Isolation, in vitro, and in vivo pathogenicity test of Tilapia lake virus (TiLV) and development of a prognostic semi-quantitative lesion scoring system for differentiating clinical/subclinical infection in farmed tilapia (Oreochromis niloticus L.).

Tilapia lake virus ('TiLV-MH-2022') was recently recovered from the naturally infected farmed tilapia. Reverse transcription-polymerase chain reaction (RT-PCR) using segment 1 specific primers, followed by Sanger sequencing, confirmed the infection. The pairwise sequence homology of segment 1 showed its close relationship with the previous isolates. The virus was successfully detected from the mucus, which emphasised the possibility of non-invasive screening of tilapia on a large scale. The virus inoculum prepared from the infected tissues was tested for in vivo and in vitro pathogenicity. Around 100-140 nm-sized electron-dense virus particles were observed in the infected OnlL cells. Based on the onset of symptoms and lesions, all RT-PCR-positive fish were categorised into two groups, 'clinical' and 'subclinical'. A lesion-scoring technique was developed for assessing the pathogenicity of the virus isolate. The external and internal gross lesions and histopathological alterations in the critical organs of the fish, such as the brain, kidney, gills, and liver, were assessed on a scale of 0 (no gross lesion) to 5 (most severe lesions). Overall lesion score was significantly high in the clinical and subclinical groups for gross and histopathology, respectively. This study is the first such attempt to standardise a semi-quantitative lesion scoring technique for TiLV infection, which establishes a clinical relevance and prognostic ability to distinguish between the apparent and inapparent infection.

A cell line derived from heart of rainbow trout is refractory to Tilapia lake virus.

Cell lines are important in vitro models to answer biological mechanisms with less genetic variations. The present study was attempted to develop a cell line from rainbow trout, where we obtained a cell line from the heart, named "RBT-H." The cell line was authenticated using karyotyping and cytochrome c oxidase subunit I (COI) gene sequencing. The karyotype demonstrated diploid chromosome number (2n) as 62 and the sequence of partial COI gene was 99.84% similar to rainbow trout COI data set, both suggesting the origin of RBT-H from the rainbow trout. The heart cell line was mycoplasma-free and found to be refractory to infection with the Tilapia lake virus. The RBT-H cell line is deposited in the National Repository of Fish Cell Line (NRFC) at ICAR-NBFGR, Lucknow, India, with Accession no. NRFC0075 for maintenance and distribution to researchers on request for R&D.

The impact of COVID-19 shelter-in-place policy responses on excess mortality.

As a way of slowing COVID-19 transmission, many countries and U.S. states implemented shelter-in-place (SIP) policies. However, the effects of SIP policies on public health are a priori ambiguous. Using an event study approach and data from 43 countries and all U.S. states, we measure changes in excess deaths following the implementation of COVID-19 shelter-in-place (SIP) policies. We do not find that countries or U.S. states that implemented SIP policies earlier had lower excess deaths. We do not observe differences in excess deaths before and after the implementation of SIP policies, even when accounting for pre-SIP COVID-19 death rates.

Widespread occurrence of Tilapia parvovirus in farmed Nile tilapia Oreochromis niloticus from India.

Tilapia parvovirus (TiPV) has been associated with heavy mortalities in tilapia as a single infection or in co-infection with Tilapia lake virus (TiLV). In this study, TiPV was detected in farmed Nile tilapia, Oreochromis niloticus, from two geographical regions of India, Maharashtra and Uttar Pradesh. TiPV-specific polymerase chain reaction (PCR) reported earlier was used in the screening. Tilapia collected from Maharashtra showed characteristic clinical signs, and TiPV was detected along with TiLV and/or Aeromonas spp. However, fish from Uttar Pradesh were apparently healthy and only TiPV could be detected in these samples. A high prevalence of TiPV was recorded from both the geographical locations, Maharashtra and Uttar Pradesh (59.6% and 95.0% respectively). The virus could be detected in tissues such as the spleen, liver, kidney, brain and mucus. The spleen appeared to be the best tissue for detecting TiPV in apparently healthy tilapia. The presence of TiPV was further confirmed through sequencing the PCR products, isolation of the virus in the cell line and electron microscopy. Sequences of the NS1 gene of the two TiPV isolates showed similarity to the earlier reported TiPV isolates. The virus could be successfully propagated in O. niloticus Liver (OnL) cell line, and cytopathic effect was observed as early as 3 days post-infection. Furthermore, the presence of non-enveloped icosahedral to round virus particles measuring about 26-35 nm could be demonstrated in the cytoplasm and nucleus of infected OnL cells in transmission electron microscopy. With this confirmation of the presence of the virus, India is the third country to report TiPV after China and Thailand. The detection of TiPV in co-infection cases with TiLV and in apparently healthy Nile tilapia suggests its wide distribution and potential synergistic effect in co-infection cases. Therefore, this emerging virus needs holistic attention to understand its virulence, host-specificity and epidemiological risk factors.

