Risk of a first clinical diagnosis of central nervous system demyelination in relation to human herpesviruses in the context of Epstein-Barr virus.

BACKGROUND AND PURPOSE: Epstein-Barr virus (EBV) is implicated in multiple sclerosis (MS) risk; evidence for other herpesviruses is inconsistent. Here, we test blood markers of infection with human herpesvirus 6 (HHV-6), varicella zoster virus (VZV), and cytomegalovirus (CMV) as risk factors for a first clinical diagnosis of central nervous system demyelination (FCD) in the context of markers of EBV infection. METHODS: In the Ausimmune case-control study, cases had an FCD, and population controls were matched on age, sex, and study region. We quantified HHV-6- and VZV-DNA load in whole blood and HHV-6, VZV, and CMV antibodies in serum. Conditional logistic regression tested associations with FCD risk, adjusting for Epstein-Barr nuclear antigen (EBNA) IgG, EBV-DNA load, and other covariates. RESULTS: In 204 FCD cases and 215 matched controls, only HHV-6-DNA load (positive vs. negative) was associated with FCD risk (adjusted odds ratio = 2.20, 95% confidence interval = 1.08-4.46, p = 0.03). Only EBNA IgG and HHV-6-DNA positivity were retained in a predictive model of FCD risk; the combination had a stronger association than either alone. CMV-specific IgG concentration modified the association between an MS risk-related human leucocyte antigen gene and FCD risk. Six cases and one control had very high HHV-6-DNA load (>1.0 × 106 copies/mL). CONCLUSIONS: HHV-6-DNA positivity and high load (possibly due to inherited HHV-6 chromosomal integration) were associated with increased FCD risk, particularly in association with markers of EBV infection. With growing interest in prevention/management of MS through EBV-related pathways, there should be additional consideration of the role of HHV-6 infection.

Varicella zoster virus quantitation in blood from symptomatic and asymptomatic individuals.

Primary infection with varicella zoster virus (VZV) occurs in immunocompromised and immunocompetent individuals. Clinical and asymptomatic reactivation with shedding of infectious virus and viremia may occur. The prevalence of VZV viremia is unknown. The aim of this study was to detect VZV viremia and quantify VZV DNA using quantitative polymerase chain reaction (qPCR) in blood from different populations. A qPCR-based method using EvaGreen® was used to quantify VZV DNA in 491 samples, including whole blood, plasma and buffy-coat, from patients hospitalized with varicella-associated disease (Group 1, n=10) and three groups with no VZV disease: individuals with a first clinical diagnosis of central nervous system demyelination (Group 2, n=213) with their age and sex-matched controls (Group 3, n=218); and HIV-infected individuals (Group 4, n=50). VZV-specific IgG antibody titres were measured in Group 3. The proportion positive for viremia and mean detectable VZV DNA load (copies/ml) were: Group 1: 100% (10/10) and 4.6 × 10(6) ± 1.4 × 10(7) ; Group 2: 4% (9/213) and 1.5 × 10(3) ± 1.8 × 10(4) ; Group 3: 8% (17/218) and 1.1 × 10(3) ± 7.8 × 10(3) ; Group 4: 12% (6/50) and 7.7 × 10(1) ± 2.8 × 10(2) . VZV DNA load and IgG titres were not significantly correlated (Group 3 only). VZV load in Group 1 was significantly elevated compared to Groups 2-4 (P<0.001); the latter were not significantly different from each other (P=0.05). VZV genotypes from clades 1-5 were identified in Group 1. VZV DNA was detected but at low frequency and viral load in both immunocompetent and immunocompromised individuals asymptomatic for VZV infection, compared to individuals with active VZV infection.

Differentiation between vaccine and wild-type varicella-zoster virus genotypes by high-resolution melt analysis of single nucleotide polymorphisms.

