Krzysztof Pyrc

Human coronavirus NL63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry

Coronavirus (CoV) infection of humans is usually not associated with severe disease. However, discovery of the severe acute respiratory syndrome (SARS) CoV revealed that highly pathogenic human CoVs (HCoVs) can evolve. The identification and characterization of new HCoVs is, therefore, an important task. Recently, a HCoV termed NL63 was discovered in patients with respiratory tract illness. Here, cell tropism and receptor usage of HCoV-NL63 were analyzed. The NL63 spike (S) protein mediated infection of different target cells compared with the closely related 229E-S protein but facilitated entry into cells known to be permissive to SARS-CoV-S-driven infection. An analysis of receptor engagement revealed that NL63-S binds angiotensin-converting enzyme (ACE) 2, the receptor for SARS-CoV, and HCoV-NL63 uses ACE2 as a receptor for infection of target cells. Potent neutralizing activity directed against NL63- but not 229E-S protein was detected in virtually all sera from patients 8 years of age or older, suggesting that HCoV-NL63 infection of humans is common and usually acquired during childhood. Here, we show that SARS-CoV shares its receptor ACE2 with HCoV-NL63. Because the two viruses differ dramatically in their ability to induce disease, analysis of HCoV-NL63 might unravel pathogenicity factors in SARS-CoV. The frequent HCoV-NL63 infection of humans suggests that highly pathogenic variants have ample opportunity to evolve, underlining the need for vaccines against HCoVs.

Croup Is Associated with the Novel Coronavirus NL63

Background The clinical relevance of infections with the novel human coronavirus NL63 (HCoV-NL63) has not been investigated systematically. We therefore determined its association with disease in young children with lower respiratory tract infection (LRTI). Methods and Findings Nine hundred forty-nine samples of nasopharyngeal secretions from children under 3 y of age with LRTIs were analysed by a quantitative HCoV-NL63-specific real-time PCR. The samples had been collected from hospitalised patients and outpatients from December 1999 to October 2001 in four different regions in Germany as part of the prospective population-based PRI.DE study and analysed for RNA from respiratory viruses. Forty-nine samples (5.2%), mainly derived from the winter season, were positive for HCoV-NL63 RNA. The viral RNA was more prevalent in samples from outpatients (7.9%) than from hospitalised patients (3.2%, p = 0.003), and co-infection with either respiratory syncytial virus or parainfluenza virus 3 was observed frequently. Samples in which only HCoV-NL63 RNA could be detected had a significantly higher viral load than samples containing additional respiratory viruses (median 2.1 × 106 versus 2.7 × 102 copies/ml, p = 0.0006). A strong association with croup was apparent: 43% of the HCoV-NL63-positive patients with high HCoV-NL63 load and absence of co-infection suffered from croup, compared to 6% in the HCoV-NL63-negative group, p < 0.0001. A significantly higher fraction (17.4%) of samples from croup patients than from non-croup patients (4.2%) contained HCoV-NL63 RNA. Conclusion HCoV-NL63 infections occur frequently in young children with LRTI and show a strong association with croup, suggesting a causal relationship.

A novel pancoronavirus RT-PCR assay: frequent detection of human coronavirus NL63 in children hospitalized with respiratory tract infections in Belgium

BackgroundFour human coronaviruses are currently known to infect the respiratory tract: human coronaviruses OC43 (HCoV-OC43) and 229E (HCoV-229E), SARS associated coronavirus (SARS-CoV) and the recently identified human coronavirus NL63 (HCoV-NL63). In this study we explored the incidence of HCoV-NL63 infection in children diagnosed with respiratory tract infections in Belgium.MethodsSamples from children hospitalized with respiratory diseases during the winter seasons of 2003 and 2004 were evaluated for the presence of HCoV-NL63 using a optimized pancoronavirus RT-PCR assay.ResultsSeven HCoV-NL63 positive samples were identified, six were collected during January/February 2003 and one at the end of February 2004.ConclusionsOur results support the notation that HCoV-NL63 can cause serious respiratory symptoms in children. Sequence analysis of the S gene showed that our isolates could be classified into two subtypes corresponding to the two prototype HCoV-NL63 sequences isolated in The Netherlands in 1988 and 2003, indicating that these two subtypes may currently be cocirculating.

The novel human coronaviruses NL63 and HKU1.

In the mid-sixties of the previous century, the first two human coronaviruses (HCoV) were identified: HCoV-229E and HCoV-OC43 (29, 50, 70). These two human coronaviruses were studied extensively from approximately 1965 to the mid-1980s (7, 37, 49, 50, 57, 70). HCoV-229E is a member of the group I coronaviruses, and HCoV-OC43 is a member of group II. Besides the human coronaviruses, there are several group I and group II animal coronaviruses that infect cattle, pigs, cats, dogs, mice, and other animals (33). There is one additional branch, the group III coronaviruses, which are found exclusively in birds (33).

Porphyromonas gingivalis Facilitates the Development and Progression of Destructive Arthritis through Its Unique Bacterial Peptidylarginine Deiminase (PAD)

Rheumatoid arthritis and periodontitis are two prevalent chronic inflammatory diseases in humans and are associated with each other both clinically and epidemiologically. Recent findings suggest a causative link between periodontal infection and rheumatoid arthritis via bacteria-dependent induction of a pathogenic autoimmune response to citrullinated epitopes. Here we showed that infection with viable periodontal pathogen Porphyromonas gingivalis strain W83 exacerbated collagen-induced arthritis (CIA) in a mouse model, as manifested by earlier onset, accelerated progression and enhanced severity of the disease, including significantly increased bone and cartilage destruction. The ability of P. gingivalis to augment CIA was dependent on the expression of a unique P. gingivalis peptidylarginine deiminase (PPAD), which converts arginine residues in proteins to citrulline. Infection with wild type P. gingivalis was responsible for significantly increased levels of autoantibodies to collagen type II and citrullinated epitopes as a PPAD-null mutant did not elicit similar host response. High level of citrullinated proteins was also detected at the site of infection with wild-type P. gingivalis. Together, these results suggest bacterial PAD as the mechanistic link between P. gingivalis periodontal infection and rheumatoid arthritis.

