Detection of human bocavirus in respiratory, fecal, and blood samples by real-time PCR.

Human bocavirus (HBoV) has been detected worldwide in respiratory samples. Two real-time PCR assays, targeting the non-structural protein (NP-1) and viral protein (VP-1) genes, were designed and validated to detect HBoV in patients with respiratory disease, gastroenteritis, or systemic illness. Sensitivity of the NP-1 and VP-1 assays were equal to the conventional PCR assay previously described by Allander et al. [2005: Proc Natl Acad Sci USA 102: 12891-12896] being 100%, and giving specificity of 94% and 93%, respectively. There was no cross-reaction identified with unrelated respiratory agents, or to human DNA. The limits of detection were 10 copies of genomic DNA equivalents per reaction for both assays. The assays were used to screen three different sample populations, combined nose, and throat swabs (n = 96) from children with acute respiratory disease, fecal samples (n = 375) from adults, and children with gastroenteritis and whole blood (n = 229) collected from 31 immunocompromised children taken over an 18-month period. In total 17 (18%) respiratory samples and 18 (4.8%) fecal samples were identified as having HBoV present. Of the pediatric whole blood specimens investigated, HBoV was detected in six (2.6%) samples from four patients. In summary, two real-time PCR assays targeting different genes were designed and validated for use as screening methods for the detection of HBoV. HBoV was found in three different specimen types: parent-collected combined nose-throat swabs, fecal samples collected from symptomatic individuals and whole blood from immunocompromised children.

Epidemics of gastroenteritis during 2006 were associated with the spread of norovirus GII.4 variants 2006a and 2006b.

BACKGROUND: Acute gastroenteritis is commonly associated with norovirus genogroup II (GII) infection. Norovirus GII has 17 classified genotypes (GII.1-GII.17), but only 1 norovirus genotype (GII.4) is associated with global epidemics of gastroenteritis. In 2006, an increase in global norovirus activity was observed. METHODS: During the period from December 2005 through August 2006, a total of 231 fecal samples were obtained from patients with acute gastroenteritis from Australia and New Zealand. Norovirus RNA was amplified and sequenced to determine norovirus genotype and relatedness to known epidemic norovirus GII.4 variants. RESULTS: Two GII.4 variants, designated 2006a and 2006b, were identified in 61.8% and 11.3%, respectively, of the 186 cases investigated. Norovirus 2006a and 2006b have also been implicated as the predominant causes of norovirus-associated gastroenteritis across Europe in 2006. CONCLUSIONS: The global increase in norovirus-associated gastroenteritis in 2006 was linked to the emergence of 2 novel GII.4 variants, 2006a and 2006b.

Connexins within the rat larynx.

OBJECTIVES/HYPOTHESIS: To determine the types and localization of connexins within the rat larynx. STUDY DESIGN: Quantitative real time polymerase chain reaction (qRT-PCR) of the epiglottis and laryngeal mucosa was used to identify connexins (Cx). Immunohistochemical labeling was then used to localize the Cxs within the larynx. METHODS: Twelve larynges from 3 to 4 month old Fisher-344 rats were used. RNA was extracted (N = 8) and cDNA produced. Primers for Cx26, Cx30, Cx32, Cx37, Cx40, and Cx43 were added and qRT-PCR performed. Others larynges were serially sectioned for immunohistochemistry. RESULTS: qRT-PCR revealed Cx43, Cx32, and Cx30 within the epiglottis and Cx43 in the vocal folds and Cx43 and Cx32 within the subglottic mucosa. Immunohistochemical staining confirmed these results. CONCLUSION: The rat epiglottis is rich in Cx43, Cx32, and Cx30 whereas the vocal folds contain Cx43 and the subglottic mucosa Cx43 and Cx32. Their localizations suggest involvement in secretion for protective purposes and they may play a key role in laryngeal pathoses.

Stereological estimates indicate that aging does not alter the capillary length density in the human posterior cricoarytenoid muscle.

