A longitudinal analysis of cytotoxic T lymphocyte precursor frequencies to the hepatitis B virus in chronically infected patients

Individuals with acute hepatitis B virus (HBV) infection characteristically mount a strong, multispecific cytotoxic T lymphocyte (CTL) response that is effective in eradicating virus. In contrast, this response in chronic carriers is usually weak or undetectable. Since it is generally acknowledged that HBV pathogenesis is immune-mediated, the occurrence of episodes of active liver disease in many carriers suggests that these individuals can mount active CTL responses to HBV. To see whether the detection of circulating CTLs is related to these flare episodes, we have determined the CTL precursor (CTLp) frequencies to HLA-A2-restricted viral peptides in seven patients over a 12-24-month period of their disease. Limiting dilution analyses (LDA) were performed longitudinally to five epitopes comprising the viral capsid (HBc), envelope (HBs) and polymerase (pol) proteins. Assays were performed against a mixture of peptides, or against each individual peptide, to measure overall CTL activity and the multispecificity of the responses, respectively. Since two of the patients were treated with recombinant human interleukin-12 (rHuIL-12) at the time, with one individual achieving complete disease remission a year later after being treated with interferon-alpha, we were also able to examine the effects of these cytokines on HBV cytotoxicity. Our results indicate that weak but detectable CTL responses do occur in chronic carriers which are generally associated with disease flares, although CTLps were also seen occasionally during minimal disease activity. The range of specificities varied between individuals and within each individual during the course of the disease. Finally, we also provide evidence that CTL reactivity is stimulated following treatment with certain cytokines, but is dependent on the time of administration.

Rapid transplacental infection with bovine pestivirus following intranasal inoculation of ewes in early pregnancy

Despite the importance of congenital viral infections in both veterinary and human medicine, only limited experimental work has been carried out to elucidate the mechanisms involved in transplacental virus infections. To further an understanding of fetal infection with pestiviruses, the distribution of bovine pestivirus in the uterine and fetal tissues of ewes in early pregnancy, following a natural route of infection, was investigated. On the 18th day of pregnancy, nine ewes were inoculated by the intranasal route with 1 X 10(5) 50% tissue culture infective doses of an Australian isolate of noncytopathic bovine pestivirus (bovine viral diarrhea virus genotype 1). All ewes were ovariohysterectomized at approximately 100 hours postinfection. Samples from the reproductive tract and conceptus were examined histologically and tested for bovine pestivirus by nested reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry and for interferon-tau mRNA expression by nonnested RT-PCR. Although no histopathologic changes were observed in the maternal or fetal tissues, virus was detected in the reproductive tract of all nine ewes and in all of the conceptuses examined. Al; the time of surgery, only two of the nine ewes were demonstrably viremic. This study demonstrates that bovine pestivirus can spread from a natural site of infection to the ovine fetus within 4 days in the absence of maternal immunity and despite the presence of interferon expression in the reproductive tract.

Significance in replication of the terminal nucleotides of the Flavivirus genome

Point mutations that resulted in a substitution of the conserved 3'-penultimate cytidine in genomic RNA or the RNA negative strand of the self-amplifying replicon of the Flavivirus Kunjin virus completely blocked in vivo replication. Similarly, substitutions of the conserved 3'-terminal uridine in the RNA negative or positive strand completely blocked replication or caused much-reduced replication, respectively. The same preference for cytidine in the 3'-terminal dinucleotide was noted in reports of the in vitro activity of the RNA-dependent RNA polymerase (RdRp) for the other genera of Flaviviridae that also employ a double-stranded RNA (dsRNA) template to initiate asymmetric semiconservative RNA positive-strand synthesis. The Kunjin virus replicon results were interpreted in the context of a proposed model for initiation of RNA synthesis based on the solved crystal structure of the RdRp of phi6 bacteriophage, which also replicates efficiently using a dsRNA template with conserved 3'-penultimate cytidines and a 3'-terminal pyrimidine. A previously untested substitution of the conserved pentanucleotide at the top of the 3'-terminal stem-loop of all Flavivirus species also blocked detectable in vivo replication of the Kunjin virus replicon RNA.

The medicine and epidemiology of bovine respiratory disease in feedlots

Bovine Respiratory Disease (BRD) results from a complex, multifactorial interaction of stressors, animal susceptibility, and respiratory pathogens. The infectious agents associated with BRD are ubiquitous among cattle populations. Typically, one or a combination of stressors are necessary to initiate BRD. Prevention of BRD should, therefore, address management procedures to minimise stressors. Administration of vaccines against BRD agents may help reduce the incidence of BRD but is unlikely to eliminate the condition. The effectiveness of antimicrobials in the treatment of BIRD depends primarily on early recognition and treatment. The use of antioxidant vitamins, minerals or other agents in the prevention and treatment of BRD warrants further research.

