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76 The Open Virology Journal 2010 4 76 84 1874 3579 10 2010 Bentham Open Open Access Understanding Human Coronavirus HCoV NL63 Sahar Abdul Rasool 1 and Burtram C Fielding 2 1 Anatomy Cluster Department of Medical Biosciences Faculty of Natural Sciences University of the Western Cape Western Cape South Africa 2 Molecular Virology Research Laboratory Medical Microbiology Cluster Department of Medical Biosciences Faculty of Natural Sciences University of the Western Cape Private Bag X17 Modderdam Road Bellville Western Cape 7535 South Africa Abstract Even though coronavirus infection of humans is not normally associated with severe diseases the identification of the coronavirus responsible for the outbreak of severe acute respiratory syndrome showed that highly pathogenic coronaviruses can enter the human population Shortly thereafter in Holland in 2004 another novel human coronavirus HCoV NL63 was isolated from a seven month old infant suffering from respiratory symptoms This virus has subsequently been identified in various countries indicating a worldwide distribution HCoV NL63 has been shown to infect mainly children and the immunocommpromised who presented with either mild upper respiratory symptoms cough fever and rhinorrhoea or more serious lower respiratory tract involvement such as bronchiolitis and croup which was observed mainly in younger children In fact HCoV NL63 is the aetiological agent for up to 10 of all respiratory diseases This review summarizes recent findings of human coronavirus HCoV NL63 infections including isolation and identification phylogeny and taxonomy genome structure and transcriptional regulation transmission and pathogenesis and detection and diagnosis Keywords Human coronavirus HCOV NL63 clinical features pathogenesis diagnosis 1 INTRODUCTION Regardless of geographic location respiratory tract infections rank among the top three killers of children under five years of age 1 A significant proportion of these respiratory tract infections have no known cause Recently however a number of novel coronaviruses have been identified as the causative agents for some of these infections 2 3 Coronaviruses CoVs belong to the family Coronaviridae in the order nidovirales Members of the Coronavirus family are positive strand RNA viruses with large genomes ranging in size from 27 33 kb The coronavirus genome encodes for a 5 replicase polyprotein ORF1a and ORF1b that in turn encodes for all the enzymes required for viral RNA replication The genome also encodes for the 3 structural proteins including spike S envelope E membrane M and nucleocapsid N which are common to all coronaviruses The structural proteins are involved in various viral processes including virus particle formation 4 Additional subgroup specific accessory genes are found interspersed among the structural genes which vary in number and location Recent studies have shown that the proteins encoded by these genes could be modulators of pathogenicity in the natural host 5 7 Address correspondence to this author at the Molecular Virology Research Laboratory Medical Microbiology Cluster Department of Medical Biosciences Faculty of Natural Sciences University of the Western Cape Private Bag X17 Modderdam Road Bellville Western Cape 7535 South Africa Tel 27 21 9593620 Fax 27 21 9593125 E mail bfielding uwc ac za These authors made equal contribution Five human coronaviruses have been identified to date four of which are known to continuously circulate in the human population especially in young children 8 9 HCoV OC43 and HCoV 229E first identified in the mid 1960s 10 11 were shown to cause the common cold 12 but rarely infections of the lower respiratory tract 3 A third human coronavirus which causes severe acute respiratory syndrome SARS CoV was identified in 2003 13 14 This virus had a worldwide spread causing acute respiratory illness with a mortality rate of 10 15 The last reported SARS CoV infections were laboratory acquired in 2004 and the virus has not been detected in the human population since 16 17 More recently two additional human coronaviruses were identified HCoV HKU1 was isolated from a 71 year old man who presented with fever and cough 3 and HCoV NL63 isolated from a seven month old baby 2 The latter is the topic of this review Several groups have studied different aspects of HCoV NL63 infections including its worldwide distribution its association with human disease and the replication characteristics of the causative virus In this review we summarize recent findings of human coronavirus HCoV NL63 infections including virus isolation and identification phylogeny and taxonomy genome structure and transcriptional regulation transmission and pathogenesis and detection and diagnosis 2 ISOLATION AND IDENTIFICATION OF HCoV NL63 HCoV NL63 was first isolated in Amsterdam in 2004 from the nasopharyngeal aspirate of a seven month old child the patient presented with symptoms suggesting