Comparison of the Amino Acid Sequence and Phylogenetic Analysis of the Peplomer,Integral Membrane and Nucleocapsid Proteins of Feline,Canine and Porcine Coronaviruses

Complete nucleotide sequences were determined by cDNA cloning of peplomer (S), integral membrane (M) and nucleocapsid (N) genes of feline infectious peritonitis virus (FIPV) type I strain KU-2, UCD1 and Black, and feline enteric coronavirus (FECV) type II strain 79–1683. Only M and N genes were analyzed in strain KU-2 and strain 79–1683, which still had unknown nucleotide sequences. Deduced amino acid sequences of S, M and N proteins were compared in a total of 7 strains of coronaviruses, which included FIPV type II strain 79–1146, canine coronavirus (CCV) strain Insavc-1 and transmissible gastroenteritis virus of swine (TGEV) strain Purdue. Comparison of deduced amino acid sequences of M and N proteins revealed that both M and N proteins had an identity of at least 90% between FIPV type I and type II. The phylogenetic tree of the M and N protein-deduced amino acid sequences showed that FIPV type I and type II form a group with FECV type II, and that these viruses were evolutionarily distant from CCV and TGEV. On the other hand, when the S protein-deduced amino acid sequences was compared, identity of only about 45% was found between FIPV type I and type II. The phylogenetic tree of the S protein-deduced amino acid sequences indicated that three strains of FIPV type I form a group, and that it is a very long distance from the FIPV type II, FECV type II, CCV and TGEV groups.     

Emergence of Pathogenic Coronaviruses in Cats by Homologous Recombination between Feline and Canine Coronaviruses

Type II feline coronavirus (FCoV) emerged via double recombination between type I FCoV and type II canine coronavirus (CCoV). In this study, two type I FCoVs, three type II FCoVs and ten type II CCoVs were genetically compared. The results showed that three Japanese type II FCoVs, M91-267, KUK-H/L and Tokyo/cat/130627, also emerged by homologous recombination between type I FCoV and type II CCoV and their parent viruses were genetically different from one another. In addition, the 3′-terminal recombination sites of M91-267, KUK-H/L and Tokyo/cat/130627 were different from one another within the genes encoding membrane and spike proteins, and the 5′-terminal recombination sites were also located at different regions of ORF1. These results indicate that at least three Japanese type II FCoVs emerged independently. Sera from a cat experimentally infected with type I FCoV was unable to neutralize type II CCoV infection, indicating that cats persistently infected with type I FCoV may be superinfected with type II CCoV. Our previous study reported that few Japanese cats have antibody against type II FCoV. All of these observations suggest that type II FCoV emerged inside the cat body and is unable to readily spread among cats, indicating that these recombination events for emergence of pathogenic coronaviruses occur frequently.     

A reverse genetics approach to study feline infectious peritonitis

Feline infectious peritonitis (FIP) is a lethal immunopathological disease caused by feline coronaviruses (FCoVs). Here, we describe a reverse genetics approach to study FIP by assessing the pathogenicity of recombinant type I and type II and chimeric type I/type II FCoVs. All recombinant FCoVs established productive infection in cats, and recombinant type II FCoV (strain 79-1146) induced FIP. Virus sequence analyses from FIP-diseased cats revealed that the 3c gene stop codon of strain 79-1146 has changed to restore a full-length open reading frame (ORF).     

Acquisition of macrophage tropism during the pathogenesis of feline infectious peritonitis is determined by mutations in the feline coronavirus spike protein

In feline coronavirus (FCoV) pathogenesis, the ability to infect macrophages is an essential virulence factor. Whereas the low-virulence feline enteric coronavirus (FECV) isolates primarily replicate in the epithelial cells of the enteric tract, highly virulent feline infectious peritonitis virus (FIPV) isolates have acquired the ability to replicate efficiently in macrophages, which allows rapid dissemination of the virulent virus throughout the body. FIPV 79-1146 and FECV 79-1683 are two genetically closely related representatives of the two pathotypes. Whereas FECV 79-1683 causes at the most a mild enteritis in young kittens, FIPV 79-1146 almost invariably induces a lethal peritonitis. The virulence phenotypes correlate with the abilities of these viruses to infect and replicate in macrophages, a feature of FIPV 79-1146 but not of FECV 79-1683. To identify the genetic determinants of the FIPV 79-1146 macrophage tropism, we exchanged regions of its genome with the corresponding parts of FECV 79-1683, after which the ability of the FIPV/FECV hybrid viruses to infect macrophages was tested. Thus, we established that the FIPV spike protein is the determinant for efficient macrophage infection. Interestingly, this property mapped to the C-terminal domain of the protein, implying that the difference in infection efficiency between the two viruses is not determined at the level of receptor usage, which we confirmed by showing that infection by both viruses was equally blocked by antibodies directed against the feline aminopeptidase N receptor. The implications of these findings are discussed.     

