Belinda L. Herring

Human immunodeficiency virus type 1 superinfection was not detected following 215 years of injection drug user exposure.

ABSTRACT Evidence for human immunodeficiency virus type 1 (HIV-1) superinfection was sought among 37 HIV-1-positive street-recruited active injection drug users (IDUs) from the San Francisco Bay area. HIV-1 sequences from pairs of samples collected 1 to 12 years apart, spanning a total of 215 years of exposure, were generated at p17 gag, the V3-V5 region of env, and/or the first exon of tat and phylogenetically analyzed. No evidence of HIV-1 superinfection was detected in which a highly divergent HIV-1 variant emerged at a frequency >20% of the serum viral quasispecies. Based on the reported risk behavior of the IDUs and the HIV-1 incidence in uninfected subjects in the same cohort, a total of 3.4 new infections would have been expected if existing infection conferred no protection from superinfection. Adjusted for risk behaviors, the estimated relative risk of superinfection compared with initial infection was therefore 0.0 (95% confidence interval, 0.00, 0.79; P = 0.02), indicating that existing infection conferred a statistically significant level of protection against superinfection with an HIV-1 strain of the same subtype, which was between 21 and 100%.

Hendra virus: an emerging paramyxovirus in Australia

Hendra virus, first identified in 1994 in Queensland, is an emerging zoonotic pathogen gaining importance in Australia because a growing number of infections are reported in horses and people. The virus, a member of the family Paramyxoviridae (genus Henipavirus), is transmitted to horses by pteropid bats (fruit bats or flying foxes), with human infection a result of direct contact with infected horses. Case-fatality rate is high in both horses and people, and so far, more than 60 horses and four people have died from Hendra virus infection in Australia. Human infection is characterised by an acute encephalitic syndrome or relapsing encephalitis, for which no effective treatment is currently available. Recent identification of Hendra virus infection in a domestic animal outside the laboratory setting, and the large range of pteropid bats in Australia, underpins the potential of this virus to cause greater morbidity and mortality in both rural and urban populations and its importance to both veterinary and human health. Attempts at treatment with ribavirin and chloroquine have been unsuccessful. Education, hygiene, and infection control measures have hitherto been the mainstay of prevention, while access to monoclonal antibody treatment and development of an animal vaccine offer further opportunities for disease prevention and control.

Frequent Hepatitis C Virus Superinfection in Injection Drug Users

The frequency of hepatitis C virus (HCV) superinfection with a divergent viral strain was determined in a cohort of recently infected young injection drug users (IDUs) with an HCV incidence rate of 25%. HCV was amplified, by use of polymerase chain reaction (PCR), from plasma samples collected from 25 HCV-infected individuals over an average period of 12 months, and their viral sequences were compared. Phylogenetic analysis identified 5 IDUs with superinfection (20%) occurring after seroconversion: 2 IDUs were superinfected with different HCV genotypes, and 3 were superinfected with divergent strains of the same genotype. The superinfecting strains were not detected as minority variants (<0.5%) in the initial plasma HCV quasi species. Extensive measures were taken to exclude PCR contamination and mix-up of samples, and superinfection results were concordant at 2 HCV genetic loci. HCV superinfection in IDUs, both intra- and intergenotype, is therefore a frequent event, with an incidence rate similar to that of de novo infections. These results suggest that no cross-protecting immunity develops during the first year of chronic infection with HCV.

Arthritogenic alphaviral infection perturbs osteoblast function and triggers pathologic bone loss

