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Consensus PCR protocols for the detection of amphibian herpesviruses (Batrachovirus).

ABSTRACT: Amphibians have been disappearing at an unprecedented rate worldwide. Among the proposed contributing factors are infectious diseases. Investigations have focused mainly on ranavirus and chytrids; however, additional agents may be relevant stressors. Two novel batrachoviruses have been discovered (ranid herpesvirus 3 [RaHV-3] and bufonid herpesvirus 1 [BfHV-1]). Their clinical role is still to be clarified; however, both have been associated with obvious skin lesions in their respective hosts. Herein we present 2 consensus PCR protocols that can be used to detect all of the known and, possibly, yet to be discovered batrachoviruses. We targeted a 200 nt long, highly conserved region of the DNA terminase gene. We established a sensitive protocol, which can detect both European batrachoviruses (European batrachovirus PCR protocol; RaHV-3 and BfHV-1) and a panbatrachovirus PCR protocol detecting all known batrachoviruses, including ranid herpesvirus 1 and 2 (RaHV-1, -2). The limit of detection (LOD) for the European batrachovirus protocol was 10¹ copies of RaHV-3 and 10² copies of BfHV-1 per reaction. The panbatrachovirus protocol could detect all known batrachoviruses with LODs of 10³ (RaHV-3, BfHV-1, RaHV-1) to 10⁴ copies (RaHV-2) per reaction. These novel detection tools can be used as a first line of detection when herpesviral infection in amphibians is suspected, followed by additional PCRs with herpesvirus-specific primers in the case of known viral species, or sequencing as in the case of novel batrachoviruses.

SUBMITTER: Licheri M

PROVIDER: S-EPMC7649549 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Equid herpesviruses (EHVs) are pathogens of equid and nonequid hosts that can cause disease and fatalities in captivity and in the wild. EHVs establish latent infections but can reactivate, and most EHVs are shed via the nasal passage. Therefore, nasal swabs are generally used for EHV monitoring. However, invasive sampling of wild equids is difficult. While feces is a commonly used substrate for detecting other pathogens, to our knowledge, EHVs have never been detected in feces of naturally infected equids. We systematically tested zebra feces for EHV presence by (i) establishing nested PCR conditions for fecal DNA extracts, (ii) controlling for environmental EHV contamination, and (iii) large-scale testing on a free-ranging zebra population. A dilution minimizing inhibition while maximizing viral DNA concentrations was determined in captive Grévy's zebra (Equus grevyi) fecal samples from individuals shedding EHV nasally. Sixteen of 42 fecal samples (38%) were EHV positive. To demonstrate that the EHV positivity was not a result of environmental contamination, rectal swabs of wild zebras were screened (n?=?18 [Equus quagga and E. zebra]), and 50% were EHV positive, indicating that the source of EHV in feces is likely the intestinal mucosa and not postdefecation contamination. Out of 270 fecal samples of wild zebras, 26% were EHV positive. Quantitative PCRs showed that the amount of virus DNA in feces was not significantly smaller than that in other samples. In summary, fecal sampling facilitates large-scale screening and may be useful to noninvasively investigate phylogenetic EHV diversity in wild and domestic equids.IMPORTANCE Equid herpesviruses (EHVs) establish latent infections, and many EHVs are shed and transmitted via nasal discharge primarily through droplet and aerosol infection. Obtaining nasal swabs and other invasive samples from wildlife is often not possible without capture and physical restraint of individuals, which are resource intensive and a health risk for the captured animals. Fecal EHV shedding has never been demonstrated for naturally infected equids. We established the conditions for fecal EHV screening, and our results suggest that testing fecal samples is an effective noninvasive approach for monitoring acute EHV shedding in equids.

Project description:SARS-CoV-2 is very contagious and has rapidly spread globally. Due to various symptomatic and asymptomatic cases and the possibility of asymptomatic transmission, there is a pressing need for a fast and sensitive detection protocol to diagnose asymptomatic people. Various SARS-CoV-2 diagnostic kits are already available from many companies and national health agencies. However, publicly available information on these diagnostic kits is lacking. In response to the growing need and the lack of information, we developed and made available a low-cost, easy-access, real-time PCR-based protocol for the early detection of the virus in a previous study. During the development of the detection protocol, we found that unoptimized primer sets could inadvertently show false-positive results, raising the possibility that commercially available diagnostic kits might also contain primer sets that produce false-positive results. Here, we provide three-step guidelines for the design and optimization of specific primer sets. The three steps include (1) the selection of primer sets for target genes (RdRP, N, E, and S) in the genome of interest (SARS-CoV-2), (2) the in silico validation of primer and amplicon sequences, and (3) the optimization of PCR conditions (i.e., primer concentrations and annealing temperatures) for specific hybridization between the primers and target genes, and the elimination of spurious primer dimers. Furthermore, we have expanded the previously developed real-time PCR-based protocol to more conventional PCR-based protocols and applied a multiplex PCR-based protocol that allows the simultaneous testing of primer sets for RdRP, N, E, and S all in one reaction. Our newly optimized protocol should be helpful for the large-scale, high-fidelity screening of asymptomatic people, even without any high-specification equipment, for the further prevention of transmission, and to achieve early intervention and treatment for the rapidly propagating virus.

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Consensus PCR protocols for the detection of amphibian herpesviruses (Batrachovirus).

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