Prescription Drug Advertising and Drug Utilization: The Role of Medicare Part D.

This paper examines how direct-to-consumer advertising (DTCA) for prescription drugs influences utilization by exploiting a large and plausibly exogenous shock to DTCA driven by the introduction of Medicare Part D. Part D led to larger increases in advertising in geographic areas with higher concentrations of Medicare beneficiaries. We examine the impact of this differential increase in advertising on non-elderly individuals to isolate advertising effects from the direct effects of Part D. We find that exposure to advertising led to large increases in treatment initiation and improved medication adherence. Advertising also had sizeable positive spillover effects on non-advertised generic drugs. Our results imply significant spillovers from Medicare Part D on the under-65 population and an important role for non-price factors in influencing prescription drug utilization.

COVID-19 Testing in Schools: Perspectives of School Administrators, Teachers, Parents, and Students in Southern California.

BACKGROUND: School-based COVID-19 testing is a potential strategy to facilitate the safe reopening of schools that have been closed due to the pandemic. This qualitative study assessed attitudes toward this strategy among four groups of stakeholders: school administrators, teachers, parents, and high school students. METHODS: Focus groups and interviews were conducted in Los Angeles from December 2020 to January 2021 when schools were closed due to the high level of COVID transmission in the community. RESULTS: Findings indicated similarities and differences in attitudes toward in-school COVID-19 testing. All groups agreed that frequent in-school COVID-19 testing could increase the actual safety and perceived safety of the school environment. School administrators expressed pessimism about the financial cost and logistics of implementing a testing program. Parents supported frequent testing but expressed concerns about physical discomfort and stigma for students who test positive. Teachers and parents noted that testing would prevent parents from sending sick children to school. Students were in favor of testing because it would allow them to return to in-person school after a difficult year of online learning. CONCLUSION: In-school COVID-19 testing could be a useful component of school reopening plans and will be accepted by stakeholders if logistical and financial barriers can be surmounted and stigma from positive results can be minimized.

Shifting the Demand for Vaccines: A Review of Strategies.

Vaccines prevent millions of deaths, and yet millions of people die each year from vaccine-preventable diseases. The primary reason for these deaths is that a significant fraction of the population chooses not to vaccinate. Why don't people vaccinate, and what can be done to increase vaccination rates besides providing free and easy access to vaccines? This review presents a conceptual framework, motivated by economic theory, of which factors shift the demand for vaccines. Next, it critically examines the literature on these demand shifters and interventions that target these demand shifters. The review concludes with offering directions for future research and lessons for public health decision making.

Establishment and characterization of a continuous cell line from caudal fin of Labeo calbasu (Hamilton, 1822).

Labeo calbasu is an important food fish and candidate species for diversification of carp aquaculture. In the present study, we have established a continuous cell line, designated as L. calbasu fin (LCF), from caudal fin of L. calbasu using explant method. The cell line has been subcultured for over 73 passages and the LCF cells show optimal growth in Leibovitz's L-15 medium supplemented with 20% fetal bovine serum at a temperature of 28°C. In karyotype analysis, the modal chromosome number of LCF cells at 35th passage was found to be 50. The amplification and sequencing of partial fragments of mitochondrial genes, namely 16S rRNA and COI from LCF cells confirmed the origin of cell line from L. calbasu. The LCF cells could be successfully transfected with GFP reporter gene, indicating suitability of these cells for expression of foreign genes. Further, following inoculation with supernatant from Tilapia lake virus (TiLV) infected cell line, no cytopathic effects were observed in the LCF cells and cell pellet was negative for TiLV in RT-PCR, indicating that LCF cells were not susceptible to TiLV. The developed cell line has been submitted to National Repository of Fish Cell Lines being maintained at ICAR-National Bureau of Fish Genetic Resources, Lucknow (accession no. NRFC063). The newly developed LCF cell line would be helpful in investigating diseases affecting this candidate species particularly the ones suspected to be of viral etiology, and for cytotoxicity and transgenic studies.

