We have determined the presence of two phylogenetic groups of glycoprotein G. One group was only found in Europe, and all the isolates in this group contain the epitope AFPL, which a common assay uses to type HSV-1. The other group was found in all tested regions, which include Africa, Asia and Europe. This group was characterized by the AVPL sequence. Y3369 is a member of this group. The two sequences differ by location statistically (χ2 = 142.8, p < 0.0001).

The identification of these two groups, as well as their localization to different parts of the world, may aid in developing strategies for clinical viral antigen assays for HSV typing. Although the isolates included in the meta analysis which have the AVPL sequence were not tested by us, they would likely fail to type as HSV-1 using this same test. It should be considered that tests for the viral antigen epitope AFLP be used with caution in Africa or Asia.

This variation may also alter the interaction of virus with host. The presence of the variations in the immunodominant region of the protein suggests these mutations could be a result of viral immune evasion. These mutations may also affect the functioning of glycoprotein G, which involves attachment and entry. Further tests are being performed to study what other effects this mutation has on the virus’s efficiency of infection.

Glycoprotein G was PCR amplified (see supplementary table) and sequenced. Examination of the sequences showed that the probable cause for the non-reactivity of the mAb assay was the presence of a valine residue in glycoprotein G at amino acid (AA) 111. This valine is near the immunodominant region of antibody binding during normal immune response. Sequencing results were deposited [GenBank:HQ833203], and compared to other isolates on GenBank. Sequencing revealed that the clinical isolate Y3369 contains an amino acid sequence consistent with a common HSV-1 sequence found in many parts of the world.

A meta analysis of three population studies which have sequenced this region of the HSV-1 US4 gene was conducted to determine the prevalence of valine at position 111, as was identified in our sample. Included were isolates from individuals from China, Japan, Kenya, South Korea, Sweden, and the United Kingdom. We discovered the valine at position 111 to be present in all HSV-1 isolates (100%, N = 141) taken from human populations from Asia and Africa. The other populations, from the UK and Sweden, contained the valine at position 111 in 36% (N = 185) of isolates. This valine at position 111 is located within the binding site for a commonly used typing mAb, which recognizes the epitope AFPL. The phenylalanine is replaced to form the sequence AVPL in this variant.

Sequences for the middle region encoding AA 110 to 164 of glycoprotein G were analyzed and a phylogenetic tree created. Phylogenetic analysis groups our isolate Y3369 as an HSV-1 with sequence V (representing valine at 111) which contains the sequence AVPL instead of AFPL, as well as other common nucleotides. All isolates from populations from Africa and Asia, as well as 36% of the European population contained the sequence AVPL, which would not be recognized by the mAb which tests for the AFPL epitope. Another study found that all isolates with a valine residue at position 111 of glycoprotein G were untypable when assaying viral antigens. This specific test would not be likely to function diagnostically in these African or Asian populations.

Herpes simplex viruses exist as two major serotypes, type 1 (HSV-1) and type 2 (HSV-2). Determination of type, either HSV-1 or HSV-2, is important in accurate diagnosis and clinical control of transmission. Several tests are available for typing HSV, including a monoclonal antibody specific for glycoprotein G and several PCR assays.

Findings

A clinical isolate was identified as herpes simplex virus, but tested negative for both HSV-1 and HSV-2 antigens using type-specific monoclonal antibody assays. The isolate was determined to be HSV-1 by PCR analysis. A mutation which likely caused the monoclonal antibody non-reactivity was found in glycoprotein G. Phylogenetic analysis revealed two groups of HSV, one with the mutation and one without. Three population studies examining mutations in HSV-1 glycoprotein G were analyzed by chi-squared test. To this point, the epitope which the monoclonal antibody recognizes was only found in HSV-1 isolates from human European populations (p < 0.0001).

Conclusions

These findings suggest that the PCR-based methods for HSV typing may be more useful than the standard monoclonal antibody test in areas of the world where the variant in glycoprotein G is more prevalent.

