Genogrouping of Vaccine Breakdown Strains (VBS) of Feline Calicivirus in Japan

Although prevention of feline calcivirus (FCV) infection by vaccination has been attempted, and isolation of FCV, development of the disease, and a few fatal cases in vaccinated cats have been reported. Fifteen FCV strains isolated from cats that had been vaccinated with commercially available FCV vaccines (F9, FCV-255, and FC-7) were genogrouped. Molecular analysis of viral genomes involved the construction of a phylogenetic tree of capsid genes using the NJ method. Cat anti-F9 serum and rabbit anti-FCV-255 serum were used for virus neutralization tests. Molecular phylogenetic analysis of the amino acid sequences of 15 virus isolates and those of the previously published and GenBank-deposited 9 global and 14 Japanese strains showed that 8 (53%) of the 15 virus isolates as well as the vaccine strains F9 and FCV-255 belonged to genogroup I (G_AI), and 7 (47%) belonged to genogroup II (G_AII). Of the 8 G_AI strains, 2 were isolated from cats that had been vaccinated with an F9 strain live vaccine, 5 from cats vaccinated with an FCV-255-derived vaccine, and 1 from a cat vaccinated with an FC-7-derived vaccine. Of the 7 G_AII strains, 5 were isolated from cats that had been vaccinated with the F9 strain live vaccine, 1 from a cat vaccinated with the FCV-255-derived vaccine, and 1 from a cat vaccinated with the FC-7-derived vaccine. These results indicate that more vaccine breakdown strains isolated from the cats vaccinated with the F9 strain-derived vaccine belong to G_AII than to G_AI, whereas more vaccine breakdown strains isolated from the cats vaccinated with the FCV-255 strain-derived vaccine belong to G_AI than to G_AII, and that when the FC-7 strain-derived vaccine is used, the vaccine breakdown strains belong almost equally to G_AI and G_AII. Thus, the genogroups of virus isolates varied with the vaccine strain used (p < 0.05). On the other hand, the neutralizing titres of feline anti-F9 serum and rabbit anti-FCV-255 serum against the 15 isolates were very low, showing no relationships between neutralizing antibody titres and genogroups. The DNA sequence identities between the virus isolates and the vaccine strains were low, at 70.6–82.9%, and no strains were found to have sequences derived from the vaccine strains. Alignment of amino acid sequences showed that the G_AI or G_AII virus isolates from the F9-vaccinated cats differed at position 428 of the 5’ hypervariable region (HVR) of capsid region of the F9 strain, whereas those from the FCV-255-vaccinated cats differed at positions 438, 453, and 460 of the 5’HVR of capsid region E of the F9 strain. We speculate that these differences influence genogrouping. The amino acid changes within the F9 linear epitopes common to G _A I and G _A II were noted at positions 450, 451, 457 of 5’HVR of the capsid region E in the isolates from F9-derived vaccine-treated cats, and 449, 450, and 451 of 5’HVR of capsid region E in the isolates from FCV-255-derived vaccine-treated cats, suggesting that these amino acid changes are involved in escapes. These results suggest that alternate vaccination with the F9 and FCV-255 strains or the use of a polyvalent vaccine containing G_AII strains serves to inhibit development.     

Full-length ORF2 sequence-based genetic and phylogenetic characterization of Korean feline caliciviruses

Feline calicivirus (FCV) is a highly infectious pathogen in cats and widely distributed worldwide with high genetic variation. Full-length open reading frame 2 of 5 from recently isolated Korean FCV isolates were sequenced and compared with those of global isolates. The results of phylogenetic analysis supported dividing global FCV isolates into two genogroups (type I and II) and demonstrated the presence of genogroup II in Korea, indicating their geographic spread in East Asia. High sequence variations in region E of the FCV isolates emphasizes that a novel vaccine needs to be developed to induce protective immunity against various FCV strains.     

