Genomic Analysis of Monkeypox Virus During the 2023 Epidemic in Korea.

We aimed to characterize the genomes of monkeypox virus isolates from the Far East, providing insights into viral transmission and evolution. Genomic analysis was conducted on 8 isolates obtained from patients with monkeypox virus disease in the Republic of Korea between May 2022 and early 2023. These isolates were classified into Clade IIb. Distinct lineages, including B.1.1, A.2.1, and B.1.3, were observed in 2022 and 2023 isolates, with only the B.1.3 lineage detected in six isolates of 2023. These genetic features were specific to Far East isolates (the Republic of Korea, Japan, and Taiwan), distinguishing them from the diverse lineages found in the Americas, Europe, Africa, and Oceania. In early 2023, the prevalence of the B.1.3 lineage of monkeypox virus identified in six patients with no overseas travel history is considered as an indicator of the potential initiation of local transmission in the Republic of Korea.

Magnitude and Duration of Serum Neutralizing Antibody Titers Induced by a Third mRNA COVID-19 Vaccination against Omicron BA.1 in Older Individuals.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant (B.1.1.529) is dominating coronavirus disease 2019 (COVID-19) worldwide. The waning protective effect of available vaccines against the Omicron variant is a critical public health issue. This study aimed to assess the impact of the third COVID-19 vaccination on immunity against the SARS-CoV-2 Omicron BA.1 strain in older individuals. MATERIALS AND METHODS: Adults aged ≥60 years who had completed two doses of the homologous COVID-19 vaccine with either BNT162b2 (Pfizer/BioNTech, New York, NY, USA, BNT) or ChAdOx1 nCoV (SK bioscience, Andong-si, Gyeongsangbuk-do, Korea, ChAd) were registered to receive the third vaccination. Participants chose either BNT or mRNA-1273 (Moderna, Norwood, MA, USA, m1273) mRNA vaccine for the third dose and were categorized into four groups: ChAd/ChAd/BNT, ChAd/ChAd/m1273, BNT/BNT/BNT, and BNT/BNT/m1273. Four serum specimens were obtained from each participant at 0, 4, 12, and 24 weeks after the third dose (V1, V2, V3, and V4, respectively). Serum-neutralizing antibody (NAb) activity against BetaCoV/Korea/KCDC03/2020 (NCCP43326, ancestral strain) and B.1.1.529 (NCCP43411, Omicron BA.1 variant) was measured using plaque reduction neutralization tests. A 50% neutralizing dilution (ND50) >10 was considered indicative of protective NAb titers. RESULTS: In total, 186 participants were enrolled between November 24, 2021, and June 30, 2022. The respective groups received the third dose at a median (interquartile range [IQR]) of 132 (125 - 191), 123 (122 - 126), 186 (166 - 193), and 182 (175 - 198) days after the second dose. Overall, ND50 was lower at V1 against Omicron BA.1 than against the ancestral strain. NAb titers against the ancestral strain and Omicron BA.1 variant at V2 were increased at least 30-fold (median [IQR], 1235.35 [1021.45 - 2374.65)] and 129.8 [65.3 - 250.7], respectively). ND50 titers against the ancestral strain and Omicron variant did not differ significantly among the four groups (P = 0.57). NAb titers were significantly lower against the Omicron variant than against the ancestral strain at V3 (median [IQR], 36.4 (17.55 - 75.09) vs. 325.9 [276.07 - 686.97]; P = 0.012). NAb titers against Omicron at V4 were 16 times lower than that at V3. Most sera exhibited a protective level (ND50 >10) at V4 (75.0% [24/32], 73.0% [27/37], 73.3% [22/30], and 70.6% [12/17] in the ChAd/ChAd/BNT, ChAd/ChAd/m1273, BNT/BNT/BNT, and BNT/BNT/m1273 groups, respectively), with no significant differences among groups (P = 0.99). CONCLUSION: A third COVID-19 mRNA vaccine dose restored waning NAb titers against Omicron BA.1. Our findings support a third-dose vaccination program to prevent the waning of humoral immunity to SARS-CoV-2.

