An outbreak of infection due to severe acute respiratory corona virus-2 in a neonatal unit from a low and middle income setting.

Introduction: The provision of kangaroo mother care (KMC) involving continuous skin-to-skin care (SSC) is an important intervention in neonatal care, which is recommended even when women are infected with severe acute respiratory syndrome coronavirus (SARS-CoV-2). We report on a nosocomial outbreak of SARS-CoV-2 infections in a KMC ward. Methods: Contact tracing was conducted following the diagnosis of SARS-CoV-2 in a mother lodging in the KMC ward. All mother-newborn dyads in the KMC and healthcare workers (HCW) were tested for SARS-CoV-2 within 24-72 h of diagnosing the index case. Nasopharyngeal swab samples were obtained and tested from contacts, with a nucleic acid amplification test (NAAT) assay. Next-generation sequencing was done on positive samples. The secondary attack rate (SAR) was calculated assuming that the mother who presented with symptoms was the source of infection. Results: Twelve (70.6%) of 17 mothers and 8 (42.1%) of 19 neonates who were in the KMC ward with the index case were found to be positive with SARS-CoV-2. Seven (87.5%) of the 8 neonates who tested positive had mothers who also tested positive. Seventy-five percent (9/12) of the mothers and 62.5% (5/8) of the neonates who tested positive were asymptomatic. Eight (27.6%) of 29 HCW were found to be positive and were all asymptomatic. One neonate died from Acinetobacter baumannii sepsis, and his post-mortem lung histopathology showed features compatible with SARS-CoV-2 pneumonia. The sequencing of 13 specimens, which included 1 mother-newborn dyad, indicated clustering to the same phylogenetic lineage with identical mutations. In assessing for factors contributing to this outbreak, it was found that spaces between beds were less than 1 m and mothers had their meals around the same table at the same time. Conclusion: We report on a nosocomial outbreak of SARS-CoV-2 in a KMC ward, affecting a high number of mothers and neonates, and to a lesser extent HCWs. Although it is difficult to point to the index case as the source of this outbreak, as asymptomatic individuals can spread infection, the inadequate adherence to non-pharmaceutical interventions was assessed to have contributed to the spread of infection. This highlights the need for awareness and adherence to mitigation strategies to avoid SARS-CoV-2 outbreaks.

The effect of inoculum dose on the genetic diversity detected within Helicoverpa armigera nucleopolyhedrovirus populations.

Environmental and infection variables may affect the genetic diversity of baculovirus populations. In this study, Helicoverpa armigera nucleopolyhedrovirus (HearNPV) was used as a model system for studying the effects of a key infection variable, inoculum dose, on the genetic diversity within nucleopolyhedrovirus populations. Diversity and equitability indices were calculated from DNA polymerase-specific denaturing gradient gel electrophoresis profiles obtained from individual H. armigera neonate larvae inoculated with either an LD5 or LD95 of HearNPV. Although the genetic diversity detected in larvae treated with an LD95 was not statistically different from the diversity detected in the HearNPV inoculum samples, there was a statistically significant difference in the genetic diversity detected in the LD5-inoculated larvae compared with the genetic diversity detected in the HearNPV samples used for the inoculations. The study suggests that inoculum dose needs to be considered carefully in experiments that evaluate HearNPV genetic diversity or in studies where differences in genetic diversity may have phenotypic consequences.

High levels of genetic variation within Helicoverpa armigera nucleopolyhedrovirus populations in individual host insects.

It has been well documented that baculovirus populations exhibit high levels of genetic variation. Due to the lack of sensitivity of the techniques currently used to study baculovirus genetic variation, relatively little is known about baculovirus genetic diversity at the individual insect level. Since denaturing gradient gel electrophoresis (DGGE) has key advantages over other methods used to study genetic variation in baculoviruses, DGGE assays were used to obtain a better understanding of the genetic variation within baculovirus populations in individual host insects. Helicoverpa armigera nucleopolyhedrovirus (HearNPV) was used as a model baculovirus system, and neonate H. armigera larvae were infected with one of two geographically distinct HearNPV isolates. DGGE assays for two lepidopteran-specific baculovirus genes, me53 and dbp1, detected many HearNPV genetic variants within individual host larvae, with up to 20 genetic variants detected in a 434-bp fragment of the dbp1 gene in a single neonate larva. High levels of HearNPV genetic diversity were detected in individual host larvae irrespective of the HearNPV isolate used to infect the larvae. This study sets a benchmark for HearNPV genetic variation in individual H. armigera larvae. The levels of HearNPV genetic diversity detected are higher than reported previously for a baculovirus population at the individual insect level.

High levels of genetic variation within core Helicoverpa armigera nucleopolyhedrovirus genes.

It is well documented that baculovirus populations contain many genotypic variants, but little is known about the degree of genetic variation in specific baculovirus genes. Helicoverpa armigera nucleopolyhedrovirus (HearNPV) was used as a model system for studying genetic variation in nucleopolyhedrovirus genes. Next generation sequencing (NGS) was used to identify single-nucleotide polymorphisms (SNPs) within a core baculovirus gene (DNA polymerase) and two core lepidopteran-specific baculovirus genes (dbp1 and me53) in HearNPV populations. Analysis of the NGS data identified 60 SNPs within the 3063 bp DNA polymerase gene, 13 SNPs within the 972 bp dbp1 gene, and 25 SNPs within the 1080 bp me53 gene. Depending on the gene, between 31 and 35% of the SNPs were non-synonymous and may, thus, affect the biological functioning of the encoded proteins. The number and homogenous distribution of SNPs found suggest that nucleotide substitution is a major contributor to HearNPV genetic diversity. Denaturing gradient gel electrophoresis (DGGE) assays were used to provide an additional method for evaluating HearNPV genetic variation. Since each of the gene-specific DGGE assays produced unique banding profiles containing numerous bands of differing relative intensities, the DGGE experiments confirmed that there was a high degree of genetic variation in core HearNPV genes and provided information on the relative frequency distribution of genetic variants in the population. The amount of gene-specific genetic variation detected in this study significantly exceeds that reported in other HearNPV studies, suggesting that NGS and DGGE may be useful techniques for studying genetic variation in other baculoviruses.

Development of highly sensitive assays for detection of genetic variation in key Helicoverpa armigera nucleopolyhedrovirus genes.

Information on the degree of genetic variation in key Helicoverpa armigera nucleopolyhedrovirus (HearNPV) genes is limited as the currently used techniques lack the detection sensitivity required to identify multiple genetic variants within a baculovirus population. To facilitate the detection and study of genetic variation within HearNPV populations, denaturing gradient gel electrophoresis (DGGE) assays were designed for a core baculovirus gene (DNA polymerase) and two core lepidopteran-specific baculovirus genes (dbp1 and me53). The gene-specific DGGE assays were capable of producing unique, sensitive and rapid genetic fingerprints of the genetic variants within a HearNPV population and were sensitive enough to detect as many as 26 genetic variants within a single portion (<500bp) of a HearNPV gene. In addition to enabling the detection of the genetic variation in key HearNPV genes, the DGGE assays allowed seven geographically distinct HearNPV populations to be differentiated on the basis of their DGGE profiles. The developed DGGE assays will be useful in studies that aim to elucidate the generation and maintenance of genetic diversity in HearNPV.