized RVFV strains, and was conditionally approved as a veterinary vaccine in the U.S. Live-attenuated RVF vaccines including MP-12 strain may form reassortant strains with other bunyavirus species. This study thus aimed to characterize the occurrence of genetic reassortment between the MP-12 strain and bunyavirus species closely related to RVFV. The Arumowot virus (AMTV) and Gouleako goukovirus (GOLV), are transmitted by mosquitoes in Africa. The results of this study showed that GOLV does not form detectable reassortant strains with the MP-12 strain in co-infected C6/36 cells. The AMTV also did not form any reassortant strains with MP-12 strain in co-infected C6/36 cells, due to the incompatibility among N, L, and Gn/Gc proteins. A lack of reassortant formation could be due to a functional incompatibility of N and L proteins derived from heterologous species, and due to a lack of packaging via heterologous Gn/Gc proteins. The MP-12 strain did, however, randomly exchange L-, M-, and S-segments with a genetic variant strain, rMP12-GM50, in culture cells. The MP-12 strain is thus unlikely to form any reassortant strains with AMTV or GOLV in nature.

A number of viruses within the Peribunyaviridae family are naturally occurring reassortants, a common phenomenon for segmented viruses. Using a minigenome-reporter and virus-like particle (VLP) production assay, we have accessed the potential of Oropouche virus (OROV), Schmallenberg virus (SBV), and other orthobunyaviruses within the Simbu serogroup to reassort. We found that the untranslated region (UTR) in the medium segment is a potential contributing factor for reassortment by the tested viruses. We demonstrate that for promoter activity to occur it was essential that the viral RNA polymerase (L) and nucleocapsid (N) proteins were from the same virus, reinforcing the hypothesis that the large and small segments that encode these proteins segregate together during genome reassortment. Our results indicate that, given the right epidemiological setting, reassortment between SBV and OROV would potentially be feasible and could contribute to the emergence of a new Simbu virus., Competing Interests: The authors declare no conflict of interest.

Previous studies have identified a functional role of PA-X for influenza viruses in mice and avian species; however, its role in swine remains unknown. Toward this, we constructed PA-X deficient virus (Sw-FS) in the background of a Triple-reassortment (TR) H1N2 swine influenza virus (SIV) to assess the impact of PA-X in viral virulence in pigs. Expression of PA-X in TR H1N2 SIV enhanced viral replication and host protein synthesis shutoff, and inhibited the mRNA levels of type I IFNs and proinflammatory cytokines in porcine cells. A delay of proinflammatory responses was observed in lungs of pigs infected by wild type SIV (Sw-WT) compared to Sw-FS. Furthermore, Sw-WT virus replicated and transmitted more efficiently than Sw-FS in pigs. These results highlight the importance of PA-X in the moderation of virulence and immune responses of TR SIV in swine, which indicated that PA-X is a pro-virulence factor in TR SIV in pigs., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Orchid fleck virus (OFV) represents a rhabdovirus with a unique bipartite genome. OFV genetic diversity at the whole genome level has not been described. Using the partial genome sequence of RNA1, we have determined that several OFV isolates derived from orchids in Japan belong to two genetically distant subgroups: subgroup I, the members of which are distributed worldwide but previously not known in Asia, and subgroup II, which is commonly distributed in Japan. However, complete genome sequence analysis of a novel Japanese subgroup I isolate revealed that although its RNA1 sequence differs considerably from those of subgroup II isolates, its RNA2 sequence is almost identical to them. Based on phylogenetic and recombination analyses, the genome reassortment events were predicted to occur between OFV subgroups including other unseen strains. Our data show that genome reassortment contributes to the genetic diversities of the bipartite rhabdoviruses and its occurrence may be geographically constrained., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Influenza A virus (IAV) RNA packaging signals serve to direct the incorporation of IAV gene segments into virus particles, and this process is thought to be mediated by segment-segment interactions. These packaging signals are segment and strain specific, and as such, they have the potential to impact reassortment outcomes between different IAV strains. Our study aimed to quantify the impact of packaging signal mismatch on IAV reassortment using the human seasonal influenza A/Panama/2007/99 (H3N2) and pandemic influenza A/Netherlands/602/2009 (H1N1) viruses. Focusing on the three most divergent segments, we constructed pairs of viruses that encoded identical proteins but differed in the packaging signal regions on a single segment. We then evaluated the frequency with which segments carrying homologous versus heterologous packaging signals were incorporated into reassortant progeny viruses. We found that, when segment 4 (HA) of coinfecting parental viruses was modified, there was a significant preference for the segment containing matched packaging signals relative to the background of the virus. This preference was apparent even when the homologous HA constituted a minority of the HA segment population available in the cell for packaging. Conversely, when segment 6 (NA) or segment 8 (NS) carried modified packaging signals, there was no significant preference for homologous packaging signals. These data suggest that movement of NA and NS segments between the human H3N2 and H1N1 lineages is unlikely to be restricted by packaging signal mismatch, while movement of the HA segment would be more constrained. Our results indicate that the importance of packaging signals in IAV reassortment is segment dependent. IMPORTANCE Influenza A viruses (IAVs) can exchange genes through reassortment. This process contributes to both the highly diverse population of IAVs found in nature and the formation of novel epidemic and pandemic IAV strains. Our study sought to determine the extent to which IAV packaging signal divergence impacts reassortment between seasonal IAVs. Our knowledge in this area is lacking, and insight into the factors that influence IAV reassortment will inform and strengthen ongoing public health efforts to anticipate the emergence of new viruses. We found that the packaging signals on the HA segment, but not the NA or NS segments, restricted IAV reassortment. Thus, the packaging signals of the HA segment could be an important factor in determining the likelihood that two IAV strains of public health interest will undergo reassortment., (Copyright © 2017 American Society for Microbiology.)

