Infection of Human B Lymphocytes with Lymphocryptoviruses Related to Epstein-Barr Virus

Lymphocryptoviruses (LCVs) naturally infecting Old World nonhuman primates are closely related to the human LCV, Epstein-Barr virus (EBV), and share similar genome organization and sequences, biologic properties, epidemiology, and pathogenesis. LCVs can efficiently immortalize B lymphocytes from the autologous species, but the ability of a given LCV to immortalize B cells from other Old World primate species is variable. We found that LCV from rhesus monkeys did not immortalize human B cells, and EBV did not immortalize rhesus monkey B cells. In this study, baboon LCV could not immortalize human peripheral blood B cells but could readily immortalize rhesus monkey B cells. Thus, efficient LCV-induced B-cell immortalization across distant Old World primate species appears to be restricted by a species-specific block. To further characterize this species restriction, we first cloned the rhesus monkey LCV major membrane glycoprotein and discovered that the binding epitope for the EBV receptor, CD21, was highly conserved. Stable infections of human B cells with recombinant amplicons packaged in rhesus monkey or baboon LCV envelopes were also consistent with a species-restricted block occurring after virus binding and penetration. Transient infections of human B cells with simian LCV resulted in latent LCV EBNA-2 gene expression and activation of cell CD23 gene expression. EBV-immortalized human B cells could be coinfected with baboon LCV, and the simian virus persisted and replicated in human B cells. Thus, several lines of evidence indicate that the species restriction for efficient LCV-induced B-cell immortalization occurs beyond virus binding and penetration. This has important implications for the study of LCV infection in Old World primate models and for human xenotransplantation where simian LCVs may be inadvertently introduced into humans.     

The Role of Influenza A Virus Hemagglutinin Residues 226 and 228 in Receptor Specificity and Host Range Restriction

Influenza A viruses can be isolated from a variety of animals, but their range of hosts is restricted. For example, human influenza viruses do not replicate in duck intestine, the major replication site of avian viruses in ducks. Although amino acids at positions 226 and 228 of hemagglutinin (HA) of the H3 subtype are known to be important for this host range restriction, the contributions of specific amino acids at these positions to restriction were not known. Here, we address this issue by generating HAs with site-specific mutations of a human virus that contain different amino acid residues at these positions. We also let ducks select replication-competent viruses from a replication-incompetent virus containing a human virus HA by inoculating animals with 10^10.5 50% egg infectious dose of the latter virus and identified a mutation in the HA. Our results showed that the Ser-to-Gly mutation at position 228, in addition to the Leu-to-Gln mutation at position 226 of the HA of the H3 subtype, is critical for human virus HA to support virus replication in duck intestine.     

Host Cell Regulation of Induction of Epstein-Barr Virus

When Epstein-Barr virus (EBV) negative cells (Raji) were treated with iododeoxyuridine, only the early antigen (EA) component was induced. There was no significant increase in EBV DNA and no virus particles were observed. Somatic-cell hybrids were prepared from the fusion of Raji and D98 cells (D98/Raji). When these cells were treated with iododeoxyuridine, early antigen EBV DNA, and virus particles were synthesized. These data suggest cellular control over the expression of the EBV genome.     

Cucumber Vein Yellowing Virus; Host Range and Virus Vector Relationships

Cucumber vein yellowing virus (CVYV) is transmitted by Bemisia tabaci, it has a narrow host range restricted to some cucurbitaceous plants including Cucumis sativus, C. melo, C. melo var. flexousus, Cucurbita pepo, C. foesti, Citrullus vulgaris, C. colocynthis and Lagenaria siceraria. Although a single whitefly can transmit the disease, the efficiency of transmission was low. At least 15–20 insects per plant were required to cause an infection of 55 % of inoculated plants. The minimum acquisition and inoculation feeding periods were 30 and 15 min, respectively. The latent period in the vector is about 75 min and the whitefly was infectious for not more than 5 h.     

Avoiding Immunity and Apoptosis: Manipulation of the Host Environment by Herpes Simplex Virus and Epstein-Barr Virus

Herpesviruses infect their hosts in early life to establish latent or persistent infection with little damage to the host. They are able to reactivate throughout the life of the host and replicate to produce an infective innoculum in the face of a fully primed immune system. They achieve this lifestyle by careful manipulation of the host environment. Here we review two aspects of host defense—apoptosis of infected cells and cytotoxic T cell recognition of infected cells. We discuss avoidance of these host responses by an α-herpesvirus, herpes simplex virus, and a γ-herpesvirus, Epstein-Barr virus.     