BACKGROUND: The analysis of single nucleotide polymorphisms (SNPs) of varicella-zoster virus (VZV) has enabled differentiation between wild-type genotypes from the Oka vaccine strain (V-Oka). OBJECTIVES: To genotype VZV strains in Australia using high-resolution melt (HRM) analysis of SNPs in five gene targets. STUDY DESIGN: Extracted DNA from 78 samples obtained from patients with chickenpox and zoster were genotyped by HRM analysis of SNPs in five open reading frames (ORFs): 1 (685 G>A), 21 (33725 C>T), 37 (66288 G>A), 60 (101464 C>A) and 62 (106262 T>C) using a double-stranded (ds) DNA saturating dye, LC Green Plus. RESULTS: For each genotype, melt curve temperature (Tm) shifts differentiated the nucleotide present at that locus (P<0.0001) with melting curve shifts between alleles ranging from 0.56 degrees C (ORF 37) to 3.34 degrees C (ORF 62). The most common genotypes detected were the European Type C (59%) and B (18%) strains. This was followed by the African/Asian Type A (14%) and Japanese J1 (9%), strains, both prevalent in the Northern Territory and Western Australia. CONCLUSIONS: HRM analysis of SNPs showed that the European B and C genotypes were most prevalent in Australia, with genotypes A and J strains also present. HRM analysis using a dsDNA dye provides a useful tool in classifying varicella-zoster viruses.

Seasonal activity, vector relationships and genetic analysis of mosquito-borne Stratford virus.

There are many gaps to be filled in our understanding of mosquito-borne viruses, their relationships with vectors and reservoir hosts, and the environmental drivers of seasonal activity. Stratford virus (STRV) belongs to the genus Flavivirus and has been isolated from mosquitoes and infected humans in Australia but little is known of its vector and reservoir host associations. A total of 43 isolates of STRV from mosquitoes collected in New South Wales between 1995 and 2013 was examined to determine the genetic diversity between virus isolates and their relationship with mosquito species. The virus was isolated from six mosquito species; Aedes aculeatus, Aedes alternans, Aedes notoscriptus, Aedes procax, Aedes vigilax, and Anopheles annulipes. While there were distinct differences in temporal and spatial activity of STRV, with peaks of activity in 2006, 2010 and 2013, a sequence homology of 95.9%-98.4% was found between isolates and the 1961 STRV prototype with 96.2%-100% identified among isolates. Temporal differences but no apparent nucleotide divergence by mosquito species or geographic location was evident. The result suggests the virus is geographically widespread in NSW (albeit only from coastal regions) and increased local STRV activity is likely to be driven by reservoir host factors and local environmental conditions influencing vector abundance. While STRV may not currently be associated with major outbreaks of human disease, with the potential for urbanisation and climate change to increase mosquito-borne disease risks, and the possibility of genomic changes which could produce pathogenic strains, understanding the drivers of STRV activity may assist the development of strategic response to public health risks posed by zoonotic flaviviruses in Australia.

Detection of knockdown resistance mutations in the common bed bug, Cimex lectularius (Hemiptera: Cimicidae), in Australia.

BACKGROUND: Pyrethroid resistance in the common bed bug, Cimex lectularius L., has been reported worldwide. An important resistance mechanism is via knockdown resistance (kdr) mutations, notably V419L and L925I. Information regarding this kdr-type resistance mechanism is unknown in Australia. This study aims to examine the status of kdr mutations in Australian C. lectularius strains. RESULTS: Several modern field-collected strains and museum-preserved reference collections of Australian C. lectularius were examined. Of the field strains (2007-2013), 96% had the known kdr mutations (L925I or both V419L/L925I). The 'Adelaide' strain (2013) and samples from the preserved reference collections (1994-2002) revealed no known kdr mutations. A novel mutation I936F was apparent in the insecticide-resistant 'Adelaide' strain, one strain from Perth (with L925I) and the majority of the reference collection specimens. The laboratory insecticide-resistant 'Sydney' strain showed a mixture of no kdr mutations (20%) and L925I (80%). CONCLUSION: The novel mutation I936F may be a kdr mutation but appeared to contribute less resistance to the pyrethroids than the V419L and L925I mutations. The detection of high frequencies of kdr mutations indicates that kdr-type resistance is widespread across Australia. Hence, there should be a reduced reliance on pyrethroid insecticides and an integrated management approach for the control of C. lectularius infestations.

