Abstract
Introduction
Felid herpesvirus 1 (FeHV-1) is widespread worldwide and is sometimes associated with chronic diseases in cats; therefore, it is clinically important. Vaccination can be used to control FeHV-1 infection. We previously isolated a strain identical to the live attenuated vaccine (LAV) strain F2 from a feline eye with a dendritic ulcer, indicating that the F2 strain may be pathogenic. The origin of the F2 strain that we previously isolated is unknown, and we surmised that the F2 strain may exist in the field. To determine whether the LAV strain F2 exists in the field, we, therefore, performed a genomic analysis of FeHV-1 isolates from cats.
Methods
FeHV-1 isolates from cats were genotyped using single-nucleotide variants (SNVs) within two marker genes related to the F2 strain as indicators. Strains with markers were subjected to next-generation sequencing to determine their whole-genome sequence (WGS).
Results
Of the nine FeHV-1 isolates, none were found to have two SNVs related to the F2 strain, whereas two isolates were found to have one SNV related to the F2 strain. Because the cats from which these strains were isolated lived together, the isolates were considered to be of the same strain, and we determined the WGS of one strain (strain A-19-5). All coding sequences of strain A-19-5 were identical to those of one of the cloned strains of the Merial vaccine (likely a variant of the F2 strain within the Merial vaccine population). The cat from which the strain was isolated had never received vaccination.
Conclusion
The results suggest that a strain identical to a variant of the F2 strain, as found in the FeHV-1-LAV, may exist in the field.
Keywords: Felid herpesvirus 1, Live attenuated vaccine, F2 strain, Variant, Cat
Introduction
Felid herpesvirus 1 (FeHV-1) belongs to the Varicellovirus genus of the Herpesviridae family. FeHV-1 is spread worldwide and is a major etiological factor for ocular and upper respiratory diseases in cats [1]. Clinical signs of FeHV-1 infection vary from mild to severe, sometimes resulting in chronic diseases such as corneal sequestration, eosinophilic keratoconjunctivitis, and chronic rhinitis [1–3]. These chronic diseases are refractory and may require lifelong treatment, so they are clinically important [1–3]. Thus, control of FeHV-1 infection is important, but the subclinical shedding of FeHV-1 and reactivation of a latent infection make infection control particularly challenging in a multi-cat household environment.
A vaccination program is currently available to control FeHV-1 infection. The FeHV-1 vaccine is recommended as a core vaccine for routine vaccination [4]. A live attenuated vaccine (LAV) stimulates an immune response that more closely mimics protection from natural infection with the virus [4]; therefore, it is often the vaccine of choice in clinical settings. The representative strain of FeHV-1-LAV is the F2 strain, which is currently available in Japan from two manufacturers: Purevax® RCP (Nippon Boehringer Ingelheim Co. Ltd., Tokyo, Japan) and Virbagen® CRP (Virbac Japan Co. Ltd., Osaka, Japan). The Purevax RCP was previously manufactured and distributed in Japan by Merial Japan Inc., which merged with Boehringer in December 2017. The contents of the vaccines from both manufacturers were the same, and the F2 strain was registered in GenBank as Merial Purevax.
We previously reported isolation of the F2 strain from a feline eye with a dendritic ulcer in 2025 [5]. Nonetheless, the origin of the F2 strain isolated in that study remains unknown; we surmised that the F2 strain had previously infected the cat in the field, causing a latent infection that was reactivated by vaccination [5]. This suggests that the F2 strain may exist in the field. If the F2 strain exists in the field and infects cats, then that is an important route of infection. To investigate whether the F2 strain exists in the field, we analyzed the genomes of the FeHV-1 strain isolated from cats clinically suspected of having an FeHV-1 infection, with markers of the F2 strain in the unique long (UL) 28 coding sequence (CDS) and UL44 CDS serving as indicators [5].