Viral nervous necrosis (VNN) certainly represents the biggest challenge for the sustainability and the development of aquaculture. A large number of economically relevant fish species have proven to be susceptible to the disease. Conversely, gilthead sea bream has generally been considered resistant to VNN, although it has been possible to isolate the virus from apparently healthy sea bream and sporadically from affected larvae and postlarvae. Unexpectedly, in 2014-2016 an increasing number of hatcheries in Europe have experienced mass mortalities in sea bream larvae. Two clinical outbreaks were monitored over this time span and findings are reported in this paper. Despite showing no specific clinical signs, the affected fish displayed high mortality and histological lesions typical of VNN. Fish tested positive for betanodavirus by different laboratory techniques. The isolates were all genetically characterized as being reassortant strains RGNNV/SJNNV. A genetic characterization of all sea bream betanodaviruses which had been isolated in the past had revealed that the majority of the strains infecting sea bream are actually RGNNV/SJNNV. Taken together, this information strongly suggests that RGNNV/SJNNV betanodavirus possesses a particular tropism to sea bream, which can pose a new and unexpected threat to the Mediterranean aquaculture.

Background: Genomic characterization of rotavirus (RoV) has not been adopted at large-scale due to the complexity of obtaining sequences for all 11 segments, particularly when feces are used as starting material., Methods: To overcome these limitations, we developed a novel RoV capture and genome sequencing method combining commercial enzyme immunoassay plates and a set of routinely used reagents., Results: Our approach had a 100% success rate, producing >90% genome coverage for diverse RoV present in fecal samples (Ct < 30)., Conclusions: This method provides a novel, reproducible and comparatively simple approach for genomic RoV characterization and could be scaled-up for use in global RoV surveillance systems., Trial Registration (prospectively Registered): Current Controlled Trials ISRCTN88101063 . Date of registration: 14/06/2012.

Segment reassortment and base mutagenesis of influenza A viruses are the primary routes to the rapid evolution of high-fitness virus genotypes. We recently described a predominant G57 genotype of avian H9N2 viruses that caused countrywide outbreaks in chickens in China during 2010 to 2013, which led to the zoonotic emergence of H7N9 viruses. One of the key features of the G57 genotype is the replacement of the earlier A/chicken/Beijing/1/1994 (BJ/94)-like M gene with the A/quail/Hong Kong/G1/1997 (G1)-like M gene of quail origin. We report here the functional significance of the G1-like M gene in H9N2 viruses in conferring increased infection severity and infectivity in primary chicken embryonic fibroblasts and chickens. H9N2 virus housing the G1-like M gene, in place of the BJ/94-like M gene, showed an early surge in viral mRNA and viral RNA (vRNA) transcription that was associated with enhanced viral protein production and with an early elevated release of progeny virus comprising largely spherical rather than filamentous virions. Importantly, H9N2 virus with the G1-like M gene conferred extrapulmonary virus spread in chickens. Five highly represented signature amino acid residues (37A, 95K, 224N, and 242N in the M1 protein and 21G in the M2 protein) encoded by the prevalent G1-like M gene were demonstrated to be prime contributors to enhanced infectivity. Therefore, the genetic evolution of the M gene in H9N2 virus increases reproductive virus fitness, indicating its contribution to the rising virus prevalence in chickens in China. IMPORTANCE We recently described the circulation of a dominant genotype (genotype G57) of H9N2 viruses in countrywide outbreaks in chickens in China, which was responsible, through reassortment, for the emergence of H7N9 viruses that cause severe human infections. A key feature of the genotype G57 H9N2 virus is the presence of the quail-origin G1-like M gene, which had replaced the earlier BJ/94-like M gene. We found that H9N2 virus with the G1-like M gene, but not the BJ/94-like M gene, showed an early surge in progeny virus production and more severe pathology and extrapulmonary virus spread in chickens. Five highly represented amino acid residues in the M1 and M2 proteins derived from the G1-like M gene were shown to mediate enhanced virus infectivity. These observations enhance what we currently know about the roles of reassortment and mutations in virus fitness and have implications for assessing the potential of variant influenza viruses that can cause a rising prevalence in chickens., (Copyright © 2017 American Society for Microbiology.)