Identification of putative kdr mutations in the tropical bed bug, Cimex hemipterus (Hemiptera: Cimicidae).

BACKGROUND: Bed bugs [both Cimex hemipterus (F.) and Cimex lectularius L.] are highly resistant to pyrethroids worldwide. An important resistance mechanism known as 'knockdown resistance' (kdr) is caused by genetic point mutations on the voltage-gated sodium channel (VGSC) gene. Previous studies have identified two point mutations (V419L and L925I) on the VGSC gene in C. lectularius that are responsible for kdr-type resistance. However, the kdr mutations in C. hemipterus have not been investigated. RESULTS: Four novel mutations, L899V (leucine to valine), M918I (methionine to isoleucine), D953G (aspartic acid to glycine) and L1014F (leucine to phenylalanine), were identified in the domain II region of the C. hemipterus VGSC gene. This region has been widely investigated for the study of kdr-type resistance to pyrethroids in other insect pests. The V419L and L925I kdr mutations as previously identified in C. lectularius were not detected in C. hemipterus. CONCLUSION: M918I and L1014F are considered to be probable kdr mutations and may play essential roles in kdr-type resistance to pyrethroids in C. hemipterus. Further studies are under way in the authors' laboratory to determine the non-kdr-type resistance mechanisms in C. hemipterus.

Diagnosis of Barmah Forest virus infection by a nested real-time SYBR green RT-PCR assay.

Barmah Forest virus (BFV) is a mosquito borne (+) ssRNA alphavirus found only in Australia. It causes rash, myalgia and arthralgia in humans and is usually diagnosed serologically. We developed a real-time PCR assay to detect BFV in an effort to improve diagnosis early in the course of infection. The limit of detection was 16 genome equivalents with a specificity of 100%. Fifty five serum samples from BFV-infected patients were tested by the PCR. 52 of 53 antibody-positive samples were PCR negative. Two culture-positive (neutralizing antibody negative) samples were positive on first round PCR, while one sample (IgM and neutralizing antibody strongly positive, IgG negative) was positive on second round PCR, suggesting that viral RNA is detectable and transiently present in early infection. PCR can provide results faster than culture, is capable of high throughput and by sequencing the PCR product strain variants can be characterized.

Changes in patterns of hospitalized children with varicella and of associated varicella genotypes after introduction of varicella vaccine in Australia.

BACKGROUND: Varicella in children, although usually mild, can cause hospitalization and rarely death. This study examined patterns of hospitalized children with varicella, and associated varicella genotypes, in 4 tertiary children's hospitals throughout Australia before and after varicella vaccine was introduced. METHODS: We obtained coded data on discharge diagnoses from each hospital before (1999 to 2001) and after (2007 to 2010) varicella vaccine introduction in 2005, adding active surveillance to capture clinical features, complications and immunization history in the latter period. Varicella vesicles were swabbed, and genotyping of varicella strains was performed by real-time polymerase chain reaction amplification. RESULTS: Overall, a 68% reduction in coded hospitalizations (varicella, 73.2% [P < 0.001]; zoster, 40% [P = 0.002]) occurred post-vaccine introduction. Of children with detailed clinical data (97 varicella and 18 zoster cases), 46 (40%) were immunocompromised. Only 6 of 32 (19%) age-eligible immunocompetent children were immunized. Complications, most commonly secondary skin infections (n = 25) and neurologic conditions (n = 14), occurred in 44% of children. There were no deaths; but 3 immunocompetent unimmunized children had severe multiple complications requiring intensive care. All strains genotyped were "wild-type" varicella, with Clade 1 (European origin) predominating. CONCLUSIONS: After the introduction of varicella vaccine, coverage of greater than 80% at 2 years of age was achieved, with varicella hospitalizations reduced by almost 70%. Of hospitalized children age-eligible for varicella vaccine, 80% were unimmunized, including all cases requiring intensive care.