Findings

Herpes simplex viruses exist as two major serotypes, type 1 (HSV-1) and type 2 (HSV-2). Determination of type, either HSV-1 or HSV-2, is important in accurate diagnosis and clinical control of transmission. Tests which can determine HSV type include viral antigen tests, serological tests of human antibodies and PCR. The importance of glycoprotein G as the test analyte is emphasized by the 2002 STD Treatment Guidelines from the CDC: “Accurate type-specific assays for HSV antibodies must be based on the HSV-specific glycoprotein G2 for the diagnosis of infection with HSV-2 and glycoprotein G1 for diagnosis of infection with HSV-1.”.

A clinical sample of a herpes simplex virus, designated Y3369 was isolated and proved refractory to typing. The isolate was obtained from an infected genital tract of a 48-year-old female patient. It was submitted to Richards Laboratories, Inc., Pleasant Grove, Utah, USA for diagnostic workup. The sample was incubated overnight, and then stained for virus-infected cells using a type-common polyclonal primary antibody and visualized by the immunoperoxidase technique using a rapid culture method. The culture showed an abundance of cells positive for antibody labeling and had HSV-typical cytopathic effects, confirming the presence of HSV in the specimen (results not shown).

The Y3369 isolate was then tested using the Wampole type-specific viral antigen test for HSV glycoprotein G. A viral stock culture was generated by inoculation of a portion of the rapid culture isolate into a culture of MV1Lu cells (mink lung, ATCC CCL-64). The specimen was also incubated in C1008 cells (Vero subline, ATCC CRL-1586) and subjected to similar serotypic analysis by staining with virus-specific monoclonal antibodies (mAbs) against HSV type 1 and type 2. These tests failed to yield a positive identification of the isolate as either HSV-1 or HSV-2 using type-specific mAb assays (Wampole Laboratories). The immunofluorescence result was negative against both reagent antisera in MV1Lu cells. The virus was also untypable in C1008 cells (not shown). The laboratory strains HSV-1 McIntyre and HSV-2 strain 333 were tested with mAb reagents and expected monotypic results were observed in these controls.

The Korean ECV 5 genome was sequenced (GenBank accession number HM775882) and its amino acid sequence was deduced. The genome is 7,430 nt in length, excluding the poly(A) tail. The 5′NCR contains 738 nt, followed by an ORF that encodes a viral polyprotein consisting of 2,196 codons, between a start codon (AUG) at position 739 and a stop codon (UGA) at position 7,326. The 3′NCR is 104 nt in length.

3.2 Genome comparison between Korean ECV5 and the Noyce strain

The Korean ECV 5 isolate genome was divided into five regions (5′NCR, P1, P2, P3, and 3′NCR) and aligned with the Noyce strain using Megalign (DNASTAR). The P1 region (85.3%) had the highest level of nucleotide identity, followed by the P3 region (84.8%), the 3′NCR (84.5%), the 5′NCR (81.8%), and the P2 region (80.0%). The P3 region (98.0%) had the highest level of amino acid identity, followed by the P1 region (97.7%), and the P2 region (96.9%).

Most of the cleavage sites were identical between Korean ECV 5 and the Noyce strain. The only exception was the cleavage site between VP1 and 2A, which was TY/GA in the Noyce strain, but TR/GA in the Korean ECV 5 isolate.
3.3 Antiviral activity of Korea ECV5

No signs of cytotoxicity were observed in Vero cells treated with any of the five antiviral drugs at a CC50 value >100 μg/mL. Amantadine and ribavirin exhibited antiviral activity against the Korean ECV 5 strain, though azidothymidine, acyclovir, and lamivudine did not. The amantadine possessed an IC50 value of 1 ± 0.42 μg/mL and a TI value of 100, while the ribavirin possessed an IC50 value of 22 ± 1.36 μg/mL and a TI value of 4.55.

4. Discussion

The ECV 5 is a rare enterovirus strain as causative aseptic meningitis and its sequence information is not common in the previous publication, including genbank database. This report describes the first complete nucleotide sequence for an ECV 5 isolated in Korea. Previous work has shown that sequence identity between different ECV serotypes is relatively high for the 5′NCR sequence, moderate for the P2 and P3 regions, and lowest for the P1 region [21]. This was not the pattern that emerged when we compared the Korean ECV 5 isolate and the Noyce strain; rather, the 5′NCR and P2 regions had relatively low nucleotide sequence identities (<81.8%), while the identities of the P1 and P3 regions were relatively high (>84.5%). Amino acid sequences for the protein coding regions had much higher sequence identities (>96.9%). Cleavage site variations have often been reported for VP2/VP3, VP3/VP1, and VP1/2A. Thus, we were not surprised to find that, though conservation was observed at most sites, there had been a substitution (TY/GA→TR/GA) at VP1/2A. There was about 20% genetic difference between prototype and the current widespread strain, and its difference was found at the cleavage site. Therefore, development and screening of antiviral drugs have to be focused on the object of the current epidemic strain.

