Patterns Of Viral Replication Research Articles

Development of a SYBR Green-Based RT-qPCR Assay for the Detection and Quantification of Lone Star Virus

Objective: Lone Star virus (LSV) is a newly characterized arbovirus with pathogenic potential. However, no detection methods are available to specifically identify and monitor LSV. Methods: Two SYBR green-based RT-qPCRs were developed and validated for the detection and quantification of LSV. Two primer sets were evaluated for reproducibility, specificity, and sensitivity. The primer sets were applied to monitor viral titers in vitro and via surveillance of LSV in collected ticks. Results: One primer set (LSV-S) was the most specific for LSV when tested against 11 other vector-borne viruses. While less specific, the LSV-M primer set detected <2 copies/μl of the viral genome and <1 copy/μL of the viral genome. Viral replication quantified with either primer set yielded similar viral replication patterns, indicating that both primer sets are sufficient to monitor viral titers in vitro. Plaque assays in human and non-human primate cell lines yielded no replicable plaques and could not be used for comparisons of viral titer quantification. LSV was not detected in the 143 ticks collected from southeast Texas. Conclusion: The SYBR green-based RT-qPCRs described herein can be utilized for the detection and monitoring of LSV for laboratory and tick surveillance purposes.

Scientific Evaluation of the Remedies Used in Turkish Folk Medicine to Treat Possible Viral Infections

Background: Plants, with their rich phytochemical treasury, are one of the main sources of drug development. However, a randomized search on plant sources for the discovery of phytochemicals with specified pharmacological activity is expensive and tedious. Therefore, it is logical to constrain the sources to increase success. In the recent four decades, the importance of traditional information has been recognized by the scientific community. Consequently, interest in field surveys for the documentation of traditional worldwide medicines has significantly increased. Aims and Methods: Infectious diseases induced by pathogenic bacteria, fungi, or viruses have been one of the most common health problems for the public, for which traditional remedies have been practiced. Since laypeople could not distinguish the microbial origin of the infection, the remedies used for treatment could not be delineated. The aim of this study was first to search the plants used in Turkish folk medicine for such infectious diseases. The second step was to find scientific evidence in the online databases for the frequently quoted plants whether they may have potential activity against virus replication. Results: A reference survey on the most frequently quoted plants revealed that 16 out of 17 were shown to possess virucide or inhibitory effects on the replication of various viruses. Conclusion: Since each virus type may have a different viral replication pattern, further detailed investigations should be carried out to reveal their exact antiviral potentials.

Abnormalities in red blood cell production and pathogenesis of anemia in the progression of rock bream iridovirus (RBIV)

Rock bream iridovirus (RBIV), belonging to Megalocytivirus, causes severe mortality in rock bream. Almost all deaths associated with RBIV are accompanied by splenic enlargement and anemia. Although red blood cells (RBCs) are involved in the immune response against viral infections, their involvement in rock bream has not yet been studied in terms of the immune response against RBIV. In this study, the viral replication patterns, blood characteristics and anemia-related factors were evaluated in rock bream post RBIV infection. The virus-infected RBCs of rock bream demonstrated similarities in the expression levels of hemoglobins (HGB) (α and β), cytokine-dependent hematopoietic cell linker (CLNK) and hematopoietic transcription factor GATA (GATA), with significantly decreasing levels from 4 days post infection (dpi) to 17 (dpi), when the viral replication was at its peak. This suggests that the expression of blood-related genes is inadequate for HGB synthesis and RBC production, thereby causing anemia leading to death. Moreover, the levels of complete blood cell count (CBC) indicators, such as RBCs, HGB and hematocrit (HCT), significantly decreased from 10 to 17 dpi. This phenomenon suggests that blood-related gene expression and/or RBC-, HGB- and HCT-related levels are critical factors in RBIV-induced anemia and disease progression. These results highlight the significance of blood-mediated immune responses against RBIV infection in rock bream. Understanding blood-related gene levels to identify blood-related immune response interactions in rock bream will be useful for development of future strategies in controlling RBIV diseases in rock bream.

