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Over 60 countries have integrated wastewater-based epidemiology (WBE) in their COVID-19 surveillance programs, focusing on wastewater treatment plants (WWTP). In this paper, we piloted the assessment of SARS-CoV-2 WBE as a complementary public health surveillance method in susceptible communities in a highly urbanized city without WWTP in the Philippines by exploring the extraction and detection methods, evaluating the contribution of physico-chemical–anthropogenic factors, and attempting whole-genome sequencing (WGS). Weekly wastewater samples were collected from sewer pipes or creeks in six communities with moderate-to-high risk of COVID-19 transmission, as categorized by the City Government of Davao from November to December 2020. Physico-chemical properties of the wastewater and anthropogenic conditions of the sites were noted. Samples were concentrated using a PEG-NaCl precipitation method and analyzed by RT-PCR to detect the SARS-CoV-2 N, RdRP, and E genes. A subset of nine samples were subjected to WGS using the Minion sequencing platform. SARS-CoV-2 RNA was detected in twenty-two samples (91.7%) regardless of the presence of new cases. Cycle threshold values correlated with RNA concentration and attack rate. The lack of a sewershed map in the sampled areas highlights the need to integrate this in the WBE planning. A combined analysis of wastewater physico-chemical parameters such as flow rate, surface water temperature, salinity, dissolved oxygen, and total dissolved solids provided insights on the ideal sampling location, time, and method for WBE, and their impact on RNA recovery. The contribution of fecal matter in the wastewater may also be assessed through the coliform count and in the context of anthropogenic conditions in the area. Finally, our attempt on WGS detected single-nucleotide polymorphisms (SNPs) in wastewater which included clinically reported and newly identified mutations in the Philippines. This exploratory report provides a contextualized framework for applying WBE surveillance in low-sanitation areas.

The COVID-19 pandemic caught the world unprepared for a global catastrophe. With the uncertainty of a vaccine in the near future, the pandemic marks the start of a new world that has to co-exist with the virus. COVID-19 has been a wake-up call to the lingering but neglected issues on environmental health and global food security but is also a timely opportunity to rise from the ashes, reboot our framework, and act with resolve for sustainable and resilient ecosystems and communities. The emerging tools and disciplines in the field of omics can be leveraged for a systems-level understanding of biological processes to accelerate advancements on knowledge, innovations, and policies towards a more sustainable utilization and management of resources in this post-pandemic age. In developing regions, omics research centers can serve as a gateway to this technology by providing access, building capacities, and facilitating collaborative and harmonized research engagements.

AND KEYWORDSBackgroundOver 50 countries have used Wastewater-Based Epidemiology (WBE) and Whole-Genome Sequencing (WGS) of SARS-CoV-2 for monitoring COVID-19 cases. COVID-19 surveillance in the Philippines relies on clinical monitoring and contact tracing, with both having limited use in early detection or prediction of community outbreaks. Thus, complementary public health surveillance methods that can provide community-level infection data faster and using lesser resources must be explored.ObjectivesThis study piloted and assessed WBE and WGS as approaches for COVID-19 surveillance in low-resource and low-sanitation communities in Davao City, Philippines.MethodsWeekly wastewater samples were collected from six barangay community sewer pipes or creeks from November to December 2020. Samples were concentrated using a PEG-NaCl precipitation method and analyzed by RT-PCR to detect the SARS-CoV-2 N, RdRP, and E genes. In addition, SARS-CoV-2 RNA-positive samples were subjected to WGS for genomic mutation surveillance. Public data from clinical surveillance were also reviewed to interpret WBE data.ResultsTwenty-two of the 24 samples (91.7%) obtained from the six barangays tested positive for SARS-CoV-2 RNA. The cycle threshold (Ct) values were correlated with RNA concentration and attack rate. Thirty-two SARS-CoV-2 mutations were detected in WGS, including novel non-synonymous mutations or indels in seven SARS-CoV-2 genes and ten mutations previously reported in the Philippines.DiscussionSARS-CoV-2 RNA was detected in community wastewater from the six barangays of Davao City, even when the barangays were classified as having a low risk of COVID-19 transmission and no new cases were reported. Despite the fragmented genome sequences analyzed, our genomic surveillance in wastewater confirmed the presence of previously reported mutations while identifying mutations not yet registered in clinical surveillance. The local context of a community must be considered when planning to adopt WBE and WGS as complementary COVID-19 surveillance methodologies, especially in low-sanitation and low-resource settings.

