Modeling the mechanisms of acute hepatitis B virus infection

Mathematical models have been used to understand the factors that govern infectious disease progression in viral infections. Here we focus on hepatitis B virus (HBV) dynamics during the acute stages of the infection and analyze the immune mechanisms responsible for viral clearance. We start by presenting the basic model used to interpret HBV therapy studies conducted in chronically infected patients. We then introduce additional models to study acute infection where immune responses presumably play an important role in determining whether the infection will be cleared or become chronic. We add complexity incrementally and explain each step of the modeling process. Finally, we validate the model against experimental data to determine how well it represents the biological system and, consequently, how useful are its predictions. In particular, we find that a cell-mediated immune response plays an important role in controlling the virus after the peak in viral load.     

Amino acid substitutions at positions 242, 255 and 268 in rabies virus glycoprotein affect spread of viral infection

Rabies virus Nishigahara strain kills adult mice after intracerebral inoculation, whereas the derivative RC‐HL strain does not. It has previously been reported by us that the R(G 242/255/268) strain, in which amino acids at positions 242, 255 and 268 on the G protein have been replaced by those from the Nishigahara strain in the genetic background of the RC‐HL strain, kills adult mice. This indicates that these three amino acids of G protein are important for pathogenicity of the Nishigahara strain. In order to obtain insights into the mechanism by which these amino acids affect pathogenicity, in this study spread of viral infection and apoptosis‐inducing ability of the attenuated RC‐HL strain and the virulent R(G 242/255/268) strain were compared. RC‐HL infection spread less efficiently in the mouse brain than did R(G 242/255/268) infection. However, the apoptosis‐inducing abilities of both viruses were almost identical, as shown by both in vitro and in vivo experiments. It was demonstrated that cell‐to‐cell spread of RC‐HL strain was less efficient than that of R(G 242/255/268) strain in mouse neuroblastoma cells. These results indicate that the three amino acid substitutions affect efficiency of cell‐to‐cell spread but not apoptosis‐inducing ability, probably resulting in the distinct distributions of RC‐HL and R(G 242/255/268) strain‐infected cells in the mouse brain and, consequently, the different pathogenicities of these strains.     

Differential usage of carbohydrate co-receptors influences cellular tropism of Theiler's murine encephalomyelitis virus infection of the central nervous system

Theiler's murine encephalomyelitis viruses (TMEV) are ubiquitous pathogens of mice, producing either rapidly fatal encephalitis (high-neurovirulence strains) or persistent central nervous system infection and inflammatory demyelination (low-neurovirulence strains). Although a protein entry receptor has not yet been identified, carbohydrate co-receptors that effect docking and concentration of the virus on the cell surface are known for both TMEV neurovirulence groups. Low-neurovirulence TMEV use α2,3-linked N-acetylneuramic acid (sialic acid) on an N-linked glycoprotein, whereas high-neurovirulence TMEV use the proteoglycan heparan sulfate (HS) as a co-receptor. While the binding of low-neurovirulence TMEV to sialic acid can be inhibited completely, only a third of the binding of high-neurovirulence TMEV to HS is inhibitable, suggesting that high-neurovirulence strains use another co-receptor or bind directly to the putative protein entry receptor. Four amino acids on the surface (VP2 puff B) of low-neurovirulence strains make contact with sialic acid through non-covalent hydrogen bonds. Since these virus residues are conserved in all TMEV strains, the capsid conformation of this region is probably responsible for sialic acid binding. A persistence determinant that maps within the virus coat using recombinant TMEV is also conformational in nature. Low-neurovirulence virus variants that do not bind to sialic acid fail to persist in the central nervous system of mice, indicating a role for sialic acid binding in TMEV persistence. Analysis of high-neurovirulence variants that do not bind HS demonstrates that HS co-receptor usage influences neuronal tropism in brain, whereas, the HS co-receptor use is not required for the infection of spinal cord anterior horn cells associated with poliomyelitis.     