Evaluation of differentially expressed genes during replication using gene expression landscape of monkeypox-infected MK2 cells: A bioinformatics and systems biology approach to understanding the genomic pattern of viral replication

PurposeThe current outbreak of monkeypox (MPX) has created colossal concerns. However, immense research gaps have been noted in our understanding of the replication process, machinery, and genomic landscape during host cell infection. To fill this gap, differentially expressed genes (DEGs) were comprehensively analyzed during viral replication in host (MK2) cells. MethodsWe used a microarray GEO dataset which was divided into three groups: control, MPXV-infected MK2 cells at 3 h, and MPXV-infected MK2 cells at 7 h. Using the dataset, DEG analysis, PPI network analysis, co-expression, and pathway analysis were conducted using bioinformatics, systems biology, and statistical approaches. ResultsWe identified 250 DEGs and 24 top-ranked genes. During the DEG analysis, we identified eight up-regulated genes (LOC695323, TMEM107, LOC695427, HIST1H2AD, LOC705469, PMAIP1, HIST1H2BJ, and HIST1H3D) and 16 down-regulated genes (HOXA9, BAMBI, LMO4, PAX6, AJUBA, CREBRF, CD24, JADE1, SLC7A11, EID2, SOX4, B4GALT5, PPARGC1A, BUB3, SOS2, and CDK19). We also developed PPI networks and performed co-expression analyses using the top-ranked genes. Furthermore, five genes were listed for co-expression pattern analysis. ConclusionsThis study will help in better understanding the replication process, machinery, and genomic landscape of the virus. This will further aid the discovery and development of therapeutics against viruses.

Evidence of Transcriptional Shutoff by Pathogenic Viral Haemorrhagic Septicaemia Virus in Rainbow Trout.

The basis of pathogenicity of viral haemorrhagic septicaemia virus (VHSV) was analysed in the transcriptome of a rainbow trout cell line inoculated with pathogenic and non-pathogenic VHSV isolates. Although both VHSV isolates showed similar viral replication patterns, the number of differentially expressed genes was 42-fold higher in cells inoculated with the non-pathogenic VHSV at 3 h post inoculation (hpi). Infection with the non-pathogenic isolate resulted in Gene Ontologies (GO) enrichment of terms such as immune response, cytokine-mediated signalling pathway, regulation of translational initiation, unfolded protein binding, and protein folding, and induced an over-representation of the p53, PPAR, and TGF-β signalling pathways. Inoculation with the pathogenic isolate resulted in the GO enrichment of terms related to lipid metabolism and the salmonella infection KEGG pathway involved in the rearrangement of the cytoskeleton. Antiviral response was evident at 12hpi in cells infected with the pathogenic isolate. Overall, the data showed a delay in the response of genes involved in immune responses and viral sensing in cells inoculated with the pathogenic isolate and suggest transcriptional shutoff and immune avoidance as a critical mechanism of pathogenicity in VHSV. These pathways offer opportunities to further understand and manage VHSV pathogenicity in rainbow trout.

A porcine alveolar macrophage cell line stably expressing CD163 demonstrates virus replication and cytokine secretion characteristics similar to primary alveolar macrophages following PRRSV infection