Enhancement and Neutralization of Feline Infectious Peritonitis Virus Infection in Feline Macrophages by Neutralizing Monoclonal Antibodies Recognizing Different Epitopes

The interaction between the enhancing and neutralizing activities of three monoclonal antibodies (MAbs) (5-6-2, 6-4-2 and 7-4-1) to the spike protein of feline infectious peritonitis virus (FIPV) strain 79-1146 was determined using feline macrophages. At a high MAb concentration, all of the three MAbs completely inhibited the FIPV infection at 37 C. However, two of them (6-4-2 and 7-4-1) enhanced FIPV infection when either the MAb concentration or reaction temperature was lowered. These MAbs also exerted an immediate infectivity-enhancing activity for up to 10 min of reaction and by 20 min, neutralizing activities were observed. Only MAb 5-6-2 consistently showed neutralizing activity regardless of the reaction conditions. Competition with sera from cats experimentally infected with FIPV strain 79-1146 or feline enteric coronavirus strain 79-1683 showed that the two epitopes recognized by MAb 5-6-2 and MAb 6-4-2, respectively, are also recognized by the natural host.     

犬冠状病毒大熊猫株纤突蛋白全基因的克隆与序列分析

在国内首次对犬冠状病毒大熊猫野毒株(CCV DXMV)纤突蛋白基因进行了克隆和序列测定.该基因全长4362bp,编码1453个氨基酸,N端前18个氨基酸为推测的信号肽序列,后1435个氨基酸构成成熟蛋白.与GenBank中已发表的11个CCV毒株S基因相比,S基因核苷酸序列同源性在40.2%～99.5%之间;推导的氨基酸序列同源性在15.9%～99.0%之间.DXMV株S基因变异区主要集中在该基因前1/2处,其中350-370、439-478、1718-1818三个区域碱基变异较大,而1060-1700区却十分保守.基于S全基因及其蛋白的聚类分析表明,DXMV株与K378、NVSL和US patent株亲源关系最近.推导的DXMV株S蛋白氨基酸序列潜在的N-联糖基化位点与CCV强毒V54相同,为34个,比Insavc-1弱毒多一个;其中第566-568位糖基化位点为多数强毒拥有而弱毒没有的.另外,DXMV株S蛋白疏水性及抗原表位与其它毒株有一定的差异,这些差异对DXMV株致病性和免疫原性等影响尚待进一步的研究.     

Peptides Corresponding to the Predicted Heptad Repeat 2 Domain of the Feline Coronavirus Spike Protein Are Potent Inhibitors of Viral Infection

Background

Feline infectious peritonitis (FIP) is a lethal immune-mediated disease caused by feline coronavirus (FCoV). Currently, no therapy with proven efficacy is available. In searching for agents that may prove clinically effective against FCoV infection, five analogous overlapping peptides were designed and synthesized based on the putative heptad repeat 2 (HR2) sequence of the spike protein of FCoV, and the antiviral efficacy was evaluated.

Methods

Plaque reduction assay and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cytotoxicity assay were performed in this study. Peptides were selected using a plaque reduction assay to inhibit Feline coronavirus infection.

Results

The results demonstrated that peptide (FP5) at concentrations below 20 μM inhibited viral replication by up to 97%. The peptide (FP5) exhibiting the most effective antiviral effect was further combined with a known anti-viral agent, human interferon-α (IFN-α), and a significant synergistic antiviral effect was observed.

Conclusion

Our data suggest that the synthetic peptide FP5 could serve as a valuable addition to the current FIP prevention methods.     

