Clinical Factors Associated with SFTS Diagnosis and Severity in Cats.

Severe fever with thrombocytopenia syndrome (SFTS) is a potentially fatal tick-borne zoonosis caused by SFTS virus (SFTSV). In addition to tick bites, animal-to-human transmission of SFTSV has been reported, but little is known about feline SFTSV infection. In this study, we analyzed data on 187 cats with suspected SFTS to identify biomarkers for SFTS diagnosis and clinical outcome. Body weight, red and white blood cell and platelet counts, and serum aspartate aminotransferase and total bilirubin levels were useful for SFTS diagnosis, whereas alanine aminotransferase, aspartate aminotransferase and serum SFTSV RNA levels were associated with clinical outcome. We developed a scoring model to predict SFTSV infection. In addition, we performed a phylogenetic analysis to reveal the relationship between disease severity and viral strain. This study provides comprehensive information on feline SFTS and could contribute to the protection of cat owners, community members, and veterinarians from the risk of cat-transmitted SFTSV infection.

Possible vertical transmission of Chikungunya virus infection detected in the cord blood samples from a birth cohort in Vietnam.

BACKGROUND: Chikungunya virus (CHIKV) is an alphavirus (genus Alphavirus, family Togaviridae) that is primarily transmitted to humans by Aedes mosquitoes, and can be transmitted from mother to child. Little is known about CHIKV transmission in Vietnam, where dengue is endemic and Aedes mosquitoes are abundant. This study aimed to determine the prevalence and characteristics of vertical CHIKV infection in a birth cohort, and seroprevalence of anti-CHIKV antibodies with or without confirmation by neutralization tests among women bearing children in Vietnam. METHODS: We collected umbilical cord blood plasma samples from each newly delivered baby in Nha Trang, Central Vietnam, between July 2017 and September 2018. Samples were subjected to molecular assay (quantitative real-time RT-PCR) and serological tests (anti-CHIKV IgM capture and IgG indirect enzyme-linked immunosorbent assay, and neutralization tests). RESULTS: Of the 2012 tested cord blood samples from newly delivered babies, the CHIKV viral genome was detected in 6 (0.3%) samples by RT-PCR, whereas, 15 samples (0.7%) were anti-CHIKV-IgM positive. Overall, 18 (0.9%, 95% CI: 0.6-1.5) samples, including three positives for both CHIKV IgM and viral genome on RT-PCR, were regarded as vertical transmission of CHIKV infection. Of the 2012 cord blood samples, 10 (0.5%, 95% CI: 0.2-0.9) were positive for both anti-CHIKV IgM and IgG. Twenty-nine (1.4%, 95% CI: 1.0-2.1) were seropositive for anti-CHIKV IgG while 26 (1.3%, 95% CI: 0.8-1.9) of them were also positive for neutralizing antibodies, and regarded as seropositive with neutralization against CHIKV infection. CONCLUSION: This is the first report of a possible CHIKV maternal-neonatal infection in a birth cohort in Vietnam. The findings indicate that follow-up and a differential diagnosis of CHIKV infection in pregnant women are needed to clarify the potential for CHIKV vertical transmission and its impact in the newborn.

Benefits of surgical treatment within 48†h of proximal femoral fracture in centenarians: a retrospective cohort study.

Introduction: Proximal femoral fractures in aging populations represent a significant concern, with an increasing prevalence among individuals aged ≥100 years. The existing research does not provide robust guidance for clinicians managing older patients aged ≥100 years with proximal femoral fractures. We investigated the safety and efficacy of surgical treatment in patients aged ≥100 years with proximal femoral fractures and evaluated the impact of early surgery on their outcomes. Methods: This retrospective cohort study involved 15 patients aged ≥100 years who underwent surgical treatment of proximal femoral fractures; the control group included 137 patients in their 90s. Data were collected between January 2010 and December 2017. Evaluation items included patient characteristics, surgical details, perioperative complication rates, length of hospital stay, the proportion of patients discharged to the same facility or home, rate of regaining walking ability, and 1-year survival rate. Results: The patients aged ≥100 years and those in their 90s had comparable outcomes. Thus, age alone does not dictate surgical success. Early surgery (≤48 h) was associated with trends toward improved perioperative complications, ambulatory ability, and return to original living environment. Discussion: This study underscores the potential benefits of surgical intervention for proximal femoral fractures in patients aged ≥100 years, indicating the relevance of early surgery (≤48 h). Our findings emphasized the importance of timely intervention and evidence-based decision-making for this demographic. Clinicians, policymakers, and patients could benefit from our insights to enhance fracture management strategies, along with future research endeavors to validate and expand our results in larger multicenter cohorts.