Messages in prescription drug advertising for four chronic diseases, 2003-2016: A content analysis.

The objective of this study was to examine the frequency and content of messages related to pharmacological and evidence-based, non-pharmaceutical treatments in direct-to-consumer advertising (DTCA) for prescription drugs treating four chronic diseases in the United States. We used content coding to identify theory-informed categories of messages appearing in a large sample of heart disease, diabetes, depression, and osteoarthritis advertisements, appearing on national and local television between 2003 and 2016 (N = 11,347,070). The data were originally accessed in 2019 and analyzed in 2020-2021. The central message in all pharmaceutical DTCA was drug efficacy. Advertisements for diabetes and heart disease, but not depression or osteoarthritis, contained general (not central) messages about diet and exercise. Advertisements for heart disease primarily portrayed diet and exercise as insufficient for controlling the target health condition. No advertisements in our sample portrayed changes in diet or physical activity as an alternative to drugs. Pharmaceutical DTCA across health conditions employ similar strategies to promote use of the advertised drug but vary widely in whether and how they describe non-pharmaceutical treatments that complement or serve as alternatives to medications. Regulators should consider the potential spillover effects of non-pharmaceutical messages in pharmaceutical DTCA when considering future regulatory endeavors.

SARS-CoV-2 seroprevalence among firefighters in Los Angeles, California.

OBJECTIVE: We estimate the seroprevalence of SARS-CoV-2 antibodies among a sample of firefighters in the Los Angeles (LA), California fire department in October 2020 and compare demographic and contextual factors for seropositivity. METHODS: We conducted a serological survey of firefighters in LA, California, USA, in October 2020. Individuals were classified as seropositive for SARS-CoV-2 if they tested positive for IgG, IgM or both. We compared demographic and contextual factors for seropositivity. RESULTS: All firefighters in LA, California, USA were invited to participate in our study, but only roughly 21% participated. Of 713 participants with valid serological data, 8.8% tested positive for SARS-CoV-2 antibodies, and among the 686 with complete survey data 8.9% tested positive for antibodies. Seropositivity was not associated with gender, age or race/ethnicity. Seropositivity was highest among firefighters who reported working in the vicinity of LA International Airport, which had a known outbreak in July 2020. CONCLUSIONS: Seroprevalence among firefighters in our sample was 8.8%, however, we lack a full workplace seroprevalence estimate to compare the relative magnitude against general population seroprevalence (15%). Workplace safety protocols, such as access to personal protective equipment and testing, can mitigate increased risk of infection at work, and may have eliminated differences in disease burden by geography and race/ethnicity in our sample.

Smoking-related psychosocial beliefs and justifications among smokers in India: Findings from Tobacco Control Policy (TCP) India Surveys.