A Case of Human Mpox with Isolated Perianal Ulcers Development in Convalescent Phase.

A 35-year-old man who returned from Germany developed fever, generalized pain, severe anal pain, and generalized skin rash, confirmed to be monkeypox (mpox). While he was previously confirmed to be human immunodeficiency virus infected, antiretroviral therapy ensured his immunocompetence. The mpox-related prodromal symptoms disappeared before isolation, and subsequent several vesicular skin lesions healed after admission. While moderate anal pain persisted for a few days, it improved during hospitalization. The mpox virus was no longer detected in samples taken from the upper respiratory tract and skin by polymerase chain reaction upon admission. However, isolated perianal ulcers developed after admission without any other mpox-related symptoms or signs, and a viable mpox virus was isolated from these ulcers. Considering the novel feature of asynchronous mucocutaneous lesion development in the current mpox epidemic, meticulous physical examination of newly developing lesions, especially in anogenital areas, should be performed during mpox management.

Different maturation of gut microbiome in Korean children.

Introduction: Gut microbiome plays a crucial role in maintaining human health and is influenced by food intake, age, and other factors. Methods: In this study based in Korea, we examined the bacterial taxonomic composition of the gut microbiota in infants (≤ 1 year), toddlers (1-<4 years), and school-aged children (4-13 years) and compared them with those of healthy adults to investigate the microbiota changes in early life and their association with the resistome. We used whole metagenome sequences obtained by Illumina HiSeq sequencing and clinical information of 53 healthy children, and sequence data of 61 adults from our previous study. Results: Our results indicate that the bacterial proportion of the gut in the population ranging from infants to adults forms three clusters: the Ruminococcus-Eubacterium (G1), Bifidobacterium-Escherichia (G2), and Bacteroides-Faecalibacterium (G3) groups. The gut microbiota of infants and toddlers (100% of infants and 85% of toddlers) constituted mostly of G2 and G3 groups, whereas 90% of adults showed G1-type gut microbiota. School-aged children showed a transitional gut microbiota composition of both infants and adults (31%, 38%, and 31% in G1, G2, and G3, respectively). Notably, the three clusters of microbiota showed significantly different patterns of bacterial diversity (p < 0.001): G2 showed the lowest Shannon index, followed by G3 and G1 (1.41, 2.08, and 2.48, respectively; median Shannon index). When combined with the adult group, alpha diversity showed a positive correlation with age (R2 = 0.3). Furthermore, clustering the composition of antibiotic resistance genes (ARG) identified two clusters (A1 and A2), and most of G1 (95%) and G3 (80%) belonged to A1. However, G2 showed the least diversity and the highest abundance of ARGs. Nine ARG families showed a significant difference among age groups; three tetracycline resistance genes, tet32, tetO, and tetW, showed a positive correlation, and six other genes, ampC, TEM, ileS, bacA, pmr transferase, and cepA, showed a negative correlation with age. Discussion: In conclusion, our results highlighted that a delayed persistence of the Bifidobacterium-dominant enterotype with a lower bacterial diversity was observed in Korean children up to 13 years of age, which suggests a different maturation process with a delayed maturation time.

Enhanced antibody responses in fully vaccinated individuals against pan-SARS-CoV-2 variants following Omicron breakthrough infection.

Omicron has become the globally dominant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant, creating additional challenges due to its ability to evade neutralization. Here, we report that neutralizing antibodies against Omicron variants are undetected following COVID-19 infection with ancestral or past SARS-CoV-2 variant viruses or after two-dose mRNA vaccination. Compared with two-dose vaccination, a three-dose vaccination course induces broad neutralizing antibody responses with improved durability against different SARS-CoV-2 variants, although neutralizing antibody titers against Omicron remain low. Intriguingly, among individuals with three-dose vaccination, Omicron breakthrough infection substantially augments serum neutralizing activity against a broad spectrum of SARS-CoV-2 variants, including Omicron variants BA.1, BA.2, and BA.5. Additionally, after Omicron breakthrough infection, memory T cells respond to the spike proteins of both ancestral and Omicron SARS-CoV-2 by producing cytokines with polyfunctionality. These results suggest that Omicron breakthrough infection following three-dose mRNA vaccination induces pan-SARS-CoV-2 immunity that may protect against emerging SARS-CoV-2 variants of concern.