Significance Persistent polyarthritis, which occurs in 30–40% of alphavirus-infected patients, has been proposed to be caused by proinflammatory mediators such as IL-6. In the present study we investigated the susceptibility and response of primary human osteoblasts to Ross River virus (RRV) infection and determined whether infection could result in bone pathology. RRV infection of osteoblasts resulted in increased receptor activator of nuclear factor-kappaB ligand (RANKL) but decreased osteoprotegerin (OPG). We are the first to report that alphavirus infection results in bone loss in an established RRV murine model and that this bone loss is prevented by IL-6 inhibition. These findings reveal that RRV can disrupt bone homeostasis and that osteoblasts play an important role in alphavirus-induced arthritis by regulating IL-6 and contribute to bone loss by disrupting the RANKL/OPG balance. Arthritogenic alphaviruses including Ross River virus (RRV), Sindbis virus, and chikungunya virus cause worldwide outbreaks of musculoskeletal disease. The ability of alphaviruses to induce bone pathologies remains poorly defined. Here we show that primary human osteoblasts (hOBs) can be productively infected by RRV. RRV-infected hOBs produced high levels of inflammatory cytokine including IL-6. The RANKL/OPG ratio was disrupted in the synovial fluid of RRV patients, and this was accompanied by an increase in serum Tartrate-resistant acid phosphatase 5b (TRAP5b) levels. Infection of bone cells with RRV was validated using an established RRV murine model. In wild-type mice, infectious virus was detected in the femur, tibia, patella, and foot, together with reduced bone volume in the tibial epiphysis and vertebrae detected by microcomputed tomographic (µCT) analysis. The RANKL/OPG ratio was also disrupted in mice infected with RRV; both this effect and the bone loss were blocked by treatment with an IL-6 neutralizing antibody. Collectively, these findings provide previously unidentified evidence that alphavirus infection induces bone loss and that OBs are capable of producing proinflammatory mediators during alphavirus-induced arthralgia. The perturbed RANKL/OPG ratio in RRV-infected OBs may therefore contribute to bone loss in alphavirus infection.

Enhanced arbovirus surveillance with deep sequencing: Identification of novel rhabdoviruses and bunyaviruses in Australian mosquitoes

Viral metagenomics characterizes known and identifies unknown viruses based on sequence similarities to any previously sequenced viral genomes. A metagenomics approach was used to identify virus sequences in Australian mosquitoes causing cytopathic effects in inoculated mammalian cell cultures. Sequence comparisons revealed strains of Liao Ning virus (Reovirus, Seadornavirus), previously detected only in China, livestock-infecting Stretch Lagoon virus (Reovirus, Orbivirus), two novel dimarhabdoviruses, named Beaumont and North Creek viruses, and two novel orthobunyaviruses, named Murrumbidgee and Salt Ash viruses. The novel virus proteomes diverged by ≥ 50% relative to their closest previously genetically characterized viral relatives. Deep sequencing also generated genomes of Warrego and Wallal viruses, orbiviruses linked to kangaroo blindness, whose genomes had not been fully characterized. This study highlights viral metagenomics in concert with traditional arbovirus surveillance to characterize known and new arboviruses in field-collected mosquitoes. Follow-up epidemiological studies are required to determine whether the novel viruses infect humans.

Wide Range of Quasispecies Diversity during Primary Hepatitis C Virus Infection

ABSTRACT Hepatitis C virus (HCV) infections may be initiated by multiple infectious particles, resulting in a genetically heterogeneous viral population, or by a single particle, leading to a clonal population in the initial stage of infection. To determine which of these scenarios is most common, we evaluated the genetic diversity of HCV quasispecies in 12 seronegative subjects with primary infection following community exposures, six acutely infected recipients of HCV-seropositive blood transfusions and six seropositive individuals with infections of undetermined durations. RNA isolated from plasma and a region of the HCV envelope gene including the first hypervariable region (HVR-1) was reverse transcription-PCR amplified and subcloned, and multiple plasmid clones were sequenced. Phylogenetic analysis indicated that all HCV variants clustered by individuals. Genetic distances among HCV variants within recently infected subjects ranged from 1 to 7.8%. On the basis of the estimated mutation rate of HCV in vivo and the Taq polymerase error rate, primary infection viral quasispecies were classified as genetically heterogeneous when the maximum sequence divergence between genetic variants in the same person was >3%. Heterogeneous quasispecies were detected in 4 of 12 preseroconversion subjects, 1 of 6 transfusion recipients, and 4 of 6 seropositive subjects. The high level of viral quasispecies genetic diversity found in at least a third of recently infected individuals is consistent with the transmission of multiple infectious particles. Community-acquired HCV infection, predominantly the result of needle sharing by injection drug users, therefore appears to be frequently initiated by the successful transmission of multiple viral variants.