Comparative mechanistic studies of de novo RNA synthesis by flavivirus RNA-dependent RNA polymerases

Flavivirus protein NS5 harbors the RNA-dependent RNA polymerase (RdRp) activity. In contrast to the RdRps of hepaci- and pestiviruses, which belong to the same family of Flaviviridae, NS5 carries two activities, a methyltransferase (MTase) and a RdRp. RdRp domains of Dengue virus (DV) and West Nile virus (WNV) NS5 were purified in high yield relative to full-length NS5 and showed full RdRp activity. Steady-state enzymatic parameters were determined on homopolymeric template poly(rC). The presence of the MTase domain does not affect the RdRp activity. Flavivirus RdRp domains might bear more than one GTP binding site displaying positive cooperativity. The kinetics of RNA synthesis by four Flaviviridae RdRps were compared. In comparison to Hepatitis C RdRp, DV and WNV as well as Bovine Viral Diarrhea virus RdRps show less rate limitation by early steps of short-product fort-nation. This suggests that they display a higher conformational flexibility upon the transition from initiation to elongation. (c) 2006 Elsevier Inc. All rights reserved.

Translation of the flavivirus Kunjin NS3 gene in cis but not its RNA sequence or secondary structure is essential for efficient RNA packaging

Our previous studies using trans-complementation analysis of Kunjin virus (KUN) full-length cDNA clones harboring in-frame deletions in the NS3 gene demonstrated the inability of these defective complemented RNAs to be packaged into virus particles (W. J. Liu, P. L. Sedlak, N. Kondratieva, and A. A. Khromykh, J. Virol. 76:10766-10775). In this study we aimed to establish whether this requirement for NS3 in RNA packaging is determined by the secondary RNA structure of the NS3 gene or by the essential role of the translated NS3 gene product. Multiple silent mutations of three computer-predicted stable RNA structures in the NS3 coding region of KUN replicon RNA aimed at disrupting RNA secondary structure without affecting amino acid sequence did not affect RNA replication and packaging into virus-like particles in the packaging cell line, thus demonstrating that the predicted conserved RNA structures in the NS3 gene do not play a role in RNA replication and/or packaging. In contrast, double frameshift mutations in the NS3 coding region of full-length KUN RNA, producing scrambled NS3 protein but retaining secondary RNA structure, resulted in the loss of ability of these defective RNAs to be packaged into virus particles in complementation experiments in KUN replicon-expressing cells. Furthermore, the more robust complementation-packaging system based on established stable cell lines producing large amounts of complemented replicating NS3-deficient replicon RNAs and infection with KUN virus to provide structural proteins also failed to detect any secreted virus-like particles containing packaged NS3-deficient replicon RNAs. These results have now firmly established the requirement of KUN NS3 protein translated in cis for genome packaging into virus particles.

Asymptomatic primary Epstein-Barr virus infection occurs in the absence of blood T-cell repertoire perturbations despite high levels of systemic viral load

Primary infection with the human herpesvirus, Epstein-Barr virus (EBV), may result in subclinical seroconversion or may appear as infectious mononucleosis (IM), a lymphoproliferative disease of variable severity. Why primary infection manifests differently between patients is unknown, and, given the difficulties in identifying donors undergoing silent seroconversion, little information has been reported. However, a longstanding assumption has been held that IM represents an exaggerated form of the virologic and immunologic events of asymptomatic infection. T-cell receptor (TCR) repertoires of a unique cohort of subclinically infected patients undergoing silent infection were studied, and the results highlight a fundamental difference between the 2 forms of infection. In contrast to the massive T-cell expansions mobilized during the acute symptomatic phase of IM, asymptomatic donors largely maintain homeostatic T-cell control and peripheral blood repertoire diversity. This disparity cannot simply be linked to severity or spread of the infection because high levels of EBV DNA were found in the blood from both types of acute infection. The results suggest that large expansions of T cells within the blood during IM may not always be associated with the control of primary EBV infection and that they may represent an overreaction that exacerbates disease. (C) 2001 by The American Society of Hematology.

The murine cytomegalovirus chemokine homolog, m131/129, is a determinant of viral pathogenicity

