Search strategy, databases, and search terms

Our research question formulated was “Is there evidence of vector competence for ZIKV for mosquito species in addition to Ae. aegypti and Ae. albopictus?”. Our review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement [33]. Articles were identified by searching electronic databases until the 15^th of March 2022. The articles were extracted from: four databases, Cochrane Library (https://www.cochranelibrary.com/), Latin American and Caribbean of Health Sciences Information System- LILACS (https://lilacs.bvsalud.org/en/), PubMed (https://pubmed.ncbi.nlm.nih.gov/) and Web of Science (https://www.webofknowledge.com); one register, World Health Organization (WHO) library catalogue (WHOLIS, https://asksource.info/resources/wholis); and Google Scholar (https://scholar.google.com/). The search strings included three general categories: 1) “ZIKV”; 2) “vector”; 3) “competence” and their combinations using free text terms and medical subject headings (MeSH) terms when applicable (e.g., PubMed) for: Zika, Aedes, Culex, vector, or competence in the title/abstract. After piloting our initial search string and determined that some relevant articles based on previous reviews were not retrieved, we added the following search terms: transmission, isolation, and feeding behavior. Our principal search strings using MeSH terms are as shown in Box 1 and were adjusted to each search engine.

We used Zotero (https://www.zotero.org/) to identify duplicate articles, and all the extracted articles were processed using the Rayyan platform (www.rayan.com) where two members of the research team (MB, CAMQ) screened each article title/abstract to identify ZIKV VC studies that represented primary research. The search was performed in English. All articles were examined by both researchers before inclusion and a third researcher and subject expert (ACM) was asked to assist with the final decision for articles without consensus. Exclusion criteria (Fig 1) were:

• Systematic or literature reviews
• Opinion papers
• Studies focused on mathematical modelling
• Vaccine development
• Case reports
• Field-based vector incrimination studies (isolation of virus from field-collected mosquitoes)
• Papers regarding workshops
• Meeting results

Inclusion criteria (Fig 1) were:

• Laboratory evaluation of vector competence
• Included vector species other than Ae. aegypti and Ae. albopictus
• Published in a peer reviewed journal

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Fig 1. PRISMA flow diagram (adapted from Page et al. [33]) describing identification of articles examining vector competence of mosquito species other than Aedes aegypti and Aedes albopictus for Zika virus transmission.

The electronic search started on 10 November 2021 and article selection was finalized on 15 March 2022.

https://doi.org/10.1371/journal.pntd.0011591.g001

Data were extracted (MB) and entered into extraction forms, including author, title, journal, publication date, and study design. Additional sections broadly followed content analysis methods, using categories as these emerged during analysis of the results [34], such as mosquito species and/or strains studied, type of mosquito used (colony or field), virus strains and doses administered, infection method used (blood feeding device, direct feeding on mouse), assays used to detect virus or RNA, strategy used to measure mosquito infection (positive bodies or midguts), dissemination (positive heads, legs, wings, salivary glands, ovaries, etc.), and most importantly, ability to transmit virus (saliva testing or transmission to mouse), as well as the number of days post-exposure that mosquitoes were processed or analyzed, results, limitation mentioned by authors, and conclusions. The data extraction forms were reviewed and checked for accuracy by ACM and CAMQ.

Quality assessment

We developed a grading tool to assess the quality of our evaluated articles, using a checklist developed from Reporting of Observational Studies in Epidemiology (STROBE) [35] and Strengthening of the Reporting of Molecular Epidemiology for Infectious Diseases (STROME-ID) [36] criteria (S1 Table). The STROBE checklist was enriched by the authors to be in accordance with the procedures followed in the papers attempting to include score categories for all relevant STROBE and STROME-ID criteria (S1 Table). The checklist was finally composed of 33 items with a possible maximum of 38 points. The evaluation, however, was heavily weighted on 20 items evaluating study methodology and a “methodology score” was additionally computed that comprised a maximum of 24 possible points. The items included a clear description of the virus and mosquito strains used in the experiments, the type of assay used to detect Zika virus or RNA in the mosquitos, if infection, dissemination, and transmission were appropriately measured, and the number of mosquitoes used to evaluate these parameters and if replicate experiments were conducted. Some items that comprised this portion of score had higher maximum points. The remaining items, not part of the methodology score emphasized proper reporting and included the term “vector competence” in the title, clear and accurate abstract and objectives, presentation of key study results in relation to study objectives, appropriate description and use of statistical analysis, transparent discussion of study limitations, and appropriate interpretation and generalizability of the study results. Two researchers (MB and ACM) scored each of the 45 included articles independently. If the scores did not match, all 38 scores were compared and where differences were observed discussed between the two researchers. For most scores that were not concordant, the article was reviewed to confirm that each researcher had correctly extracted the information necessary to provide a score (e.g., type of laboratory assay, mosquito colony history, virus passage history, number of virus or mosquito strains used). Scores were adjusted based on careful review of the manuscript and the score justification provided by each researcher. For some scores that were more subjective (e.g., clearly written abstract, adequate discussion of study limitation) the researchers discussed their scores, compared them to other articles to standardize their approach to each question. Each score was discussed until both researchers agreed on a single score. No studies were excluded after the quality assessment, but the analysis and the report of the results consider this quality assessment.