Human coronavirus NL63, a new respiratory virus

Abstract From the mid‐1960s onwards, it was believed that only two human coronavirus species infect humans: HCoV‐229E and HCoV‐OC43. Then, in 2003, a novel member of the coronavirus family was introduced into the human population: SARS‐CoV, causing an aggressive lung disease. Fortunately, this virus was soon expelled from the human population, but it quickly became clear that the human coronavirus group contains more members then previously assumed, with HCoV‐NL63 identified in 2004. Despite its recent discovery, ample results from HCoV‐NL63 research have been described. We present an overview of the publications on this novel coronavirus.

Mosaic structure of human coronavirus NL63, one thousand years of evolution.

Abstract Before the SARS outbreak only two human coronaviruses (HCoV) were known: HCoV-OC43 and HCoV-229E. With the discovery of SARS-CoV in 2003, a third family member was identified. Soon thereafter, we described the fourth human coronavirus (HCoV-NL63), a virus that has spread worldwide and is associated with croup in children. We report here the complete genome sequence of two HCoV-NL63 clinical isolates, designated Amsterdam 57 and Amsterdam 496. The genomes are 27,538 and 27,550 nucleotides long, respectively, and share the same genome organization. We identified two variable regions, one within the 1a and one within the S gene, whereas the 1b and N genes were most conserved. Phylogenetic analysis revealed that HCoV-NL63 genomes have a mosaic structure with multiple recombination sites. Additionally, employing three different algorithms, we assessed the evolutionary rate for the S gene of group Ib coronaviruses to be ∼3×10−4 substitutions per site per year. Using this evolutionary rate we determined that HCoV-NL63 diverged in the 11th century from its closest relative HCoV-229E.

Culturing the Unculturable: Human Coronavirus HKU1 Infects, Replicates, and Produces Progeny Virions in Human Ciliated Airway Epithelial Cell Cultures

ABSTRACT Culturing newly identified human lung pathogens from clinical sample isolates can represent a daunting task, with problems ranging from low levels of pathogens to the presence of growth suppressive factors in the specimens, compounded by the lack of a suitable tissue culture system. However, it is critical to develop suitable in vitro platforms to isolate and characterize the replication kinetics and pathogenesis of recently identified human pathogens. HCoV-HKU1, a human coronavirus identified in a clinical sample from a patient with severe pneumonia, has been a major challenge for successful propagation on all immortalized cells tested to date. To determine if HCoV-HKU1 could replicate in in vitro models of human ciliated airway epithelial cell cultures (HAE) that recapitulate the morphology, biochemistry, and physiology of the human airway epithelium, the apical surfaces of HAE were inoculated with a clinical sample of HCoV-HKU1 (Cean1 strain). High virus yields were found for several days postinoculation and electron micrograph, Northern blot, and immunofluorescence data confirmed that HCoV-HKU1 replicated efficiently within ciliated cells, demonstrating that this cell type is infected by all human coronaviruses identified to date. Antiserum directed against human leukocyte antigen C (HLA-C) failed to attenuate HCoV-HKU1 infection and replication in HAE, suggesting that HLA-C is not required for HCoV-HKU1 infection of the human ciliated airway epithelium. We propose that the HAE model provides a ready platform for molecular studies and characterization of HCoV-HKU1 and in general serves as a robust technology for the recovery, amplification, adaptation, and characterization of novel coronaviruses and other respiratory viruses from clinical material.

Human Parechovirus Type 1, 3, 4, 5, and 6 Detection in Picornavirus Cultures

ABSTRACT Picornavirus cultures that could not be typed in neutralization assays were analyzed by VP1 reverse transcription-PCR (RT-PCR) and a virus discovery tool (VIDISCA). Human parechoviruses (HPeVs) were frequently identified, among which were the uncommon isolates HPeV-4, HPeV-5, and HPeV-6. The HPeV-5 isolate could be amplified only by VIDISCA and not by VP1 RT-PCR.

Inhibition of Human Coronavirus NL63 Infection at Early Stages of the Replication Cycle

ABSTRACT Human coronavirus NL63 (HCoV-NL63), a recently discovered member of the Coronaviridae family, has spread worldwide and is associated with acute respiratory illness in young children and elderly and immunocompromised persons. Further analysis of HCoV-NL63 pathogenicity seems warranted, in particular because the virus uses the same cellular receptor as severe acute respiratory syndrome-associated coronavirus. As there is currently no HCoV-NL63-specific and effective vaccine or drug therapy available, we evaluated several existing antiviral drugs and new synthetic compounds as inhibitors of HCoV-NL63, targeting multiple stages of the replication cycle. Of the 28 compounds that we tested, 6 potently inhibited HCoV-NL63 at early steps of the replication cycle. Intravenous immunoglobulins, heptad repeat 2 peptide, small interfering RNA1 (siRNA1), siRNA2, β-d-N4-hydroxycytidine, and 6-azauridine showed 50% inhibitory concentrations of 125 μg/ml, 2 μM, 5 nM, 3 nM, 400 nM, and 32 nM, respectively, and low 50% cytotoxicity concentrations (>10 mg/ml, >40 μM, >200 nM, >200 nM, >100 μM, and 80 μM, respectively). These agents may be investigated further for the treatment of coronavirus infections.