Methods
Animals and Specimen Collection
Of the cats that underwent ophthalmic examination at three veterinary clinics from May 2019 to June 2022, 47 cats were suspected of having an FeHV-1 infection; therefore, specimens were collected to diagnose this infection via viral culture. The public importance of a genomic analysis of the FeHV-1 strain was fully explained, and then the owners approved the use of these specimens for that purpose. The owners were interviewed regarding their cats’ vaccination and medication history and living conditions. After receiving an affirmative response and permission from each owner, an antibody test for feline immunodeficiency virus (FIV) and an antigen test for feline leukemia virus (FeLV) were performed using the SNAP FIV/FeLV Combo Test (IDEXX Laboratories Inc., Tokyo, Japan). A sterile Dacron swab (Texwipe®, Kernersville, NC, USA) was used to collect the swab by wiping tears from the conjunctiva. As previously indicated [5], the swab was placed in a sterile plastic tube with 0.5 mL of Eagle’s minimal essential medium (MEM; Sigma-Aldrich Japan K.K., Tokyo, Japan) supplemented with gentamicin at a final concentration of 50 μg/mL. The tubes were refrigerated (2–6°C) and used for virus isolation.
Virus Isolation and DNA Extraction
The Crandell-Rees feline kidney (CRFK) cell line was used for virus isolation [6]. Virus culture was performed as previously described [5]. MEM supplemented with 10% fetal bovine serum was used as the growth medium, and the serum concentration was reduced to 4% to serve as a maintenance medium. The swab was centrifuged at 700×g for 5 min, and 100 μL of the supernatant was inoculated into a monolayer culture of CRFK cells in six-well plates. After adsorption at 37°C for 30 min in a 5% CO2, the cell surface was washed with MEM. The cells were cultured at 37°C in 5% CO2 in maintenance medium for up to 1 week. Viral DNA was extracted as the total DNA from virus-infected cells and used for PCR [7]. Viral DNA for next-generation sequencing (NGS) was extracted from nucleocapsids in virus-infected cells to obtain a high concentration of viral DNA that was as free of cell-derived DNA as possible [8].
PCR Primers, PCR, and NGS
Molecular detection of FeHV-1 in viral isolates was performed with PCR targeting the FeHV-1 glycoprotein B (gB) gene, as described by Vögtlin et al. [9]. Primers specific for the nucleotide sequences of UL28 and UL44 CDS were used for simplified genotyping of FeHV-1 clinical isolates as the F2 strain [5]. PCR was performed using KOD-Plus-Neo (Toyobo Co. Ltd., Osaka, Japan), and its products were purified using the QIAquick PCR & Gel Cleanup Kit (QIAGEN K.K., Tokyo, Japan). Sequencing was performed by Hokkaido System Science Co. Ltd. (Hokkaido, Japan), and the resulting sequences were analyzed using the software SnapGene (www.snapgene.com). The obtained sequences were registered in the DNA Data Bank of Japan (accession numbers: LC864352–LC864369). NGS was performed by Macrogen Japan Corp. (Tokyo, Japan) to determine the whole-genome sequence of clinical isolates, in which markers of the F2 strain were detected by simplified genotyping. Sequencing was performed on the NovaSeq 6000 platform (Illumina K.K., Tokyo, Japan). All confirmed sequences were registered in GenBank (accession number: PV209842), and a detailed genomic analysis was performed using Geneious Prime version 2023.1.2 [5].
Analysis of the LAV Cloned Strain
Using the plaque method, FeHV-1 clones were previously obtained from Purevax RCP (lot number: F18579, expiration date: July 2024), which contained the F2 strain [5], and they were then analyzed for UL28 and UL44 CDS.
Results
Virus Isolation and Identification
In specimens from 9 of the 47 cats, a clear cytopathic effect on CRFK cells, characterized by herpesvirus-like rounding of cells, was observed by day 2. PCR targeting the FeHV-1 gB gene resulted in the detection of a DNA fragment of approximately 740 bp in length, which was the same size as the expected amplified fragment of FeHV-1 gB (data not shown), so the nine virus isolates were identified as FeHV-1.