respiratory Understanding Human Coronavirus HCoV NL63 The Open Virology Journal 2010 Volume 4 77 tract infection coryza conjunctivitis and fever while his chest X ray showed typical features of bronchiolitis The aspirate tested negative for all known respiratory viruses A group of Dutch scientists found that the virus initiated a cytopathic effect when inoculated onto tertiary monkey kidney cells The group used a new technique VIDISCA to clone and amplify the viral genome VIDISCA is a novel approach that provides a fast and effective tool for amplification of unknown genomes based on cDNA amplified fragment length polymorphism 2 18 The virus was identified as a member of the Coronaviridae family It was shown to be a novel member of Group I coronaviruses because of the similarity of its genome sequence to HCoV 229E Another group in the Netherlands 19 reported the independent isolation and identification of essentially the same virus at about the same time 3 PHYLOGENY AND TAXONOMY OF HCoV NL63 Based on antigenicity genome organization and sequence homology coronaviruses are divided into three distinct groups 20 Group 1 contains transmissible gastroenteritis virus TGEV porcine epidemic diarrhea virus PDEV feline infectious peritonitis virus FIPV canine coronavirus and HCoV 229E among others Group 2 contains mouse hepatitis virus MHV bovine coronavirus haemagglutinating encephalomyelitis virus HCoV HKU1 and HCoV OC43 to name a few with Bat SARS CoV and SARS CoV considered distantly related Group 2b coronaviruses Group 3 contains the avian coronaviruses 21 23 Based on phylogenetic analysis HCoV NL63 belongs to the Group I coronaviruses 2 24 Interestingly evidence of recombination during the evolution of HCoV NL63 has been reported and viral isolates have in fact a mosaic genome structure The authors speculate that HCoV NL63 diverged from a HCoV 229E ancestor in the past followed by a separation into two distinct HCoV NL63 lineages These two lineages recombined during co infection giving rise to the two currently observed genotypic subgroups 24 27 In fact recombination between different HCoV NL63 isolates has been suggested resulting in a mixture of clinical virus variants circulating in the human population 8 27 30 3 1 Genome Structure and Transcriptional Regulation HCoV NL63 has a single stranded RNA genome that is capped and polyadenylated 27 The genome is 27553 bases in size with the genome order 5 ORF1a ORF1b S ORF3 E M N polyT 3 Fig 1 Seven distinct ORFs are produced from six distinct mRNAs which include the full length genomic RNA and a nested set of five subgenomic sg mRNAs 4 Coronavirus mRNAs are generated in the membrane associated replication centers 31 The five sg mRNAs encode for the viral structural and accessory proteins S ORF3 E M and N With the exception of ORF E a common transcription regulatory sequence TRS with core sequence AACUAAA is located upstream of all the ORFs this TRS is crucial for sg mRNA formation 27 32 HCoV NL63 uses a discontinuous replication strategy to generate sg mRNAs during the minus strand synthesis 4 27 32 which are then copied into plus strand mRNAs All plus strand mRNAs share a common 70 nucleotide leader sequence at their 5 ends that is identical to the sequence at the 5 end of the genomic RNA 27 Fig 1 Schematic comparison of the genome organization of coronaviruses infecting humans Genomic maps shown are based on the complete genome sequences NCBI accession numbers are shown in brackets HCoV NL63 Human coronavirus HCOV NL63 NC 005831 HCoV 229E Human coronavirus 229E NC 002645 SARS CoV Severe acute respiratory syndrome coronavirus NC 004718 HKU1 CoV Human coronavirus HKU1 NC 006577 HCoV OC43 Human coronavirus OC43 NC 005147 ORFs S 1 E 2 M 3 and N 4 are shown and open reading frames encoding for accessory genes are shaded in grey ORF4 of HCoV 229E is shown as a single open reading frame 34 5 3 1a 1b 30 kb20 kb HCoV NL63 HCoV 229E SARS CoV HCoV OC43 HKU1 CoV 1 1 1 1 1 2 3 4 2 3 4 2 3 4 2 3 4 2 3 4 78 The Open Virology Journal 2010 Volume 4 Abdul Rasool and Fielding 3 1 1 Protein 1a 1b HCoV NL63 ORF1a 1b contains a putative elaborated pseudoknot structure that triggers a 1 ribosomal frameshift to translate the complete 1ab polyprotein for a mini review of the putative ORF1a 1b products and functions see Pryc et al 2007 27 and Van der Hoek et al 2006 8 Chen and colleagues identified processed products nsp3 and nsp4 of the HCoV NL63 replicase polyprotein which could be detected at 24 hours post infection These products localize in the peri nuclear sites of virus infected cells Also the group identified and characterized two viral papain like proteases PLP1 and PLP2 which process the viral replicase polyprotein Interestingly the PLP2 protease has deubiquitinating activity 9 although the function of this is not clear in viral replication 3 1 2 Spike Protein The species tropism and virulence of a particular coronavirus are largely determined by the spike S glycoprotein Coronavirus S proteins