Chemokines are important mediators of the early inflammatory response to infection and modify a wide range of host immune responses. Functional homologs of cellular chemokines have been identified in a number of herpesviruses, suggesting that the subversion of the host chemokine response contributes to the pathogenesis of these viruses. Transcriptional and reverse transcription-PCR analyses demonstrated that the murine cytomegalovirus (MCMV) chemokine homolog, m131, was spliced at the 3' end to the adjacent downstream open reading frame, m129, resulting in a predicted product of 31 kDa, which is significantly larger than most known chemokines. The in vivo impact of m131/129 was investigated by comparing the replication of MCMV mutants having m131/129 deleted (Delta m131/129) with that of wild-type (wt) MCMV. Our studies demonstrate that both wt and Delta m131/129 viruses replicated to equivalent levels during the first 2 to 3 days following in vivo infection. However, histological studies demonstrated that the early inflammatory response elicited by Delta m131/129 was reduced compared with that of wt MCMV. Furthermore, the Delta m131/129 mutants failed to establish a high-titer infection in the salivary glands, These results suggest that m131/129 possesses proinflammatory properties in vivo and is important for the dissemination of MCMV to or infection of the salivary gland. Notably, the Delta m131/129 mutants were cleared more rapidly from the spleen and liver during acute infection compared with wt MCMV. The accelerated clearance of the mutants was dependent on NK cells and cells of the CD4(+) CD8(+) phenotype. These data suggest that m131/129 may also contribute to virus mechanisms of immune system evasion during early infection, possibly through the interference of NK cells and T cells.

Gag-Pol supplied in trans is efficiently packaged and supports viral function in human immunodeficiency virus type 1

The intracellular trafficking and subsequent incorporation of Gag-Pol into human immunodeficiency virus type 1 (HIV-1) remains poorly defined. Gag-Pol is encoded by the same mRNA as Gag and is generated by ribosomal frameshifting. The multimerization of Gag and Gag-Pol is an essential step in the formation of infectious viral particles. In this study, we examined whether the interaction between Gag and Gag-Pol is initiated during protein translation in order to facilitate the trafficking and subsequent packaging of Gag-Pol into the virion. A conditional cotransfection system was developed in which virion formation required the coexpression of two HIV-1-based plasmids, one that produces both Gag and Gag-Pol and one that only produces Gag-Pol. The Gag-Pol proteins were either immunotagged with a His epitope or functionally tagged with a mutation (K65R) in reverse transcriptase that is associated with drug resistance. Gag-Pol packaging was assessed to determine whether the Gag-Pol incorporated into the virion was preferentially packaged from the plasmid that expressed both Gag and Gag-Pol or whether it could be packaged from either plasmid. Our data show that translation of Gag and Gag-Pol from the same mRNA is not critical for virion packaging of the Gag-Pol polyprotein or for viral function.

The influence of viral genotypes and rejection episodes on the recurrence of hepatitis C after liver transplantation

Purpose. The aim of this study was to report the influence of hepatitis C virus (HCV) genotype and rejection episodes on the outcome of orthotopic liver transplantation (OLT), hepatitis recurrence, and progression to graft cirrhosis after OLT. Methods. Fifty-three patients who all had undergone OLT for end-stage liver cirrhosis were selected for this study. Hepatitis C genotype was determined. Recurrent hepatitis and rejection were diagnosed based on elevated liver function tests and a liver biopsy. Results. The patients were followed up for a mean of 51.9 +/- 34.3 months. The cumulative survival rate was no different in OLT for hepatitis C and OLT for all other liver diseases. After OLT, serum HCV RNA was detected in 93%. Histological recurrence occurred in 85% of all patients. The 1-, 3-, and 5-year recurrence rates were 48%, 77%, and 85%, respectively. Of the 41 patients with recurrent hepatitis C, 4 (10%) had cirrhosis, 18 (44%) had hepatitis with fibrosis, and 91 (46%) had hepatitis without fibrosis at the end of follow-up. A total of 32% of the patients were infected by HCV genotype 1b and 68% by other HCV genotypes. The recurrence rates were significantly higher in patients infected with genotype 1b than in those with other genotypes (p = 0.04). Twenty of 48 patients (42%) experienced acute rejection. There was a strong association between the number of rejection episodes and the incidence of HCV-related cirrhosis (p < 0.01). Conclusion. Our findings showed the genotype 1b to result in a higher recurrence rate after OLT. On the other hand, rejection episodes were associated with a more rapid progression to graft cirrhosis.

Contemporaneous fluctuations in T cell responses to persistent herpes virus infections

The classical paradigm for T cell dynamics suggests that the resolution of a primary acute virus infection is followed by the generation of a long-lived pool of memory T cells that is thought to be highly stable. Very limited alteration in this repertoire is expected until the immune system is re-challenged by reactivation of latent viruses or by cross-reactive pathogens. Contradicting this view, we show here that the T cell repertoire specific for two different latent herpes viruses in the peripheral blood displayed significant contemporaneous co-fluctuations of virus-specific CD8(+) T cells. The coordinated responses to two different viruses suggest that the fluctuations within the T cell repertoire may be driven by sub-clinical viral reactivation or a more generalized 'bystander' effect. The later contention was supported by the observation that, while absolute number of CD3(+) T cells and their subsets and also the cell surface phenotype of antigen-specific T cells remained relatively constant, a loss of CD62L expression in the total CD8(+) T cell population was coincident with the expansion of tetramer-positive virus-specific T cells. This study demonstrates that the dynamic process of T cell expansion and contractions in persistent viral infections is not limited to the acute phase of infection, but also continues during the latent phase of infection.