The in vitro investigation of cytokine secretion induced by porcine reproductive and respiratory syndrome virus (PRRSV) requires porcine alveolar macrophages (PAMs) and their interaction with immunocytes. However, immortalized monoclonal PAMs (mPAMs) are non-permissive for PRRSV infection. The porcine CD163 receptor isolated from primary PAMs (pPAMs) confers susceptibility to PRRSV infection; thus, this approach could be used to establish a novel cell line to facilitate the exploration of PRRSV infection kinetics. Here, we amplified the coding region of the CD163 gene from pPAMs and integrated it into an mPAM line using a lentivirus expression system. After verification, the monoclonal PAM cell line stably expressing CD163 (mPAM-CD163-GFP) was infected with either the highly pathogenic PRRSV strain JXA1 or the classical PRRSV strain SD1, which produced high infectious titers of progeny virus reaching > 109 copies/mL or a 50 % tissue culture infective dose of 105.5 over at least 100 cell generations. We also investigated cytokine and Toll-like receptor expression in infected mPAM-CD163-GFP cells and pPAMs. The mPAM-CD163-GFP cell line showed similar patterns of viral replication and cytokine secretion compared with pPAMs, so it may be extremely useful for replacing primary cells for in vitro investigations of the mechanisms of cytokine secretion and interactions between PRRSV-infected PAMs and immunocytes.

The Oncolytic Herpes Simplex Virus Talimogene Laherparepvec Shows Promising Efficacy in Neuroendocrine Cancer Cell Lines

Metastatic neuroendocrine cancer still constitutes a palliative situation, lacking promising treatment options. Oncolytic virotherapy, a novel type of virus-based immunotherapy, lyses tumor cells using genetically engineered viruses thereby activating the immune system to induce an optimized antitumor response which could bring down tumor masses to a stage of minimal residual tumor disease. The oncolytic vector talimogene laherparepvec (T-VEC, herpes simplex virus [HSV] type 1) has already shown excellent safety profiles in clinical studies and has become the first ever FDA/EMA-approved oncolytic virus (OV). This work presents a first preclinical assessment of this state-of-the-art OV, using a panel of human neuroendocrine tumor/neuroendocrine carcinoma (NET/NEC) cell lines. Cytotoxicity, transgene expression, and viral replication patterns were studied. Furthermore, the antiproliferative activity was compared to the one of mTOR inhibitor Everolimus and also interactions between the OV and Everolimus were evaluated. Moreover, virostatic effects of ganciclovir (GCV) on replication of T-VEC were assessed and electron microscopic pictures were taken to comprehend viral envelopment and details of the replication cycle of T-VEC in human neuroendocrine cancer. It could be shown that T-VEC infects, replicates in, and lyses human NET/NEC cells exhibiting high oncolytic efficiencies already at quite low virus concentrations. Interestingly, Everolimus was not found to have any relevant impact on rates of viral replication, but no additive effects could be proved using a combinatorial therapy regimen. On the other hand, GCV was shown to be able to limit replication of T-VEC, thus establishing an important safety feature for future treatments of NET/NEC patients. Taken together, T-VEC opens up a promising novel treatment option for NET/NEC patients, warranting its further preclinical and clinical development.

Early Expansion of CD56dim NKG2Alow with Late Surge and Persistence of CD56dimNKG2AnegNKG2Cbright NK Cells Attenuate Cytomegalovirus (CMV) Replication and Recurrence As Well As Leukemia Relapse Following Haploidentical HSCT with T Cell Co-Stimulation Blockade and Ptcy