Functional differences in the peplomer glycoproteins of feline coronavirus isolates.

The feline coronaviruses can be divided into two distinct antigenic groups on the basis of antigenic differences found on the peplomer (E2) glycoprotein of the virus. Because the E2 glycoprotein is responsible for many of the biological functions of coronaviruses, experiments were done to determine whether there were any E2 functional differences between these two antigenic groups. The avirulent feline infectious peritonitis virus (FIPV) isolate FIPV-UCD-2, which has one antigenic type of E2, was less rapidly internalized and could not spread from cell to cell in the presence of neutralizing antibody. Two virulent isolates, FIPV-DF2 and FIPV-79-1146, as well as the non-FIP-causing feline enteric coronavirus (FECV) isolate FECV-79-1683, all of which have the second antigenic type of E2, were very rapidly internalized and were able to spread from cell to cell despite the presence of neutralizing antibody. The avirulent FIPV-UCD-2 and FECV-79-1683 isolates were more labile at 37 degrees C at pHs of 6.5 and above than were the virulent FIPV-DF2 and FIPV-79-1146 isolates.     

Genome organization and reverse genetic analysis of a type I feline coronavirus

In this study we report the complete sequence and genome organization of the serotype I feline coronavirus (FCoV) strain Black. Furthermore, a reverse genetic system was established for this FCoV strain by cloning a full-length cDNA copy into vaccinia virus. This clone served as basis for the generation of recombinant FCoV (recFCoV) that was shown to bear the same features in vitro as the parental FCoV. Using this system, accessory 3abc genes in the FCoV genome were replaced by green fluorescent protein (recFCoV-GFP) and Renilla luciferase genes (recFCoV-RL). In addition, we showed that feline CD14⁺ blood monocytes and dendritic cells can be easily detected after infection with recFCoV-GFP. Thus, our established reverse genetic system provides a suitable tool to study the molecular biology of serotype I FCoV.     

从健康狐狸、貉粪中检出犬冠状病毒的两种基因型

取某养殖场健康狐狸 (6 1份 )、貉 (2 4份 )粪样 ,用套式PCR(RT-nPCR)方法检测犬冠状病毒 (CCV)并进行基因型鉴定。结果显示 :狐狸粪样中有 4 3份 (70 . 5 % )为CCVⅡ型阳性 ,2 9份 (47. 5 % )为CCVⅠ型阳性 ,两型混合感染2 5份 ,占 4 1 0 % ,两型总感染率为 77 .0 %。 2 4份貉粪样中 ,2 2份为CCVⅡ型阳性 (91 .7% ) ,其中 16份为CCVⅠ型、Ⅱ型混合感染 (6 6 . 7% )。分别取狐狸和貉粪样中CCVⅠ、Ⅱ型阳性PCR产物各 2份 (共 8份 )进行测序 ,均与CCV的M基因序列相符 ,证实PCR结果非假阳性。序列及系统进化分析表明 ,CCVⅠ型与源于意大利腹泻犬的CCVⅠ型 (AF5 0 2 5 83)同源性最高 (96 . 7%～ 98. 1% ) ;CCVⅠ、Ⅱ两种基因型同源性为 88. 3%～ 89. 7% ,在进化树中处于两个大的分支上 ;同为CCVⅡ型 ,狐狸源与貉源序列也存在多个位点差异。首次在貉粪中检出CCV ,证实在健康狐狸、貉群体中存在CCV流行 ,且CCVⅠ、Ⅱ型并存。     

Rapid and sensitive identification of RNA from the emerging pathogen,coxsackievirus A6