NS1 protein secretion during the acute phase of West Nile virus infection

The West Nile virus (WNV) nonstructural protein NS1 is a protein of unknown function that is found within, associated with, and secreted from infected cells. We systematically investigated the kinetics of NS1 secretion in vitro and in vivo to determine the potential use of this protein as a diagnostic marker and to analyze NS1 secretion in relation to the infection cycle. A sensitive antigen capture enzyme-linked immunosorbent assay (ELISA) for detection of WNW NS1 (polyclonal-ACE) was developed, as well as a capture ELISA for the specific detection of NS1 multimers (4G4-ACE). The 4G4-ACE detected native NS1 antigens at high sensitivity, whereas the polyclonal-ACE had a higher specificity for recombinant forms of the protein. Applying these assays we found that only a small fraction of intracellular NS1 is secreted and that secretion of NS1 in tissue culture is delayed compared to the release of virus particles. In experimentally infected hamsters, NS1 was detected in the serum between days 3 and 8 postinfection, peaking on day 5, the day prior to the onset of clinical disease; immunoglobulin M (IgM) antibodies were detected at low levels on day 5 postinfection. Although real-time PCR gave the earliest indication of infection (day 1), the diagnostic performance of the 4G4-ACE was comparable to that of real-time PCR during the time period when NS1 was secreted. Moreover, the 4G4-ACE was found to be superior in performance to both the IgM and plaque assays during this time period, suggesting that NS1 is a viable early diagnostic marker of WNV infection.

A systematic review and meta-analysis of the diagnostic accuracy of rapid immunochromatographic assays for the detection of dengue virus IgM antibodies during acute infection

A meta-analysis of rapid (

Frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections

Viruses are the major cause of pediatric acute respiratory tract infection (ARTI) and yet many suspected cases of infection remain uncharacterized. We employed 17 PCR assays and retrospectively screened 315 specimens selected by season from a predominantly pediatric hospital-based population. Before the Brisbane respiratory virus research study commenced, one or more predominantly viral pathogens had been detected in 15.2% (n = 48) of all specimens. The Brisbane study made an additional 206 viral detections, resulting in the identification of a microbe in 67.0% of specimens. After our study, the majority of microbes detected were RNA viruses (89.9%). Overall, human rhinoviruses (HRVs) were the most frequently identified target (n=140) followed by human adenoviruses (HAdVs; n = 25), human metapneumovirus (HMPV; n=18), human bocavirus (HBoV; n = 15), human respiratory syncytial virus (HRSV; n = 12), human coronaviruses (HCoVs; n = 11), and human herpesvirus-6 (n = 11). HRVs were the sole microbe detected in 37.8% (n = 31) of patients with suspected lower respiratory tract infection (LRTI). Genotyping of the HRV VP4/VP2 region resulted in a proposed subdivision of HRV type A into sublineages A1 and A2. Most of the genotyped HAdV strains were found to be type C. This study describes the high microbial burden imposed by HRVs, HMPV, HRSV, HCoVs, and the newly identified virus, HBoV on a predominantly paediatric hospital population with suspected acute respiratory tract infections and proposes a new formulation of viral targets for future diagnostic research studies.

Seropositivity to Rabbit Haemorrhagic Disease Virus in non-target mammals during periods of viral activity in a population of wild rabbits in New Zealand

As part of a longitudinal study of the epidemiology of rabbit haemorrhagic disease virus (RHDV) in New Zealand, serum samples were obtained from trapped feral animals that may have consumed European rabbit (Oryctolagus cuniculus) carcasses (non-target species). During a 21-month period when RHDV infection was monitored in a defined wild rabbit population, 16 feral house cats (Felis catus), 11 stoats (Mustela erminea), four ferrets (Mustela furo) and 126 hedgehogs (Erinaceus europaeus) were incidentally captured in the rabbit traps. The proportions of samples that were seropositive to RHDV were 38% for cats, 18% for stoats, 25% for ferrets and 4% for hedgehogs. Seropositive non-target species were trapped in April 2000, in the absence of an overt epidemic of rabbit haemorrhagic disease (RHD) in the rabbit population, but evidence of recent infection in rabbits was shown. Seropositive non-target species were found up to 2.5 months before and 1 month after this RHDV activity in wild rabbits was detected. Seropositive predators were also trapped on the site between 1 and 4.5 months after a dramatic RHD epidemic in February 2001. This study has shown that high antibody titres can be found in non-target species when there is no overt evidence of RHDV infection in the rabbit population, although a temporal relationship could not be assessed statistically owning to the small sample sizes. Predators and scavengers might be able to contribute to localised spread of RHDV through their movements.