Studies of some human skeletal muscles demonstrate an age-related capillarity decrease. An age-related decrease in blood flow to the posterior cricoarytenoid muscle (PCA) in rats has been reported, as well as a decreased ability to abduct the vocal folds. We, therefore, hypothesized that decreased muscle capillarity may contribute to PCA dysfunction in the elderly. Using immunological and stereological techniques, human PCAs (ages 18-98 yr; 28 men, 23 women) were examined for age-related changes in muscle fiber-type-specific and/or total capillary length density. While analysis shows no age-related changes in total muscle or fiber-type-specific capillary length densities (L(V cap)), there are significant age-related increases in L(V cap) within the interstitial tissue (P = 0.001) and in the ratio of the type I L(V cap) to type I surface (P = 0.002), with a strong trend for type II L(V cap) (P = 0.055). There is also an age-related decrease in the muscle fiber surface density for both type I and II fibers (P < 0.001 and 0.04, respectively). Data also show that women have a significantly higher type II L(V cap) (P = 0.039), regardless of age. In addition, with the exception of female type I L(V cap), all measured variables are significantly higher for type I fibers (P < 0.001), independent of age or sex. While data indicate there are age-related changes of capillary-muscle fiber relationships within the PCA, they do not support the hypothesis of an age-related loss of capillarity.

Age-related blood flow and capillary changes in the rat utricular macula: a quantitative stereological and microsphere study.

Vascular change may contribute to age-related vestibular dysfunction. Previously, we reported a significant age-related decrease in blood flow (BF) and mean capillary diameter (D(cap)) in the rat posterior canal crista. The purpose of this study was to examine an otolith organ, the utricle, for similar changes. Old male Fischer 344 rats (O; 28-31 mos) were anesthetized, and the left cardiac ventricle was transcutaneously injected with radioactive microspheres to determine BF. The temporal bones were removed, fixed, and decalcified. The utricles were dissected free and placed into a gamma counter with the reference samples. The specimens were then plastic embedded and serially sectioned at 1 microm according to the vertical section technique. Microsphere surface counts were made and neuroepithelial BF calculated. A systematic random set of sections was sampled and analyzed using stereological techniques for estimates of D(cap), capillary surface area/unit volume (S(v,cap)), capillary length/ unit volume (L(v,cap)), and volume of utricular neuroepithelium (V(ut)). Using these data, total capillary surface (S(cap)) and total length (L(cap)) were calculated. Statistical comparisons were made with data from our previous study of young animals (Y; 3-6 mos). Results indicate a significant age-related decrease in BF (Y = 0.125 microL/min, O = 0.062 microL/min; P = 0.003), D(cap) (Y = 5.95 micro, O = 4.57 microm; P = 0.0002), S(vcap) (Y = 12.33 mm2/mm3, = 9.87 mm2/mm3, P = 0.016), S(cap) (Y = 0.178 mm2, O = 0.129 mm2; p = 0.01), and V(ut) (Y = 0.014 mm3, O = 0.013 mm3; P = 0.04) with no significant change in L(v,cap) (Y = 655 mm/mm3, O = 686 mm/mm3, P = 0.41) or L(cap) (Y = 9.47 mm, O = 8.96 mm; P = 0.49). These age-related vascular changes are likely to have a significant impact on utricular physiological and thus, dysequilibrium.

Evidence of a gustatory-vestibular pathway for protein transport.

OBJECTIVE: To demonstrate anatomically a pathway for protein transport from the palate to the vestibular system. METHOD: The vestibulofacial anastomosis and associated ganglion cells were identified in a collection of 160 horizontally sectioned human temporal bones that had been stained with hematoxylin and eosin. Wheat germ agglutinin-horseradish peroxidase (HRP) was applied to the greater superficial petrosal nerve in 4 Sprague-Dawley rats. After 30 hours, the rats were killed by intracardiac perfusion, and the seventh and eighth nerves with adjacent brainstem removed. Frozen sections cut at 30 mum through this block were then reacted for HRP, counterstained with neutral red, and mounted on slides for examination in the light microscope. RESULTS: Thirty-two of the 160 human temporal bones contained sections through the vestibulofacial anastomosis and its ganglion. In all cases, the ganglion was incorporated into the vestibular ganglion (VG) adjacent to the nervus intermedius. In all 4 experimental rats, HRP reaction product labeled a small number of ganglion cells in the VG adjacent to the nervus intermedius and facial nerve. CONCLUSION: These observations support the presence of a pathway from receptors in the palate to the VG.

Age-related blood flow changes in the rat intrinsic laryngeal muscles.