A Novel, Comprehensive A129 Mouse Model for Investigating Dengue Vaccines and Evaluating Pathogenesis.

In search of a mouse model for use in evaluating dengue vaccines, we assessed A129 mice that lacked IFN-α/ß receptors, rendering them susceptible to dengue virus (DENV) infection. To our knowledge, no reports have evaluated dengue vaccine efficiency using A129 mice. A129 mice were given a single intraperitoneal (IP) or subcutaneous (SC) injection of the vaccine, Dengvaxia. After 14 days of immunization via the IP or SC injection of Dengvaxia, the A129 mice exhibited notably elevated levels of anti-DENV immunoglobulin G and neutralizing antibodies (NAb) targeting all four DENV serotypes, with DENV-4 displaying the highest NAb levels. After challenge with DENV-2, Dengvaxia and mock-immunized mice survived, while only the mock group exhibited signs of morbidity. Viral genome levels in the serum and tissues (excluding the brain) were considerably lower in the immunized mice compared to those in the mock group. The SC administration of Dengvaxia resulted in lower viremia levels than IP administration did. Therefore, given that A129 mice manifest dengue-related morbidity, including viremia in the serum and other tissues, these mice represent a valuable model for investigating novel dengue vaccines and antiviral drugs and for exploring dengue pathogenesis.

Clinical, Virological, and Immunological Features in Cosmopolitan Genotype DENV-2-Infected Patients during a Large Dengue Outbreak in Sri Lanka in 2017.

In 2017, Sri Lanka experienced its largest dengue epidemic and reported severe and unusual presentations of dengue with high morbidity. This outbreak was associated with the reemergence of dengue virus-2 (DENV-2), with the responsible strain identified as a variant of the previously circulating DENV-2 cosmopolitan genotype. In this study, we characterized the DENV-2 cosmopolitan genotype from patients during this epidemic. Also, we identified host factors that contributed to the severity of dengue infection in patients infected with this particular virus. Ninety-one acute serum samples from patients at the National Hospital in Kandy were randomly selected. Of these, 40.2% and 48.9% were positive for dengue IgM and IgG, respectively. NS1 antigen levels were significantly higher in primary infections. The severe dengue (SD) and dengue with warning signs (DWWS) groups exhibited significantly higher viral genome and infectivity titers than the dengue without warning signs (DWoWS) group. The highest viremia level was observed in SD patients. As for host cytokine response, interferon α (IFN-α) levels were significantly higher in the DWoWS group than in the DWWS and SD groups, whereas interleukin (IL)-12p40 and tumor necrosis factor α (TNF-α) levels in SD patients were significantly higher than in the other two groups. The TNF-α, IL-4, and monocyte chemoattractant protein-1 concentrations were positively correlated with NS1 antigen levels. From whole-genome analysis, NS4 had the highest frequency of amino acid variants, followed by the E gene. Our study suggests that viremia levels and immune responses contributed to SD outcomes, and these findings may help in identifying an effective therapeutic strategy against SD infection.

Burden of Chikungunya Virus Infection during an Outbreak in Myanmar.