Animals
The clinical data on cats from which FeHV-1 was isolated are shown in Table 1. Eight cats were domestic shorthairs, and one was a Maine coon. There were five females (two neutered and three intact) and four males (one neutered and three intact). The cats ranged in age from approximately 1–156 months (mean, approximately 48 months). An FIV antibody test and FeLV antigen test were performed on 4 cats (Cat Nos. 5, 6, 8, and 9), and both were negative. Seven of the 9 cats (Cat Nos. 1–6 and 9) had never been vaccinated. Cat Nos. 6 and 9 were approximately 1 month of age and were seen immediately after being picked up from the outdoors; therefore, they were deemed to have no history of vaccination or medication. The vaccination history for Cat No. 7 was unknown, and Cat No. 8 was first vaccinated with Felivac® L-3 (Kyoritsu Seiyaku Corp., Tokyo, Japan), which contained the KSH02 strain of the FeHV-1-LAV, approximately 1 month before observation at this clinic. Two cats (Cat Nos. 2 and 8) received medications at other clinics. Cat No. 2 received an injection of Intercat® (Kyoritsu), a recombinant feline interferon, and was administered oral famciclovir (manufacturer and dose unknown) 1 day before observation. The treatment of Cat No. 8 was initiated 10 days before virus isolation, with Intercat (Kyoritsu) being injected once a day for 4 days, chloramphenicol and colistin sodium methanesulfonate eye drops (Ophthalon Ophthalmic Solution, Wakamoto Pharm. Corp., Osaka, Japan) being administered to both eyes 3–4 times a day for 10 days, and additional nonsteroidal anti-inflammatory eye drops (Tearose®; Senju Pharm. Co. Ltd., Osaka, Japan) being administered to both eyes 3–4 times a day, 2 days before observation. Cat Nos. 6 and 9 were kept outdoors; Cat No. 3 was allowed indoors and outdoors; and the remaining cats (Cat Nos. 1, 2, 4, 5, 7, and 8) were kept indoors. Cat No. 1 was living with 3 other cats that had no history of vaccination. Cat No. 2 was living with 10 other cats with no known vaccination history. Cat Nos. 3, 6, and 9 did not live with other cats. Cat Nos. 4 and 5 lived together, along with 5 other cats, only one of which had been vaccinated 11 years earlier with a vaccine from an unknown manufacturer. Cat No. 7 lived with 3 other cats, none of which had been previously vaccinated. Cat No. 8 lived with 3 other cats, two of which had been vaccinated with the same vaccine (Kyoritsu) as Cat No. 8.
Table 1.
Clinical data on cats from which felid herpesvirus 1 (FeHV-1) was isolated
| Attributes | Cat No. | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | |
| FeHV-1 isolate | A-19-1 | B-19-1 | C-19-1 | A-19-5 | A-19-6 | A-19-7 | A-20-1 | A-21-1 | A-22-1 |
| Date specimen collected (month and year) | May 2019 | June 2019 | June 2019 | Nov 2019 | Nov 2019 | Nov 2019 | Oct 2020 | June 2021 | May 2022 |
| Breed | DSH | Maine coon | DSH | DSH | DSH | DSH | DSH | DSH | DSH |
| Sex | Male | Female | Female | Male | Female | Male | Female | Female | Male |
| Neutering status | Intact | Intact | Intact | Neutered | Neutered | Intact | Neutered | Intact | Intact |
| Age, months | 6 | 1 | 24 | 96 | 144 | 1 | 156 | 2 | 1 |
| FIV/FeLV | ND | ND | ND | ND | Both negative | Both negative | ND | Both negative | Both negative |
| Vaccination history | None | None | None | None | None | None | Unknown | Yesa | None |
| Medication history | None | Intercat, famciclovir | None | None | None | None | None | Intercat, chloramphenicol, NSAIDs | None |
| Living conditions | Indoors | Indoors | Outdoors and indoors | Indoors | Indoors | Outdoors | Indoors | Indoors | Outdoors |
| Cats living together, n | 3 | 10 | 0 | 6 | 6 | 0 | 3 | 3 | 0 |
| Vaccination history of cats living together | None | Unknown | – | Yesa (1 cat) | Yesa (1 cat) | – | None | Yesb (2 cat) | – |
DSH, domestic shorthair; FIV, feline immunodeficiency virus; FeLV, feline leukemia virus; ND, not determined; NSAIDs, nonsteroidal anti-inflammatory drugs.
aVaccine manufacturer unknown.
bKSH02 strain (Felivac L-3; Kyoritsu Seiyaku Corp).