恒河猴离乳幼猴B病毒动态监测

目的调查恒河猴离乳幼猴BV感染的动态变化,探讨早期控制的可能。方法采用BV全病毒为抗原的ELISA试剂盒,对两批离乳后群养的54只恒河猴幼猴每月采血检测BV感染情况,连续检测13个月。结果随月龄的增加,BV感染率上升,其中12月、1月、2月、3月、4月BV阳性猴数量相对增加较明显。结论幼猴性成熟期之前BV感染率超过70％且呈现出一定的变化规律,春、冬季BV的感染率相对较高,应早期加强BV的有效预防控制,降低BV的感染,提高猴群质量。     

Incidence of potato virus Y infection in seed and ware tubers of the potato cv. Record

The incidence of potato virus Y (PVY) infection was assessed in samples of potato tubers, cv. Record, taken from Scottish seed stocks and English ware crops grown from some of these seed stocks. PVY was readily detected by ELISA of tuber sprouts. PVY-infected tubers were found in 10 seed stocks of 84 tested. The mean level of virus infection was 0.23%, 0.76% and 0.56% in Super Elite, Elite and AA stocks respectively. In 46 commercial ware crops grown from some of these seed stocks, a substantial proportion of the harvested tubers in all but one of the crops were infected with PVY, the mean percentage of infected tubers was 58.5%. Ware crops grown from seven seed stocks in which PVY had been detected (mean 6.2% infection in seed) contained a mean of 70% infected tubers, compared with 56% infection in crops grown from 39 stocks in which PVY was not detected in the seed tubers. The predominant PVY strain detected in the ware crops was the veinal necrosis strain (PVY^vn).     

Effect of virus infection on symplastic transport of fluorescent tracers in Nicotiana clevelandii leaf epidermis

The molecular weight exclusion limit of plasmodesmata in subveinal epidermal cells of Nicotiana clevelandii (Gray) leaves was estimated by microinjection and fluorescence microscopy using fluorescein isothiocyanate-peptide conjugates, carboxyfluorescein and Lucifer Yellow CH. The largest fluorochrome which moved symplastically between cells had a molecular weight of 749, although movement did not appear to depend purely on molecular weight parameters. Systemic infection of plants by tobacco rattle tobravirus, tomato black ring nepovirus or potato Y potyvirus did not alter the limits of plasmodesmatal conductance of the fluorochromes. However, carrot mottle umbravirus and groundnut rosette umbravirus diminished the symplastic mobility of some fluorescent tracers. These results imply that intercellular movement of these viruses does not involve a long-lasting increase in the plasmodesmatal molecular size exclusion limit.Abbreviations CMotV carrot mottle umbravirus - GRV groundnut rosette umbravirus - Glu l-glutamate - GluGlu -glutamyl glutamate - FITC fluorescein isothiocyanate - Ala₆ hexa-l-alanine - Gly₆ hexa-l-glycine - PVY potato Y potyvirus - TBRV tomato black ring nepovirus - TRY tobacco rattle tobravirus - TyrGlyGly tyrosylglycylglycine     

The role of volunteer potatoes in the spread of potato virus YN in ware crops of cv. Record

Surveys were made for the presence of potato virus Y (PVY) in the planted seed and harvested tubers in ware potato crops of cv. Record grown at three sites in England in 1994 (survey 1) and seven sites in 1995 (survey 2). PVY was not found in samples of planted seed, but high levels of infection were found in many, but not all, harvested crops. However, plants of volunteer potatoes (VP) (i.e. plants arising from tubers or true seed derived from previous crops and surviving in the soil) were frequently found to be infected. Infection in tubers harvested from crops in the first survey ranged from 2–52%. In 1995, VP were collected from two of the three English sites where potato crops had been grown the previous season and also from a site in Scotland where PVY infection in an experimental crop of cv. Record had been monitored in 1994. The percentages of infected VP ranged from 2–54%. PVY^N was the predominant strain found in sampled VP, with only two plants (out of 300 infected) containing PVY^O. In the second survey, VP were assessed within the 1995 ware crops and were found at four sites, at which they comprised between 4–8% of emerged potato plants. Between 31–93% of VP were infected. Again, PVY^N was the predominant strain with one plant containing PVY^O and another PVY^C (out of 189 infected). A sample of harvested tubers from each site was also tested for PVY. At those sites which had many infected VP, the harvested crop contained a large percentage of infected tubers, ranging from 60–97%. Two sites which had not previously been used for cropping potatoes had no VP and a very low incidence of PVY infection in the harvested tubers (1% and 2%). However, although no VP were found at one site, 31% of harvested tubers were infected, suggesting that alternative inoculum sources may be important.     