Rotavirus A (RVA) strains, a leading cause of severe gastroenteritis in children worldwide, commonly possess the Wa or DS-1 genotype constellations. During a hospital-based study conducted in Hanoi, Vietnam, in the 2012-2013 rotavirus season, G1P[8] strains with a virtually identical short RNA migration pattern were detected in 20 (14%) of 141 rotavirus-positive samples. Two representatives of these strains were shown by whole-genome sequencing to be double-gene reassortants possessing the genotype constellation of G1-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Sequencing and a database search revealed that these Vietnamese G1P[8] double-gene reassortant strains shared an immediate ancestor with a locally circulating G2P[4] strain in all of the inner-capsid and non-structural protein genes, whereas they were more closely related in the VP7 and VP4 genes to a Chinese G1P[8] strain and a Chinese G3P[8] strain, respectively, than to locally circulating G1P[8] strains. Despite the marked similarity between Japanese and Thai G1P[8] double-gene reassortant strains, phylogenetic analysis suggested that the Vietnamese and Japanese/Thai G1P[8] double-gene reassortant strains originated from independent reassortment events. Clinically, children infected with Vietnamese G1P[8] double-gene reassortant strains experienced severe diarrhoea, but it was not more severe than that in children infected with ordinary G1P[8] strains. In conclusion, Vietnamese G1P[8] double-gene reassortant strains originated from a locally circulating G2P[4] strain and caused severe diarrhoea, but there was no evidence of increased virulence.

Genetic lineages of swine influenza A viruses (SIVs) have recently been established in Turkeys in the United States. To identify molecular determinants that are involved in virulence and transmission of SIVs to Turkeys, we sequentially passaged two triple reassortant H3N2 SIV isolates from Minnesota in ten day old specific-pathogen free (SPF) Turkey embryos and tested them in seven-day old Turkey poults. We found that SIV replication in Turkey embryos led to minimal mutations in and around the receptor binding and antigenic sites of the HA molecule, while other gene segments were unchanged. The predominant changes associated with Turkey embryo passage were A223V, V226A and T248I mutations in the receptor-binding and glycosylation sites of the HA molecule. Furthermore, Turkey embryo propagation altered receptor specificity in SIV strain 07-1145. Embryo passaged 07-1145 virus showed a decrease in α2, 6 sialic acid receptor binding compared to the wild type virus. Intranasal infection of wild type SIVs in one-week-old Turkey poults resulted in persistent diarrhea and all the infected birds seroconverted at ten days post infection. The 07-1145 wild type virus also transmitted to age matched in-contact birds introduced one-day post infection. Turkeys infected with embryo passaged viruses displayed no clinical signs and were not transmitted to in-contact poults. Our results suggest that Turkey embryo propagation attenuates recent TR SIVs for infectivity and transmission in one week old Turkeys. Our findings will have important implications in identifying molecular determinants that control the transmission and virulence of TR SIVs in Turkeys and other species., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Quail is considered as an intermediate host for generation of the novel reassortant influenza A viruses (IAVs). In this study, we evaluated the replication ability of the three novel H3N1 reassortant viruses recovered from pandemic H1N1 2009 (pH1N1) and duck H3N2 (dkH3N2) co-infected quail generated from our previous study in embryonated chicken eggs, mammalian (MDCK) and human lung derived (A549) cells. Our study demonstrated that all of the reassortant viruses replicated efficiently in avian and mammalian cells, albeit with slightly lower titers than the parental viruses. Of note, all of the reassortant viruses showed enhanced replication in human lung derived A549 cells compared to their parental viruses. Interestingly, among the reassortant viruses tested, a reassortant virus (P(NA,NS)-DK) containing NA and NS genes derived from pH1N1 and the other genes from dkH3N2 exhibited the highest replication ability in all in vitro models, indicating a high level of gene compatibility of this reassortant virus. Our results highlight the potential role of quail as intermediate hosts for the generation of the viable reassortant viruses with ability to replicate efficiently in avian, mammalian, and particularly human lung derived cells. These findings emphasize the need for the continuous IAV surveillance in quail to prevent the risk of the emergence of the novel viable reassortant viruses., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Reassortment is one of the main mechanisms of evolution in dsRNA viruses with segmented genomes. It contributes to generate genetic diversity and plays an important role in the emergence and spread of new strains with altered virulence. Natural reassorment has been demonstrated among infectious pancreatic necrosis-like viruses (genus Aquabirnavirus, Birnaviridae). In the present study, coinfections between different viral strains, and genome sequencing by the Sanger and Illumina methods were applied to analyze the frequency of reassortment of this virus in vitro, the possible mechanisms involved, and its effect on virulence. Results have demonstrated that reassortment is a cell-dependent and non-random process, probably through differential expression of the different mRNA classes in the ribosomes of a specific cell, and by specific associations between the components to construct the ribonucleoprotein (RNP) complexes and/or RNP cross-inhibition. However, the precise mechanisms involved, known in other viruses, still remain to be demonstrated in birnaviruses., (Copyright © 2016 Elsevier B.V. All rights reserved.)