Feline infectious peritonitis virus (FIPV) and feline enteric coronavirus (FECV) are two important coronaviruses of domestic cat worldwide. Although FCoV is prevalent among cats; the fastidious nature of type I FCoV to grow on cell culture has limited further studies on tissue tropism and pathogenesis of FCoV. While several studies reported serological evidence for FCoV in Malaysia, neither the circulating FCoV isolated nor its biotypes determined. This study for the first time, describes the isolation and biotypes determination of type I and type II FCoV from naturally infected cats in Malaysia. Of the total number of cats sampled, 95% (40/42) were RT-PCR positive for FCoV. Inoculation of clinical samples into Crandell feline kidney cells (CrFK), and Feline catus whole fetus-4 cells (Fcwf-4), show cytopathic effect (CPE) characterized by syncytial cells formation and later cell detachment. Differentiation of FCoV biotypes using RT-PCR assay revealed that, 97.5% and 2.5% of local isolates were type I and type II FCoV, respectively. These isolates had high sequence homology and phylogenetic similarity with several FCoV isolates from Europe, South East Asia and USA. This study reported the successful isolation of local type I and type II FCoV evident with formation of cytopathic effects in two types of cell cultures namely the CrFK and Fcwf-4 , where the later cells being more permissive. However, the RT-PCR assay is more sensitive in detecting the antigen in suspected samples as compared to virus isolation in cell culture. The present study indicated that type I FCoV is more prevalent among cats in Malaysia.     

Methylation of H3 K4 and K79 is not strictly dependent on H2B K123 ubiquitylation

Covalent modifications of histone proteins have profound consequences on chromatin structure and function. Specific modification patterns constitute a code read by effector proteins. Studies from yeast found that H3 trimethylation at K4 and K79 is dependent on ubiquitylation of H2B K123, which is termed a “trans-tail pathway.” In this study, we show that a strain unable to be ubiquitylated on H2B (K123R) is still proficient for H3 trimethylation at both K4 and K79, indicating that H3 methylation status is not solely dependent on H2B ubiquitylation. However, additional mutations in H2B result in loss of H3 methylation when combined with htb1-K123R. Consistent with this, we find that the original strain used to identify the trans-tail pathway has a genomic mutation that, when combined with H2B K123R, results in defective H3 methylation. Finally, we show that strains lacking the ubiquitin ligase Bre1 are defective for H3 methylation, suggesting that there is an additional Bre1 substrate that in combination with H2B K123 facilitates H3 methylation.     

Determination of the cell tropism of serotype 1 feline infectious peritonitis virus using the spike affinity histochemistry in paraffin‐embedded tissues

Unlike for serotype II feline coronaviruses (FCoV II), the cellular receptor for serotype I FCoV (FCoV I), the most prevalent FCoV serotype, is unknown. To provide a platform for assessing the pattern by which FCoV I attaches to its host receptor(s), HEK293 cell lines that stably express the ectodomains of the spike (S) proteins derived from a FCoV I feline enteric coronavirus strain UU7 (FECV UU7) and a feline infectious peritonitis virus strain UU4 (FIPV UU4) were established. Using the recombinant S proteins as probes to perform S protein affinity histochemistry in paraffin‐embedded tissues, although no tissue or enteric binding of FECV UU7 S protein was detected, it was found that by immunohistochemistry that the tissue distribution of FIPV UU4 S protein‐bound cells correlated with that of FIPV antigen‐positive cells and lesions associated with FIP and that the affinity binding of FIPV UU4 S protein on macrophages was not affected by enzymatic removal of host cell‐surface sialic acid with neuraminidase. These findings suggest that a factor(s) other than sialic acid contribute(s) to the macrophage tropism of FIPV strain UU4. This approach allowed obtaining more information about both virus–host cell interactions and the biological characteristics of the unidentified cellular receptor for FCoV I.

     

Antigenic comparison of feline coronavirus isolates: evidence for markedly different peplomer glycoproteins.

The antigenic relationships among seven feline coronavirus isolates were investigated by using a panel of 26 monoclonal antibodies (MAbs). The MAbs were categorized into five immunoreactive groups which were used to delineate two antigenic types of feline coronaviruses. One antigenic type included the more virulent feline infectious peritonitis virus (FIPV) isolates (FIPV-UCD-1, FIPV-UCD-4, FIPV-TN406, FIPV-DF2, and FIPV-79-1146), whereas the second antigenic type was composed of the avirulent isolate FIPV-UCD-2. The feline enteric coronavirus isolate FECV-79-1683 shared some characteristics of both of the major antigenic groups. Epitopes on the nucleocapsid and envelope polypeptides were in general highly conserved among both antigenic types, although a few type-specific antigenic sites were discriminated. The most striking finding was the marked antigenic difference in the peplomer (E2) glycoproteins between the two antigenic types. Seven anti-E2 MAbs reacted with one antigenic type of E2, whereas seven other anti-E2 MAbs recognized a different antigenic form of E2. None of the 14 anti-E2 MAbs reacted with all of the isolates.     