Phylogenetic Analysis of Field Isolates of Feline Calcivirus (FCV) in Japan by Sequencing Part of Its Capsid Gene

The molecular epidemiology of the infectious disease caused by feline calcivirus (FCV) in Japan was investigated by analysing the phylogenetic relationship among 21 Japanese field isolates, including the F4 strain, and 30 global isolates. Parts of the capsid gene (B–F) of the isolates were amplified by RT-PCR, and the amino acid sequences were compared with those from the global isolates. Thirty-seven and 14 out of a total of 51 isolates were clustered into two distinct genogroups, I and II respectively, by UPGMA and NJ analysis. Seven of the 21 Japanese isolates (33%) fell into group I together with 30 global isolates, while the other 14 Japanese isolates (67%) belonged to group II. The bootstrap repetition analysis of groups I and II formed by the NJ method gave a value of 99.0%. The 14 latter Japanese isolates were clearly separated from the isolates in group I, and they were different from any previously known FCV, forming a new genogroup, which implies that this lineage has been confined to Japan. Comparing the amino acid sequences shared by groups I and II, the amino acid at position 377 in B region was asparagine (Asn or Asp (NH₂)) in group I, while it was lysine (Lys) in all the strains in group II. Similarly, the amino acid at position 539 in the F region was alanine (Ala) or proline (Pro) in group I, while it was valine (Val) in group II; glycine (Gly) at position 557 in group I was serine (Ser) in Group II; and phenylalanine (Phe) or leucine (Leu) at position 566 in genogroup I was tyrosine (Tyr) in group II.     

1株猫杯状病毒的分离鉴定及其ORF2序列的遗传演化分析

为了解猫杯状病毒形态特征及遗传演化情况,采用F81细胞从患病宠物猫的鼻拭子样品中分离获得1株猫杯状病毒(feline calicivirus,FCV),命名为SH1。经电镜观察,病毒粒子呈球形,无囊膜,符合FCV的形态特征。采用RT-PCR方法扩增了该毒株的全基因组,并进行了序列测定和衣壳蛋白基因(ORF2)序列的分析。结果显示:分离株与国内外参考株的ORF2序列的核苷酸和氨基酸同源性分别为74.1%~79.7%和84.5%~90.9%;ORF2基因的遗传演化分析显示,30株FCV毒株形成两大分支,即基因群Ⅰ和Ⅱ,分离株属于基因群Ⅰ;进一步分析发现,基因群Ⅰ和Ⅱ主要在377、539和557氨基酸位点存在差异,基因群Ⅰ和Ⅱ分别为N、A、G和K、V、S。研究结果为FCV感染的防控提供科学依据。     

Sequence variation within the capsid protein of Australian isolates of feline calicivirus.

The capsid protein of Australian feline calicivirus (FCV) isolates is demonstrably different from the prototype strain F9. Five Australian isolates of FCV, dating from 1970 to 1989, were analysed by western blotting and immunoprecipitation. Varying reactivity to a panel of F9 specific monoclonal antibodies (MAbs) was observed. DNA sequencing of RT-PCR generated clones supported the observation of variation between capsid proteins. Predicted amino acid sequences varied by 11 to 17.5% across the whole capsid when compared to the published F9 sequence. Differences in amino acid sequence were most apparent in previously described hypervariable regions (C and E). Within hypervariable region E differences of 22 to 34% were observed compared to F9. The observed lack of reactivity to F9 MAbs correlated with amino acid changes within previously characterized binding sites within region E.     

Genetic characterization of 2 novel feline caliciviruses isolated from cats with idiopathic lower urinary tract disease

Feline caliciviruses (FCVs) are potential etiologic agents in feline idiopathic lower urinary tract disease (I-LUTD). By means of a modified virus isolation method, we examined urine obtained from 28 male and female cats with nonobstructive I-LUTD, 12 male cats with obstructive I-LUTD, and 18 clinically healthy male and female cats. All cats had been routinely vaccinated for FCV. Two FCVs were isolated; I (FCV-U1) from a female cat with nonobstructive I-LUTD, and another (FCV-U2) from a male cat with obstructive I-LUTD. To determine the genetic relationship of FCV-U1 and FCV-U2 to other FCVs. capsid protein gene RNA was reverse transcribed into cDNA, amplified, and sequenced. Multiple amino acid sequence alignments and phylogenetic trees were constructed for the entire capsid protein, hypervariable region E, and the more conserved (nonhypervariable) regions A, B, D, and F. When compared to 23 other FCV isolates with known biotypes, the overall amino acid sequence identity of the capsid protein of FCV-U1 and FCV-U2 ranged from 83 to 96%; identity of hypervariable regions C and E ranged from 58 to 85%. Phylogenetically, FCV-U1 clearly separated from other FCV strains in phenograms based on nonhypervariable regions. In contrast, FCV-U2 consistently segregated with the Urbana strain in all phenograms. Clustering of isolates by geographic origin was most apparent in phenograms based on nonhypervariable regions. No clustering of isolates by biotype was apparent in any phenograms. Our results indicate that FCV-UI and FCV-U2 are genetically distinct from other known vaccine and field strains of FCV.     