Antibody Responses to SARS-CoV-2 in Children With COVID-19.

BACKGROUND: The immunologic features of children with coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are not clearly delineated. This study was conducted to evaluate SARS-CoV-2-specific antibody responses in children with COVID-19. METHODS: The levels of anti-spike (S) IgG, anti-SARS-CoV-2 IgG, and neutralizing antibody (NAb) were measured during various time points in children <19 years of age with COVID-19 in South Korea from February 2020 to September 2020. RESULTS: One hundred sixty-five blood samples from 114 children with COVID-19 (43.9% asymptomatic and 56.1% mildly symptomatic) were analyzed. In both asymptomatic and mildly symptomatic children, the positive rates of anti-S IgG, anti-SARS-CoV-2 IgG, and NAb were low within 7 days after onset, but they soon reached 100% 14 to <28 days after onset. In symptomatic children, the geometric mean titers (GMTs) of antibodies were all below the positive cutoff during the first 2 weeks from onset and peaked at 28 to <56 days (5.6 for anti-S IgG, 383.6 for anti-SARS-CoV-2 IgG, and 55.0 for NAb, P < .001, respectively). Antibody levels remained detectable up to 3 months after infection. The antibody GMTs during the period 14 to <56 days after symptom onset were highest in children aged 0-4 years. CONCLUSIONS: These results collectively present the humoral immune responses during SARS-CoV-2 infection in children. A further longitudinal study is needed to thoroughly understand the immune system and for effective vaccine development in children during the COVID-19 pandemic.

Booster BNT162b2 COVID-19 Vaccination Increases Neutralizing Antibody Titers Against the SARS-CoV-2 Omicron Variant in Both Young and Elderly Adults.

Concerns about the effectiveness of current vaccines against the rapidly spreading severe acute respiratory syndrome-coronavirus-2 omicron (B.1.1.529) variant are increasing. This study aimed to assess neutralizing antibody activity against the wild-type (BetaCoV/Korea/KCDC03/2020), delta, and omicron variants after full primary and booster vaccinations with BNT162b2. A plaque reduction neutralization test was employed to determine 50% neutralizing dilution (ND50) titers in serum samples. ND50 titers against the omicron variant (median [interquartile range], 5.3 [< 5.0-12.7]) after full primary vaccination were lower than those against the wild-type (144.8 [44.7-294.0]) and delta (24.3 [14.3-81.1]) variants. Furthermore, 19/30 participants (63.3%) displayed lower ND50 titers than the detection threshold (< 10.0) against omicron after full primary vaccination. However, the booster vaccine significantly increased ND50 titers against BetaCoV/Korea/KCDC03/2020, delta, and omicron, although titers against omicron remained lower than those against the other variants (P < 0.001). Our study suggests that booster vaccination with BNT162b2 significantly increases humoral immunity against the omicron variant.

Clinical Characteristics of 40 Patients Infected With the SARS-CoV-2 Omicron Variant in Korea.

Since severe acute respiratory syndrome-coronavirus-2 variant B.1.1.529 (omicron) was first reported to the World Health Organization on November 24, 2021, the cases of the omicron variant have been detected in more than 90 countries over the last month. We investigated the clinical and epidemiological characteristics of the first 40 patients with the omicron variant who had been isolated at the National Medical Center in South Korea during December 4-17, 2021. The median age of the patients was 39.5 years. Twenty-two patients (55%) were women. Seventeen patients (42.5%) were fully vaccinated, and none were reinfected with the omicron. Eighteen (45%) had recent international travel history. Half of the patients (19, 47.5%) were asymptomatic, while the others had mild symptoms. Six patients (15%) showed lung infiltrations on chest image; however, none required supplemental oxygen. These mild clinical features are consistent with recent case reports on the omicron variant from other countries.