The technical limitations of capillary sequencing in providing insights on phylogeny have been greatly aided in recent years by the implementation of next generation sequencing platforms which can generate whole mitochondrial genome (mitogenome) sequences. In this study, enriched mitochondrial DNA of

Rotavirus A is one of the leading etiological agents of porcine gastroenteritis, a condition which results to stunted growth among piglets. Moreover, there is increasing evidence for zoonosis of rotavirus A (RVA), which is also the principal cause of diarrhea in children. In the absence of rigorous animal health monitoring in Philippine backyard farms, there is therefore a strong need for RVA surveillance. In this study, 30 randomly selected backyard farms were subjected to surveillance for RVA for 12 months. Results show that RVA detection at a monthly farm-level rate ranged from 0 to 52%, with an overall average of 23%. RVA had higher detection rates in adult pigs compared to young piglets and was most prevalent in non-diarrheic stools, indicating asymptomatic circulation of the virus. Spatiotemporal analysis demonstrated that the viral circulation exhibits a seasonal pattern that peaks and forms geographical clusters during the cooler months of the year, suggesting farm-to-farm transmission. Risk factor analysis identified specific farm conditions that increase the likelihood of RVA circulation: presence of gilts, larger herd size, presence of other animals, and abiotic factors such as low relative humidity and low altitude. The same analysis also revealed three major management practices that can help reduce the pressure of infection in these farms: sanitation and waste disposal, animal grouping, and diet. This new perspective on porcine RVA circulation will benefit the underprivileged backyard farmers and help empower them to protect both animal and public health.

Emerging infectious diseases due to coronavirus (CoV) infections have received significant global attention in the past decade and have been linked to bats as the original source. The diversity, distribution, and host associations of bat CoVs were investigated to assess their potential for zoonotic transmission. Phylogenetic, network, and principal coordinate analysis confirmed the classification of betacoronaviruses (BetaCoVs) into five groups (2A to 2E) and a potentially novel group, with further division of 2D into five subgroups. The genetic co-clustering of BetaCoVs among closely related bats reflects host taxon-specificity with each bat family as the host for a specific BetaCoV group, potentially a natural barrier against random transmission. The divergent pathway of BetaCoV and host evolution suggests that the viruses were introduced just prior to bat dispersal and speciation. As such, deviant patterns were observed such as for 2D-IV, wherein cross-taxon transmission due to overlap in bat habitats and geographic range among genetically divergent African bat hosts could have played a strong role on their shared CoV lineages. In fact, a few bat taxa especially the subfamily Pteropodinae were shown to host diverse groups of BetaCoVs. Therefore, ecological imbalances that disturb bat distribution may lead to loss of host specificity through cross-taxon transmission and multi-CoV infection. Hence, initiatives that minimize the destruction of wildlife habitats and limit wildlife-livestock-human interfaces are encouraged to help maintain the natural state of bat BetaCoVs in the wild.ImportanceBat Betacoronaviruses (BetaCoVs) pose a significant threat to global public health and have been implicated in several epidemics such as the recent pandemic by severe acute respiratory syndrome coronavirus 2. Here, we show that bat BetaCoVs are predominantly host-specific, which could be a natural barrier against infection of other host types. However, a strong overlap in bat habitat and geographic range may facilitate viral transmission to unrelated hosts, and a few bat families have already been shown to host multi-CoV variants. We predict that continued disturbances on the ecological balance may eventually lead to loss of host specificity. When combined with enhanced wildlife-livestock-human interfaces, spillover to humans may be further facilitated. We should therefore start to define the ecological mechanisms surrounding zoonotic events. Global surveillance should be expanded and strengthened to assess the complete picture of bat coronavirus diversity and distribution and their potential to cause spillover infections.

Rabies is a lethal viral disease and dogs are the major disease reservoir in the Philippines. Spatio-temporal variations in environmental factors are known to affect disease dynamics. Some rabies-affected countries considered investigating the role of weather components in driving rabies cases and it has helped them to strategize their control efforts. In this study, cointegration analysis was conducted between the monthly reported rabies cases and the weather components, such as temperature and precipitation, to verify the effect of weather components on rabies incidence in Davao City, Philippines. With the Engle-Granger cointegration tests, we found that rabies cases are cointegrated into each of the weather components. It was further validated, using the Granger causality test, that each weather component predicts the rabies cases and not vice versa. Moreover, we performed the Johansen cointegration test to show that the weather components simultaneously affect the number of rabies cases, which allowed us to estimate a vector-error correction model for rabies incidence as a function of temperature and precipitation. Our analyses showed that canine rabies in Davao City was weather-sensitive, which implies that rabies incidence could be projected using established long-run relationship among reported rabies cases, temperature, and precipitation. This study also provides empirical evidence that can guide local health officials in formulating preventive strategies for rabies control and eradication based on weather patterns.