Dengue virus therapeutic intervention strategies based on viral, vector and host factors involved in disease pathogenesis

Dengue virus (DV) is the most widespread arbovirus, being endemic in over 100 countries, and is estimated to cause 50 million infections annually. Viral factors, such as the genetic composition of the virus strain can play a role in determining the virus virulence and subsequent clinical disease severity. Virus vector competence plays an integral role in virus transmission and is a critical factor in determining the severity and impact of DV outbreaks. Host genetic variations in immune-related genes, including the human leukocyte antigen, have also been shown to correlate with clinical disease and thus may play a role in regulating disease severity. The hosts immune system, however, appears to be the primary factor in DV pathogenesis with the delicate interplay of innate and acquired immunity playing a crucial role. Although current research of DV pathogenesis has been limited by the lack of an appropriate animal model, the development of DV therapeutics has been a primary focus of research groups around the world. In the past decade advances in both the development of vaccines and anti-virals have increased in dramatically. This review summarises the current understanding of viral, vector and host factors which contribute to dengue virus pathogenesis and how this knowledge is critically important in the development of pharmaceutical interventions.

Segregation of Human Immunodeficiency Virus Type 1 Subtypes by Risk Factor in Australia

ABSTRACT The aim of this study was to determine which human immunodeficiency virus type 1 (HIV-1) subtypes were circulating in Australia and to correlate the subtypes with risk factors associated with the acquisition of HIV-1 infection. DNA was extracted from peripheral blood mononuclear cells, and HIV-1 env genes were amplified and subtyped using heteroduplex mobility analysis, with selected samples sequenced and phylogenetic analysis performed. The HIV-1 env subtypes were determined for 141 samples, of which 40 were from female patients and 101 were from male patients; 13 samples were from children. Forty-seven patients were infected by homosexual or bisexual contact, 46 were infected through heterosexual contact, 21 were infected from injecting drug use (IDU), 13 were infected by vertical transmission, 8 were infected from nosocomial exposure, and 6 were infected by other modes of transmission, including exposure to blood products, ritualistic practices, and two cases of intrafamilial transmission. Five subtypes were detected; B (n = 104), A (n = 5), C (n = 17), E (CRF01_AE; n = 13), and G (n = 2). Subtype B predominated in HIV-1 acquired homosexually (94% of cases) and by IDU (100%), whereas non-subtype B infections were mostly seen in heterosexually (57%) or vertically (22%) acquired HIV-1 infections and were usually imported from Africa and Asia. Subtype B strains of group M viruses predominate in Australia in HIV-1 transmitted by homosexual or bisexual contact and IDU. However, non-B subtypes have been introduced, mostly acquired via heterosexual contact.

Macrophage Migration Inhibitory Factor Receptor CD74 Mediates Alphavirus-Induced Arthritis and Myositis in Murine Models of Alphavirus Infection

OBJECTIVE Arthrogenic alphaviruses such as Ross River virus (RRV) and chikungunya virus (CHIKV) circulate worldwide. This virus class causes debilitating illnesses that are characterized by arthritis, arthralgia, and myalgia. In previous studies, we identified macrophage migration inhibitory factor (MIF) as a critical inflammatory factor in the pathogenesis of alphaviral diseases. The present study was undertaken to characterize the role of CD74, a cell surface receptor of MIF, in both RRV- and CHIKV-induced alphavirus arthritides. METHODS Mouse models of RRV and CHIKV infection were used to investigate the immunopathogenesis of arthritic alphavirus infection. The role of CD74 was assessed using histologic analysis, real-time polymerase chain reaction, flow cytometry, and plaque assay. RESULTS In comparison to wild-type mice, CD74-/- mice developed only mild clinical features and had low levels of tissue damage. Leukocyte infiltration, characterized predominantly by inflammatory monocytes and natural killer cells, was substantially reduced in the infected tissue of CD74-/- mice, but production of proinflammatory cytokines and chemokines was not decreased. CD74 deficiency was associated with increased monocyte apoptosis, but had no effect on monocyte migratory capacity. Consistent with these findings, alphaviral infection resulted in a dose-dependent up-regulation of CD74 expression in human peripheral blood mononuclear cells, and serum MIF levels were significantly elevated in patients with RRV or CHIKV infection. CONCLUSION CD74 appears to regulate immune responses to alphaviral infection through its effects on cellular recruitment and survival. These findings suggest that both MIF and CD74 play a critical role in mediating alphaviral disease, and blocking these factors with novel therapeutic agents could substantially ameliorate the pathologic manifestations.

Dengue vaccine efficacy trial: does interference cause failure?