CMV related morbidity and mortality remains a major limiting factor in Haploidentical HSCT. We have developed a novel approach to Haploidentical PBSCT combining T cell Costimulation Blockade (T-COSB) with PTCy and Cyclosporine or Sirolimus (BBMT 2018). We studied the pattern of CMV reactivation (CMV-R) and its progression in 60 patients undergoing Haploidentical HSCT in this protocol in relation to the kinetics of Immune Reconstitution (IR) and pattern of leukemia relapse. PCR based CMV monitoring was carried out until 180 days with preemptive anti-CMV therapy. All patients and donors were CMV seropositive. CMV-R was 54.2% with median onset at 32 days (14-75) in the study group [compared to 52.5% in 42 who had received PTCy alone (p=0.8)]. The median peak viral load was 5.7 × 103/ml (1.8-25.8). The median duration of anti-CMV therapy was 16 days (7-60 days). There was no recurrence noted before100 days and only 2/32 (6.2%) beyond 100 days with no CMV disease. CMV-R was higher in those transplanted for malignant diseases compared to non-malignant diseases (62.5% vs 8.2%, p=0.01). Analysis of IR in relation to CMV-R showed no difference in CD3 or CD4+T cell reconstitution. However, CD56dimCD16+ NK cells at D+60 were significantly higher in patients without CMV-R (median 95 vs 45 cells/µl, p=0.02) and the same population of cells were associated with lower peak of viral load (p=0.04). In those with a lower viral peak and lower duration of viral replication, CD56dimCD16+ NK cells had a higher KIR repertoire with lower NKG2A expression. In those with CMV-R, a subsequent surge in absolute NK cells was observed (median 188 vs 112 cells/µl, p=0.01). This was primarily contributed by massive expansion of CD56dimNKG2AnegNKG2Cbright NK cells (median 7.8% [5-14.6%] at day +30 to 35% [28-65%] at day +90, p=0.01, Fig 1). This phenomenon was not witnessed in patients treated with PTCy alone. Peak viral load inversely correlated with NKG2C+ NK cells and CD45RO+CD8+ T cells at day +90 (p=0.03). The incidence of aGVHD (10.2%), cGVHD (18%), NRM (9.5%), and overall survival (75.8%) were not influenced by CMV-R. Incidence of relapse was 23.5 % at 28-144 days (median 85 days). However, none of the patients who relapsed before 100 days had proliferation of NKG2C+ NK cells and only 1/38 patients with proliferation and persistence of this subpopulation beyond 100 days experienced leukemia relapse at a median follow-up of 18 months (p=0.01). The kinetics of NK cell reconstitution in relation to the pattern of viral replication suggests that an early surge of CD56dimNKG2Alow NK cells with cytotoxic potential is responsible for the initial containment of viral replication followed by late surge and persistence of memory type NKG2C+NK cells preventing late recurrence as well as leukemia relapse. This unique synaptic response was possibly responsible for improved outcome with this novel approach of T-COSB based haploidentical HSCT.

Alternate routes of influenza A virus infection in Mallard (Anas platyrhynchos)

The natural reservoir for all influenza A viruses (IAVs) is wild birds, particularly dabbling ducks. During the autumn, viral prevalence can be very high in dabbling ducks (> 30%) in the Northern Hemisphere, and individuals may be repeatedly infected. Transmission and infection is through the fecal–oral route, whereby birds shed viruses in feces and conspecifics are infected though feeding in virus-contaminated water. In this study we wanted to assess two alternative infection routes: cloacal drinking and preening. Using experimental infections, we assessed patterns of infection using a combination of virus shedding, as assessed by real-time PCR from cloacal swabs, and patterns of viral replication using virus-immunohistochemistry of gastrointestinal tissues. The cloacal drinking experiment consisted of two trials using cloacal inoculation at two different time points to account for age differences, as well as a trial whereby ducks were allowed to take up virus-laden water through the cloaca. All ducks became infected, and rather than the bursa of Fabricius being the main site of replication, the colon had the highest intensity of replication, as inferred through immunohistochemistry. In experiments assessing preening, feathers were contaminated with virus-laden water and all ducks became infected, regardless of whether they were kept individually or together. Further, naive contacts were infected by the individuals whose feathers were virus-contaminated. Overall, we reinforce that IAV transmission in dabbling ducks is multifactorial—if exposed to virus-contaminated water ducks may be infected through dabbling, preening of infected feathers, and cloacal drinking.