Sequence analysis of E2 glycoprotein genes of classical swine fever viruses: identification of a novel genogroup in Thailand

Thirty classical swine fever viruses (CSFV) isolated in Thailand between 1988 and 1996 were characterised by genetic sequence analysis of a part of their E2 coding regions, comparing the new data with that for representative reference viruses from other countries and continents. Thai isolates were divided into three distinct genogroups, indicating multiple origins for the outbreaks. Eighteen isolates from 1988-1995 form a new genogroup not previously described from any other geographical region. Eleven isolates from 1988-1995 are in the same genogroup as old US and European strains represented by reference strains Alfort 187 and Brescia. The viruses of this group seem to have died out in Europe but still persist in Thailand. One recent isolate from 1996 represents another previously described genogroup being closely related to Italian viruses isolated in the same year.     

Ability of antibodies to two new caliciviral vaccine strains to neutralise feline calicivirus isolates from the UK

This study examined a panel of 110 UK field isolates of feline calicivirus (FCV) for susceptibility to cross-neutralisation by a panel of eight antisera raised in cats infected with FCV strains F9, 255, FCVG1 and FCV431. The pairs of antisera raised against F9 or 255, neutralised 20 and 21 per cent or 37 and 56 per cent of field strains of virus respectively. In contrast, the pairs of antisera raised against the newer vaccine strains FCVG1 or FCV431 neutralised 29 and 70 per cent or 67 and 87 per cent of field strains respectively. Antisera raised against the two newer strains, namely FCVG1 and FCV431, neutralised a greater proportion of field strains of calicivirus than antisera raised against the older FCV vaccine strains F9 and 255.     

Neutralizing feature of commercially available feline calicivirus (FCV) vaccine immune sera against FCV field isolates.

Four types of commercially available feline calicivirus (FCV) vaccine were compared in terms of their efficacy on the basis of the ability of the sera of specific-pathogen-free cats immunized by two injections of each type of vaccine to neutralize FCV field isolates. Each vaccine immune serum neutralized relatively well strains F4, F9, and 255, which were FCV laboratory strains. As to 36 strains of field isolates, however, vaccines A, B, C, and D immune sera did not neutralize 18-20 of the strains (50.0%-55.6%), 19-22 of the strains (52.8%-61.1%), 22-25 of the strains (61.1%-69.4%), and 8-16 of the strains (22.2%-44.4%), respectively. These results indicate that there is much difference in neutralizing antigenicity between the existing vaccine strains and the FCV strains that are prevalent in Japan, suggesting the need for improvement of FCV vaccines.     

Genetic analysis of Akabane virus isolates from cattle in Korea

Bayesian Inference (BI) and Neighbor Joining (NJ) analyses of the phylogenetic relationships between the nucleotide sequences of the N gene of Akabane virus revealed an unclear topology among genogroups I–III, which was probably caused by genetic reassortment or recombination between these genogroups. In contrast, nucleotide and amino acid phylogenetic tree analyses of the M RNA segment agreed with the topologies obtained by using the BI and NJ methods. Therefore, distinct genogrouping of Akabane virus isolates should be performed using the M RNA segment. Four Korea isolates were classified into genogroup II together with Akabane virus strains isolated from all areas of Japan, including Okinawa Island. However, more nationwide isolates and more clinical data from Korean cattle farms will be required in the future to confirm the precise relationships between genotypes and pathogenicity.     

Comparison of the ability of feline calicivirus (FCV) vaccines to neutralise a panel of current UK FCV isolates

Feline calicivirus (FCV) comprises a large number of strains which are related antigenically to varying degrees. The antigenic variability creates problems for choosing antigens to include in vaccines. Historically, these have been selected for use based on their cross-reactivity with a high proportion of field strains. However, it is important to determine the current level of cross-reactivity of vaccines and whether or not this may be decreasing owing to widespread vaccine use. In this in vitro study, we have compared the ability of antisera to two vaccine viruses (FCV strain F9 and FCV strain 255) to neutralise a panel of 40 recent UK field isolates. These 40 isolates were obtained by randomised, cross-sectional sampling of veterinary practices in different geographical regions of the UK so as to ensure they were representative of viruses circulating in the veterinary-visiting population of cats in the UK. Virus neutralisation assays showed that both vaccine strains are still broadly cross-reactive, with F9 antiserum neutralising 87.5% and 255 antiserum 75% of isolates tested with antiserum dilutions of 1 in 2 or greater. However, when antibody units were used, in order to take account of differences in homologous titres between antisera, fewer isolates were neutralised, with F9 antiserum showing a slightly higher proportion of isolates neutralised than 255. Multivariable analysis of the sample population of 1206 cats from which the 40 isolates were derived found that vaccinated cats were at a decreased risk of being positive for FCV, whereas cats from households with more than one cat, and cats with mouth ulcers were at increased risk. In addition as cats became older their risk of shedding FCV decreased.     

