Reverse Genetic Analysis of Adaptive Mutations within the Capsid Proteins of Enterovirus 71 (EV-A71) Strains Necessary for Infection of CHO-K1 Cells

正Dear Editor,Previous studies had described the adaptation of enterovirus 71 (EV-A71) strains that enabled entry and viral replication in Chinese Hamster Ovary (CHO) cell line(Zaini and Mc Minn 2012; Zaini et al. 2012). These adapted     

Glycan Analysis and Influenza A Virus Infection of Primary Swine Respiratory Epithelial Cells: THE IMPORTANCE OF NeuAc��2�C6 GLYCANS*

To better understand influenza virus infection of pigs, we examined primary swine respiratory epithelial cells (SRECs, the primary target cells of influenza viruses in vivo), as a model system. Glycomic profiling of SRECs by mass spectrometry revealed a diverse range of glycans terminating in sialic acid or GalαGal. In terms of sialylation, α2–6 linkage was more abundant than α2–3, and NeuAc was more abundant than NeuGc. Virus binding and infection experiments were conducted to determine functionally important glycans for influenza virus infection, with a focus on recently emerged swine viruses. Infection of SRECs with swine and human viruses resulted in different infectivity levels. Glycan microarray analysis with a high infectivity “triple reassortant” virus ((A/Swine/MN/593/99 (H3N2)) that spread widely throughout the North American swine population and a lower infectivity human virus isolated from a single pig (A/Swine/ONT/00130/97 (H3N2)) showed that both viruses bound exclusively to glycans containing NeuAcα2–6, with strong binding to sialylated polylactosamine and sialylated N-glycans. Treatment with mannosamine precursors of sialic acid (to alter NeuAc/NeuGc abundances) and linkage-specific sialidases prior to infection indicated that the influenza viruses tested preferentially utilize NeuAcα2–6-sialylated glycans to infect SRECs. Our data indicate that NeuAcα2–6-terminated polylactosamine and sialylated N-glycans are important determinants for influenza viruses to infect SRECs. As NeuAcα2–6 polylactosamine glycans play major roles in human virus infection, the importance of these receptor components in virus infection of swine cells has implications for transmission of viruses between humans and pigs and for pigs as possible adaptation hosts of novel human influenza viruses.     

In vitro and in vivo mutational analysis of the 3'-terminal regions of hepatitis e virus genomes and replicons

Hepatitis E virus (HEV) replication is not well understood, mainly because the virus does not infect cultured cells efficiently. However, Huh-7 cells transfected with full-length genomes produce open reading frame 2 protein, indicative of genome replication (6). To investigate the role of 3'-terminal sequences in RNA replication, we constructed chimeric full-length genomes with divergent 3'-terminal sequences of genotypes 2 and 3 replacing that of genotype 1 and transfected them into Huh-7 cells. The production of viral proteins by these full-length chimeras was indistinguishable from that of the wild type, suggesting that replication was not impaired. In order to better quantify HEV replication in cell culture, we constructed an HEV replicon with a reporter (luciferase). Luciferase production was cap dependent and RNA-dependent RNA polymerase dependent and increased following transfection of Huh-7 cells. Replicons harboring the 3'-terminal intergenotypic chimera sequences were also assayed for luciferase production. In spite of the large sequence differences among the 3' termini of the viruses, replication of the chimeric replicons was surprisingly similar to that of the parental replicon. However, a single unique nucleotide change within a predicted stem structure at the 3' terminus substantially reduced the efficiency of replication: RNA replication was partially restored by a covariant mutation. Similar patterns of replication were obtained when full-length genomes were inoculated into rhesus macaques, suggesting that the in vitro system could be used to predict the effect of 3'-terminal mutations in vivo. Incorporation of the 3'-terminal sequences of the swine strain of HEV into the genotype 1 human strain did not enable the human strain to infect swine.     