Chikungunya virus (CHIKV) infection is a re-emerging arboviral disease with no approved vaccine, although numerous options are in development. Before vaccine implementation, disease burden, affected age group, and hospitalization rate information should be documented. In 2019, a sizeable outbreak of the East Central South African genotype of CHIKV occurred in Myanmar, and during this period, a cross-sectional study was conducted in two regions, Mandalay and Yangon, to examine the molecular and seropositivity rate of the CHIKV infection. The participants (1124) included dengue-suspected pediatric patients, blood donors, and healthy volunteers, who were assessed using molecular assays (quantitative real-time RT-PCR), serological tests (anti-CHIKV IgM capture and IgG indirect enzyme-linked immunosorbent assays), and neutralization tests. The tests confirmed the following positivity rates: 11.3% (127/1124) for the molecular assay, 12.4% (139/1124) for the anti-CHIKV IgM Ab, 44.5% (500/1124) for the anti-CHIKV IgG Ab, and 46.3% (520/1124) for the CHIKV neutralizing Ab. The highest rate for the molecular test occurred with the dengue-suspected pediatric patients. The seroprevalence rate through natural infection was higher in the healthy volunteers and blood donors than that in the pediatric patients. The results of this study will help stakeholders determine the criteria for choosing appropriate recipients when a CHIKV vaccine is introduced in Myanmar.

Epidemiological evidence of acute transmission of Zika virus infection in dengue suspected patients in Sri-Lanka.

BACKGROUND: Zika Virus (ZIKV) is a re-emerging, arthropod-borne flavivirus transmitted by Aedes mosquitoes (Ae. aegypti and Ae. albopictus). The coexistence of dengue virus (DENV) and ZIKV concurrently has been associated with a wide array of neurological complications, which may influence the clinical outcomes of infections. Sri Lanka witnessed a severe dengue epidemic in 2017, characterized by extraordinary and severe disease manifestations with considerable morbidity. Therefore, this study assessed the potential occurrence of ZIKV infection during DENV outbreak in Sri Lanka from 2017 to 2019, which could bear substantial implications for public health. METHODS: Five hundred ninety-five serum samples were procured from individuals suspected of dengue and admitted to Kandy National Hospital between 2017 and 2018 and the Negombo District General Hospital between 2018 and 2019. These samples underwent quantitative real-time RT-PCR (qRT-PCR) to identify the presence of the ZIKV gene, while enzyme-linked immunosorbent assay was employed to detect ZIKV-specific IgM and IgG antibodies. Focus reduction neutralization tests were subsequently conducted to confirm ZIKV infection. RESULTS: Among the 595 serum samples, 6 (1.0%) tested positive for ZIKV using qRT-PCR. Anti-ZIKV IgM and IgG were identified in 18.0% and 38.6% patients. Sixty-six (11.0%) samples demonstrated the presence of anti-ZIKV IgM and IgG. Within ZIKV IgM-positive samples, 2.2% exhibited neutralizing antibodies against ZIKV. Through the implementation of qRT-PCR, ZIKV IgM detection, and neutralization testing, 2% and 3.7% cases of ZIKV infections were confirmed in the Kandy and Negombo regions, respectively. CONCLUSION: This study is the inaugural endeavor to substantiate the existence of ZIKV infection in Sri Lanka utilizing molecular and serological analysis. The findings of this investigation imply that ZIKV was circulating throughout the 2017-2019 DENV outbreak. These results underscore the necessity for improved preparedness for future outbreaks, fortifying governmental policies on public health, and establishing effective early warning systems regarding the emergence of these viruses.

Structure and its transformation of elliptical nege-like virus Tanay virus.

Negeviruses that infect insects are recently identified virus species that are phylogenetically related to several plant viruses. They exhibit a unique virion structure, an elliptical core with a short projection. Negeviruses encode two structural proteins, a glycoprotein that forms a short projection, and an envelope protein that forms an elliptical core. The glycoprotein has been reported only in the negeviruses' genes, and not in phylogenetically related plant viruses' genes. In this report, we first describe the three-dimensional electron cryo-microscopy (cryo-EM) structure of Tanay virus (TANAV), one of the nege-like viruses. TANAV particle demonstrates a periodical envelope structure consisting of three layers surrounding the centred viral RNA. The elliptical core dynamically changes its shape under acidic and even low detergent conditions to form bullet-like or tubular shapes. The further cryo-EM studies on these transformed TANAV particles reveal their overall structural rearrangement. These findings suggest putative geometries of TANAV and its transformation in the life cycle, and the potential importance of the short projection for enabling cell entry to the insect hosts.