Identification of FAM111A as an SV40 Host Range Restriction and Adenovirus Helper Factor

The small genome of polyomaviruses encodes a limited number of proteins that are highly dependent on interactions with host cell proteins for efficient viral replication. The SV40 large T antigen (LT) contains several discrete functional domains including the LXCXE or RB-binding motif, the DNA binding and helicase domains that contribute to the viral life cycle. In addition, the LT C-terminal region contains the host range and adenovirus helper functions required for lytic infection in certain restrictive cell types. To understand how LT affects the host cell to facilitate viral replication, we expressed full-length or functional domains of LT in cells, identified interacting host proteins and carried out expression profiling. LT perturbed the expression of p53 target genes and subsets of cell-cycle dependent genes regulated by the DREAM and the B-Myb-MuvB complexes. Affinity purification of LT followed by mass spectrometry revealed a specific interaction between the LT C-terminal region and FAM111A, a previously uncharacterized protein. Depletion of FAM111A recapitulated the effects of heterologous expression of the LT C-terminal region, including increased viral gene expression and lytic infection of SV40 host range mutants and adenovirus replication in restrictive cells. FAM111A functions as a host range restriction factor that is specifically targeted by SV40 LT.     

Herpes Simplex Virus 1 Counteracts Tetherin Restriction via Its Virion Host Shutoff Activity

The interferon-inducible membrane protein tetherin (Bst-2, or CD317) is an antiviral factor that inhibits enveloped virus release by cross-linking newly formed virus particles to the producing cell. The majority of viruses that are sensitive to tetherin restriction appear to be those that acquire their envelopes at the plasma membrane, although many viruses, including herpesviruses, envelope at intracellular membranes, and the effect of tetherin on such viruses has been less well studied. We investigated the tetherin sensitivity and possible countermeasures of herpes simplex virus 1 (HSV-1). We found that overexpression of tetherin inhibits HSV-1 release and that HSV-1 efficiently depletes tetherin from infected cells. We further show that the virion host shutoff protein (Vhs) is important for depletion of tetherin mRNA and protein and that removal of tetherin compensates for defects in replication and release of a Vhs-null virus. Vhs is known to be important for HSV-1 to evade the innate immune response in vivo. Taken together, our data suggest that tetherin has antiviral activity toward HSV-1 and that the removal of tetherin by Vhs is important for the efficient replication and dissemination of HSV-1.     

EB病毒相关胃癌中病毒分型的研究

收集236例胃癌组织标本及135份健康人群咽漱液(throat washings,TWs)标本,采用原位杂交、PCR-Southern blot筛选出17例EB病毒相关胃癌(EBVaGC)(7.2%)和33例EBV阳性的TWs标本(24.4%);应用PCR-RFLP、巢式PCR及DNA测序等方法,检测EBV阳性标本病毒1/2分型、F/f分型、I/i分型及LMP1XhoI(+)/(-)等四种基因变异。EBVaGC及健康对照均为F型变异,未检测到f型变异。EBVaGC中1/2型、I/i型及LMP1XhoI(+)/(-)型的例数及比例分别为:17(100%)/0(0)、1(5.9%)/16(94.1%)及0(0)/15(88.2%);而TWs中上述分型的相应数据为25(75.8%)/8(24.2%)、11(33.3%)/19(57.6%)及12(36.4%)/18(54.5%),各位点两种基因型在EBVaGC和健康人中的分布不同(1/2:P=0.047;I/i:P=0.048;XhoI(+)/(-):P=0.012)。综合分析表明在3种基因多态性均能确定的标本中,EBVaGC均为1/i/XhoI(-)亚型(15/1...     

Analysis of Host Range Restriction Determinants in the Rabbit Model: Comparison of Homologous and Heterologous Rotavirus Infections