Transcriptomic analysis of spleen infected with infectious salmon anemia virus reveals distinct pattern of viral replication on resistant and susceptible Atlantic salmon (Salmo salar)

The infectious salmon anemia virus (ISAv) produces a systemic infection in salmonids, causing large losses in salmon production. However, little is known regarding the mechanisms exerting disease resistance. In this paper, we perform an RNA-seq analysis in Atlantic salmon challenged with ISAv (using individuals coming from families that were highly susceptible or highly resistant to ISAv infection). We evaluated the differential expression of both host and ISAv genes in a target organ for the virus, i.e. the spleen. The results showed differential expression of host genes related to response to stress, immune response and protein folding (genes such as; atf3, mhc, mx1-3, cd276, cd2, cocs1, c7, il10, il10rb, il13ra2, ubl-1, ifng, ifngr1, hivep2, sigle14 and sigle5). An increased protein processing activity was found in susceptible fish, which generates a subsequent unfolded protein response. We observed extreme differences in the expression of viral segments between susceptible and resistant groups, demonstrating the capacity of resistant fish to overcome the virus replication, generating a very low viral load. This phenomenon and survival of this higher resistant fish seem to be related to differences in immune and translational process, as well as to the increase of HIV-EP2 (hivep2) transcript in resistant fish, although the causal mechanism is yet to be discovered. This study provides valuable information about disease resistance mechanisms in Atlantic salmon from a host-pathogen interaction point of view.

The Transcription and Translation Landscapes during Human Cytomegalovirus Infection Reveal Novel Host-Pathogen Interactions.

Viruses are by definition fully dependent on the cellular translation machinery, and develop diverse mechanisms to co-opt this machinery for their own benefit. Unlike many viruses, human cytomegalovirus (HCMV) does suppress the host translation machinery, and the extent to which translation machinery contributes to the overall pattern of viral replication and pathogenesis remains elusive. Here, we combine RNA sequencing and ribosomal profiling analyses to systematically address this question. By simultaneously examining the changes in transcription and translation along HCMV infection, we uncover extensive transcriptional control that dominates the response to infection, but also diverse and dynamic translational regulation for subsets of host genes. We were also able to show that, at late time points in infection, translation of viral mRNAs is higher than that of cellular mRNAs. Lastly, integration of our translation measurements with recent measurements of protein abundance enabled comprehensive identification of dozens of host proteins that are targeted for degradation during HCMV infection. Since targeted degradation indicates a strong biological importance, this approach should be applicable for discovering central host functions during viral infection. Our work provides a framework for studying the contribution of transcription, translation and degradation during infection with any virus.

Molecular characteristics and functional analysis of full-length hepatitis B virus quasispecies from a patient with chronic hepatitis B virus infection

It has been hypothesized that naturally occurring mutations in HBV may play a role in the pathogenesis of HBV-related disease. We determined the molecular characteristics of naturally occurring HBV isolates and performed a functional analysis of full-length hepatitis B virus quasispecies from a patient with chronic hepatitis B. The 10 HBV clones isolated were identified as HBV genotype B4 and subtype adw. In most clones, amino acid substitutions and nucleotide changes occurred in a specific region of the core protein, X protein and in the core promoter. In the core protein, cI3L, cL60M, and cI97L were detected in 8 of 10 clones and cP130T was detected in all 10 clones. In the X protein, xI127M was detected in 5 clones. In the basal core promoter, the A1762T/G1764A mutation was found only in 1 clone. An 11-bp nucleotide insertion between nucleotides 1772 and 1773, was found in 2 clones. Six clones that were replication-competent exhibited variation in the level of replicating capacity in vitro even though the average genetic distance between the HBV clones was only 0.5% (range: 0.3–0.7%). Among the replication-competent 6 clones, 5 clones showed higher replication competency compared with clone B9 (reference clone used in this study) in Huh7 cells. However, 4 clones showed lower replication competency compared with clone B9 in HepG2 cells. In conclusion, the HBV virus exhibits genetic variation in the form of quasispecies with different mutation patterns, and these quasispecies may be recognized by distinct viral replication patterns even in patients with subtle genetic mutation.