Coinfection and circulation of chikungunya virus and dengue virus in pediatric patients in Myanmar, 2019.

Myanmar is an endemic country for arboviruses, and outbreaks occur frequently. A cross-sectional analytical study was conducted during the peak season of the chikungunya virus (CHIKV) outbreak in 2019. A total of 201 patients with acute febrile illness who were admitted to the 550-bedded Mandalay Children Hospital in Myanmar were enrolled in the study, and virus isolation, serological tests, and molecular tests for the dengue virus (DENV) and CHIKV were performed for all samples. Out of 201 patients, 71 (35.3%) were only DENV-infected, 30 (14.9%) were only CHIKV-infected and 59 (29.4%) were coinfected with DENV and CHIKV. The viremia levels of the DENV- and CHIKV- mono-infected groups were significantly higher than those of the group coinfected with DENV and CHIKV. Genotype I of DENV-1, genotypes I and III of DENV-3, genotype I of DENV-4 and the East/Central/South African genotype of CHIKV were co-circulating during the study period. Two novel epistatic mutations of CHIKV (E1:K211E and E2:V264A) were noted. This study highlighted that there were many coinfection cases during the outbreak and that the co-circulation of both viruses in DENV-endemic regions warrants effective monitoring of these emerging pathogens via comprehensive surveillance to facilitate the implementation of effective control measures.

Intended preoperative trans-arterial embolization for large hepatocellular carcinoma: a retrospective cohort study.

BACKGROUND: Generally, a large tumor size of hepatocellular carcinoma (HCC) is associated with poor visibility and uncertainty in the surgical field which results in increased surgical difficulty as well as unfavorable postoperative outcomes. We performed intended preoperative trans-arterial embolization (TAE) in patients with a large HCC. In this study, we investigated the oncological significance of intended preoperative TAE for a large HCC, using a comparison between patients with and without TAE, and detailed analyses for pre- and post-TAE status. METHODS: A total of 411 patients who underwent hepatectomy for primary HCC at the University of Yamanashi Hospital between January 2007 and December 2018 were included in this study. The patients were divided into two groups: patients with larger HCCs (≥50 mm, n=51) and those with smaller HCC (<50 mm, n=360) according to the size of their HCCs. Comparison of clinicopathological features between these groups and clinical outcomes between the TAE and non-TAE groups were compared. In addition, a detailed analysis of each case in the TAE group was conducted, comparing clinicopathological factors between pre- and post-TAE status. RESULTS: The clinical unfavorable short- and long-term outcomes of patients with large HCCs (≥50 mm) were revealed compared to those with small HCCs (<50 mm). The prognostic analyses showed that a large tumor size and increased tumor markers, multiple tumor numbers, and others were adverse prognostic factors, and vascular invasions and residual tumors were included in the multivariate analysis. Further detailed analyses revealed that the average rates of change in tumor size and tumor shrinkage after TAE were - 48.6±35.6 mm and - 30.7±17.0%, respectively. Pathological high necrotic changes in the tumor, after multiple-times TAE aiming to a better effect, were related to a better prognosis in patients with large HCC. Poor prognostic factors became less common in patients who underwent intended preoperative TAE, and these patients had better prognoses. CONCLUSIONS: The large tumor size of HCC is associated with unfavorable outcomes; the intended preoperative TAE for large HCC patients performed multiple times aiming to affect the tumor as much as possible might improve their prognoses.