The main limitation of both the rabbit and mouse models of rotavirus infection is that human rotavirus (HRV) strains do not replicate efficiently in either animal. The identification of individual genes necessary for conferring replication competence in a heterologous host is important to an understanding of the host range restriction of rotavirus infections. We recently reported the identification of the P type of the spike protein VP4 of four lapine rotavirus strains as being P[14]. To determine whether VP4 is involved in host range restriction in rabbits, we evaluated infection in rotavirus antibody-free rabbits inoculated orally with two P[14] HRVs, PA169 (G6) and HAL1166 (G8), and with several other HRV strains and animal rotavirus strains of different P and G types. We also evaluated whether the parental rhesus rotavirus (RRV) (P5B[3], G3) and the derived RRV-HRV reassortant candidate vaccine strains RRV × D (G1), RRV × DS-1 (G2), and RRV × ST3 (G4) would productively infect rabbits. Based on virus shedding, limited replication was observed with the P[14] HRV strains and with the SA11 Cl3 (P[2], G3) and SA11 4F (P6[1], G3) animal rotavirus strains, compared to the homologous ALA strain (P[14], G3). However, even limited infection provided complete protection from rotavirus infection when rabbits were challenged orally 28 days postinoculation (DPI) with 10³ 50% infective doses of ALA rabbit rotavirus. Other HRVs did not productively infect rabbits and provided no significant protection from challenge, in spite of occasional seroconversion. Simian RRV replicated as efficiently as lapine ALA rotavirus in rabbits and provided complete protection from ALA challenge. Live attenuated RRV reassortant vaccine strains resulted in no, limited, or productive infection of rabbits, but all rabbits were completely protected from heterotypic ALA challenge. The altered replication efficiency of the reassortants in rabbits suggests a role for VP7 in host range restriction. Also, our results suggest that VP4 may be involved in, but is not exclusively responsible for, host range restriction in the rabbit model. The replication efficiency of rotavirus in rabbits also is not controlled by the product of gene 5 (NSP1) alone, since a reassortant rotavirus with ALA gene 5 and all other genes from SA11 was more severely replication restricted than either parental rotavirus strain.Rotaviruses are the leading cause of acute viral gastroenteritis in humans and animals throughout the world. Rotaviruses belong to the Reoviridae family and are characterized by a genome consisting of 11 segments of double-stranded RNA (dsRNA), enclosed in a triple-layered protein capsid (28). Serotype designations are based on independent neutralization determinants on the two outer capsid proteins VP4 (P serotypes, for protease-sensitive protein) and VP7 (G serotypes, for glycoprotein) (28). Serotype specificity determined by cross-neutralization assays using hyperimmune sera against the whole virus is mainly defined by VP7, and 14 G serotypes have been identified (28). Recently, antisera or monoclonal antibodies raised to VP4 and sequence analysis of VP4 identified 12 P serotypes and 20 P genotypes, respectively (28, 39). Rotavirus VP4 protein is responsible for a number of important biological functions, such as the enhancement of infectivity by proteolytic cleavage of VP4 into VP8* and VP5*, hemagglutination, restricted growth in cell culture, virulence, initial virus attachment to cells, and protease sensitivity associated with plaque formation (1, 4, 25, 34, 40, 51).The use of animal models, including the rabbit and mouse models, has been essential to the understanding of rotavirus infection, pathology, disease, immunity, and testing of prospective vaccines in children (21). The limitations of the rabbit and adult mouse models of rotavirus infection for vaccine testing are as follows: (i) human rotavirus (HRV) strains do not efficiently replicate in either animal, (ii) clinical disease is not observed, and (iii) only homologous virus strains (isolated from the same species) replicate efficiently and spread horizontally to uninoculated control animals, whereas heterologous virus strains (isolated from a different species) do not (6, 15, 16, 29, 31, 35, 37, 44, 50, 55). We and others developed a rabbit model of rotavirus infection that is useful for defining basic parameters of active immunity, immunogenicity, and protective efficacy of vaccines (12, 15–21, 36, 61). Rabbits are productively infected with homologous lapine rotavirus strains up to at least the age of 5 years, which allows examination of active and long-term immunity for vaccine studies (13, 15–17, 36, 61). Group A lapine rotavirus strains have been isolated in Canada, Japan, Italy, and the United States, and those that have been characterized are serotype G3 (8, 11, 15, 53, 56, 61). Recently, the P type of four different strains was identified as genotype P[14] (11). Previously, limited infection of rabbits with a heterologous strain had been obtained only with SA11 Cl3 (P[2], G3) (15).Attempts to identify host range and virulence determinants for rotavirus have implicated different constellations of genes, including genes 2, 3, 4, 5, 8, 9, 10, and 11 (5, 23, 30, 33, 37, 38, 41, 43, 44, 60, 62, 65). Although host range restriction and virulence may be multigenic, two genes, 4 and 5, are of interest because they cluster according to species of origin, suggesting a role in host range restriction. The finding that genome segment 5 (NSP1) sequences cluster according to species of origin (24, 39, 65) and that, in the mouse model, gene 5 segregates with transmission of virus among littermates (5), led to the hypothesis that NSP1 is involved in host range restriction. VP4 sequence analyses of rotavirus strains isolated from different species revealed that specific VP4 types also generally correlate with the species of origin of each rotavirus strain (43, 60). Therefore, once we identified the P type of four lapine rotaviruses as P[14], we tested two P[14] HRV strains, PA169 (G6) and HAL1166 (G8) (32) to determine if VP4 is involved in host range restriction. We also tested several other HRV strains, live attenuated reassortant candidate vaccine strains [rhesus rotavirus (RRV) × D (G1), RRV × DS-1 (G2), and RRV × ST3 (G4)], and animal rotavirus strains of different P and G types to determine if they could productively infect rabbits. In addition, to evaluate whether the single rotavirus gene 5 is responsible for replication efficiency in rabbits, rabbits were inoculated with a reassortant rotavirus with the lapine ALA gene 5 and all the other genes from the simian rotavirus SA11 Cl3 strain.     