Establishment of mousemodel for human metapneumovirus infection

：Objective To establish amouse model for human metapneumovirus (hMPV) infec-tion, and to explore the course ofviral replication and pathology and lay ground for vaccine development. Methods BALB/cmice were infected intranasally with hMPV, and sacrificed on day 1,3, 5, 7, 9 and 16 postinoculation. Lungs were used for viral isolation, viral titration by an improved plaqueassays, pulmonary histopathology and detection of hMPV mRNA expression by RT-PCR. ResultsLive viruses were successful-ly isolated from the lungs of infected mice, hMPV replicatewell in the lungs and viral titers peaked on the 5th day post inoculation (5.16 ± 1.09)× 105PFU/g, remaining to be detectable on the 9th day post inocu- lation (2.79 ± 1.22) ×102PFU/g. Genomic RNA of hMPV was able to be detected on the 16th day post in-oculation byRT-PCR targeting F gene. Histopathology of lungs was characterized by interstitialpneumonia in particular during 3 ～ 7 days post inoculation. Conclusion hMPV infected BALB/cmice model was success-fully established, hMPV infected mice showed similar patterns ofviral replication and pulmonary histopathol-ogy as in infected humans. Key words: Human metapneumovirus; BALB/c mice; Animal model

Cellular and cytokine responses in feathers of chickens vaccinated against Marek's disease

In Marek's disease virus infection, feather follicle epithelium (FFE) constitutes the site of formation of infectious virus particles and virus shedding. The objective of this study was to characterize cellular and cytokine responses as indicators of cell-mediated immune response in FFE and associated feather pulp following immunization against Marek's disease. Analysis of feather tips collected between 4 and 28 days post-immunization (d.p.i.) from chickens vaccinated post-hatch with either CVI988/Rispens or herpesvirus of turkeys revealed that replication of these vaccine viruses started at 7 d.p.i., peaked by 21 d.p.i., and subsequently, showed a declining trend. This pattern of viral replication, which led to viral genome accumulation in feather tips, was associated with infiltration of T cell subsets particularly CD8+ T cells into the feather pulp area and the expression of cytokine genes such as interferon-γ, which is an indication of elicitation of cell-mediated immune responses at the site of virus shedding.

AcMNPV ac143 ( odv-e18) is essential for mediating budded virus production and is the 30th baculovirus core gene

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac143 ( odv-e18) is a late gene that encodes for a predicted 9.6 kDa structural protein that locates to the occlusion derived viral envelope and viral induced intranuclear microvesicles [Braunagel, S.C., He, H., Ramamurthy, P., and Summers, M.D. (1996). Transcription, translation, and cellular localization of three Autographa californica nuclear polyhedrosis virus structural proteins: ODV-E18, ODV-E35, and ODV-EC27. Virology 222, 100–114.]. In this study we demonstrate that ac143 is actually a previously unrecognized core gene and that it is essential for mediating budded virus production. To examine the role of ac143 in the baculovirus life cycle, we used the AcMNPV bacmid system to generate an ac143 knockout (KO) virus (AcBAC ac142 REP -ac143KO ). Fluorescence and light microscopy showed that infection by AcBAC ac142 REP- ac143KO is limited to a single cell and titration assays confirmed that AcBAC ac142 REP- ac143KO was unable to produce budded virus (BV). Progression to very late phases of the viral infection was evidenced by the development of occlusion bodies in the nuclei of transfected cells. This correlated with the fact that viral DNA replication was unaffected in AcBAC ac142 REP- ac143KO transfected cells. The entire ac143 promoter, which includes three late promoter motifs, is contained within the ac142 open reading frame. Different deletion mutants of this region showed that the integrity of the ac142-ac143 core gene cluster was required for the bacmids to display wild-type patterns of viral replication, BV production and RNA transcription.