Manipulation of the Porcine Epidemic Diarrhea Virus Genome Using Targeted RNA Recombination

Porcine epidemic diarrhea virus (PEDV) causes severe economic losses in the swine industry in China and other Asian countries. Infection usually leads to an acute, often lethal diarrhea in piglets. Despite the impact of the disease, no system is yet available to manipulate the viral genome which has severely hampered research on this virus until today. We have established a reverse genetics system for PEDV based on targeted RNA recombination that allows the modification of the 3′-end of the viral genome, which encodes the structural proteins and the ORF3 protein. Using this system, we deleted the ORF3 gene entirely from the viral genome and showed that the ORF3 protein is not essential for replication of the virus in vitro. In addition, we inserted heterologous genes (i.e. the GFP and Renilla luciferase genes) at two positions in the viral genome, either as an extra expression cassette or as a replacement for the ORF3 gene. We demonstrated the expression of both GFP and Renilla luciferase as well as the application of these viruses by establishing a convenient and rapid virus neutralization assay. The new PEDV reverse genetics system will enable functional studies of the structural proteins and the accessory ORF3 protein and will allow the rational design and development of next generation PEDV vaccines.     

Cleavage of the Feline Calicivirus Capsid Precursor Is Mediated by a Virus-Encoded Proteinase

Feline calicivirus (FCV), a member of the Caliciviridae, produces its major structural protein as a precursor polyprotein from a subgenomic-sized mRNA. In this study, we show that the proteinase responsible for processing this precursor into the mature capsid protein is encoded by the viral genome at the 3′-terminal portion of open reading frame 1 (ORF1). Protein expression studies of either the entire or partial ORF1 indicate that the proteinase is active when expressed either in in vitro translation or in bacterial cells. Site-directed mutagenesis was used to characterize the proteinase Glu-Ala cleavage site in the capsid precursor, utilizing an in vitro cleavage assay in which mutant precursor proteins translated from cDNA clones were used as substrates for trans cleavage by the proteinase. In general, amino acid substitutions in the P1 position (Glu) of the cleavage site were less well tolerated by the proteinase than those in the P1′ position (Ala). The precursor cleavage site mutations were introduced into an infectious cDNA clone of the FCV genome, and transfection of RNA derived from these clones into feline kidney cells showed that efficient cleavage of the capsid precursor by the virus-encoded proteinase is a critical determinant in the growth of the virus.     

Influenza A Virus PB1-F2 Protein Expression Is Regulated in a Strain-Specific Manner by Sequences Located Downstream of the PB1-F2 Initiation Codon

Translation of influenza A virus PB1-F2 occurs in a second open reading frame (ORF) of the PB1 gene segment. PB1-F2 has been implicated in regulation of polymerase activity, immunopathology, susceptibility to secondary bacterial infection, and induction of apoptosis. Experimental evidence of PB1-F2 molecular function during infection has been collected primarily from human and avian viral isolates. As the 2009 H1N1 (H1N1pdm09) strain highlighted, some swine-derived influenza viruses have the capacity to infect human hosts and emerge as a pandemic. Understanding the impact that virulence factors from swine isolates have on both human and swine health could aid in early identification of viruses with pandemic potential. Studies examining PB1-F2 from swine isolates have focused primarily on H1N1pdm09, which does not encode PB1-F2 but was engineered to carry a full-length PB1-F2 ORF to assess the impact on viral replication and pathogenicity. However, experimental evidence of PB1-F2 protein expression from swine lineage viruses has not been demonstrated. Here, we reveal that during infection, PB1-F2 expression levels are substantially different in swine and human influenza viruses. We provide evidence that PB1-F2 expression is regulated at the translational level, with very low levels of PB1-F2 expression from swine lineage viruses relative to a human isolate PB1-F2. Translational regulation of PB1-F2 expression was partially mapped to two independent regions within the PB1 mRNA, located downstream of the PB1-F2 start site. Our data suggest that carrying a full-length PB1-F2 ORF may not be predictive of PB1-F2 expression in infected cells for all influenza A viruses.     