Adaptive Gene Amplification As an Intermediate Step in the Expansion of Virus Host Range

The majority of recently emerging infectious diseases in humans is due to cross-species pathogen transmissions from animals. To establish a productive infection in new host species, viruses must overcome barriers to replication mediated by diverse and rapidly evolving host restriction factors such as protein kinase R (PKR). Many viral antagonists of these restriction factors are species specific. For example, the rhesus cytomegalovirus PKR antagonist, RhTRS1, inhibits PKR in some African green monkey (AGM) cells, but does not inhibit human or rhesus macaque PKR. To model the evolutionary changes necessary for cross-species transmission, we generated a recombinant vaccinia virus that expresses RhTRS1 in a strain that lacks PKR inhibitors E3L and K3L (VVΔEΔK+RhTRS1). Serially passaging VVΔEΔK+RhTRS1 in minimally-permissive AGM cells increased viral replication 10- to 100-fold. Notably, adaptation in these AGM cells also improved virus replication 1000- to 10,000-fold in human and rhesus cells. Genetic analyses including deep sequencing revealed amplification of the rhtrs1 locus in the adapted viruses. Supplying additional rhtrs1 in trans confirmed that amplification alone was sufficient to improve VVΔEΔK+RhTRS1 replication. Viruses with amplified rhtrs1 completely blocked AGM PKR, but only partially blocked human PKR, consistent with the replication properties of these viruses in AGM and human cells. Finally, in contrast to AGM-adapted viruses, which could be serially propagated in human cells, VVΔEΔK+RhTRS1 yielded no progeny virus after only three passages in human cells. Thus, rhtrs1 amplification in a minimally permissive intermediate host was a necessary step, enabling expansion of the virus range to previously nonpermissive hosts. These data support the hypothesis that amplification of a weak viral antagonist may be a general evolutionary mechanism to permit replication in otherwise resistant host species, providing a molecular foothold that could enable further adaptations necessary for efficient replication in the new host.     

The Host Restriction of Haemoproteus columbae

SYNOPSIS. Sporozoites of Haemoproteus columbae failed to produce an erythrocytic infection in 4 Columba palumbus , tho 2 C. livia (the natural host of H. columbae ), inoculated with part of the same material, developed parasitemia. H. columbae formed oocysts in all of 6 Ornithomyia avicularia (the natural vector of the haemoproteid of C. palumbus ), but most of the oocysts appeared degenerate and no sporozoites were seen in the 4 O. avicularia which survived for 10–19 days.     

综述: 病毒与宿主细胞的糖基化修饰及相关功能

病毒的复制和对宿主的入侵与自身结构蛋白的糖基化修饰密切相关．对于宿主而言,在病毒感染宿主和宿主抗病毒的过程中,宿主的糖基化过程一方面可抑制病毒的复制和入侵,另一方面可促进病毒对宿主的感染,抑制宿主糖苷酶可抑制病毒的复制．从病毒方面来看,由于病毒自身缺乏糖基化修饰系统,病毒的糖基化过程是借宿主细胞内的合成系统对自身进行糖基化修饰．病毒的糖基化过程对病毒蛋白的折叠与稳定、病毒的感染和入侵、参与识别宿主细胞受体和参与病毒的免疫逃逸等过程起着重要的作用．随着糖基化研究技术的发展,以糖基化为基础的功能应用也越来越深入：如新型病毒疫苗和新型抗病毒药物的研制,以糖蛋白质组学研究为基础的质谱技术和生物信息学方法的发展,以及利用糖基化对病毒性疾病的诊断和治疗等,这些均为糖基化深入研究发展奠定了基础．本文就病毒与宿主细胞糖基化过程、相关功能以及研究应用等进展作一综述．     

Host Restriction and Transduction in Rhizobium meliloti

A host restriction difference exists between Rhizobium meliloti Rm41 and SU47 exists as indicated by the reduce plating efficiency of transducing phage ΦM12h1. Restriction can be attenuated by incubating cells at 42°C for 3 h; this procedure overcomes a block to transduction from SU47 to Rm41.