Descriptive results

We retrieved 8,986 articles from four databases, one register, and one searchable website. We identified 6,637 as duplicates that were removed prior to screening (Fig 1). We screened the title and abstract of the remaining 2,349 articles and from those 2,246 were excluded based on the inclusion/exclusion criteria: of the 103 articles retrieved, 58 only evaluated Ae. aegypti or Ae. albopictus, finally leaving 45 papers that were included in our analysis of secondary vector species for ZIKV.

The 45 included peer-reviewed articles, summarized in Table 1, came from 20 journals, most with a focus on emerging infections (Emerging Infectious Diseases [3], Emerging Microbes and Infections [5], Eurosurveillance [3]), infectious and tropical diseases (American Journal of Tropical Medicine and Hygiene [2], BMC Infectious Diseases [1], Mem. Insti. Oswaldo Cruz [1], PLOS Neglected Tropical Diseases [9], Parasites and Vectors [4], Vector-borne and Zoonotic Diseases [1]), virology or microbiology (Frontiers in Microbiology [1], Journal of General Virology [1], Mbio [1], MDPI-pathogens [2], Virology Journal [1], Viruses [2]), entomology (Journal of the American Mosquito Association [1], Journal of Medical Entomology [3]), and three general journals (Nature [2], PeerJ [1], Proceedings B [1]).

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Table 1. Evidence table, in chronological order from 2014 to 2021.

ZIKV = Zika virus, USUV = Usutu virus, VC = vector competence, VD = Virus/RNA detection in field collected mosquitoes, IT = Intrathoracic inoculations (examining upstream barriers to transmission), dpi = days post infection (includes manuscripts reporting dpe = days post exposure), RT = Room temperature. Infection Rate (IR, [#inf./#tested]) is defined as the percentage of mosquitoes containing virus in bodies or midguts (number positive/number tested). Terminology used for dissemination and transmission are expressed as cumulative (C) or stepwise (S) rates depending on the experimental design. For dissemination, we use cumulative dissemination rate (CDR, [#dissem./#tested]) or stepwise dissemination rate (SDR, [#dissem./#inf]), defined as the percentage of mosquitoes containing virus in head, legs+wings, or salivary glands/ovaries (number positive/number of engorged mosquitoes tested for infection [CDR] or number positive /number of infected mosquitoes [SDR]). For transmission, we use cumulative transmission rate (CTR, [#transm./#tested]) or stepwise transmission rate (STR, [#transm./#dissem.]), defined as the number of mosquitoes with virus in saliva or transmitting ZIKV to a mouse (number positive/number of engorged mosquitoes tested [CTR] or number positive/number disseminated infections [STR]). For authors that emphasized salivary gland testing, CSGR = cumulative salivary gland positivity rate is used. For the quality assessment TS = Total Score, MS = Methodology Score, QAS = percentile in quantile analysis among all articles scored.

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Quality grading tool assessment

Our quality grading tool scores (QAS) showed a positively skewed distribution ranging from 20 to 35 out of a possible 38 maximum points. The mean (± SD) score was 29.0 + 4.2, with the lowest quartile representing scores from 20–25 and the top quartile ranging from 33–35 points out of a maximum of 38 points. Methodology scores (MS), a component representing 63% of the total score (TS) showed a similar distribution with a mean (± SD) score of 17.2 + 3.2, with the bottom quartile scores ranging from 11–14 and top quartile from 21–22, out of a possible 24 methodology points. It is notable that the article scoring lowest was also the only article published before 2015 which does not include an infectious virus assay or assess transmission. Four of five of the lowest scores were from papers where no infectious assay was used. Interestingly, of the seven publications in short format (letters, dispatches, short communications), five (71%) scored in the lowest 25%, in large part because important metadata was excluded.

