Mycobacterium avium subsp. paratuberculosis, the etiological agent of paratuberculosis, affects a wide range of domestic ruminants and has been suggested to be involved in Crohn's disease in humans. Most available methods for identifying and differentiating strains of this difficult species are technically demanding and have limited discriminatory power. Here, we report the identification of novel PCR-based typing markers consisting of variable-number tandem repeats (VNTRs) of genetic elements called mycobacterial interspersed repetitive units (MIRUs). Eight markers were applied to 183 M. avium subsp. paratuberculosis isolates from bovine, caprine, ovine, cervine, leporine, and human origins from 10 different countries and to 82 human isolates of the closely related species M. avium from France. Among the M. avium subsp. paratuberculosis isolates, 21 patterns were found by MIRU-VNTR typing, with a discriminatory index of 0.751. The predominant R01 IS900 restriction fragment length polymorphism type, comprising 131 isolates, was divided into 15 MIRU-VNTR types. Among the 82 M. avium isolates, the eight MIRU-VNTR loci distinguished 30 types, none of which was shared by M. avium subsp. paratuberculosis isolates, resulting in a discriminatory index of 0.889. Our results suggest that MIRU-VNTR typing is a fast typing method that, in combination with other methods, might prove to be optimal for PCR-based molecular epidemiological studies of M. avium/M. avium subsp. paratuberculosis pathogens. In addition, presumably identical M. avium subsp. paratuberculosis 316F vaccine strains originating from the Weybridge laboratory and from different commercial batches from Mérial actually differed by one or both typing methods. These results indicate a substantial degree of genetic drift among different vaccine preparations, which has important implications for prophylactic approaches.

Following an increase in detection of enterovirus 68 (EV68) in community surveillance of respiratory infections in The Netherlands in 2010, epidemiological and virological analyses were performed to investigate the possible public health impact of EV68 infections. We retrospectively tested specimens collected from acute respiratory infections surveillance and through three children cohort studies conducted in The Netherlands from 1994 through 2010. A total of 71 of 13,310 (0.5%) specimens were positive for EV68, of which 67 (94%) were from symptomatic persons. Twenty-four (34%) of the EV68 positive specimens were collected during 2010. EV68-positive patients with respiratory symptoms showed significantly more dyspnea, cough and bronchitis than EV68-negative patients with respiratory symptoms. Phylogenetic analysis showed an increased VP1 gene diversity in 2010, suggesting that the increased number of EV68 detections in 2010 reflects a real epidemic. Clinical laboratories should consider enterovirus diagnostics in the differential diagnosis of patients presenting with respiratory symptoms.

Enterovirus D68 (EV-D68) is an emerging pathogen that can cause severe respiratory disease and is associated with cases of paralysis, especially among children. Heretofore, information on host factor requirements for EV-D68 infection is scarce. Haploid genetic screening is a powerful tool to reveal factors involved in the entry of pathogens. We performed a genome-wide haploid screen with the EV-D68 prototype Fermon strain to obtain a comprehensive overview of cellular factors supporting EV-D68 infection. We identified and confirmed several genes involved in sialic acid (Sia) biosynthesis, transport, and conjugation to be essential for infection. Moreover, by using knockout cell lines and gene reconstitution, we showed that both α2,6- and α2,3-linked Sia can be used as functional cellular EV-D68 receptors. Importantly, the screen did not reveal a specific protein receptor, suggesting that EV-D68 can use multiple redundant sialylated receptors. Upon testing recent clinical strains, we identified strains that showed a similar Sia dependency, whereas others could infect cells lacking surface Sia, indicating they can use an alternative, nonsialylated receptor. Nevertheless, these Sia-independent strains were still able to bind Sia on human erythrocytes, raising the possibility that these viruses can use multiple receptors. Sequence comparison of Sia-dependent and Sia-independent EV-D68 strains showed that many changes occurred near the canyon that might allow alternative receptor binding. Collectively, our findings provide insights into the identity of the EV-D68 receptor and suggest the possible existence of Sia-independent viruses, which are essential for understanding tropism and disease.

Mycobacterium avium subsp. paratuberculosis is an emerging pathogen of mammals and is being actively investigated as a possible zoonotic agent. The lack of reliable diagnostic assays has hampered rational assessment of the prevalence of this organism in humans and animals. We have used a comparative genomic approach to reveal genomic differences between M. avium subsp. paratuberculosis and its close relative M. avium subsp. avium, a highly prevalent environmental organism. From computational and DNA microarray-based study of two prototype strains, M. avium subsp. avium strain 104 and M. avium subsp. paratuberculosis strain K10, we have uncovered two types of large sequence polymorphisms (LSPs): those present in the former but missing in the latter (LSP A s) and those only present in the latter (LSP P s). We examined the distribution of 3 LSP A s and 17 LSP P s across a panel of 383 M. avium complex isolates in order to determine their potential utility for the development of accurate diagnostic tests. Our results show that the absence of LSP A 8 is 100% specific for the identification of M. avium subsp. paratuberculosis. Of the 17 LSP P s, 10 regions were not specific for M. avium subsp. paratuberculosis while 7 were shown to be highly specific (>98%) and, in some cases, highly sensitive as well (up to 95%). These data highlight the need to evaluate these regions across a diverse panel of clinical and environmental isolates and indicate the LSPs best suited for M. avium subsp. paratuberculosis diagnostics.Mycobacterium avium subsp. paratuberculosis is a serious pathogen of domestic ruminants, in which it causes paratuberculosis (Johne's disease), a chronic and eventually fatal inflammatory bowel disease. Its rising incidence in many regions of the world has resulted in significant economic losses to the livestock industry (13). This organism also infects free-ranging wildlife species (16). The impact of M. avium subsp. paratuberculosis in nonruminant wildlife species is largely unknown; however, the potential for interspecies transmission has important implications for paratuberculosis control programs. M. avium subsp. paratuberculosis is also being actively investigated as the possible cause of a debilitating human inflammatory bowel disease, Crohn's disease (4, 5, 18).Effective control of Johne's disease and investigation of the potential link to Crohn's disease have been hampered by the lack of effective assays to easily and accurately diagnose M. avium subsp. paratuberculosis infections. Commercially available serological assays for bovine disease are convenient but offer poor sensitivity, especially during subclinical disease. Moreover, owing to the high degree of genetic similarity between M. avium subsp. paratuberculosis and the other members of the M. avium complex, many of the proteins that are recognized during the early stages of infection are not specific for M. avium subsp. paratuberculosis (14). Since M. avium subsp. avium is highly prevalent in the environment but not associated with a similar cli...

Enteroviruses are a major cause of human disease. Adipose-specific phospholipase A2 (PLA2G16) was recently identified as a pan-enterovirus host factor and potential drug target. In this study, we identify a possible mechanism of PLA2G16 evasion by employing a dual glycan receptor-binding enterovirus D68 (EV-D68) strain. We previously showed that this strain does not strictly require the canonical EV-D68 receptor sialic acid. Here, we employ a haploid screen to identify sulfated glycosaminoglycans (sGAGs) as its second glycan receptor. Remarkably, engagement of sGAGs enables this virus to bypass PLA2G16. Using cryo-EM analysis, we reveal that, in contrast to sialic acid, sGAGs stimulate genome release from virions via structural changes that enlarge the putative openings for genome egress. Together, we describe an enterovirus that can bypass PLA2G16 and identify additional virion destabilization as a potential mechanism to circumvent PLA2G16.