Simplified Genotyping
Single-nucleotide variants (SNVs) in UL28 (1280A) and UL44 (397G) were used for simplified genotyping of the F2 strain [5]. A genomic analysis of these two regions in nine isolates revealed no strains with the SNVs in either CDSs, but strains with the SNV in UL44 but not in UL28 (strains A-19-5 and A-19-6) were found (Table 2). A GenBank search for strains with nucleotides identical to the A-19-5 and A-19-6 strains at these locations yielded five field strains (LOU-17, LOU-18, MM-2, MM-3, and MM-7).
Table 2.
Nucleotide differences and locations in the F2 strain marker genes, unique long (UL) 28 and UL44, and other genes, UL8, UL41, UL44, and UL48 in each felid herpesvirus-1 strain
| Virus strain | F2 strain marker genes | Other genes | GenBank | ||||
|---|---|---|---|---|---|---|---|
| UL28 (1280a) | UL44 (397a) | UL8 (1393a) | UL38 (716a) | UL41 (737a) | UL48 (395a) | ||
| C-27 | C | A | C | A | T | T | NC_013590 |
| F2 (Virbac) | A | G | C | A | T | T | KR296657 |
| F2 (Intervet) | A | G | C | A | T | T | KR381803 |
| F2 (Merial) | A | G | C | R | Y | Y | OL410296 |
| A-19-1 | C | A | ND | ND | ND | ND | This study |
| B-19-1 | C | A | ND | ND | ND | ND | This study |
| C-19-1 | C | A | ND | ND | ND | ND | This study |
| A-19-5 | C | G | C | G | T | C | This study |
| A-19-6 | C | G | ND | ND | ND | ND | This study |
| A-19-7 | C | A | ND | ND | ND | ND | This study |
| A-20-1 | C | A | ND | ND | ND | ND | This study |
| A-21-1 | C | A | ND | ND | ND | ND | This study |
| A-22-1 | C | A | ND | ND | ND | ND | This study |
| LOU-17 | C | G | T | G | T | C | OQ756208 |
| LOU-18 | C | G | T | G | T | C | OQ756209 |
| MM-2 | C | G | C | G | T | T | OL410287 |
| MM-3 | C | G | C | G | T | T | OL410288 |
| MM-7 | C | G | C | G | T | T | OL410292 |
ND, not determined.
aLocations represent the number of nucleotides, with the first nucleotide in each coding sequence counted as 1.
Next-Generation Sequence
Cat No. 4, from which the A-19-5 strain was isolated, and Cat No. 5, from which the A-19-6 strain was isolated, were living together. Both cats developed clinical signs suggesting an FeHV-1 infection at almost the same time so the strains were presumed to be identical. Thus, NGS was performed to determine the whole-genome sequence of the A-19-5 strain, as shown in Table 2, and to compare it to the prototype C-27 strain, three F2 strains (Virbac, Intervet, and Merial), and five field strains (LOU-17, LOU-18, MM-2, MM-3, and MM-7) that have the same SNVs in UL28 and UL44 as the A-19-5 strain. The sequence was 134,931 bp in length (GenBank accession number: PV209842). All CDSs of the A-19-5 strain were compared to those of the three F2 strains. In addition to the absence of the SNV in UL28, the A-19-5 strain had nucleotides in UL38 and UL48 that differed from those in the two F2 strains (Virbac and Intervet), and the nucleotide differences in UL38 and UL48 were identical to the locations of the undetermined nucleotides in the Merial vaccine (Table 2). All other CDSs of the A-19-5 strain were identical to those of the two F2 strains, including UL41, for which the Merial vaccine had an undetermined nucleotide. All CDSs of the A-19-5 strain were compared to those of the five field strains. With the exception of one nucleotide (1393C) in UL8, all CDSs of the A-19-5 strain were identical to those of the LOU-17 and LOU-18 strains (Table 2). With the exception of one nucleotide (395C) in UL48, all CDSs of the A-19-5 strain were identical to those of the MM-2, MM-3, and MM-7 strains (Table 2). These findings indicate that strains with the same SNVs in the markers of the F2 strain as the A-19-5 strain (lacking the SNV in UL28 and having the SNV in UL44) and CDSs that are very similar to those of the A-19-5 strain have been isolated.