Simian immunodeficiency viruses replication dynamics in African non‐human primate hosts: common patterns and species‐specific differences

To define potential common features of simian immunodeficiency virus (SIV) infections in different naturally infected host species, we compared the dynamics of viral replication in 31 African green monkeys (10 sabeus, 15 vervets and seven Caribbean AGMs), 14 mandrills and three sooty mangabeys (SMs) that were experimentally infected with their species-specific viruses. After infection, these SIVs replicated rapidly reaching viral loads (VLs) of 10(5)-10(9) copies/ml of plasma between days 9-14 post-infection (p.i). Set point viremia was established between days 42 and 60 p.i., with levels of approximately 10(5)-10(6) copies/ml in SM and mandrills, and lower levels (10(3)-10(5) copies/ml) in AGMs. VL during the chronic phase did not correlate with viral genome structure: SIVmnd-2 (a vpx-containing virus) and SIVmnd-1 (which does not contain vpu or vpx) replicated to similar levels in mandrills. VL was dependent on virus strain: vervets infected with three different viral strains showed different patterns of viral replication. The pattern of viral replication of SIVagm.sab, which uses both CCR5 and CXCR4 co-receptors was similar to those of the other viruses. Our results show a common pattern of SIV replication in naturally and experimentally infected hosts. This is similar overall to that observed in pathogenic SIV infection of macaques. This result indicates that differences in clinical outcome between pathogenic and non-pathogenic infections rely on host responses rather than the characteristics of the virus itself.

Longitudinal Assessment of Human Immunodeficiency Virus Type 1 (HIV-1)-Specific Gamma Interferon Responses during the First Year of Life in HIV-1-Infected Infants

Human immunodeficiency virus type 1 (HIV-1) infection results in different patterns of viral replication in pediatric compared to adult populations. The role of early HIV-1-specific responses in viral control has not been well defined, because most studies of HIV-1-infected infants have been retrospective or cross-sectional. We evaluated the association between HIV-1-specific gamma interferon (IFN-gamma) release from the cells of infants of 1 to 3 months of age and peak viral loads and mortality in the first year of life among 61 Kenyan HIV-1-infected infants. At 1 month, responses were detected in 7/12 (58%) and 6/21 (29%) of infants infected in utero and peripartum, respectively (P = 0.09), and in approximately 50% of infants thereafter. Peaks of HIV-specific spot-forming units (SFU) increased significantly with age in all infants, from 251/10(6) peripheral blood mononuclear cells (PBMC) at 1 month of age to 501/10(6) PBMC at 12 months of age (P = 0.03), although when limited to infants who survived to 1 year, the increase in peak HIV-specific SFU was no longer significant (P = 0.18). Over the first year of life, infants with IFN-gamma responses at 1 month had peak plasma viral loads, rates of decline of viral load, and mortality risk similar to those of infants who lacked responses at 1 month. The strength and breadth of IFN-gamma responses at 1 month were not significantly associated with viral containment or mortality. These results suggest that, in contrast to HIV-1-infected adults, in whom strong cytotoxic T lymphocyte responses in primary infection are associated with reductions in viremia, HIV-1-infected neonates generate HIV-1-specific CD8+-T-cell responses early in life that are not clearly associated with improved clinical outcomes.

Delayed central nervous system virus suppression during highly active antiretroviral therapy is associated with HIV encephalopathy, but not with viral drug resistance or poor central nervous system drug penetration.