Understanding the mechanism of virus removal by Q sepharose fast flow chromatography during the purification of CHO‐cell derived biotherapeutics

During production of therapeutic monoclonal antibodies (mAbs) in mammalian cell culture, it is important to ensure that viral impurities and potential viral contaminants will be removed during downstream purification. Anion exchange chromatography provides a high degree of virus removal from mAb feedstocks, but the mechanism by which this is achieved has not been characterized. In this work, we have investigated the binding of three viruses to Q sepharose fast flow (QSFF) resin to determine the degree to which electrostatic interactions are responsible for viral clearance by this process. We first used a chromatofocusing technique to determine the isoelectric points of the viruses and established that they are negatively charged under standard QSFF conditions. We then determined that virus removal by this chromatography resin is strongly disrupted by the presence of high salt concentrations or by the absence of the positively charged Q ligand, indicating that binding of the virus to the resin is primarily due to electrostatic forces, and that any non‐electrostatic interactions which may be present are not sufficient to provide virus removal. Finally, we determined the binding profile of a virus in a QSFF column after a viral clearance process. These data indicate that virus particles generally behave similarly to proteins, but they also illustrate the high degree of performance necessary to achieve several logs of virus reduction. Overall, this mechanistic understanding of an important viral clearance process provides the foundation for the development of science‐based process validation strategies to ensure viral safety of biotechnology products. Biotechnol. Bioeng. 2009; 104: 371–380 © 2009 Wiley Periodicals, Inc.     

Lymphocyte and neutrophil dysfunction associated with hepatitis B virus and hepatitis non-A, non-B virus infection in the chimpanzee.

Chimpanzees were examined for the effect of viral hepatitis infections on specific and nonspecific immune response mechanisms. The data suggest that infection with either hepatitis B virus or hepatitis non-A, non-B virus may result in suppression of cellular immune response components. Mitogen-induced lymphocyte proliferation was lower in virus-infected chimpanzees than in naive animals. Neutrophils from virus infected animals exhibited decreased or altered chemiluminescence kinetics.     

Selective spread of herpes simplex virus in the central nervous system after ocular inoculation.

The spread of herpes simplex virus (HSV) was studied in the mouse central nervous system (CNS) after ocular inoculation. Sites of active viral replication in the CNS were identified by autoradiographic localization of neuronal uptake of tritiated thymidine. Labeled neurons were first noted in the CNS at 4 days postinoculation in the Edinger-Westphal nucleus, ipsilateral spinal trigeminal nucleus, pars caudalis, pars interpolaris, and ipsilateral dorsal horn of the rostral cervical spinal cord. By 5 days postinoculation, additional sites of labeling included the seventh nerve nucleus, nucleus locus coeruleus, and the nuclei raphe magnus and raphe pallidus. None of these sites are contiguous to nuclei infected at 4 days, but all are synaptically related to these nuclei. By 7 days postinoculation, no new foci of labeled cells were noted in the brain stem, but labeled neurons were noted in the amygdala, hippocampus, and somatosensory cortex. Neurons in both the amygdala and hippocampus receive axonal projections from the locus coeruleus. On the basis of these findings, we conclude that the spread of HSV in the CNS after intracameral inoculation is not diffuse but is restricted to a small number of noncontiguous foci in the brain stem and cortex which become infected in a sequential fashion. Since these regions are synaptically related, the principal route of the spread of HSV in the CNS after ocular infection appears to be along axons, presumably via axonal transport rather than by local spread.     