Associations between Chest CT Abnormalities and Clinical Features in Patients with the Severe Fever with Thrombocytopenia Syndrome.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by the SFTS virus. It involves multiple organ systems, including the lungs. However, the significance of the lung involvement in SFTS remains unclear. In the present study, we aimed to investigate the relationship between the clinical findings and abnormalities noted in the chest computed tomography (CT) of patients with SFTS. The medical records of 22 confirmed SFTS patients hospitalized in five hospitals in Nagasaki, Japan, between April 2013 and September 2019, were reviewed retrospectively. Interstitial septal thickening and ground-glass opacity (GGO) were the most common findings in 15 (68.1%) and 12 (54.5%) patients, respectively, and lung GGOs were associated with fatalities. The SFTS patients with a GGO pattern were elderly, had a disturbance of the conscious and tachycardia, and had higher c-reactive protein levels at admission (p = 0.009, 0.006, 0.002, and 0.038, respectively). These results suggested that the GGO pattern in patients with SFTS displayed disseminated inflammation in multiple organs and that cardiac stress was linked to higher mortality. Chest CT evaluations may be useful for hospitalized patients with SFTS to predict their severity and as early triage for the need of intensive care.

[Two Cases of Carcinoma en Cuirasse, as Cutaneous Metastasis of Breast Cancer].

We present 2 cases of carcinoma en cuirasse, an uncommon clinical manifestation of metastatic cutaneous breast cancer. Case 1, a 70-year-old woman, presented with diffuse erythematous, indurated skin lesions that covered her entire anterior chest wall. Skin biopsy revealed tumor cells in the dermis which were ER and PgR positive and HER2 negative. CT showed pleural and pericardial effusion which led to a final diagnosis of cutaneous metastasis from breast cancer. Fulvestrant monotherapy was initiated and maintained a good clinical effect for 40 months. She died of multiple liver metastasis after 53 months from her first visit. Case 2 was a 71-year-old woman, with a 24 month history of a left breast tumor that gradually accompanied erythematous skin indurations and erosion, which spread to her entire left chest wall and contralateral breast. Following skin biopsy and CT, she was diagnosed to have triple negative breast cancer with multiple lymph node and cutaneous metastasis. After 4 cycles of EC, capecitabine was administrated and her skin lesions improved rapidly, including the lymph nodes. She is currently alive after 12 months since her first visit and under chemotherapy against new cutaneous metastasis.

Soluble podoplanin as a biomarker in diffuse­type gastric cancer.

Diffuse­type gastric cancer, also known as scirrhous gastric cancer, is characterized by a larger number of stromal cells, referred to as cancer­associated fibroblasts (CAFs), than the number of cancer cells in the tissue. The present study focused on CAFs in gastric cancer and examined their potential as a blood biomarker. A total of 46 and 84 patients with gastric cancer were respectively included in a development and an independent validation cohort to assess the clinicopathological characteristics of plasma podoplanin (PDPN) levels. The prognostic impact of plasma PDPN was also investigated in the validation cohort. The cut­off value of the plasma­PDPN concentration was set to the median plasma PDPN concentration in the development cohort that was then divided into the high­PDPN and low­PDPN groups. The high­PDPN group tended to have more diffuse­type disease (P=0.079), which was further confirmed through logistic regression analysis (P=0.008). Kaplan­Meier survival estimates indicated that the recurrence­free survival rate was significantly lower in the high­PDPN group (P=0.029). In conclusion, plasma soluble PDPN was demonstrated to be a marker for diffuse gastric cancer and may reflect the prognosis of this disease.

Seroepidemiological surveys of tick-borne encephalitis virus and novel tick-borne viruses in wild boar in Nagasaki, Japan.

In Japan, tick-borne viruses such as tick-borne encephalitis virus (TBEV) and severe fever with thrombocytopenia syndrome virus have been identified in humans, animals, and ticks. In addition, novel tick-borne viruses have been isolated from ticks in Japan. This study aimed to determine the seroprevalence of TBEV and novel viruses, particularly Tofla virus (TFLV), Kabuto Mountain virus (KAMV), and Muko virus (MUV) in wild boar in Nagasaki, Japan. Enzyme-linked immunosorbent assays and neutralization tests were performed to detect antibodies against each virus. Wild boar serum tested positive for antibodies against KAMV, TFLV, and TBEV, but not MUV. This study revealed the seroprevalence of newly identified tick-borne viruses and TBEV in animals residing in the Nagasaki area. The seroprevalence of these viruses in sentinel animals may inform policies aimed at preventing tick-borne virus disease outbreaks.