Longitudinal Analysis of Memory T-Cell Responses in Survivors of Middle East Respiratory Syndrome.

BACKGROUND: Middle East respiratory syndrome (MERS) is a highly lethal respiratory disease caused by a zoonotic betacoronavirus. The development of effective vaccines and control measures requires a thorough understanding of the immune response to this viral infection. METHODS: We investigated cellular immune responses up to 5 years after infection in a cohort of 59 MERS survivors by performing enzyme-linked immunospot assay and intracellular cytokine staining after stimulation of peripheral blood mononuclear cells with synthetic viral peptides. RESULTS: Memory T-cell responses were detected in 82%, 75%, 69%, 64%, and 64% of MERS survivors from 1-5 years post-infection, respectively. Although the frequency of virus-specific interferon gamma (IFN-γ)-secreting T cells tended to be higher in moderately/severely ill patients than in mildly ill patients during the early period of follow-up, there was no significant difference among the different clinical severity groups across all time points. While both CD4+ and CD8+ T cells were involved in memory T-cell responses, CD4+ T cells persisted slightly longer than CD8+ T cells. Both memory CD4+ and CD8+ T cells recognized the E/M/N proteins better than the S protein and maintained their polyfunctionality throughout the period examined. Memory T-cell responses correlated positively with antibody responses during the initial 3-4 years but not with maximum viral loads at any time point. CONCLUSIONS: These findings advance our understanding of the dynamics of virus-specific memory T-cell immunity after MERS-coronavirus infection, which is relevant to the development of effective T cell-based vaccines.

Seroprevalence of scrub typhus, murine typhus and spotted fever groups in North Korean refugees.

OBJECTIVES: The aim of this study was to investigate the seroprevalence of antibodies against scrub typhus, murine typhus and spotted fever groups among North Korean refugees within 1 year of their arrival in South Korea. METHODS: We recruited North Korean refugees who had settled in South Korea after a short stay in a third country and did not have any health problems. The antibody titer was measured using a commercial indirect fluorescence assay immunoglobulin G antibody kit. RESULTS: The seroprevalence of antibodies against scrub typhus, murine typhus, and spotted fever groups among the 99 participants was 22.2%, 17.2%, and 10.1%, respectively, with 8.1% of participants testing positive for both spotted fever and murine typhus. CONCLUSIONS: Refugees may be exposed to rickettsial infections in North Korea and their journey from North Korea. This study is the first to report the seroprevalence of antibodies against the 3 common rickettsial diseases among North Korean refugees. The findings suggest that rickettsial infections should be added to the list of differential diagnoses for North Koreans with fever after entering South Korea.

Critical role of neutralizing antibody for SARS-CoV-2 reinfection and transmission.

Cases of laboratory-confirmed SARS-CoV-2 reinfection have been reported in a number of countries. Further, the level of natural immunity induced by SARS-CoV-2 infection is not fully clear, nor is it clear if a primary infection is protective against reinfection. To investigate the potential association between serum antibody titres and reinfection of SARS-CoV-2, ferrets with different levels of NAb titres after primary SARS-CoV-2 infection were subjected to reinfection with a heterologous SARS-CoV-2 strain. All heterologous SARS-CoV-2 reinfected ferrets showed active virus replication in the upper respiratory and gastro-intestinal tracts. However, the high NAb titre group showed attenuated viral replication and rapid viral clearance. In addition, direct-contact transmission was observed only from reinfected ferrets with low NAb titres (<20), and not from other groups. Further, lung histopathology demonstrated the presence of limited inflammatory regions in the high NAb titre groups compared with control and low NAb groups. This study demonstrates a close correlation between a low NAb titre and SARS-CoV-2 reinfection in a recovered ferret reinfection model.

Evidence of Severe Acute Respiratory Syndrome Coronavirus 2 Reinfection After Recovery from Mild Coronavirus Disease 2019.