Association between the frequency of class II HLA antigens and the susceptibility to intrauterine infection of hepatitis B virus

Multiple factors determine the susceptibility to intrauterine hepatitis B virus (HBV) infection. These factors include the HBV structure, HBV mutation, HBV DNA level, placental barrier, the immune status of the mother, and the genetic make-ups of the newborn infants. Since HLA system is an integral component of the immune response, we hypothesized that the highly polymorphic HLA genes are the key determinants of intrauterine HBV infection. In this study, we selected newborn infants of HBsAg-positive mothers, and divided the infants into 2 groups: intrauterine infection group and non-intrauterine infection group according to the status whether or not they were infected at birth. Each infected infant was compared with 2 controls from the same birth cohort. HLA-DR allele typing was performed using a PCR-sequence specific primer (PCR-SSP) for 24 subjects with intrauterine infection and 48 controls without infection. We found that, among the fifteen (15) HLA-DR alleles assessed, HLA-DRB1*07 was the one, and the only one, significantly in excess (OR = 6.66, P = 0.004) in the intrauterine infection group compared to the non-intrauterine infection group. Our findings thus suggest that high frequency of HLA class II molecules, e.g. HLA-DRB1*07, is associated with the susceptibility of the infants to intrauterine HBV infection.     

Synthesis and comparison of substituted 1,2,3-dithiazole and 1,2,3-thiaselenazole as inhibitors of the feline immunodeficiency virus (FIV) nucleocapsid protein as a model for HIV infection

We report the first biological evaluation the 1,2,3-thiaselenazole class of compound and utilising a concise synthetic approach of sulfur extrusion, selenium insertion of the 1,2,3-dithiazoles. We created a small diverse library of compounds to contrast the two ring systems. This approach has highlighted new structure activity relationship insights and lead to the development of sub-micro molar anti-viral compounds with reduced toxicity. The 1,2,3-thiaselenazole represents a new class of potential compounds for the treatment of FIV and HIV.     

Mechanistic insights into viral clearance during the chromatography steps in antibody processes by using virus surrogates

Viral safety is required for biological products to treat human diseases, and the burden of inactivation and or virus removal lies on the downstream purification process. Minute virus of mice (MVM) is a nonenveloped parvovirus commonly used as the worst-case model virus in validation studies because of its small size and high chemical stability. In this study, we investigated the use of MVM-mock virus particle (MVP) and bacteriophage ΦX174 as surrogates for MVM to mimic viral clearance studies, with a focus on chromatography operations. Based on structural models and comparison of log reduction value among MVM, MVP, and ΦX174, it was demonstrated that MVP can be used as a noninfectious surrogate to assess viral clearance during process development in multiple chromatography systems in a biosafety level one (BSL-1) laboratory. Protein A (ProA) chromatography was investigated to strategically assess the impact of the resin, impurities, and the monoclonal antibody product on virus removal.     

Crystal Structure of the Vaccinia Virus Uracil-DNA Glycosylase in Complex with DNA

Vaccinia virus polymerase holoenzyme is composed of the DNA polymerase catalytic subunit E9 associated with its heterodimeric co-factor A20·D4 required for processive genome synthesis. Although A20 has no known enzymatic activity, D4 is an active uracil-DNA glycosylase (UNG). The presence of a repair enzyme as a component of the viral replication machinery suggests that, for poxviruses, DNA synthesis and base excision repair is coupled. We present the 2.7 Å crystal structure of the complex formed by D4 and the first 50 amino acids of A20 (D4·A20_1–50) bound to a 10-mer DNA duplex containing an abasic site resulting from the cleavage of a uracil base. Comparison of the viral complex with its human counterpart revealed major divergences in the contacts between protein and DNA and in the enzyme orientation on the DNA. However, the conformation of the dsDNA within both structures is very similar, suggesting a dominant role of the DNA conformation for UNG function. In contrast to human UNG, D4 appears rigid, and we do not observe a conformational change upon DNA binding. We also studied the interaction of D4·A20_1–50 with different DNA oligomers by surface plasmon resonance. D4 binds weakly to nonspecific DNA and to uracil-containing substrates but binds abasic sites with a K_d of <1.4 μm. This second DNA complex structure of a family I UNG gives new insight into the role of D4 as a co-factor of vaccinia virus DNA polymerase and allows a better understanding of the structural determinants required for UNG action.     