Release of Genotype 1 Hepatitis E Virus from Cultured Hepatoma and Polarized Intestinal Cells Depends on Open Reading Frame 3 Protein and Requires an Intact PXXP Motif

Hepatitis E virus genotype 1 strain Sar55 replicated in subcloned Caco-2 intestinal cells and Huh7 hepatoma cells that had been transfected with in vitro transcribed viral genomes, and hepatitis E virions were released into the culture medium of both cell lines. Virus egress from cells depended on open reading frame 3 (ORF3) protein, and a proline-rich sequence in ORF3 was important for egress from cultured cells and for infection of macaques. Both intracellular ORF3 protein accumulation and virus release occurred at the apical membrane of polarized Caco-2 cells. ORF3 protein and lipids were intimately associated with virus particles produced in either cell line; ORF2 epitopes were masked in these particles and could not be immunoprecipitated with anti-ORF2.Hepatitis E virus (HEV) remains enigmatic in spite of recent advances (see references 7 and 16 for reviews). HEV is a major cause of acute hepatitis in numerous developing countries, but hepatitis E is infrequently detected in industrialized countries even though seroprevalence rates of anti-HEV as high as 20% in these countries have been reported. Although hepatitis E normally is a self-limited acute disease, recent studies have identified it as an emerging cause of chronic hepatitis in immunocompromised patients. Whereas contaminated drinking water is the source of most infections in developing countries, the sources in industrialized countries are not fully evaluated, but many, if not most, infections appear linked to eating undercooked meat, especially pork. These differences in epidemiology may reflect the fact that most infections in developing countries are caused by genotypes 1 and 2 while those in industrialized countries are mainly due to genotypes 3 and 4.HEV was initially classified as a calicivirus, but subsequent sequence analysis suggested that it was more closely related to the enveloped rubella virus. However, although HEV may be associated with lipids under some conditions (22), HEV virions do not possess an envelope. Four genotypes of HEV that infect humans have been identified (4). Genotypes 1 and 2 infect primates exclusively, whereas genotypes 3 and 4 are zoonotic and commonly also infect swine and rarely other nonprimates. Recent identification of a strain infecting farmed rabbits in China suggests that other reservoirs may exist (32).The capsid protein encoded by open reading frame 2 (ORF2) is able to form infectious virus particles, but these particles remain cell associated. The crystal structure of a truncated recombinant protein has been solved, but the size of the protein in mature virions is unknown (11, 15, 28, 31). The virus is not cytopathic, and it is unclear how it gets out of cells.The 7.2-kb genome of HEV is a capped mRNA that contains three ORFs that encode proteins involved in replication (ORF1), a capsid protein (ORF2), and a small protein of only 113 to 114 amino acids (ORF3). All but the 5′ terminus of ORF3 is overlapped by ORF2, and both proteins are translated from the same bicistronic subgenomic RNA (10). When overexpressed in cell culture, ORF2 is glycosylated, and ORF3 is phosphorylated (26); this phosphorylated ORF3 protein binds to nonglycosylated ORF2 protein in cell culture, but phosphorylation is not required for infection of macaques (9). The virus has been exceedingly difficult to propagate in cell culture, but recently Okamoto and colleagues reported the successful adaptation of both a genotype 3 and a genotype 4 strain to efficient growth in cultures of PLC/PRF/5 hepatoma or A549 lung cells (23, 24).The tiny ORF3 protein is particularly intriguing because it has a significant impact on virus propagation through mechanisms that have yet to be defined. Data from experiments performed with overexpressed ORF3 protein have suggested that, among other things, ORF3 may interact with cellular proteins, including signaling proteins containing Src homology 3 domains (14), bikunin (27), hemopexin (21), and microtubule proteins (13), and it may function to modulate the acute-phase disease response (3), protect cells from mitochondrial depolarization (18), and enhance expression of glycolytic pathway enzymes (17). Yet within transfected hepatoma cells in culture, virions of an ORF3 null mutant of genotype 1 were assembled in the absence of ORF3 protein and were infectious for naïve hepatoma cells (6) although this same ORF3 null mutant was unable to mount a detectable infection in rhesus monkeys (8). Also, swine transfected with genotype 3 mutant genomes encoding a truncated ORF3 protein did not get infected, indicating that an intact ORF3 protein is needed for infectivity in vivo (12). This lack of infectivity in vivo is possibly explained by the recent demonstration that the ORF3 protein of genotype 3 virus is important for export of virions out of cultured cells in vitro (30); however, this dependence on ORF3 for virion egress has not been confirmed in vivo or for strains of the other three genotypes.The four major genotypes of human HEV appear to segregate naturally into two distinct groups. One group contains genotype 1 and 2 strains that lack a zoonotic component and are spread mainly via contaminated water; in contrast, the second group contains genotype 3 and 4 strains which are able to cross species boundaries and are zoonotic since humans have been infected as a result of eating undercooked meat (16, 25). The molecular basis for the two groupings is unknown, and much more extensive comparative analyses are required to determine which variables are epidemiologically relevant. Here, for lack of an efficient cell culture system for genotype 1 or 2 strains, we have utilized an infectious cDNA clone of a genotype 1 strain in order to explore the role of the ORF3 protein in this group.     