BACKGROUND: Positive results from real-time reverse-transcription polymerase chain reaction (rRT-PCR) in recovered patients raise concern that patients who recover from coronavirus disease 2019 (COVID-19) may be at risk of reinfection. Currently, however, evidence that supports reinfection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has not been reported. METHODS: We conducted whole-genome sequencing of the viral RNA from clinical specimens at the initial infection and at the positive retest from 6 patients who recovered from COVID-19 and retested positive for SARS-CoV-2 via rRT-PCR after recovery. A total of 13 viral RNAs from the patients' respiratory specimens were consecutively obtained, which enabled us to characterize the difference in viral genomes between initial infection and positive retest. RESULTS: At the time of the positive retest, we were able to acquire a complete genome sequence from patient 1, a 21-year-old previously healthy woman. In this patient, through the phylogenetic analysis, we confirmed that the viral RNA of positive retest was clustered into a subgroup distinct from that of the initial infection, suggesting that there was a reinfection of SARS-CoV-2 with a subtype that was different from that of the primary strain. The spike protein D614G substitution that defines the clade "G" emerged in reinfection, while mutations that characterize the clade "V" (ie, nsp6 L37F and ORF3a G251V) were present at initial infection. CONCLUSIONS: Reinfection with a genetically distinct SARS-CoV-2 strain may occur in an immunocompetent patient shortly after recovery from mild COVID-19. SARS-CoV-2 infection may not confer immunity against a different SARS-CoV-2 strain.

Development of a reverse transcription-loop-mediated isothermal amplification as a rapid early-detection method for novel SARS-CoV-2.

The previous outbreaks of SARS-CoV and MERS-CoV have led researchers to study the role of diagnostics in impediment of further spread and transmission. With the recent emergence of the novel SARS-CoV-2, the availability of rapid, sensitive, and reliable diagnostic methods is essential for disease control. Hence, we have developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the specific detection of SARS-CoV-2. The primer sets for RT-LAMP assay were designed to target the nucleocapsid gene of the viral RNA, and displayed a detection limit of 102 RNA copies close to that of qRT-PCR. Notably, the assay has exhibited a rapid detection span of 30 min combined with the colorimetric visualization. This test can detect specifically viral RNAs of the SARS-CoV-2 with no cross-reactivity to related coronaviruses, such as HCoV-229E, HCoV-NL63, HCoV-OC43, and MERS-CoV as well as human infectious influenza viruses (type B, H1N1pdm, H3N2, H5N1, H5N6, H5N8, and H7N9), and other respiratory disease-causing viruses (RSVA, RSVB, ADV, PIV, MPV, and HRV). Furthermore, the developed RT-LAMP assay has been evaluated using specimens collected from COVID-19 patients that exhibited high agreement to the qRT-PCR. Our RT-LAMP assay is simple to perform, less expensive, time-efficient, and can be used in clinical laboratories for preliminary detection of SARS-CoV-2 in suspected patients. In addition to the high sensitivity and specificity, this isothermal amplification conjugated with a single-tube colorimetric detection method may contribute to the public health responses and disease control, especially in the areas with limited laboratory capacities.

Infection and Rapid Transmission of SARS-CoV-2 in Ferrets.

The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in China and rapidly spread worldwide. To prevent SARS-CoV-2 dissemination, understanding the in vivo characteristics of SARS-CoV-2 is a high priority. We report a ferret model of SARS-CoV-2 infection and transmission that recapitulates aspects of human disease. SARS-CoV-2-infected ferrets exhibit elevated body temperatures and virus replication. Although fatalities were not observed, SARS-CoV-2-infected ferrets shed virus in nasal washes, saliva, urine, and feces up to 8 days post-infection. At 2 days post-contact, SARS-CoV-2 was detected in all naive direct contact ferrets. Furthermore, a few naive indirect contact ferrets were positive for viral RNA, suggesting airborne transmission. Viral antigens were detected in nasal turbinate, trachea, lungs, and intestine with acute bronchiolitis present in infected lungs. Thus, ferrets represent an infection and transmission animal model of COVID-19 that may facilitate development of SARS-CoV-2 therapeutics and vaccines.