Analysis of the LAV Cloned Strain
The three undetermined nucleotides in the Merial F2 strain (UL38, UL41, and UL48 CDS) were previously analyzed in our laboratory using 12 clones obtained by cloning the Merial vaccine [5]. Comparison of the sequences of these clones to those of the A-19-5 strain identified two clones (Merial clones 1 and 8) with the same SNVs as the A-19-5 strain (Table 3). Therefore, the locations of SNVs in UL28 and UL44 were analyzed in these two clones. The nucleotides in Merial clone 8 and the A-19-5 strain were identical (Table 3). Sequencing of UL28 and UL44 in the Merial vaccine prior to cloning indicated that the location of the SNV in UL28 predominantly had an A waveform, but a low C waveform was also observed. The waveforms for the SNV in UL44 did not overlap (data not shown).
Table 3.
Nucleotide differences and location in the F2 strain marker genes, unique long (UL) 28 and UL44, and other genes, UL38, UL41, and UL48 in the A-19-5 strain, the Merial F2 strain, and clones of the Merial F2 strain
| Virus strain [reference] | F2 strain marker genes | Other genes | |||
|---|---|---|---|---|---|
| UL28 (1280a) | UL44 (397a) | UL38 (716a) | UL41 (737a) | UL48 (395a) | |
| A-19-5 | C | G | G | T | C |
| F2 (Merial) | A | G | R | Y | Y |
| Merial clone 1 [5] | A | G | G | T | C |
| Merial clone 8 [5] | C | G | G | T | C |
aLocations represent the number of nucleotides, with the first nucleotide in each coding sequence counted as 1.
Discussion
In the present study, a strain with all CDSs identical to one of the clones of the Merial vaccine (likely a variant of the F2 strain within the Merial vaccine population) was isolated from an unvaccinated cat. This finding indicates that a variant of the F2 strain may exist in the field and be shed by infected cats.
In the past, an LAV strain was surmised to be transmitted via incidental exposure to a cat through a cat’s mouth, nose, or other means [10]. In contrast, the cats from which a variant of the F2 strain was isolated in the present study had never been vaccinated; therefore, their clinical signs were not associated with vaccination with an LAV, and they may have been infected with a variant of the LAV strain existing in the field. Given these considerations, the possibility of an incidental infection with a variant of the LAV strain in the field or reactivation of an existing infection with a variant of the LAV strain due to some form of stress must be considered a route of transmission when a clinical isolate is identified as a variant of the LAV strain.
A previous study indicated that SNVs specific to the F2 strain are present in UL28 and UL44 [5, 11]. However, the A-19-5 strain isolated in the present study had the SNV in UL44 but did not have the SNV in UL28. In contrast, the Merial vaccine contained several FeHV-1 strains with SNVs in UL38, UL41, and UL48 [5], and two of the clones of the vaccine strain had SNVs identical to those in the A-19-5 strain. Analysis of the nucleotides in UL28 of these clones revealed that one clone did not have the SNV in UL28, one of the markers of the F2 strain, and that the clone was identical to the A-19-5 strain.