Of the five antiviral drugs tested here (azidothymidine, acyclovir, amantadine, lamivudine, and ribavirin), only amantadine (IC50: 1 μg/mL) and ribavirin (IC50: 22 μg/mL) had antiviral activity against Korean ECV 5, with amantadine showing stronger effects than ribavirin. Therefore, the amantadine and ribavirin could be applied to patients infected with ECV 5. It was reported that the amantadine could suppress the IRES mediated translation, and ribavirin is a nucleoside analogue with broad-spectrum antiviral activity by decreasing viral replication in EV71.

In conclusion, this manuscript is the first report of the complete nucleotide sequence of the Korean ECV strain, as well as the first examination of its response to various antiviral drugs. This data should be useful in preventing future outbreaks of ECV5 and in treating patients infected with the strain. Accordingly, it is necessary to test more of the same kind of antiviral drugs and various enterovirus serotypes in future studies.

One day before infection, Vero cells were seeded onto a 96-well culture plate at a concentration of 2 × 104 cells per well. Next day, the medium was removed and then washed with 1 × phosphate buffered saline (PBS). Infectivity of the virus stock was determined by the SRB method and was determined as infectivity of the virus by SRB ID50 (50% infective dose). Following this, 0.09 mL of diluted virus suspension of ECV containing CCID50 (50% cell culture infective dose) of the virus stock to produce an appropriate cytopathic effects within 2 days after infection and 0.01 mL of medium containing an appropriate concentration of the compounds were added. The antiviral activity of each test material was determined with a 10-fold diluted concentration ranging from 0.1 to 100 μL/mL. Three wells were used as virus controls (virus-infected non-drug-treated cells) while three wells were used as cell controls (non-infected non-drug-treated cells). The culture plates were incubated at 37°C in 5% CO2 for 2 days. After washing once with 1 × PBS, 100 μL of cold (-20°C), 70% acetone was added to each well and left for 30 min at -20°C. 70% acetone was removed and 96-well plates were left at dry oven for 30 min. 100 μL of 0.4% (w/v) SRB in 1% acetic acid solution was added to each well and left at room temperature for 30 min. Unbound SRB was removed and the plates were washed 5 times with 1% acetic acid before oven drying and were then left in a dry oven for 1 day. Bound SRB was solubilized with 100 μL of 10 mM unbuffered tris-base solution and plates were left on a table for 30 min. The absorbance was read at 540 nm by using a VERSAmax microplate reader (Molecular Devices, Palo Alto, CA, USA) with a reference absorbance at 620 nm. To calculate the IC50 (50% inhibitory concentration) values, the results were transformed to percentage of controls and the IC50 values were graphically obtained from the dose-response curves.

2.4 Cytotoxicity assay

Vero cells were seeded into a 96-well culture plate at a concentration of 2 × 104 cells per well. Next day, the medium was removed and the 96-well plates were replaced with media containing the serially diluted compounds, and the cells were further incubated for 48 hrs. The culture medium was removed and washed with 1 × PBS. The next step was conducted by antiviral activity assay described above. To calculate the CC50 values, the results were transformed to percentage of controls and the CC50 values were graphically obtained from the dose-response curves.

In this study, the molecular biological characteristics and genetic diversity of Korean ECV 5 that do not exist the using antiviral agents for it but widespread currently were analyzed through the complete nucleotide sequencing and compared with ECV 5 prototype strain (Noyce). We also selected 5 kinds of antiviral drugs (azidothymidine, acyclovir, amantadine, lamivudine, and ribavirin) that were used as inhibitors of other viruses and examined for anti-viral activity for Korean ECV 5.