Live-attenuated vaccines replicate at low concentrations and elicit protective immunity without causing disease. This strategy has proven to be successful when the vacc ine targets one pathogen, as is the case for vaccines against yellow fever and Japanese encephalitis viruses. Translation of this straightforward idea to target dengue has proven frustrating, because dengue is a complex fl aviviral disease that is caused by not one, but four antigenically distinct dengue viruses (DENV-1, 2, 3, and 4). Extension of the liveattenuated vaccine approach to dengue by mixing four monovalent attenuated dengue vaccine viruses into a tetravalent formulation was assessed by investigators at Mahidol University, Thailand, in a tetravalent dengue live-attenuated vaccine trial. With this approach, viral interference was reported, in which antibody responses to particular dengue serotypes were reduced or obliterated. Data from Sanofi ’s recent phase 2b trial of its tetravalent chimeric yellow fever-based dengue (CYD) vaccine in Thailand suggest that viral interference is a possible factor contributing to its low effi cacy. Viral interference in the tetravalent CYD vaccine was fi rst reported in 2009, when it was tested in monkeys as a precursor to human trials. In this study, neutralising antibody titres diff ered between CYD serotypes, with CYD-4 titres dominating. This dominance was associated with its higher replication potential and the predominance of CYD-4 viraemia, the basis for which is unknown. To confi rm the occurrence of interference in human beings unequivocally, ideally data for all four monovalent CYD vaccines individually and in tetravalent combination are needed in one trial. Although such data are unavailable, comparisons can be made between monovalent CYD-2 and tetravalent CYD vaccine trials reported by Sanofi in the past few years. Sanofi ’s CYD-2 monovalent DENV-2 vaccine showed excellent immunogenicity in a phase 1 trial. One injection of 5 log10 plaque-forming units of CYD-2 produced plaque reduction neutralisation test50 (PRNT50) geometric mean titres higher than 350 against DENV-2 strain 16 681, in both fl avivirus-naive (n=13) and fl avivirus-immune (n=14) adult volunteers. This result was durable for up to 1 year. By contrast, a CYD tetravalent formulation, containing 5 log10 plaque-forming units of each vaccine virus, tested in fl avivirus-seropositive adults (n=12) in a phase 1 trial in the dengue-endemic Philippines, produced somewhat lower PRNT50 titres to DENV-2 (246), 1 month after completion of a three-dose immunisation schedule spread across 1 year. In another phase 1 trial in Mexico City, where dengue is not endemic, a similar CYD tetravalent formulation elicited much lower DENV-2 neutralising titres (about 50), regardless of previous yellow fever vaccination. In another study of the CYD tetravalent vaccine in fl avivirus-naive adult volunteers (n=23), which resulted in low geometric mean titres against DENV-2 and detectable CYD-4 viraemia after one dose, the investigators emphasised the need for a three-dose immunisation schedule to reduce interference. In fact, in all these human studies and the monkey study, imbalance in neutralising antibody titres and dominance of DENV-4 viraemia and immunogenicity after the priming dose were recurring results consistent with the occurrence of interference in tetravalent CYD formulations. The absence of protective effi cacy against DENV-2 infection in Sanofi ’s phase 2b trial strongly suggests that interference limits CYD-2 replication and vaccine take. The high DENV-2 PRNT50 titres in this study, measured roughly 1 month after immunisation, probably represent cross-reactive antibodies elicited by the CYD-4 vaccine virus. Although these scored as DENV-2 neutralising antibodies in vitro, they may have contributed to the uptake of the DENV-2 strain (circulating in Thailand at the time of the phase 2b trial) in vivo, resulting in DENV-2 viraemia and compromising overall effi cacy. Although the fact that 2 year follow-up has not revealed any sign of 8 Heddini A, Cars O, Qiang S, Tomson G. Antibiotic resistance in China—a major future challenge. Lancet 2009; 373: 30. 9 Timothy DH, Owens CR, McGowan EJ Jr, et al. Infectious Diseases Society of America and the Society for Healthcare Epidemiology of America guidelines for developing an institutional program to enhance antimicrobial stewardship. Clin Infect Dis 2007; 44: 159–77. 10 Ministry of Health, People’s Republic of China. Notifi cation for the special campaign of antibitics rational use in hospitals. http://www.moh.gov.cn/ mohyzs/s3586/201203/54251.shtml (accessed Mar 16, 2012).