Feeling manipulated: cytomegalovirus immune manipulation

Background

ORF59 DNA polymerase processivity factor of the human rhadinovirus, Kaposi's sarcoma-associated herpesvirus (KSHV), is required for efficient copying of the genome during virus replication. KSHV ORF59 is antigenic in the infected host and is used as a marker for virus activation and replication.

Results

We cloned, sequenced and expressed the genes encoding related ORF59 proteins from the RV1 rhadinovirus homologs of KSHV from chimpanzee (PtrRV1) and three species of macaques (RFHVMm, RFHVMn and RFHVMf), and have compared them with ORF59 proteins obtained from members of the more distantly-related RV2 rhadinovirus lineage infecting the same non-human primate species (PtrRV2, RRV, MneRV2, and MfaRV2, respectively). We found that ORF59 homologs of the RV1 and RV2 Old World primate rhadinoviruses are highly conserved with distinct phylogenetic clustering of the two rhadinovirus lineages. RV1 and RV2 ORF59 C-terminal domains exhibit a strong lineage-specific conservation. Rabbit antiserum was developed against a C-terminal polypeptide that is highly conserved between the macaque RV2 ORF59 sequences. This anti-serum showed strong reactivity towards ORF59 encoded by the macaque RV2 rhadinoviruses, RRV (rhesus) and MneRV2 (pig-tail), with no cross reaction to human or macaque RV1 ORF59 proteins. Using this antiserum and RT-qPCR, we determined that RRV ORF59 is expressed early after permissive infection of both rhesus primary fetal fibroblasts and African green monkey kidney epithelial cells (Vero) in vitro. RRV- and MneRV2-infected foci showed strong nuclear expression of ORF59 that correlated with production of infectious progeny virus. Immunohistochemical studies of an MneRV2-infected macaque revealed strong nuclear expression of ORF59 in infected cells within the differentiating layer of epidermis corroborating previous observations that differentiated epithelial cells are permissive for replication of KSHV-like rhadinoviruses.

Conclusion

The ORF59 DNA polymerase processivity factor homologs of the Old World primate RV1 and RV2 rhadinovirus lineages are phylogenetically distinct yet demonstrate similar expression and localization characteristics that correlate with their use as lineage-specific markers for permissive infection and virus replication. These studies will aid in the characterization of virus activation from latency to the replicative state, an important step for understanding the biology and transmission of rhadinoviruses, such as KSHV.     

Antiviral Responses by Swine Primary Bronchoepithelial Cells Are Limited Compared to Human Bronchoepithelial Cells Following Influenza Virus Infection

Swine generate reassortant influenza viruses because they can be simultaneously infected with avian and human influenza; however, the features that restrict influenza reassortment in swine and human hosts are not fully understood. Type I and III interferons (IFNs) act as the first line of defense against influenza virus infection of respiratory epithelium. To determine if human and swine have different capacities to mount an antiviral response the expression of IFN and IFN-stimulated genes (ISG) in normal human bronchial epithelial (NHBE) cells and normal swine bronchial epithelial (NSBE) cells was evaluated following infection with human (H3N2), swine (H1N1), and avian (H5N3, H5N2, H5N1) influenza A viruses. Expression of IFNλ and ISGs were substantially higher in NHBE cells compared to NSBE cells following H5 avian influenza virus infection compared to human or swine influenza virus infection. This effect was associated with reduced H5 avian influenza virus replication in human cells at late times post infection. Further, RIG-I expression was lower in NSBE cells compared to NHBE cells suggesting reduced virus sensing. Together, these studies identify key differences in the antiviral response between human and swine respiratory epithelium alluding to differences that may govern influenza reassortment.     