CONCLUSIONS: These results indicate that a deceased laryngeal blood flow (BF) could be one contributing factor to age-related phonatory and airway dysfunction. OBJECTIVE: Studies of non-laryngeal muscles suggest that decreased BF may contribute to an age-related decline in muscle performance. We hypothesized that there is an age-related BF decrease to the intrinsic laryngeal muscles. MATERIALS AND METHODS: Intrinsic laryngeal muscle BF was measured in young (3-6 months old; n=11) and old (28-30 months old; n=21) male Fischer 344 rats during quiet respiration using the radiolabeled microsphere technique. RESULTS: BF to the posterior cricoarytenoid (PCA) was very high even during this submaximal recruitment, consistent with its specialization for oxidative metabolism and fatigue resistance. The results demonstrated significant (p<0.05) age-related BF decreases in the thyroarytenoid (young, 163; old, 64 ml/min/100 g), cricothyroid (young, 104; old, 52 ml/min/100 g), and PCA (young, 404; old, 235 ml/min/100 g).

Norovirus GII.4 variant 2006b caused epidemics of acute gastroenteritis in Australia during 2007 and 2008.

BACKGROUND: Over the last decade, four epidemics of norovirus-associated gastroenteritis have been reported in Australia. These epidemics were characterized by numerous outbreaks in institutional settings such as hospitals and nursing homes, as well as increases in requests for NoV testing in diagnostic centers. During 2007 and 2008, widespread outbreaks of acute gastroenteritis were once again seen across Australia, peaking during the winter months. OBJECTIVES: The primary objective of this study was to characterize two winter epidemics of NoV-associated gastroenteritis in 2007 and 2008 in Australia. Following this, we aimed to determine if these epidemics were caused by a new GII.4 variant or a previously circulating NoV strain. STUDY DESIGN: NoV-positive fecal samples (n=219) were collected over a 2-year period, December 2006 to December 2008, from cases of acute gastroenteritis in Australia. NoV RNA was amplified from these samples using a nested RT-PCR approach targeting the 5' end of the capsid gene, termed region C. Further, characterization was performed by sequence analysis of the RdRp and capsid genes and recombination was identified using SimPlot. RESULTS: From 2004 to 2008, peaks in the numbers of NoV-positive EIA tests from the Prince of Wales Hospital Laboratory correlated with the overall number of gastroenteritis outbreaks reported to NSW Health, thereby supporting recent studies showing that NoV is the major cause of outbreak gastroenteritis. The predominant NoV GII variant identified during the 2007-2008 period was the GII.4 pandemic variant, 2006b (71.51%, 128/179), which replaced the 2006a variant identified in the previous Australian epidemic of 2006. Four novel GII variants were also identified including the three GII.4 variants: NoV 2008, NoV Osaka 2007 and NoV Cairo 2007, and one novel recombinant NoV designated GII.e/GII.12. CONCLUSION: The increase in acute gastroenteritis outbreaks in 2007 and 2008 were associated with the spread of the NoV GII.4 variant 2006b.

Epidemic viral gastroenteritis in Queensland coincides with the emergence of a new norovirus variant.

Norovirus infections cause widespread morbidity and have significant economic impact on the community. An increase in outbreaks of norovirus gastroenteritis in hospitals, nursing homes and in the community was observed in Queensland in 2004. Molecular analysis of positive samples indicated the emergence of a single strain of norovirus. A 252 nucleotide sequence from the polymerase region (POL) was compared to sequences of the new variant genotype GII.4 that has caused epidemics in the Northern Hemisphere in 2002 and 2003. Sequence analysis indicated greater than 95 per cent similarity in the POL between the Queensland strain and the Northern Hemisphere 2002/3 GII.4 variant. Phylogenetic analysis revealed that the Queensland strain forms a branch within the GII.4 genotype separate from the 2002 variant from Europe and North America. Although norovirus genotype GII.4 had circulated in Queensland in the past, the 2004 strain was characterised specifically by three nucleotides not present in any other sequences held in our database covering the years 2002-June 2004.

A measles outbreak in the Whitsundays, Queensland: the shape of things to come?

This report describes a small outbreak of measles that occurred in the Whitsunday region, north Queensland, in July to August 2002. With one exception, all the cases were deliberately unvaccinated because their parents were conscientious objectors to vaccination. It is suggested that this pattern of measles outbreaks, with most cases being not preventable because of conscientious objection, will become increasingly recognised in the future.