mediate attachment to the cellular receptors and subsequent fusion of the virus and cell membrane 20 The N terminal portion of HCoV NL63 S contains a unique 179 amino acid domain not present in other coronaviruses This region represents the most variable region of the HCoV NL63 genome and a role in immune evasion for this region has been proposed 8 27 HCoV NL63 S is a single chain glycoprotein and consists of an N terminal receptor binding domain S1 and a C terminal transmembrane fusion domain S2 S2 consists of two highly conserved heptad repeat HR sequences that are larger than the corresponding regions for the group II and group III coronaviruses 20 27 33 Initial proteolytic studies of the S2 fusion core identified an helical domain consisting of a trimer of the HR segments N57 and C42 The resolved crystal structure of this trimeric complex shows distinctive high affinity conformations of interacting cross sectional layers of six helices It has been suggested that the larger HR regions of the group I coronaviruses may be required to prime the S proteins for their fusion activating conformational changes during entry of the virus 20 3 1 3 Accessory Protein ORF3 Coronavirus genomes contain accessory genes found interspersed among the structural genes which vary in number and location between the different coronavirus groups Fig 1 Both HCoV NL63 and HCoV 229E are group I coronaviruses and encode for only one accessory gene product between the S and E genes 34 Accessory genes are poorly characterized and the functions of the gene products are not well understood Initial research into the functions of these genes has shown that they are non essential and dispensable for virus growth in cell culture 5 6 35 More recent studies have shown that the accessory genes are required for in vivo infection and pathogenecity in the natural host 6 7 36 38 Unlike the SARS CoV genome the HCoV NL63 genome encodes for only one accessory protein ORF3 Fig 1 ORF3 is expressed from distinct subgenomic sg mRNA 3 which is one of at least six distinct mRNAs 4 The ORF3 gene encodes for a putative 225 amino acid protein about 25 6 kDa in size Pyrc et al 2004 reports that the HCoV NL63 ORF3 gene has a unique nucleotide composition and appears as a U rich and A poor region within the genome indicating a recent gene transfer event from another viral or cellular origin 4 The ORF3 protein of HCoV NL63 is homologous to proteins of the other Group 1 coronaviruses with an amino acid sequence most similar 43 identity and 62 similarity to HCoV 229E ORF4 Based on comparative in silico analysis of HCoV NL63 ORF3 and other human coronavirus ORF3 like homologues Fielding and Suliman speculate that the protein could contribute to pathogenesis in the natural host 39 More recently studies have shown that HCoV NL63 localizes along the secretory pathway ERGIC Golgi plasma membrane and co localizes with the structural proteins M and E in the ERGIC Also studies have shown that this N glycosylated protein is incorporated into virions during assembly This further suggests an important function for HCoV NL63 ORF3 particularly in virus assembly and or budding from the infected cell 40 4 TRANSMISSION AND PATHOGENESIS 4 1 Mode of Entry into the Cell It is well documented that SARS CoV and HCoV NL63 use the same receptor angiotensin converting enzyme ACE 2 for entry into the host cell 41 42 However the consequences following the entry are very different SARS CoV causes severe respiratory distress while HCoV NL63 might lead to a mild respiratory infection This was attributed to the ability of each virus receptor binding protein spike or S protein to bind to ACE 2 on the cell surface It was found that the HCoV NL63 S protein has a weaker interaction with ACE 2 than the SARS CoV S protein This lower affinity interaction with ACE 2 might partly explain the different pathological consequences of infection by SARS CoV and HCoV NL63 43 Nevertheless high pathogenicity is expected to evolve with coronaviruses As for HCoV NL63 the possibility of the development of a recombinant virus variant is high because of its high prevalence and the possibility of a recombination event through co infection 24 Moreover the virus is able to survive for up to seven days in an aqueous solution and respiratory secretions and remains infective at room temperature In heavily populated regions direct person to person transmission is considered the major route of HCoV NL63 spread 42 44 4 2 Seasonal Incidence The virus has been shown to have a worldwide distribution and was observed primarily in the winter season in temperate climates On the other hand countries with extreme weather like Canada have also shown virus activity around January to March although milder symptoms were reported 45 Interestingly seasonal variations have been reported in China where infection with HCoV NL63 appeared mainly in spring and summer 26 Also a recent study of coronaviruses in Thailand did not show any seasonal predilection 