Coronaviruses (CoVs) are common agents of respiratory and gastrointestinal tract infections but are also considered as important emerging infectious disease (EID) agents that have led to global epidemics of severe respiratory disease. Human CoV EIDs have been proposed to have originated from bat CoVs (BtCoV). This study reports the detection of CoV in bats from the University of the Philippines (UP) Mindanao, Davao City using an optimized reverse transcription nested polymerase chain reaction (RT-nPCR) for bat CoV RNA-dependent RNA polymerase (RdRp) gene. Out of 49 bats, only one was positive for the virus (2.04% detection rate). Phylogenetic analysis demonstrated the genetic relatedness of the CoV, which was detected in the fruit bat Macroglossus minimus, to other bat and mammalian CoVs of betacoronavirus under sub-group 2d. The CoV also exhibited 94.55% identity and close genetic relationship with BtCoV 2265/Philippines/2010 from Luzon, Philippines, suggesting a common lineage of these viruses that may have pre-existed prior to biogeographic movements of bats in the country. This is the first reported detection of CoV in bats from Mindanao, hence further surveillance of circulating viruses in wildlife is recommended to expand the understanding of BtCoV evolution and potential for zoonosis. These findings also emphasize the need to limit potential zoonotic transmissions through bat-animal or human interactions by preserving natural habitats.

Rabies is a fatal zoonotic and neglected tropical disease caused by the rabies virus (RABV) and is associated with neuronal dysfunction and death, with dogs as the predominant carrier. The Philippines plans to eradicate rabies by 2022, but this is challenged with sub-optimal coverage of vaccination programs coupled with sustained transmission chains, making it unable to eradicate the disease. We investigated the dynamics of canine rabies in the highly urbanized Davao City of the Philippines and its neighboring localities by assessing genetic relationships, transmission patterns, selection pressure, and recombination events using the whole genome sequence of 49 RABV cases from June 2018 to May 2019, majority of which (46%) were from the district of Talomo, Davao City. Although phylogeographic clustering was observed, local variants also exhibited genetic sub-lineages. Phylogenetic and spatial transmission analysis provided evidence for intra- and inter-city transmission predominantly through the Talomo district of Davao City. Around 84% of the cases were owned dogs, but the genetic similiarity of RABVs from stray and owned dogs further alluded to the role of the former as transmission vectors. The high rate of improper vaccination among the affected dogs (80%) was also a likely contributor to transmission. The RABV population under Investigation is generally under strong purifying selection with no evidence of vaccine evasion due to the genetic homogeneity of viruses from vaccinated and improperly vaccinated dogs. However, some homologous recombination (HR) events were identified along the G and L genes, also predominantly associated with viruses from Talomo. The complementary findings on epidemiology, transmission, and recombination for Talomo suggest that high incidence areas can be seeds for virus dispersal and evolution. We recommend further Investigations on the possibility of HR in future large-scale genome studies. Finally, districts associated with these phenomena can be targeted for evidence-based local strategies that can help break RABV transmission chains and prevent emergence of novel strains in Davao City.

An antiviral containment strategy for foot-and-mouth disease (FMD) outbreaks could support or replace current contingency plans in case of an outbreak in Europe and could spare many healthy animals from being pre-emptively culled. Recently, substantial progress has been made towards the development of small molecule drugs that inhibit FMD virus (FMDV) replication in vitro. For the initial in vivo evaluation of antiviral lead molecules, a refined FMDV-infection model in guinea pigs (GP) is herewith described. This GP model was validated by demonstrating the antiviral effect of T-1105 (an influenza virus inhibitor with reported activity against FMDV). Sixteen animals were orally administered with T-1105 twice daily (400 mg/kg/day) for five consecutive days and inoculated intraplantarly with 100 GPID50 of the GP-adapted FMDV strain O1 Manisa 1 h after the first administration. The efficacy of T-1105 was compared with that of prophylactic vaccination with a highly potent double-oil emulsion-inactivated O1 Manisa vaccine. Ten animals received a single, full (2 ml) cattle vaccine dose and were inoculated 3 weeks later. Fourteen T-1105-treated and all vaccinated GP were completely protected from generalization of vesicular lesions. At 2 dpi, viral RNA was detected in serum of 9/16 T-1105-treated and of 6/10 vaccinated animals. At 4 dpi, viral RNA was detected in serum, organs and oral swabs of half of the T-1105-treated animals and only in the serum of 1/10 of the vaccinated animals. Mean viral RNA levels in serum and organs of T-1105-treated and vaccinated animals were reduced compared to untreated controls (P