2. Materials and methods

2.1 Virus isolation and identification

The Korean ECV 5 strain was isolated from cerebrospinal fluid sampled from a male patient with aseptic meningitis who had been admitted to the Department of Pediatrics at the Soonchunhyang University Cheonan Hospital, Korea, in June 2006. The sample was inoculated into Vero cells and then incubated at 37°C with 5% CO2 until the appearance of cytopathic effects (CPE) took place. The identification of the Korean isolate was verified by a Basic Local Alignment Search Tool (BLAST) search in VP1 sequences; that is, the VP1 sequences of Korean isolate had the highest nucleotide similarity with ECV5 serotype strains.
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2.2 Nucleotide sequencing and sequence analysis

The complete nucleotide sequence of the Korean ECV 5 strain was determined using a primer walking strategy; the sequences of the genome termini were determined by random amplification of cDNA ends (RACE) system (Invitrogen, Carlsbad, CA, USA). The PCR products were purified using a QIAquick PCR Purification Kit (Qiagen, Hamburg, Germany). The purified DNA was added to a reaction mixture containing 2 μL of Big Dye terminator reaction mix (ABI Prism BigDye Terminator Cycle Sequencing Kit; Applied Biosystems, Foster, CA, USA) and 2 pmoles of each primer. Sequencing reactions were subjected to an initial denaturation at 96°C for 1 min and 25 cycles consisting of 96°C for 10 sec, 50°C for 5 sec, and 60°C for 4 min in a Gene Amp PCR system 2700 (Applied Biosystems). The products were purified by precipitation with 100% cold ethanol and 3 M sodium-acetate (pH 5.8), then loaded on an automated 3100 Genetic Analyzer (Applied Biosystems).

Nucleotide sequences of enterovirus isolates were constructed to contig and were compared with a reference, the Noyce strain, which was originally identified in the USA in 1954, and the nucleotide sequence of which was obtained from Genbank databases. Sequence analyses were performed using computer software included in the Lasergene package (DNASTAR, Inc., Madison, WI, USA).

An outbreak of echovirus 5 (ECV 5) occurred in Korea in 2006, marking the first time this virus had been identified in the country since enterovirus surveillance began in 1993. Using a sample isolated from a young male patient with aseptic meningitis, we performed sequencing of the Korean ECV 5 strain and compared it with a prototype strain (Noyce). At the nucleotide level, the P1 region (85.3%) had the highest identity value; at the amino acid level, the P3 region (98.0%) had the highest identity value. The two strains shared all cleavage sites, with the exception of the VP1/2A site, which was TY/GA in the Noyce strain but TR/GA in the Korean ECV 5 isolate. In Vero cells infected with the Korean ECV 5 isolate, no cytotoxicity was observed in the presence of azidothymidine, acyclovir, amantadine, lamivudine, or ribavirin, when the drugs were administered at a CC50 value >100 μg/mL. Of the five drugs, only amantadine (IC50: 1 ± 0.42 μg/mL, TI: 100) and ribavirin (IC50: 22 ± 1.36 μg/mL, TI: 4.55) had any antiviral activity against the Korean ECV 5 isolate.

1. Introduction

Human enteroviruses (HEV) are RNA viruses from the Picornaviridae family. The 80 immunologically-distinct serotypes that are known to cause infections in humans can be grouped as follows: polioviruses (PV), echoviruses (ECV), coxsackieviruses A (CVA), coxsackieviruses B (CVB), and enterovirus (EV) types 68-71. These viruses are also classified genetically into five species (HEV-A to HEV-D and PVs). HEV-B group containing ECV 5 are CBV 1 to 6, CVA9, ECV 1 to ECV 7, ECV 9, ECV 11 to ECV 21, ECV 24 to ECV 27, ECV 29 to 33, EV 69, EV 73.

ECV cause the same types of infections in humans as the CVB group, but are given a distinct classification primarily because they lack pathogenicity in newborn mice. There are, however, strains of ECV that are pathogenic in mice. ECV 5 infections have been associated with a wide variety of neurological and exanthematic diseases. The prototype strain of ECV 5 was isolated from a patient with aseptic meningitis and was later grouped as the fifth enterovirus serotype. An outbreak of aseptic meningitis caused by ECV 5 occurred in Korea in 2006, marking the first time that ECV 5 had been identified in the country since enterovirus surveillance began in 1993.