46 while Wu et al 2007 reported that the virus is detected during the autumn season in Taiwan 47 It is evident that the virus has no predilection to a particular season and is not affected by temperature variations as infections can occur throughout the year Table 1 Understanding Human Coronavirus HCoV NL63 The Open Virology Journal 2010 Volume 4 79 4 3 Prevalence The human coronaviruses account for a significant number of hospitalization for children under 18 years of age the elderly and immunocompromised individuals In fact a one year study of children hospitalized in Hong Kong China has shown that respiratory tract infection with coronaviruses accounts for 4 4 of all admissions for acute respiratory infections Of these HCoV NL63 was the most common coronavirus identified with an incidence of 2 6 48 Moreover a study in Japan has shown that out of 419 specimens that tested negative for common respiratory viruses five 1 2 were positive for human coronavirus HCoV NL63 49 Another Japanese report has indicated that out of 118 nasopharyngeal swab samples obtained from hospitalized children younger than two years of age three 2 5 were positive for HCoV NL63 50 In Europe high prevalence was also noted In Italy a study conducted on 322 infants suffering from acute respiratory disease has shown that 8 7 of the cases examined were caused by coronaviruses with HCoV NL63 accounting for 21 4 of the latter 51 In France 300 respiratory specimens were checked for HCoV NL63 presence of the 300 samples 28 9 3 were positive 29 Likewise seven cases were reported in a one year study in Belgium 28 Interestingly the Dutch group that first described the virus has obtained 949 samples from a German group who conducted a population based study on lower respiratory tract infection in children under three years of age 52 the group re analyzed the samples and detected a 5 2 incidence of HCoV NL63 53 In Australia the virus was detected in Melbourne where a study conducted on 543 patients with respiratory symptoms has shown 18 cases 3 3 of human coronavirus HCoV NL63 infection 54 Moreover in the USA and Canada the virus was reported in 79 8 8 out of 895 and 19 3 6 out of 525 patients respectively Currently the accuracy of the percentage of detection is hampered by two main problems firstly the suitability of the samples examined a recent study has shown that there are differences between respiratory samples collected by nose throat swabs and by nasopharyngeal aspirates specifically regarding their potential to detect and identify respiratory pathogens 55 The second problem is that diagnostic tests for HCoVs are not frequently used in the routine testing for viruses which probably results in the percentage of HCoVs infections being greatly underestimated 56 Moreover throughout the years several methods of variable sensitivity were used to determine the incidence of the virus 57 59 4 4 Co Infection Many groups have reported that the occurrence of co infections with HCoV NL63 and other respiratory viruses including other human coronaviruses influenza A virus respiratory syncytial virus RSV parainfluenza virus and human metapneumovirus hMPV are common 26 30 46 47 51 53 54 60 61 Also co infected patients are more likely to be hospitalized indicating the severity of this kind of superinfection In a study from Germany RSV A and HCoV NL63 was the most common co infection indentified in children less than three years of age This is probably due to the high incidence of RSV A in winter and the overlap in seasonality of the viruses 53 Also in Italy HCoV NL63 circulates as a mixture of variant strains and is often associated with other viral infections 30 In South Africa co infection of patients with HCoV NL63 and bocavirus in hospitalized children is reported Nasopharyngeal and bronchoalveolar lavage samples from 341 patients were screened for common respiratory viruses and the co presence of HCoV NL63 and bocavirus in at least one sample was reported 62 Interestingly the viral load of HCoV NL63 is lower in co infected patients than in patients infected with HCOV NL63 only There are various possible explanations for this phenomenon 53 1 HCoV NL63 might be the initial infection that weakens the immune system enough for a second infection to gain a foothold By the time this second infection shows symptoms the HCoV NL63 infection might have already have been brought under control by the host immune system 2 the two viruses may be in competition for the same receptor or target cell in the respiratory organs 3 the elevated activation of the innate immune response triggered by the second respiratory virus may cause inhibition of HCoV NL63 or 4 prolonged persistence of HCoV NL63 at low levels The high prevalence of co infections of HCoV NL63 and other respiratory viruses increases the chances of genetic recombination with these human or zoonotically transmitted viruses In fact Pryc et al 2006 states HCoV NL63 resulted from a recombination event between PEDV and an an