Immune Responses to Middle East Respiratory Syndrome Coronavirus During the Acute and Convalescent Phases of Human Infection.

BACKGROUND: An understanding of immune responses against the Middle East respiratory syndrome (MERS) is important for the development of treatments and preventive measures. Here, we investigated the spectrum of immune responses occurring in patients with MERS during the early period of infection. METHODS: We obtained peripheral blood samples from 27 hospitalized patients recruited during the epidemic that occurred in 2015 in South Korea. Plasma cytokines/chemokines and antibodies were quantified. Virus-specific T cells were examined by intracellular cytokine staining after stimulation of peripheral blood mononuclear cells with overlapping peptides spanning whole virus structural proteins. RESULTS: At the acute phase of infection, elevated levels of plasma proinflammatory cytokines/chemokines were detected in proportion to the severity of the disease. Distinctively high frequencies of MERS coronavirus-reactive CD8+ T cells were also observed in patients with severe/moderate illness, whereas antibody and CD4+ T-cell responses were minimally detected at this stage. At the convalescent phase, disease severity-dependent antibody responses emerged and antigen-reactive cells were identified in both T-cell subsets. These T cells belonged to the T-helper 1 or type 1 cytotoxic T cell subtypes. While CD8+ T cells responded preferentially to the viral S protein compared with E/M/N proteins, especially at the acute stage, slightly more CD4+ T cells recognized E/M/N proteins compared with S protein at the convalescent phase. CONCLUSIONS: Our findings show an association between the early CD8+ T-cell response and the severity of the infection, and also provide basic information that may help to prepare effective control strategies for MERS in humans.

Correlation between Pneumonia Severity and Pulmonary Complications in Middle East Respiratory Syndrome.

This nationwide, prospective cohort study evaluated pulmonary function and radiological sequelae according to infection severity in 73 survivors from the 2015 Middle East respiratory syndrome (MERS) outbreak in Korea. Patients with severe pneumonia in MERS-coronavirus infection had more impaired pulmonary function than those with no or mild pneumonia at the 1-year follow-up, which was compatible with the radiological sequelae. Severe pneumonia significantly impairs pulmonary function and makes long radiological sequelae in MERS.

Development and evaluation of an anti-rabies virus phosphoprotein-specific monoclonal antibody for detection of rabies neutralizing antibodies using RFFIT.

BACKGROUND: Rabies is a major public health problem with a fatality rate close to 100%; however, complete prevention can be achieved through pre- or post-exposure prophylaxis. The rapid fluorescent focus inhibition test (RFFIT) is one of the recommended testing methods to determine the production of neutralizing antibodies after vaccination. Here, we report the development of a new monoclonal antibody (mAb) designed to react specifically with Rabies virus (RABV) phosphoprotein (P protein), and the evaluation of its applicability to the RFFIT and its effectiveness as a diagnostic reagent for human rabies. METHODOLOGY/PRINCIPAL FINDINGS: The mAb KGH P 16B8 was produced to target the P protein of the Korean KGH RABV strain. An indirect immunofluorescence assay (IFA) was conducted to detect various strains of RABV in various cell lines. Alexa-conjugated KGH P 16B8 (16B8-Alexa) was developed for the RFFIT. The IFA test could detect RABV up to a 1:2,500 dilution, with a detection limit comparable to that of a commercial diagnostic reagent. The sensitivity, specificity, positive predictive value, and negative predictive value of the RFFIT using 16B8-Alexa in 414 clinical specimens were 98.67%, 99.47%, 99.55%, and 98.42%, respectively. The results of the RFFIT with 16B8-Alexa were strongly correlated with those obtained using an existing commercial diagnostic reagent (r = 0.995, p<0.001). CONCLUSIONS/SIGNIFICANCE: The mAb developed in this study shows high sensitivity and specificity, confirming its clinical utility with the RFFIT to measure the rabies neutralizing antibody titer and establish a diagnosis in human. Thus, 16B8-Alexa is expected to serve as an alternative diagnostic reagent that is widely accessible, with potentially broad applications beyond those of the RFFIT in Korea. Further studies with 16B8-Alexa should provide insight into the immunological mechanism of the P protein of Korean RABV.