HIV-1 encephalopathy (HIVE) is associated with high levels of viral RNA in the central nervous system (CNS). Highly active antiretroviral therapy (HAART) effectively reduces HIV replication in both plasma and cerebrospinal fluid (CSF). Some individuals, however, exhibit delayed CSF HIV RNA suppression in the presence of rapid plasma responses. We investigated the reasons for this discrepancy. CSF and plasma were collected prospectively in paired samples before and once or several times during HAART in 40 HIV-positive subjects. Ten had HIVE and 30 patients were neurologically asymptomatic or had non-HIVE neurological manifestations. The slopes of viral RNA decay during HAART were compared between the compartments. The presence of HIVE was defined by clinical standards and its severity categorized according to the Memorial Sloan Kettering score. CSF and plasma levels of antiretroviral drugs were measured. Viral drug resistance during HAART in CSF and plasma was analysed both genotypically and phenotypically. Slow CSF viral decay and a high degree of compartmental discordance (slopeCSF/slopeplasma) were both significantly correlated with HIVE (P < 0.00002). There was no correlation of a rapid CSF response with Centers for Disease Control and Prevention stage, CD4 cell count, or with the number of antiretroviral compounds and their known CSF penetration. Slow CSF viral decay was associated with neither low levels of antiretroviral drugs in the CSF or plasma, nor with viral drug resistance. None of the treatment-associated variables, but only the presence of HIVE, was associated with delayed virus elimination during HAART in the CSF. This suggests a distinct pattern of viral replication in the CNS in HIVE.

In vivo transmission and dynamics of deleted genomes after experimental infection of woodchuck hepatitis B virus in adult animals.

The presence of Deleted Genomes has been shown in a number of viral models including Hepadnaviridae. The analysis of woodchuck hepatitis B virus (WHV) population after experimental infection of woodchuck 197 (W197) with WHV7-PI inoculum revealed the presence of two Deleted Genomes: DG600 lacking a 1330 bp region (Core/Polymerase/PreS1) and DG900 showing a deletion of 869 nts (Pol/PreS/S). These mutants were also present in WHV7-PI. The successive WHV experimental infections in adult animals were performed using W197-w7 inoculum containing DG600 and DG900. Infections were divided into three groups presenting different patterns of viral replication, different presence of markers, occurrence of variants and persistence of infection. The first group displayed 2-3 weeks viremic phase and WHV-DNA titres of 10-30 ng/ml; the second a longer viremic phase (8-9 weeks) and higher WHV-DNA titres (up to 78 ng/ml). In contrast, the third group exhibited lifetime presence of WHV-DNA and WHVeAg in serum and viral replication in liver. The Deleted Genomes were transmitted in the newly infected animals with the same genomic organization. DG600 was persistently found only in chronically infected woodchuck, whereas a different pattern of presence was described for DG900. The characterization of these classes of deleted mutants in woodchuck-WHV model raises new questions on the link between DGs and persistent infections.

Mutations in the NS3 gene and 3'-NCR of Japanese encephalitis virus isolated from an unconventional ecosystem and implications for natural attenuation of the virus.

The T1P1 strain of Japanese encephalitis (JE) virus was recently isolated from paddy-free Liu-Chiu Islet in which natural JE antibody has been prevalent. In mouse neuroblastoma-derived Neuro-2a cells, T1P1 appeared significantly lower in virus productivity than another local isolate, CH1392. It implied that this new isolate possesses a characteristic viral replication pattern other than that of CH1392. T1P1 has also shown lower neurovirulence, which was reflected by a significantly higher LD(50) (2.44 x 10(6) PFU) than CH1392 (2.87 x 10(2) PFU). In comparison of the full-length RNA sequences between T1P1 and CH1392, a total of 7 nucleotides, including 1 in preM/M and 2 each in NS3, NS5, and the 3'-end noncoding region (NCR), appeared different. Of them, only the changes in NS3 (position 325, T for CH1392, A for T1P1; and position 364, G for CH1392 and A for T1P1) resulted in substitutions of deduced amino acids. There were two additional nucleotide changes appearing in the 3'-NCR. The amino acids 109 Phe and 122 Glu in NS3 of CH1392 were substituted by Ile and Lys, respectively, in T1P1. The unique growth properties and low virulence of T1P1 presented in this report were likely related to abnormal enzymatic activity due to mutations of the NS3 gene (especially position 364) and possibly to the mutations in the 3'-NCR. The natural attenuation of T1P1 that has been circulating in paddy-free Liu-Chiu Islet may account for the absence of clinical JE cases in past years.