A viral mechanism in acute exacerbations of chronic type B hepatitis: Hepatitis B virus reinfection and subsequent reactivation of two viral strains

Chronic hepatitis B virus (HBV) infections are frequently associated with exacerbations of hepatitis of which the majority are due to reactivation of viral activity. Variation in a viral genome during persistent infection has been shown to be a possible cause for reactivation. In this study, we have found another possible mechanism. HBV in a patient with repeated exacerbations was isolated at six different times during follow-up and was characterized by polymerase chain reaction and DNA sequencing. The first episode of exacerbation was accompanied with increased replication of an HBV strain. The second episode, however, was associated with the sudden appearance of an HBV strain that displayed enough sequence variations to warrant the designation as a separate strain. The results suggested a reinfection event by another independent HBV. Subsequent exacerbations were then related to coactivation of both viral stains. These observations provide significant information toward understanding the acute exacerbations of chronic type B hepatitis.     

Enhanced expression of matrix metalloproteinase-9 by hepatitis B virus infection in liver cells

The hepatitis B virus (HBV) is a major cause of human liver disease, including hepatocellular carcinoma (HCC). The prognosis for HCC is largely dependent on the clinicopathological characteristics regarding invasion and metastasis. Enhanced matrix metalloproteinase-9 (MMP-9) expression has been implicated as playing an important role in metastasis and invasion of HCC. However, the relationship between HBV infection and MMP-9 expression in HCC is currently poorly understood. We report here on a study of the levels of MMP-9 and MMP-2 expression in human fetal liver tissue, rat liver tissue, and Chang, HepG2, and Hep3B cells by gelatin zymography. Among these sources, Hep3B cells, which contain the integrated hepatitis B viral genome, continuously secrete the hepatitis B viral surface antigen, and express HBV genomic RNA, expressed high levels of proMMP-9, and a small amount of active MMP-9 was detected in Hep3B cells as assayed by zymography. We investigated the issue of whether HBV infection affects MMP-9 expression, which is known to play an important role in HCC invasion and metastasis. As a first step, human fetal hepatocyte (HFH) and HepG2 (HCC origin, HBV not detected) cells were subjected to infection with HBV, and the resulting infected cells successfully established are hereafter referred to as HFH-T2 and HepG2-HBV. The expression of MMP-9 was upregulated by the infected HBV in HFH-T2 and HepG2-HBV cells, as assayed by zymography, Northern blot, and Western blot analysis, and small amounts of active MMP-9 were detected in HFH-T2 and HepG2-HBV cells as assayed by zymography. The activation of the immature proMMP-9 to the mature MMP-9 could be induced by plasmin treatment. The activation of proMMP-9 was increased to a greater extent with plasmin treatment than without plasmin in HFH-T2 and HepG2-HBV cells but the addition of recombinant TIMP-1 inhibited the activation of proMMP-9. Finally, the addition of plasmin to the invasion assay using Matrigel resulted in an increase in invasiveness of HFH-T2 and HepG2-HBV cells, as well as MMP-9 activation, but the treatment with TIMP-1 inhibited the invasiveness of HFH-T2 and HepG2-HBV cells as well as MMP-9 activation. We conclude from these findings that HBV infection of hepatocytes and HepG2 cells affected the upregulation of MMP-9 expression and MMP-9 activation and, thus, increased the invasion potential by plasmin. To our knowledge, this is a first report showing that an HBV infection is linked to the upregulation of MMP-9 in HCC.