BACKGROUND: Previous research in high-income countries (HICs) has shown that smokers reduce their cognitive dissonance through two types of justifications over time: risk minimizing and functional beliefs. To date, however, the relationship between these justifications and smoking behaviors over time has limited evidence from low- and middle-income countries. This study examines these of justifications and their relation to quitting behavior and intentions among smoking tobacco users in India. METHODS: The data are from the Tobacco Control Policy (TCP) India Survey, a prospective cohort of nationally representative sample of tobacco users. The respondents include smoked tobacco (cigarettes and bidi) users (n = 1112) who participated in both Wave 1 (W1; 2010-2011) and Wave 2 (W2; 2012-2013) surveys. Key measures include questions about psychosocial beliefs such as functional beliefs (e.g., smoking calms you down when you are stressed or upset) and risk-minimizing beliefs (e.g., the medical evidence that smoking is harmful is exaggerated) and quitting behavior and intentions at Wave 2. FINDINGS: Of the 1112 smokers at W1, 78 (7.0%) had quit and 86 (7.8%) had intentions to quit at W2. Compared to W1, there was a significant increase in functional beliefs at W2 among smokers who transitioned to mixed use (using both smoking and smokeless tobacco) and a significant decrease among those who quit. At W2, smokers who quit held significantly lower levels of functional beliefs, than continuing smokers, and mixed users ((M = 2.96, 3.30, and 3.93, respectively, p < .05). In contrast, risk-minimizing beliefs did not change significantly between the two waves. Additionally, higher income and lower functional beliefs were significant predictors of quitting behavior at W2. CONCLUSION: These results suggest that smokers in India exhibit similar patterns of dissonance reduction as reported in studies from HICs: smokers who quit reduced their smoking justifications in the form of functional beliefs, not risk-minimizing beliefs. Smokers' beliefs change in concordance with their smoking behavior and functional beliefs tend to play a significant role as compared to risk-minimizing beliefs. Tobacco control messaging and interventions can be framed to target these functional beliefs to facilitate quitting.

Did prioritizing essential workers help to achieve racial/ethnic equity in early COVID-19 vaccine distribution? The LA pandemic surveillance cohort study.

BACKGROUND: Most US states and counties prioritized essential workers for early access to COVID-19 vaccines due to their heightened occupational risk. Racial/ethnic groups most impacted by COVID-19 are overrepresented among essential workers. This study estimates the effects of prioritizing essential workers on racial/ethnic equity in COVID-19 vaccination. METHODS: Survey data were collected from 5500 Los Angeles County adult residents in March and April 2021. Multivariate regression models were used to assess marginal changes in probabilities of vaccination attributable to essential worker status by race/ethnicity. These probabilities were multiplied by population proportions of essential workers in each racial/ethnic group to estimate the effects of prioritizing essential workers on vaccine equity in the population. RESULTS: While Latinos (24.9%), Blacks (22.4%), and Asians (21.4%) were more likely to be prioritized essential workers than Whites (14.3%), their marginal gains in vaccine uptake due to their essential worker status did not significantly differ from that of Whites. At the population-level, prioritizing vaccines for essential workers increased the probabilities of vaccination by small and similar amounts among Asians (5.3%; 95% confidence interval [CI]: 3.3%, 7.5%), Blacks (4.0%; 95% CI: 1.7%, 6.5%), Latinos (3.7%; 95% CI: 2.3%, 5.1%), and Whites (2.9%; 95% CI :1.9%, 3.9%). CONCLUSIONS: Prioritizing essential workers did not provide proportionally greater early vaccine uptake benefits to racial/ethnic groups that were disproportionately affected by COVID-19. Early prioritization of essential workers during vaccine campaigns is an important but insufficient strategy for reducing racial/ethnic disparities in early vaccine uptake. Additional strategies addressing access and trust are needed to achieve greater equity in vaccine distribution.

Transcriptome analysis reveals immune pathways underlying resistance in the common carp Cyprinus carpio against the oomycete Aphanomyces invadans.

Infection with Aphanomyces invadans is a serious fish disease with major global impacts. Despite affecting over 160 fish species, some of the species like the common carp Cyprinus carpio are resistant to A. invadans infection. In the present study, we investigated the transcriptomes of head kidney of common carp experimentally infected with A. invadans. In time course analysis, 5288 genes were found to be differentially expressed (DEGs), of which 731 were involved in 21 immune pathways. The analysis of immune-related DEGs suggested that efficient processing and presentation of A. invadans antigens, enhanced phagocytosis, recognition of pathogen-associated molecular patterns, and increased recruitment of leukocytes to the sites of infection contribute to resistance of common carp against A. invadans. Herein, we provide a systematic understanding of the disease resistance mechanisms in common carp at molecular level as a valuable resource for developing disease management strategies for this devastating fish-pathogenic oomycete.

The Effects of Accountable Care Organizations Forming Preferred Skilled Nursing Facility Networks on Market Share, Patient Composition, and Outcomes.