Tumor necrosis factor (TNF-alpha) triggers biphasic activation of the NF-kappaB transcriptional regulator. This process consists of an initial, IkappaBalpha-mediated transient phase and a later, persistent phase dependent on IkappaBbeta degradation. To presumably interfere with the fulfillment of this immunity-associated event in cells infected with the molluscum contagiosum virus (MCV), this pathogen produces the intracellular MC159 protein. To define the mode of action of MC159, the impact of TNF-alpha on HEK 293T cells ectopically expressing the MC159 protein was examined. In this regard, TNF-alpha-induced expression of an NF-kappaB-regulated luciferase reporter gene was partially inhibited by the MC159 protein. This ability was attributed to blockage of the persistent phase of TNF-alpha-induced NF-kappaB activation for the following reasons: (1) the initial phase of NF-kappaB transcriptional activation was not affected by the MC159 protein; (2) the MC159 protein inhibited TNF-alpha-directed degradation of IkappaBbeta, but not IkappaBalpha; and (3) expression of the late NF-kappaB-regulated cell genes, TNF-alpha and CCL2, was decreased in the presence of the MC159 protein while transcription of the early NF-kappaB-regulated cell gene, CXCL1, was not altered. Previously reported MC159-RIP interactions appear to be irrelevant for the MC159 inhibitory function. In contrast, MC159-TRAF2 associations are more relevant for inhibitory function since mutant MC159 proteins unable to bind TRAF2 also cannot inhibit TNF-mediated NF-kappaB activation. In vivo, the MC159 protein may act to prolong virus survival by preventing the infected cell from responding to TNF-alpha, ultimately preventing the cellular production of proinflammatory and immunoattractant molecules.

Foot-and-mouth disease virus (FMDV) is a highly pathogenic member of the genus Aphthovirus (family Picornaviridae) that is only to be manipulated in high-containment facilities, thus complicating research on and discovery of antiviral strategies against the virus. Bovine rhinitis B virus (BRBV) and equine rhinitis A virus (ERAV), phylogenetically most closely related to FMDV, were explored as surrogates for FMDV in antiviral studies. Although no efficient cell culture system has been reported so far for BRBV, we demonstrate that infection of primary bovine kidney cells resulted in an extensive but rather poorly-reproducible induction of cytopathic effect (CPE). Madin-Darby bovine kidney cells on the other hand supported viral replication in the absence of CPE. Antiviral tests were developed for ERAV in Vero A cells employing a viral RNA-reduction assay and CPE-reduction assay; the latter having a Z' factor of 0.83±0.07. The BRBV and ERAV models were next used to assess the anti-aphthovirus activity of two broad-spectrum antiviral agents 2'-C-methylcytidine (2CMC) and ribavirin, as well as of the enterovirus-specific inhibitor enviroxime. The effects of the three compounds in the CPE-reduction (ERAV) and viral RNA-reduction assays (BRBV and ERAV) were comparable. Akin to 2CMC, compound A, a recently-discovered non-nucleoside pan-serotype FMDV inhibitor, also inhibited the replication of both BRBV and ERAV, whereas enviroxime was devoid of activity. The BRBV and ERAV surrogate models reported here can be manipulated in BSL-2 laboratories and may facilitate studies to unravel the mechanism of action of novel FMDV inhibitors.

Summary Recent European contingency plans envisage emergency vaccination as an animal-friendly control strategy for foot-and-mouth disease (FMD). Anti-viral drugs may be used as an alternative or complementary measure. We here demonstrate that the nucleoside analogue 2′-C-methylcytidine (2′CMC) protects severe combined immunodeficient (SCID) mice against lethal FMD virus infection. In brief, SCID mice were inoculated with serotype A FMD virus and treated for five consecutive days with 2′CMC. All 15 treated mice remained healthy until the end of the study at 14 days post-infection (dpi). At that time, viral RNA was no longer detected in 13 of 15 treated mice. All eight untreated mice suffered from an acute generalized disease and were euthanized for ethical reasons on average at 4 dpi. These results illustrate the potential of small molecules to control FMD.