Evidence of Chikungunya virus seroprevalence in Myanmar among dengue-suspected patients and healthy volunteers in 2013, 2015, and 2018.

INTRODUCTION: Chikungunya virus (CHIKV) is a mosquito-borne virus known to cause acute febrile illness associated with debilitating polyarthritis. In 2019, several institutions in Myanmar reported a CHIKV outbreak. There are no official reports of CHIKV cases between 2011 and 2018. Therefore, this study sought to determine the seroprevalence of CHIKV infection before the 2019 outbreak. METHODS: A total of 1,544 serum samples were collected from healthy volunteers and patients with febrile illnesses in Yangon, Mandalay, and the Myeik district in 2013, 2015, and 2018. Participants ranged from one month to 65 years of age. Antibody screening was performed with in-house anti-CHIKV IgG and IgM ELISA. A neutralization assay was used as a confirmatory test. RESULTS: The seroprevalence of anti-CHIKV IgM and anti-CHIKV IgG was 8.9% and 28.6%, respectively, with an overall seropositivity rate of 34.5%. A focus reduction neutralization assay confirmed 32.5% seroprevalence of CHIKV in the study population. Age, health status, and region were significantly associated with neutralizing antibodies (NAbs) and CHIKV seropositivity (p < 0.05), while gender was not (p = 0.9). Seroprevalence in 2013, 2015, and 2018 was 32.1%, 28.8%, and 37.3%, respectively. Of the clinical symptoms observed in participants with fevers, arthralgia was mainly noted in CHIKV-seropositive patients. CONCLUSION: The findings in this study reveal the circulation of CHIKV in Myanmar's Mandalay, Yangon, and Myeik regions before the 2019 CHIKV outbreak. As no treatment or vaccine for CHIKV exists, the virus must be monitored through systematic surveillance in Myanmar.

Direct Viral RNA Detection of SARS-CoV-2 and DENV in Inactivated Samples by Real-Time RT-qPCR: Implications for Diagnosis in Resource Limited Settings with Flavivirus Co-Circulation.

The RT-qPCR method remains the gold standard and first-line diagnostic method for the detection of SARS-CoV-2 and flaviviruses, especially in the early stage of viral infection. Rapid and accurate viral detection is a starting point in the containment of the COVID-19 pandemic and flavivirus outbreaks. However, the shortage of diagnostic reagents and supplies, especially in resource-limited countries that experience co-circulation of SARS-CoV-2 and flaviviruses, are limitations that may result in lesser availability of RT-qPCR-based diagnostic tests. In this study, the utility of RNA-free extraction methods was assessed for the direct detection of SARS-CoV-2 and DENV-2 in heat-inactivated or chemical-inactivated samples. The findings demonstrate that direct real-time RT-qPCR is a feasible option in comparison to conventional real-time RT-qPCR based on viral genome extraction-based methods. The utility of heat-inactivation and direct real-time RT-qPCR for SARS-CoV-2, DENV-2 viral RNA detection was demonstrated by using clinical samples of SARS-CoV-2 and DENV-2 and spiked cell culture samples of SARS-CoV-2 and DENV-2. This study provides a simple alternative workflow for flavivirus and SARS-CoV-2 detection that includes heat inactivation and viral RNA extraction-free protocols, with aims to reduce the risk of exposure during processing of SARS-CoV-2 biological specimens and to overcome the supply-chain bottleneck, particularly in resource limited settings with flavivirus co-circulation.

Serological Evidence of Zika Virus Infection in Febrile Patients and Healthy Blood Donors in Sabah, Malaysian Borneo, 2017-2018.