Identification of a feline coronavirus type I strain from a cat with feline infectious peritonitis by RT-pCR and phylogenetic analysis

A case of feline infectious peritonitis (FIP) in an 11-month-old European shorthair cat is reported. The infected cat displayed loss of weight, respiratory distress, ascitis, anemia and died within 15 days after the first appearance of clinical signs. Lesions typical of a mixed form (effusive and non-effusive) of FIP were observed and by RT-PCR a feline coronavirus (FCoV) type I strain was detected in several tissues. The RT-PCR results were confirmed by sequence analysis of the amplified products. Phylogeny carried out on fragments of the M and S genes showed that the FCoV strain segregates with typical type I FCoVs.     

Gain, preservation, and loss of a group 1a coronavirus accessory glycoprotein

Coronaviruses are positive-strand RNA viruses of extraordinary genetic complexity and diversity. In addition to a common set of genes for replicase and structural proteins, each coronavirus may carry multiple group-specific genes apparently acquired through relatively recent heterologous recombination events. Here we describe an accessory gene, ORF3, unique to canine coronavirus type I (CCoV-I) and characterize its product, glycoprotein gp3. Whereas ORF3 is conserved in CCoV-I, only remnants remain in CCoV-II and CCoV-II-derived porcine and feline coronaviruses. Our findings provide insight into the evolutionary history of coronavirus group 1a and into the dynamics of gain and loss of accessory genes.     

Tracking the Evolution of Dengue Virus Strains D2S10 and D2S20 by 454 Pyrosequencing

Dengue virus is the most prevalent mosquito-borne virus worldwide. In this study, we used pyrosequencing to analyze the whole viral genome of two mouse-adapted strains, D2S10 and D2S20, that induce a dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS)-like lethal disease in mice lacking the type I and/or type II interferon receptors. Previous experiments with D2S10 indicated that N124D and K128E mutations in the envelope protein were responsible for the severe disease induced in mice compared to its parental strain PL046. Here we demonstrate that D2S20 is more virulent than D2S10 and captured the presence of five key amino acid mutations – T70I, N83D, and K122I in envelope (E), and A62T in nonstructural protein 2A (NS2A) and G605V in nonstructural protein 5 (NS5) – that may account for this. These findings set the foundation for further dissection of the viral determinants responsible for dengue disease manifestations in mouse models.     

Epstein-Barr Virus Nuclear Antigen 1 Sequences in Endemic and Sporadic Burkitt’s Lymphoma Reflect Virus Strains Prevalent in Different Geographic Areas

The Epstein-Barr virus (EBV) nuclear antigen EBNA1 is the only viral protein detectably expressed in virus genome-positive Burkitt’s lymphoma (BL); recent work has suggested that viral strains with particular EBNA1 sequence changes are preferentially associated with this tumor and that, within a patient, the tumor-associated variant may have arisen de novo as a rare mutant of the dominant preexisting EBV strain (K. Bhatia, A. Raj, M. J. Gutierrez, J. G. Judde, G. Spangler, H. Venkatesh, and I. T. Magrath, Oncogene 13:177–181, 1996). In the present work we first study 12 BL patients and show that the virus strain in the tumor is identical in EBNA1 sequence and that it is matched at several other polymorphic loci to the dominant strain rescued in vitro from the patient’s normal circulating B cells. We then analyze BL-associated virus strains from three different geographic areas (East Africa, Europe, and New Guinea) alongside virus isolates from geographically matched control donors by using sequence changes in two separate regions of the EBNA1 gene (N-terminal codons 1 to 60 and C-terminal codons 460 to 510) to identify the EBNA1 subtype of each virus. Different geographic areas displayed different spectra of EBNA1 subtypes, with only limited overlap between them; even type 2 virus strains, which tended to be more homogeneous than their type 1 counterparts, showed geographic differences at the EBNA1 locus. Most importantly, within any one area the EBNA1 subtypes associated with BL were also found to be prevalent in the general population. We therefore find no evidence that Burkitt lymphomagenesis involves a selection for EBV strains with particular EBNA1 sequence changes.