Closely related archaeal Haloarcula hispanica icosahedral viruses HHIV-2 and SH1 have nonhomologous genes encoding host recognition functions

Studies on viral capsid architectures and coat protein folds have revealed the evolutionary lineages of viruses branching to all three domains of life. A widespread group of icosahedral tailless viruses, the PRD1-adenovirus lineage, was the first to be established. A double β-barrel fold for a single major capsid protein is characteristic of these viruses. Similar viruses carrying genes coding for two major capsid proteins with a more complex structure, such as Thermus phage P23-77 and haloarchaeal virus SH1, have been isolated. Here, we studied the host range, life cycle, biochemical composition, and genomic sequence of a new isolate, Haloarcula hispanica icosahedral virus 2 (HHIV-2), which resembles SH1 despite being isolated from a different location. Comparative analysis of these viruses revealed that their overall architectures are very similar except that the genes for the receptor recognition vertex complexes are unrelated even though these viruses infect the same hosts.     

Amino acid 226 in the hemagglutinin of H4N6 influenza virus determines binding affinity for alpha2,6-linked sialic acid and infectivity levels in primary swine and human respiratory epithelial cells

Avian lineage H4N6 influenza viruses previously isolated from pigs differ at hemagglutinin amino acids 226 and 228 from H4 subtype viruses isolated from birds. Using a parental H4N6 swine isolate and hemagglutinin mutant viruses (at residues 226 and/or 228), we determined that viruses which contain L226 had a higher affinity for sialic acid α2,6 galactose (SAα2,6Gal) and a higher infectivity level for primary swine and human respiratory epithelial cells, whereas viruses which contain Q226 had lower SAα2,6Gal affinity and lower infectivity levels for both types of cells. Using specific neuraminidases, we found that irrespective of their relative binding preferences, all of the influenza viruses examined utilized SAα2,6Gal to infect swine and human cells.     

Endoplasmic Reticulum Stress Induced Synthesis of a Novel Viral Factor Mediates Efficient Replication of Genotype-1 Hepatitis E Virus

Hepatitis E virus (HEV) causes acute hepatitis in many parts of the world including Asia, Africa and Latin America. Though self-limiting in normal individuals, it results in ~30% mortality in infected pregnant women. It has also been reported to cause acute and chronic hepatitis in organ transplant patients. Of the seven viral genotypes, genotype-1 virus infects humans and is a major public health concern in South Asian countries. Sporadic cases of genotype-3 and 4 infection in human and animals such as pigs, deer, mongeese have been reported primarily from industrialized countries. Genotype-5, 6 and 7 viruses are known to infect animals such as wild boar and camel, respectively. Genotype-3 and 4 viruses have been successfully propagated in the laboratory in mammalian cell culture. However, genotype-1 virus replicates poorly in mammalian cell culture and no other efficient model exists to study its life cycle. Here, we report that endoplasmic reticulum (ER) stress promotes genotype-1 HEV replication by inducing cap-independent, internal initiation mediated translation of a novel viral protein (named ORF4). Importantly, ORF4 expression and stimulatory effect of ER stress inducers on viral replication is specific to genotype-1. ORF4 protein sequence is mostly conserved among genotype-1 HEV isolates and ORF4 specific antibodies were detected in genotype-1 HEV patient serum. ORF4 interacted with multiple viral and host proteins and assembled a protein complex consisting of viral helicase, RNA dependent RNA polymerase (RdRp), X, host eEF1α1 (eukaryotic elongation factor 1 isoform-1) and tubulinβ. In association with eEF1α1, ORF4 stimulated viral RdRp activity. Furthermore, human hepatoma cells that stably express ORF4 or engineered proteasome resistant ORF4 mutant genome permitted enhanced viral replication. These findings reveal a positive role of ER stress in promoting genotype-1 HEV replication and pave the way towards development of an efficient model of the virus.     

Cell cycle dependence of foamy retrovirus infection.