Prevalence of varicella-zoster virus genotypes in Australia characterized by high-resolution melt analysis and ORF22 gene analyses.

DNA sequence variation analysis has divided varicella-zoster virus (VZV; Human herpesvirus 3) into distinct geographical clades: European, Asian, African and Japanese. These genotypes are becoming increasingly prevalent within regions atypical to their original source and there has been the suggestion of recombination between genotypes. Seventy-eight clinical isolates from hospitalized patients with varicella were collected in New South Wales, the Northern Territory, Western Australia and Victoria from 2006 to 2009. The wild-type strains and the vaccine strain (vOka) were differentiated by single nucleotide polymorphism detection using high-resolution melt analysis of five target genes (ORF1, -21, -37, -60 and -62), and by DNA sequence analysis of a 484 bp region of ORF22. Phylogenetic analysis showed that 46 % (36/78) of the clinical isolates were European clade 1 (C/E1) strains, 21 % (16/78) were European clade 3 (B/E2) strains, 12 % (9/78) were Asian/African clade 5 (A/M1) strains, 10 % (8/78) were clade 4 (J2/M2), 6 % (5/78) were clade 2 (J/J) and 5 % (4/78) belonged to the novel clade VI. No significant association was shown between VZV genotype and region, age or gender. Although European strains were most common, the results suggest an increase in African/Asian, Japanese and clade VI genotypes circulating in Australia.

Prolonged varicella viraemia and streptococcal toxic shock syndrome following varicella vaccination of a health care worker.

A 49-year-old health care worker received varicella vaccine in accordance with current Australian guidelines. She developed streptococcal toxic shock syndrome, complicated by acute atraumatic dislocation of the right wrist secondary to poststreptococcal reactive arthritis - to our knowledge, the first report of spontaneous wrist dislocation as a complication in this condition. Vaccination was accompanied by prolonged viraemia with the varicella vaccine strain - also, we believe, the first report of this in an immunocompetent patient.

Pancreatic stellate cells: partners in crime with pancreatic cancer cells.

Pancreatic stellate cells (PSC) produce the stromal reaction in pancreatic cancer, but their role in cancer progression is not fully elucidated. We examined the influence of PSCs on pancreatic cancer growth using (a) an orthotopic model of pancreatic cancer and (b) cultured human PSCs (hPSC) and human pancreatic cancer cell lines MiaPaCa-2 and Panc-1. Athymic mice received an intrapancreatic injection of saline, hPSCs, MiaPaCa-2 cells, or hPSCs + MiaPaCa-2. After 7 weeks, tumor size, metastases, and tumor histology were assessed. In vitro studies assessed the effect of cancer cell secretions on PSC migration and the effect of hPSC secretions on cancer cell proliferation, apoptosis, and migration. Possible mediators of the effects of hPSC secretions on cancer cell proliferation were examined using neutralizing antibodies. Compared with mice receiving MiaPaCa-2 cells alone, mice injected with hPSCs + MiaPaCa-2 exhibited (a) increased tumor size and regional and distant metastasis, (b) fibrotic bands (desmoplasia) containing activated PSCs within tumors, and (c) increased tumor cell numbers. In vitro studies showed that, in the presence of pancreatic cancer cells, PSC migration was significantly increased. Furthermore, hPSC secretions induced the proliferation and migration, but inhibited the apoptosis, of MiaPaCa-2 and Panc-1 cells. The proliferative effect of hPSC secretions on pancreatic cancer cells was inhibited in the presence of neutralizing antibody to platelet-derived growth factor. Our studies indicate a significant interaction between pancreatic cancer cells and stromal cells (PSCs) and imply that pancreatic cancer cells recruit stromal cells to establish an environment that promotes cancer progression.