Several Zika virus (ZIKV) seroprevalence studies have been conducted in Africa, Asia, Oceania, the Americas, and the Caribbean. However, studies on ZIKV seroprevalence are limited in Malaysia though several studies have shown that the disease is endemic in the Malaysian state of Sabah. To evaluate the seroprevalence of ZIKV infection, 818 serum samples were collected from febrile patients and healthy blood donors from the Kudat and Kota Kinabalu districts in Sabah from 2017 to 2018. They were screened for ZIKV infection by IgM and IgG ELISA, and positive ZIKV IgM samples were subjected to a 90% neutralization test for confirmation. Twenty-four (6% [95% CI 4 to 8]) confirmed and two (0.5% [95% CI 0.13 to 1.8]) probable ZIKV infections were detected among 400 febrile illness patients. Of 418 healthy blood donor samples, six (1.4% [95% CI 0.65 to 3]) were determined as confirmed ZIKV infections and six (1.4% [95% CI 0.65 to 3]) indicated probable ZIKV infection. This is the first study on the seroprevalence of ZIKV infections among patients and healthy blood donors in Sabah. Compared with previous studies in Malaysia, this study shows that the incidence of ZIKV infection has increased. It also suggests that a sero-surveillance system is essential to determine the circulation of ZIKV in Sabah, Malaysia.

Development and Evaluation of Quantitative Immunoglobulin G Enzyme-Linked Immunosorbent Assay for the Diagnosis of Coronavirus Disease 2019 Using Truncated Recombinant Nucleocapsid Protein as Assay Antigen.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Real-time RT-PCR is the most commonly used method for COVID-19 diagnosis. However, serological assays are urgently needed as complementary tools to RT-PCR. Hachim et al. 2020 and Burbelo et al. 2020 demonstrated that anti-nucleocapsid(N) SARS-CoV-2 antibodies are higher and appear earlier than the spike antibodies. Additionally, cross-reactive antibodies against N protein are more prevalent than those against spike protein. We developed a less cross-reactive immunoglobulin G (IgG) indirect ELISA by using a truncated recombinant SARS-CoV-2 N protein as assay antigen. A highly conserved region of coronaviruses N protein was deleted and the protein was prepared using an E. coli protein expression system. A total of 177 samples collected from COVID-19 suspected cases and 155 negative control sera collected during the pre-COVID-19 period were applied to evaluate the assay's performance, with the plaque reduction neutralization test and the commercial SARS-CoV-2 spike protein IgG ELISA as gold standards. The SARS-CoV-2 N truncated protein-based ELISA showed similar sensitivity (91.1% vs. 91.9%) and specificity (93.8% vs. 93.8%) between the PRNT and spike IgG ELISA, as well as also higher specificity compared to the full-length N protein (93.8% vs. 89.9%). Our ELISA can be used for the diagnosis and surveillance of COVID-19.

Discrepancies in Infectivity of Flavivirus and SARS-CoV-2 Clinical Samples: An Improved Assay for Infectious Virus Shedding and Viremia Assessment.

Infectivity and neutralizing antibody titers of flavivirus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are frequently measured using the conventional plaque assay. While the assay is useful in the determination of infectivity, conventional plaque assays generally possess lower sensitivity and are time-consuming compared to nucleic acid amplification tests. In this study, a microcrystalline cellulose (MCC), Avicel, was evaluated as an alternative to the conventional virus overlay medium, methylcellulose, for a plaque assay. The plaque assay was performed using dengue and COVID-19 clinical samples and laboratory-established flavivirus and SARS-CoV-2 strains. In virus titration of clinical samples, the plaques were significantly larger, and the virus titers were higher when Avicel MCC-containing overlay medium was used than with conventional methylcellulose overlay medium. In addition, for some clinical samples and laboratory virus strains, infectious particles were detected as plaques in the Avicel MCC-containing medium, but not in the conventional methylcellulose medium. The results suggest that the viremia titer determined using the new overlay medium containing Avicel MCC may better reflect the innate infectious and plaque-forming capabilities of clinical samples and better reflect virus infectivity.