In common with oncoviruses but unlike the lentivirus human immunodeficiency virus type 1, foamy (spuma) viruses require host cell proliferation for productive infection. We show that human immunodeficiency virus type 1 replicates in RD-CD4 cells regardless of the growth arrest condition of the cells, while murine leukemia virus is unable to infect growth-arrested RD-CD4 cells or cells progressing through a partial cell cycle that includes S phase but not mitosis. Human foamy virus, like murine leukemia virus, does not productively infect G1/S or G2 growth-arrested cells. Two other foamy viruses, simian foamy virus type 1, isolated from a macaque, and simian foamy virus type 6, isolated from a chimpanzee, also fail to establish productive infection in G1/S-arrested cells.     

C-Terminal Amino Acids 471-507 of Avian Hepatitis E Virus Capsid Protein Are Crucial for Binding to Avian and Human Cells

The infection of chickens with avian Hepatitis E virus (avian HEV) can be asymptomatic or induces clinical signs characterized by increased mortality and decreased egg production in adult birds. Due to the lack of an efficient cell culture system for avian HEV, the interaction between virus and host cells is still barely understood. In this study, four truncated avian HEV capsid proteins (ORF2-1 – ORF2-4) with an identical 338aa deletion at the N-terminus and gradual deletions from 0, 42, 99 and 136aa at the C-terminus, respectively, were expressed and used to map the possible binding site within avian HEV capsid protein. Results from the binding assay showed that three truncated capsid proteins attached to avian LMH cells, but did not penetrate into cells. However, the shortest construct, ORF2-4, lost the capability of binding to cells suggesting that the presence of amino acids 471 to 507 of the capsid protein is crucial for the attachment. The construct ORF2-3 (aa339-507) was used to study the potential binding of avian HEV capsid protein to human and other avian species. It could be demonstrated that ORF2-3 was capable of binding to QT-35 cells from Japanese quail and human HepG2 cells but failed to bind to P815 cells. Additionally, chicken serum raised against ORF2-3 successfully blocked the binding to LMH cells. Treatment with heparin sodium salt or sodium chlorate significantly reduced binding of ORF2-3 to LMH cells. However, heparinase II treatment of LMH cells had no effect on binding of the ORF2-3 construct, suggesting a possible distinct attachment mechanism of avian as compared to human HEV. For the first time, interactions between avian HEV capsid protein and host cells were investigated demonstrating that aa471 to 507 of the capsid protein are needed to facilitate interaction with different kind of cells from different species.     

Conditional rather than absolute requirements of the capsid coding sequence for initiation of methionine-independent translation in Plautia stali intestine virus

The positive-stranded RNA genome of Plautia stali intestine virus (PSIV) has an internal ribosome entry site (IRES) in an intergenic region (IGR). The IGR-IRES of PSIV initiates translation of the capsid protein by using CAA, the codon for glutamine. It was previously reported (J. Sasaki and N. Nakashima, J. Virol. 73:1219-1226, 1999) that IGR-IRES extended by several nucleotides into the capsid open reading frame (ORF). Despite the fact that the secondary structure model of the IGR-IRES is highly conserved, we were unable to find structural similarities in the 5' region of the capsid ORFs in related viruses. Therefore, we reevaluated the role of the capsid ORF in IGR-IRES-mediated translation in PSIV. Mutation of the CAA codon with various triplets did not inhibit IGR-IRES-mediated translation. N-terminal amino acid analyses of mutated products showed that the IGR-IRES could initiate translation by using various elongator tRNAs. By replacement of the capsid ORF with exogenous coding sequences having AUG deleted, translation products were produced in most cases, but capsid-exogenous fusion proteins were produced more efficiently than were the translation products. These data indicate that the 5' part of the capsid ORF is not an absolute requirement for the IGR-IRES-mediated translation. RNA structure probing analyses showed that the 5' part of the capsid ORF was a single strand, while that of exogenous reading frames was structured. Exogenous sequences also caused structural distortion in the 3' part of the IGR-IRES. We hypothesize that the single-stranded capsid ORF helps to form the tertiary structure of the IGR-IRES and facilitates precise positioning of ribosomes.     

trans-Encapsidation of a Poliovirus Replicon by Different Picornavirus Capsid Proteins