BACKGROUND: Through participation in payment reforms such as bundled payment and accountable care organizations (ACOs), hospitals are increasingly financially responsible for health care use and adverse health events occurring after hospital discharge. To improve management and coordination of postdischarge care, ACO hospitals are establishing a closer relationship with skilled nursing facilities (SNFs) through the formation of preferred SNF networks. RESEARCH DESIGN: We evaluated the effects of preferred SNF network formation on care patterns and outcomes. We included 10 ACOs that established preferred SNF networks between 2014 and 2015 in the sample. We first investigated whether hospitals "steer" patients to preferred SNFs by examining the percentage of patients sent to preferred SNFs within each hospital before and after network formation. We then used a difference-in-difference model with SNF fixed effects to evaluate the changes in patient composition and outcomes of preferred SNF patients from ACO hospitals after network formation relative to patients from other hospitals. RESULTS: We found that preferred network formation was not associated with higher market share or better outcomes for preferred SNF patients from ACO hospitals. However, we found a small increase in the average number of Elixhauser comorbidities for patients from ACO hospitals after network formation, relative to patients from non-ACO hospitals. CONCLUSIONS: After preferred SNF network formation, there is some evidence that ACO hospitals sent more complex patients to preferred SNFs, but there was no change in the volume of patients received by these SNFs. Furthermore, preferred network formation was not associated with improvement in patient outcomes.

Transcriptome analysis of liver elucidates key immune-related pathways in Nile tilapia Oreochromis niloticus following infection with tilapia lake virus.

Nile tilapia (Oreochromis niloticus) is one of the most important aquaculture species farmed worldwide. However, the recent emergence of tilapia lake virus (TiLV) disease, also known as syncytial hepatitis of tilapia, has threatened the global tilapia industry. To gain more insight regarding the host response against the disease, the transcriptional profiles of liver in experimentally-infected and control tilapia were compared. Analysis of RNA-Seq data identified 4640 differentially expressed genes (DEGs), which were involved among others in antigen processing and presentation, MAPK, apoptosis, necroptosis, chemokine signaling, interferon, NF-kB, acute phase response and JAK-STAT pathways. Enhanced expression of most of the DEGs in the above pathways suggests an attempt by tilapia to resist TiLV infection. However, upregulation of some of the key genes such as BCL2L1 in apoptosis pathway; NFKBIA in NF-kB pathway; TRFC in acute phase response; and SOCS, EPOR, PI3K and AKT in JAK-STAT pathway and downregulation of the genes, namely MAP3K7 in MAPK pathway; IFIT1 in interferon; and TRIM25 in NF-kB pathway suggested that TiLV was able to subvert the host immune response to successfully establish the infection. The study offers novel insights into the cellular functions that are affected following TiLV infection and will serve as a valuable genomic resource towards our understanding of susceptibility of tilapia to TiLV infection.

Co-infection of Lactococcus garvieae and Tilapia lake virus (TiLV) in Nile tilapia Oreochromis niloticus cultured in India.

Tilapia lake virus (TiLV) and Lactococcus garvieae are 2 major pathogens of cultured Nile tilapia Oreochromis niloticus. In June-July 2018, a disease outbreak was reported in Nile tilapia cultured in brackish water floating cages in Kerala, India. Affected fish died gradually, and cumulative mortality reached ~75% within 1 mo. In the present study, TiLV and L. garvieae were isolated from the infected fish and confirmed. Nucleotide analysis of the partial sequence of segment 3 revealed that the present TiLV isolate showed 100% similarity with TiLV MF574205 and 97.65% similarity with TiLV KU552135 isolated in Israel. The partial 16S rDNA nucleotide sequence of L. garvieae shared 99% similarity with the 16S rDNA nucleotide sequence of L. garvieae isolated from Nile tilapia in Brazil. Eight virulence genes (hly1, hly2, hly3, NADH oxidase, adhPav, LPxTG-1, LPxTG-4, adhC1) were amplified in the present isolate. In the experimental challenge study, the onset of mortality started earlier in fish co-infected with TiLV and L. garvieae (3 d post-infection [dpi]) compared to other groups. Cumulative mortality (90% at 12 dpi) was significantly higher in the co-infected group than in fish infected with TiLV (60% at 12 dpi) and L. garvieae (40% at 12 dpi) alone. This study reveals that synergistic co-infection with TiLV and other bacteria may increase mortality in disease outbreaks. To the best of our knowledge, this is the first reported co-infection of L. garvieae with TiLV associated with mass mortality in Nile tilapia in India.