Cat No. 4, from which the variant of the F2 strain was isolated, was born in the same household, where it is currently maintained and has four other littermates. None of these cats had lived outdoors or had a history of vaccination. In addition, the mother cat of Cat No. 4 was purchased from a pet shop and vaccinated 11 years earlier with a vaccine from an unknown manufacturer. At 3 years of age, the mother cat ran away for 2 days. She became pregnant, gave birth in the household, and did not go outdoors for approximately 8 years. Cat No. 5 lived with Cat No. 4 and lived outdoors until she was brought indoors at approximately 3 months of age. At 4 years of age, she ran away, became pregnant, underwent an abortion, and did not go outdoors for approximately the next 8 years. Therefore, all cats in the household of Cat No. 4 were kept entirely indoors for approximately 8 years. Cats kept indoors are generally considered to have a low risk of exposure to pathogens. However, even indoor cats may be exposed to pathogens from outdoor routes of transmission by fomites (the owner’s body, clothing, or shoes or cats brought to a veterinary clinic), and from indoor routes of transmission by other cats in the household (subclinically infected or carrier cats) [4]. In the present study, all cats in Cat No. 4’s household developed, to varying degrees, clinical signs suggestive of an FeHV-1 infection at about the same time; therefore, the cat that was the origin of the infection could not be identified. That said, the owner of Cat No. 4 did not have an occupation involving contact with vaccines or cats and had no known contact with cats outside of the household. Therefore, as a possible route of outdoor fomite transmission of a pathogen, the variant of the F2 strain could have been attached to the owner or cats at a veterinary clinic and then subsequently brought into the household, leading to the spread of the infection. The last time Cat No. 4 was seen at the veterinary clinic before the virus was isolated was approximately 5 months earlier for a wellness examination, when one littermate and the mother cat also underwent a wellness examination. During the period between this wellness examination and the isolation of the virus from Cat No. 4, none of the other cats in the household of Cat No. 4 had been seen at any other veterinary clinic. The maximum survival time of FeHV-1 is 18 h at room temperature, and its incubation period is 2–6 days [1]. Therefore, the variant of the F2 strain is unlikely to have adhered at a veterinary clinic and to have been brought into the household this time. Therefore, a pathogen being transmitted from the outdoors by a fomite is unlikely. As a route of transmission of a pathogen indoors, the spread of infection from a subclinically infected cat in the household is unlikely because all cats in the household had clinical signs. That said FeHV-1 causes a latent infection in 80% of infected cats [12], so the variant of the F2 strain that latently infected a carrier cat may have been reactivated and spread within the household, causing clinical signs. However, the cat that was the origin of the infection could not be identified and which cats in the household were carriers and when they were infected were unknown. The carrier cat could have been infected when it was brought to a veterinary clinic in the past, when the mother cat of Cat No. 4 was kept in a pet shop, or when Cat No. 5 was living outdoors. Another possibility is that the vaccine administered to the mother cat of Cat No. 4 may contained a variant of the F2 strain. An LAV strain used to vaccinate against the varicella-zoster virus, which belongs to the same Varicellovirus genus as FeHV-1, is known to cause latent infection when subcutaneously injected [13]. Therefore, if the variant of the F2 strain was used to vaccinate the mother cat of Cat No. 4, it might have caused a latent infection and the cat could have potentially become a carrier. Thus, a cat carrying the variant of the F2 strain might be present in the household of Cat No. 4. However, the exact route of infection in Cat No. 4 remains unknown.