Nucleotide sequence of UK and Australian isolates of feline calicivirus (FCV) and phylogenetic analysis of FCVs.

We have determined the first complete genome sequence and capsid gene sequences of feline calicivirus (FCV) isolates from the UK and Australia. These were compared with other previously published sequences. The viruses used in the comparisons were isolated between 1957 and 1995 from various geographical locations and obtained from cats showing a range of clinical signs. Despite these diverse origins, comparisons between all strains showed a similar degree of sequence variation within both ORF1 (non-structural polyprotein) and ORF2 (major capsid protein) (amino acid distances of 7.7-13.0% and 8.8-18.6%, respectively). In contrast, ORF3 (putative minor structural protein) sequences indicated a more heterogenous distribution of FCV relatedness (amino acid distances of 1.9-17.9%). Phylogenetic analysis suggested that, unlike some other caliciviruses, FCV isolates within the current data set fall into one diverse genogroup. Within this group, there was an overall lack of geographic or temporal clustering which may be related to the epidemiology of FCV infection in cats. Analysis of regions of variability in the genome has shown that, as well as the previously identified variable regions in ORF2, similar domains exist within ORFs 1 and 3 also, although to a lesser extent. In ORF1, these variable domains largely fall between the putative non-structural protein functional domains.     

Molecular characterization and genogrouping of VP1 of aquatic birnavirus GC1 isolated from rockfish Sebastes schlegeli in Korea

The cDNA nucleotide sequence of genome segment B encoding the VP1 protein was determined for the aquatic birnavirus GC1 isolated from the rockfish Sebastes schlegeli in Korea. The VP1 protein of GC1 contains a 2,538 bp open reading frame, which encodes a protein comprising 846 amino acid residues that has a predicted MW of 94 kDa. The sequence contains 6 potential Asn-X-Ser/Thr motifs. Eight potential Ser phosphorylation sites and 1 potential Tyr phophorylation site were also identified. GC1 contains the Leu-Lys-Asn (LKN) motif instead of the typical Gly-Asp-Asp (GDD) motif found in other aquatic birnaviruses. We also identified the GLPYIGKT motif, the putative GTP-binding site at amino acid position 248. In total, the VP1 regions of 22 birnavirus strains were compared for analyzing the genetic relationship among the family Birnaviridae. Based on the deduced amino acid sequences, GC1 was observed to be more closely related to the infectious pancreatic necrosis virus (IPNV) from the USA, Japan, and Korea than the IPNV from Europe. Further, aquatic birnaviruses containing GC1 and IPNV have genogroups that are distinct from those in the genus Avibirnaviruses and Entomo-birnaviruses. The birnavirusstrains were clustered into 5 genogroups based on their amino acid sequences. The marine aquatic birnaviruses (MABVs) containing GC1 were included in the MABV genogroup; the IPNV strains isolated from Korea, Japan, and the USA were included in genogroup 1 and the IPNV strains isolated primarily from Europe were included in genogroup 2. Avibirnaviruses and entomobirnaviruses were included in genogroup 3 and 4, respectively.     

Genetic variation analyses of porcine epidemic diarrhea virus isolated in mid-eastern China from 2011 to 2013