Infectious spleen and kidney necrosis virus-associated large-scale mortality in farmed giant gourami, Osphronemus goramy, in India.

Megalocytivirus cause diseases that have serious economic impacts on aquaculture, mainly in East and South-East Asia. Five primary genotypes are known: infectious spleen and kidney necrosis virus (ISKNV), red sea bream iridovirus (RSIV), turbot reddish body iridovirus (TRBIV), threespine stickleback iridovirus (TSIV) and scale drop disease virus (SDDV). ISKNV-mediated infectious spleen and kidney necrosis disease (ISKND) is a major viral disease in both freshwater and marine fish species. In this study, we report the isolation of ISKNV from diseased giant gourami, Osphronemus goramy, in India. Transmission electron microscopy of ultrathin sections of kidney and spleen revealed the presence of numerous polygonal naked viral particles having an outer nucleocapsid layer within the cytoplasm of enlarged cells (115-125 nm). Molecular and phylogenetic analyses confirmed the presence of ISKNV and the major capsid protein (MCP) (1,362 bp) gene in the infected fish had a high similarity to the other ISKNV-I isolates. Moreover, ISKNV was propagated in the Astronotus ocellatus fin (AOF) cell line and further confirmed genotypically. A high mortality rate (60%) was observed in gourami fish injected with ISKNV-positive tissue homogenate through challenge studies. Considering the lethal nature of ISKNV, the present study spotlights the implementation of stringent biosecurity practices for the proper control of the disease in the country.

Impact of lifestyle diseases on income and household consumption: evidence from an emerging economy.

With increase in lifestyle diseases such as diabetes, hypertension and heart disease in emerging economies, it is important to understand the impact of acquiring these health issues on household income and consumption. Using a panel data of Indian households, we show that acquiring diabetes, hypertension or heart disease results in increased medical and non-medical expenses for high-income households. However, affected low-income households, to meet their increased medical expenses, had to divert resources from non-medical expenses. Our findings add to the extant marketing literature on impact of crises on consumption behaviour and the marketing implications of such changes in consumption behaviour.

Trends in the Use of Skilled Nursing Facility and Home Health Care Under the Hospital Readmissions Reduction Program: An Interrupted Time-series Analysis.

BACKGROUND: Medicare's Hospital Readmission Reduction Program (HRRP) penalizes hospitals with elevated 30-day readmission rates for acute myocardial infarction (AMI), heart failure (HF), or pneumonia. To reduce readmissions, hospitals may have increased referrals to skilled nursing facilities (SNFs) and home health care. RESEARCH DESIGN: Outcomes included 30-day postdischarge utilization of SNF and home health care, including any use as well as days of use. Subjects included Medicare fee-for-service beneficiaries aged 65 years and older who were admitted with AMI, HF, or pneumonia to hospitals subject to the HRRP. Using an interrupted time-series analysis, we compared utilization rates observed after the announcement of the HRRP (April 2010 through September 2012) and after the imposition of penalties (October 2012 through September 2014) with projected utilization rates that accounted for pre-HRRP trends (January 2008 through March 2010). Models included patient characteristics and hospital fixed effects. RESULTS: For AMI and HF, utilization of SNF and home health care remained stable overall. For pneumonia, observed utilization of any SNF care increased modestly (1.0%, P<0.001 during anticipation; 2.4%, P<0.001 after penalties) and observed utilization of any home health care services declined modestly (-0.5%, P=0.008 after announcement; -0.7%, P=0.045 after penalties) relative to projections. Beneficiaries with AMI and pneumonia treated at penalized hospitals had higher rates of being in the community 30 days postdischarge. CONCLUSIONS: Hospitals might be shifting to more intensive postacute care to avoid readmissions among seniors with pneumonia. At the same time, penalized hospitals' efforts to prevent readmissions may be keeping higher proportions of their patients in the community.