Cat No. 4 (the cat from which the A-19-5 strain was isolated) and Cat No. 5 (the cat from which the A-19-6 strain was isolated) lived together. The primary clinical signs in both cats were sneezing and loss of appetite, which are consistent with a typical FeHV-1 infection [1]. Moreover, a strain with the same nucleotides in two markers of the F2 strain as the A-19-5 strain was isolated from Cat No. 5, which developed clinical signs at approximately the same time as Cat No. 4, suggesting that the variant of the F2 strain may be contagious. Moreover, the A-19-5 strain may cause latent infections, as mentioned earlier. Thus, the variant of the F2 strain may be pathogenic. However, the F2 strain may be attenuated by serial passage [14]. Comparison of all CDSs in the A-19-5 and three F2 strains revealed differences in the presence or absence of three SNVs (one in UL28, one in UL38, and one in UL48); in the A-19-5 strain, the SNV in UL28 was absent, but the SNVs in UL38 and UL48 were present. All SNVs were nonsynonymous variants. The SNVs in UL28 (encoding DNA packaging terminase subunit 2) and UL44 (encoding envelope glycoprotein C) were used for simplified genotyping of the F2 strain from field isolates [5, 11], and they may be responsible for the attenuation of the F2 strain. However, deletion of UL44 has not been found to substantially reduce the virulence of FeHV-1 [15], and the SNV in UL44 may not be responsible for the attenuation of the F2 strain. Therefore, the SNV in UL28 may be involved in the attenuation of the F2 strain. The A-19-5 strain lacks the SNV in UL28, which may be related to the reversion to the virulence of the A-19-5 strain. However, no functional data indicating that UL28 is potentially associated with the attenuation of FeHV-1 have been reported to date so the association of the SNV in UL28 with the virulence of FeHV-1 remains speculative. That said, Cat No. 4 lived in an environment where 7 cats were predicted to be in close contact, and the viral load to which these cats were exposed could have been relatively high, possibly resulting in cross-contamination among the cats [16]. Co-infection with feline calicivirus, Mycoplasma felis, and Chlamydia felis, which cause clinical signs similar to those of an FeHV-1 infection, may also increase the severity of clinical signs [17]. Although feline calicivirus was not isolated in the present study, genetic testing for M. felis and C. felis was not performed. If either or both of these pathogens co-infected Cat No. 4 and Cat No. 5, they may have exacerbated the clinical signs of sneezing and loss of appetite. Therefore, evaluating the virulence of a variant of the F2 strain is difficult, and the lack of testing for both pathogens represents a limitation of the present study. Further studies are required to evaluate the virulence of a variant of the F2 strain.
Among the clinical isolates from cats that were registered in GenBank, two strains (LOU-17 and LOU-18) that differed from the A-19-5 strain in only one SNV (in UL8) in all CDSs were found, and these two strains could also be a variant of the F2 strain. These two strains were isolated from cats with clinical signs in the USA [18]; therefore, a variant of the F2 strain may also exist in the USA. In addition, three strains (MM-2, MM-3, and MM-7) isolated from cheetahs kept in the same facility in the USA have been registered in GenBank. These three strains differed from the A-19-5 strain in only one SNV (in UL48) in all CDSs. These strains are also very similar to the FeHV-1-LAV strain, as described by Marino et al. [16], and may be a variant of the F2 strain.
Only nine isolates were analyzed in the present study. To estimate the prevalence of FeHV-1-LAV strains in the field, broader surveillance should, therefore, be conducted by performing a genomic analysis of other FeHV-1-LAV strains and by increasing the number of isolates analyzed. In conclusion, a strain identical to a variant of the F2 strain may exist in the field and be shed by infected cats.
Statement of Ethics
An ethics statement is not applicable because the specimens used in this study were used to diagnose viral infections, and all owners provided approval for the use of these specimens for a genomic analysis of felid herpesvirus 1.
Conflict of Interest Statement
The authors have no conflicts of interest to declare.
Funding Sources
This study was not supported by any sponsor or funding.
Author Contributions
Yasuharu Suga: conceptualization, data curation, resources, and writing – original draft; Akihiko Saito and Yasunari Kitamura: conceptualization and resources; and Rikio Kirisawa: conceptualization, data curation, investigation supervision, and writing – review, and editing. All the authors approved the final manuscript.
Funding Statement
This study was not supported by any sponsor or funding.
Data Availability Statement
The nucleotide sequence data reported here are available in the DDBJ and GenBank databases under accession numbers LC864352–LC864369 and PV209842.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The nucleotide sequence data reported here are available in the DDBJ and GenBank databases under accession numbers LC864352–LC864369 and PV209842.