This study sought to improve an existing live attenuated influenza vaccine (LAIV) by including nucleoprotein (NP) from wild-type virus rather than master donor virus (MDV). H7N9 LAIV reassortants with 6:2 (NP from MDV) and 5:3 (NP from wild-type virus) genome compositions were compared with regard to their growth characteristics, induction of humoral and cellular immune responses in mice, and ability to protect mice against homologous and heterologous challenge viruses. Although, in general, the 6:2 reassortant induced greater cell-mediated immunity in C57BL6 mice than the 5:3 vaccine, mice immunized with the 5:3 LAIV were better protected against heterologous challenge. The 5:3 LAIV-induced CTLs also had better in vivo killing activity against target cells loaded with the NP 366 epitope of recent influenza viruses. Modification of the genome of reassortant vaccine viruses by incorporating the NP gene from wild-type viruses represents a simple strategy to improve the immunogenicity and cross-protection of influenza vaccines., Competing Interests: statement All the authors have declared that have no conflict of interest., (Copyright © 2016 Elsevier Inc. All rights reserved.)

This study investigated the pathogenicity and transmissibility of a reverse-genetics-derived highly pathogenic avian influenza (HPAI) H5N1 lineage influenza A virus that was isolated from a human, A/Iraq/755/06. We also examined surface gene reassortant viruses composed of the haemagglutinin and neuraminidase from A/Iraq/755/06 and the internal genes of a 2009 pandemic H1N1 virus, A/New York/18/2009 (2Iraq/06 : 6NY/09 H5N1), and haemagglutinin and neuraminidase from A/New York/18/2009 with the internal genes of A/Iraq/755/06 (2NY/09 : 6Iraq/06 H1N1). The parental A/Iraq/755/06 caused little to no lesions in swine, limited virus replication was observed in the upper respiratory and lower respiratory tracts and transmission was detected in 3/5 direct-contact pigs based on seroconversion, detection of viral RNA or virus isolation. In contrast, the 2Iraq/06 : 6NY/09 H5N1 reassortant caused mild lung lesions, demonstrated sustained virus replication in the upper and lower respiratory tracts and transmitted to all contacts (5/5). The 2NY/09 : 6Iraq/06 H1N1 reassortant also caused mild lung lesions, there was evidence of virus replication in the upper respiratory and lower respiratory tracts and transmission was detected in all contacts (5/5). These studies indicate that an HPAI-derived H5N1 reassortant with pandemic internal genes may be more successful in sustaining infection in swine and that HPAI-derived internal genes were marginally compatible with pandemic 2009 H1N1 surface genes. Comprehensive surveillance in swine is critical to identify a possible emerging HPAI reassortant in all regions with HPAI in wild birds and poultry and H1N1pdm09 in pigs or other susceptible hosts.