Porcine diarrhea outbreaks caused by porcine epidemic diarrhea virus (PEDV) has occurred in China with significant losses of piglets since 2010. In this study, the complete S and ORF3 genes of 15 field PEDV isolates in mid-eastern China from 2011 to 2013 were detected and compared with other reference strains. Based on S gene, all of the PEDV strains could be assigned to 3 genogroups. Only 1 isolate, {"type":"entrez-nucleotide","attrs":{"text":"JS120103","term_id":"364436861","term_text":"JS120103"}}JS120103, belonged to genogroup 1 and showed a close relationship with previous Chinese strains DX and JS-2004-2, European strain CV777, and Korean strain DR13. The other 14 isolates belonged to genogroup 3 and showed a close relationship with other Chinese strains isolated after 2010. The S genes of those isolates were 9 nucleotides longer in length than {"type":"entrez-nucleotide","attrs":{"text":"JS120103","term_id":"364436861","term_text":"JS120103"}}JS120103 and the other reference strains in genogroup 1, with 15 bp insertion and 6 bp deletion. Homology analyses revealed that all of the Chinese field isolates, except {"type":"entrez-nucleotide","attrs":{"text":"JS120103","term_id":"364436861","term_text":"JS120103"}}JS120103, are 97.6% to 100% (95.8% to 100%) identical in nucleotide (deduced amino acid) sequence to each other. Meanwhile, based on the ORF3 gene, all of the PEDV isolates could be separated into 3 genogroups. Eleven of the 15 field isolates in this study belonged to genogroup 3 and were 95.8% to 100% identical in nucleotide sequence or 95.6% to 100% in deduced amino acid sequence to each other. Our results indicate that the variant PEDV strain spread wildly in mid-eastern China. This will be useful to take into consideration in the control and prevention of this disease.     

Mapping neutralizing and non-neutralizing epitopes on the capsid protein of feline calicivirus

Neutralizing epitopes on feline calicivirus (FCV) capsid protein were mapped using chimeric capsid proteins recombinant between two FCV isolates that do not show any cross-neutralization. The three chimeric proteins examined were expressed in murine L929 cells employing an MVA/T7 vaccinia virus expression system and inoculated into major histocompatibility complex haplotype-matched C3/HN mice. Based on the neutralizing antibody titre the neutralizing epitope(s) could be mapped to the 5' hypervariable region of the E region or potentially to the C region. The epitopes of some non-neutralizing antibodies were mapped with the same chimeric proteins to the regions B or D and F of the FCV capsid protein.     

Mapping Neutralizing and Non‐Neutralizing Epitopes on the Capsid Protein of Feline Calicivirus

Neutralizing epitopes on feline calicivirus (FCV) capsid protein were mapped using chimeric capsid proteins recombinant between two FCV isolates that do not show any cross‐neutralization. The three chimeric proteins examined were expressed in murine L929 cells employing an MVA/T7 vaccinia virus expression system and inoculated into major histocompatibility complex haplotype‐matched C3/HN mice. Based on the neutralizing antibody titre the neutralizing epitope(s) could be mapped to the 5′ hypervariable region of the E region or potentially to the C region. The epitopes of some non‐neutralizing antibodies were mapped with the same chimeric proteins to the regions B or D and F of the FCV capsid protein.     

Properties of a calicivirus isolated from cats dying in an agitated state

In June 1993, two of five pet cats kept in Yokohama city in Japan suddenly became agitated and died. Feline calicivirus (FCV) was isolated from them. One strain (FCV-S) was isolated from the spinal cord, lung and tonsil of cat 1, another (FCV-B) from the ileum, medulla oblongata and cervical spinal cord of cat 2, and a third (FCV-SAKURA) from the oral cavity of one of the three surviving cats which showed no clinical signs. These three strains were equally resistant to pH 3.0 and serologically similar to each other, but distinct from strain F9. A genetic analysis, using a 208 base pair fragment from region E of the capsid, showed that FCV-Ari had a 70.4 per cent nucleotide and 77.3 per cent amino acid homology and FCV-F9 had a 68.6 per cent nucleotide and 73.9 per cent amino acid homology with the three strains, indicating that these two strains were genetically distinct from the three new isolates. Unvaccinated cats and cats which had been vaccinated against FCV-F9 developed watery diarrhoea but did not become agitated after the administration of FCV-S. The FCV-S strain did not induce signs of excitability after it was administered orally to specific pathogen-free cats.     

Genotyping of infectious pancreatic necrosis virus isolates from Mexico state

The infectious pancreatic necrosis virus (IPNV; genus Aquabirnavirus) affects salmon and trout, causing high mortality in first-feeding fry. The classification of this virus includes nine serotypes and seven genogroups. In Mexico, two different isolates were identified in 2000 and 2008, respectively. Both isolates were classified into genogroup I according to the RNA genome of this virus. As Mexico is importing rainbow trout Oncorhynchus mykiss eggs from different countries, the aim of this study was to genotype IPNV isolates obtained from four rainbow trout producer regions within the state of Mexico. We utilized a fragment of the VP2* (outer capsid protein) gene sequence of Mexican IPNV isolates as a molecular marker to determine the genogroup to which they belong. Although all Mexican IPNV isolates were grouped into genogroup I, we identified genetic diversity among these isolates, and 14 unique nucleotide sequence types were associated with the four producer regions in Mexico State.