A trans-encapsidation assay was established to study the specificity of picornavirus RNA encapsidation. A poliovirus replicon with the luciferase gene replacing the capsid protein-coding region was coexpressed in transfected HeLa cells with capsid proteins from homologous or heterologous virus. Successful trans-encapsidation resulted in assembly and production of virions whose replication, upon subsequent infection of HeLa cells, was accompanied by expression of luciferase activity. The amount of luciferase activity was proportional to the amount of trans-encapsidated virus produced from the cotransfection. When poliovirus capsid proteins were supplied in trans, >2 × 10⁶ infectious particles/ml were produced. When coxsackievirus B3, human rhinovirus 14, mengovirus, or hepatitis A virus (HAV) capsid proteins were supplied in trans, all but HAV showed some encapsidation of the replicon. The overall encapsidation efficiency of the replicon RNA by heterologous capsid proteins was significantly lower than when poliovirus capsid was used. trans-encapsidated particles could be completely neutralized with specific antisera against each of the donor virus capsids. The results indicate that encapsidation is regulated by specific viral nucleic acid and protein sequences.     

Tissue tropism of swine influenza viruses and reassortants in ex vivo cultures of the human respiratory tract and conjunctiva

The 2009 pandemic influenza H1N1 (H1N1pdm) virus was generated by reassortment of swine influenza viruses of different lineages. This was the first influenza pandemic to emerge in over 4 decades and the first to occur after the realization that influenza pandemics arise from influenza viruses of animals. In order to understand the biological determinants of pandemic emergence, it is relevant to compare the tropism of different lineages of swine influenza viruses and reassortants derived from them with that of 2009 pandemic H1N1 (H1N1pdm) and seasonal influenza H1N1 viruses in ex vivo cultures of the human nasopharynx, bronchus, alveoli, and conjunctiva. We hypothesized that virus which can transmit efficiently between humans replicated well in the human upper airways. As previously reported, H1N1pdm and seasonal H1N1 viruses replicated efficiently in the nasopharyngeal, bronchial, and alveolar epithelium. In contrast, representative viruses from the classical swine (CS) (H1N1) lineage could not infect human respiratory epithelium; Eurasian avian-like swine (EA) (H1N1) viruses only infected alveolar epithelium and North American triple-reassortant (TRIG) viruses only infected the bronchial epithelium albeit inefficiently. Interestingly, a naturally occurring triple-reassortant swine virus, A/SW/HK/915/04 (H1N2), with a matrix gene segment of EA swine derivation (i.e., differing from H1N1pdm only in lacking a neuraminidase [NA] gene of EA derivation) readily infected and replicated in human nasopharyngeal and bronchial epithelia but not in the lung. A recombinant sw915 with the NA from H1N1pdm retained its tropism for the bronchus and acquired additional replication competence for alveolar epithelium. In contrast to H1N1pdm, none of the swine viruses tested nor seasonal H1N1 had tropism in human conjunctiva. Recombinant viruses generated by swapping the surface proteins (hemagglutinin and NA) of H1N1pdm and seasonal H1N1 virus demonstrated that these two gene segments together are key determinants of conjunctival tropism. Overall, these findings suggest that ex vivo cultures of the human respiratory tract provide a useful biological model for assessing the human health risk of swine influenza viruses.     

An Attenuated Variant of the GDVII Strain of Theiler’s Virus Does Not Persist and Does Not Infect the White Matter of the Central Nervous System

The DA strain of Theiler’s virus causes a persistent and demyelinating infection of the white matter of spinal cord, whereas the GDVII strain causes a fatal gray-matter encephalomyelitis. Studies with recombinant viruses showed that this difference in phenotype is controlled mainly by the capsid. However, conflicting results regarding the existence of determinants of persistence in the capsid of the GDVII strain have been published. Here we show that a GDVII virus whose neurovirulence has been attenuated by an insertion in the 5′ noncoding region does not persist in the central nervous systems of mice. Furthermore, this virus infects the gray matter efficiently, but not the white matter. These results confirm the absence of determinants of persistence in the GDVII capsid. They suggest that the DA capsid controls persistence by allowing the virus to infect cells in the white matter of the spinal cord.