Development of infectious clones of a wild-type Korean rabies virus and evaluation of their pathogenic potential.

Most reverse genetic (RG) systems for rabies viruses (RVs) have been constructed on the genome background of laboratory-adapted strains. In this study, we developed an RG system using a Korean wild type (KGH) strain to investigate the pathogenic potential of different strains. We developed a RG system with the KGH strain for the first time. Following the complete genome sequencing of the KGH strain, pKGH infectious clones were constructed using the CMV/T7 promoter, and HamRz and HdvRz were introduced to allow self-cleavage of the synthesized RNA. We successfully recovered the rescued virus by constructing chimeric RVs in which we replaced a part of the construct with the partial gene from the fixed RC-HL strain. The rescued viruses formed clearer and countable plaques in an immunostaining plaque assay, with a distinct plaque morphology. Furthermore, compared with the chimeric RVs, the pKGH/RCinsΔ4 strain containing the KGH strain G protein exhibited a decreased efficiency of cell-to-cell spreading in BHK-21 cells and significantly reduced (100-1000 fold) replication kinetics. However, pKGH/RCinsΔ4 strain-infected mice revealed 100% morbidity at 11days post-infection, whereas other chimeric RV strains showed no mortality. Our RG system is a useful tool for studying differences in the cell-to-cell spreading efficiency and replication with respect to the different internalization patterns of street and fixed laboratory-adapted viruses.

Immunostained plaque assay for detection and titration of rabies virus infectivity.

The fluorescent antibody test (FAT) is the most commonly used method for detection of the rabies virus (RV). The plaque assay can only be applied to fixed RVs, and cannot be used for street RVs. In this study, plaque formation allowing the determination of both fixed and street RVs was achieved using the immune plaque assay. The immune plaque assay carried out using both fixed and street RVs showed the formation of clear and countable plaques after immunostaining with anti-RV P monoclonal antibody and HRP-conjugated anti-mouse IgG. Plaque size increased with incubation time, and the plaque morphology differed according to viral strain. Fixed RVs had the dot-shaped regular plaque morphology and street RVs had the small irregular-shape plaque morphology. In addition, no significant differences were observed between the growth kinetics of the KGH strain when the virus was titrated using the FAT and the immune plaque assay. It allowed the successful detection and quantification of both street and fixed RVs through the production of clear, countable plaques, making it easy to obtain objective results. The assay is an applicable tool for the detection of RVs in various investigations, including virus neutralizing antibody testing, cell-to-cell spread, and viral drug sensitivity testing.

Alternative application of Tau protein in Creutzfeldt-Jakob disease diagnosis: Improvement for weakly positive 14-3-3 protein in the laboratory.

The 14-3-3 protein has been used as a biomarker for the diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD). However, weakly positive 14-3-3 leads to false positive results and an incorrect diagnosis. We attempted to use quantitative data for tau protein to provide an accurate diagnosis based on weak 14-3-3 protein. Sixty-two patients with sCJD, including pathologically confirmed, clinically definite, and probable cases, and 89 non-CJD patients were investigated based on a Korean population. Among them, 20 sCJD and 14 non-CJD showed weakly positive 14-3-3. The total tau (t-tau) and phosphorylated tau (p-tau) protein levels were measured by ELISA, and the p-tau to t-tau ratio (p/t ratio) was calculated. The combined use of the 14-3-3 protein assay, t-tau levels, and p/t ratio improved the specificity of diagnosis compared with the use of the 14-3-3 protein assay alone (47% for 14-3-3 alone; 85.94% for 14-3-3 combined with t-tau; 90.62% for 14-3-3 combined with the p/t ratio). In addition, 18 of 20 sCJD and 12 of 14 non-CJD who were weakly positive for 14-3-3 were positive for the p/t ratio and negative for the p/t ratio, respectively. When used in combination with the 14-3-3 protein, the tau protein is useful as a biomarker for the precise diagnosis of sCJD.