The ECV 5 genome contains approximately 7,500 nucleotide-long single-stranded RNA molecules with polarity and carries a small viral peptide (VPg) covalently attached to its 5′ end. The 5′ untranslated region (UTR) of the RNA is approximately 700 nt in length and is unusually long compared with the homologous region of cellular mRNA. The internal ribosomal entry site (IRES) was discovered in the 5′ UTR of the HEVs. In these viruses, the IRES can fold to be a functional secondary RNA structure and drive translation initiation [10]. The coding region encompasses a single open reading frame that encodes a polyprotein divided into three sub-regions, P1, P2, and P3. The P1 region encodes the genetic information of four structural proteins, VP4, VP2, VP3, and VP1. The non-structural proteins are encoded in the P2 (2A-2C) and P3 (3A-3D) regions. A short 3′ UTR of approximately 100 nt separates the coding region from the poly (A) tail.

Analysis of the phenotype of individual clones from the person who infected subject 17, obtained by isolation under limiting-dilution conditions, revealed a simultaneous presence ofNSI and SI clones in the PBMC of the donor at the putative moment of transmission (not shown). Therefore, selective transmission ofNSI clones cannot be totally excluded as an alternative explanation for the absence of SI viruses in subject 17. However, evidence for suppression of transmitter SI virus in the recipient has been observed in mother-child pairs. The relative inability ofSI viruses to infect monocytes may explain why these variants are preferentially cleared early in infection. Indeed, in the asymptomatic phase after seroconversion, monocytotropic NSI variants were found to be predominant, indicating that only viruses able to establish a low-grade persistent infection of monocytes can survive the vigorous anti-Hlv-I immune response at that time. The temporary disappearance ofSI isolates observed in patient 7 may reflect inadequate suppression of these variants by the immune system. Also in favor of this mechanism are two recent studies providing evidence for an important role ofthe immune system in the initial containment ofHIVI infection. Thus overt persistence ofSI isolates from the beginning of infection onward would be found only in individuals infected with SI viruses in whom the primary anti-HIV-l immune response is defective, as is suggested by the relatively low numbers of CD8+ T cells and the more transient rise in numbers of activated CD8+ T cells observed in individuals with SI viruses. Such an inefficient anti-HIV-1 immune response may be due to a compromised immune system at the time ofexposure, caused possibly by concurrent infections as noted in patient 7.

Discussion
In this study we analyzed immunologic parameters and the biologic phenotype of HIV-l during primary HIV-l infection in 19 individuals. In agreement with other studies, we observed a transient CD4+ and CD8+ T lymphocytopenia about the time ofseroconversion in 6 of8 individuals from whom frequent PBMC samples were available directly after infection. A similar dip in T lymphocyte numbers occurred during acute influenza virus infection (unpublished data). After seroconversion in 10 of 19 individuals, CD8+ T cell numbers increased. Transient rises in numbers of CD8+ T cells and activated CD8+ T cells have also been seen in acute infection with other viruses (e.g., cytomegalovirus, Epstein-Barr virus, influenza, and rubella [unpublished data]. Therefore, the CD8+ T lymphocytosis and increased numbers of activated CD8+ T cells in HIV-1 infection most likely reflect an antiviral immune response rather than immunopathologic events typical for HIV-1 infection. In a previous longitudinal study on HIV-I-infected individuals, we observed only NSI isolates in stable asymptomatic persons. In about half of the individuals progressing to AIDS, SI isolates were detected and this detection was associated with rapid CD4+ T cell decline. Similarly, in this study, NSI isolates were found exclusively in 16 of 19 individuals with primary HIV-1 infection. In 15 of 16 of these individuals, normal CD4+ T cell numbers were observed until the end of follow-up (mean, 391 days ± 259). The occurrence of Kaposi’s sarcoma with normal CD4+ T cell numbers in two of these individuals is in agreement with our previous findings that this AIDS indicator diagnosis is observed more frequently in patients with NSI isolates. In the present study rapid CD4+ T cell decline, however, was only observed in the three persons with SI isolates, resulting in AIDS with opportunistic infections in two of them, 6 and 19 months after seroconversion.