Description of the included studies

Time and geographical clustering of studies.

The included papers could be categorized in three-time periods representing the period prior to, during, and after the 2015–2018 ZIKV epidemic in the western hemisphere. Among our selected articles, one was published before 2015, 27 were published between 2015 and 2018, and 17 published after 2018 (Fig 2). The earliest study was on Ae. hensilli, the most abundant Aedes species found on the island of Yap, in the Federated States of Micronesia, the location of the only described ZIKV outbreak reported prior to 2015, prompting interest in the vector capacity of this species [37].

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Fig 2. Geographic and temporal distribution of 45 vector competence studies of mosquito species other than Aedes aegypti and Aedes albopictus for Zika virus transmission included from an electronic search finalized on 15 March 2022 [78].

Figure created in ARCGIS using the following map which the information in the links claim is open source. https://www.arcgis.com/home/item.html?id=30e5fe3149c34df1ba922e6f5bbf808f https://services.arcgisonline.com/ArcGIS/rest/services/World_Topo_Map/MapServer/0 https://www.arcgis.com/apps/mapviewer/index.html?layers=30e5fe3149c34df1ba922e6f5bbf808f.

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Among the papers published during the 2015–2018 ZIKV epidemic, the majority (21 of 27) were laboratory studies principally from the USA and Europe that examined 1) laboratory colony mosquitoes that were rapidly available for VC studies; or 2) assessed local mosquito species in those areas that could represent risk of transmission to populations in those regions. For example, laboratory colonies of An. gambiae, An. stephensi, and Cx. quinquefasciatus were tested for competence for ZIKV [58]. Although the laboratory methodology was sound, they did not include an Ae. aegypti comparator, and this study is illustrative of the rush to publish this type of work at the time. There were also articles from Brazil [31] and China [30] that argued the role of Cx. quinquefasciatus as a possible secondary vector of Zika virus, especially in Brazil, where its role in the ongoing and devastating outbreak was of great public health concern.

Starting in 2019, the rational for published studies continued with the evaluation of local species [62,65,67,68,70,71,73,76,77] but also expanded to examining the role of sylvatic species [64], clarify the role of Cx. quinquefasciatus [13,63,66,72,74], and ask more specific questions about the mechanisms of ZIKV transmission within their respective vectors [69,71].

Of the total number of the articles included in this review, 38% (17 articles) were focused in North and Central America, and the Caribbean, (Panama, Puerto Rico, Mexico, Guadeloupe, Canada and USA), 10 papers focused on Europe (Switzerland-France, Netherlands, Spain, Germany, Italy, United Kingdom and Reunion Islands), five on Asia (China and Japan), four on Oceania (French Polynesia- New Caledonia—Samoa-Wallis and Futuna, Papua New Guinea, Australia and French Polynesia), four on South America (Brazil), two on Africa (Tunisia and Senegal), and one was not identified. Two papers tested mosquitoes from different continents: one from Oceania (New Caledonia) and South America (Brazil) and another from North America (USA) and Africa (Tunisia).

Mosquito species tested for vector competence

Table 2 summarizes the mosquito species undergoing VC assays for ZIKV. A total of 27 Aedes species other than Ae. aegypti and Ae. albopictus were studied. Seven, two, and one species from the genus Culex, Anopheles, and Coquillettidia were studied, respectively. Finally, Li et al. [75] examined the vector competence of Armigeres subalbatus as a mosquito species associated with human waste pools and potential infection through exposure to urine. Culex quinquefasciatus was the focus of almost half of VC examining species other than Ae. aegypti and Ae. albopictus (22 articles). Other species studies by multiple authors were Cx. pipiens (9 articles), Ae. japonicus (5 articles), and Ae. vexans, Ae. caspius, and Cx. tarsalis (3 articles each), and Ae. triseriatus, Ae. polynesiensis, and Ae. notoscriptus (2 articles each).

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Table 2. Summary of species studied country of origin.

https://doi.org/10.1371/journal.pntd.0011591.t002

Mosquitoes used in vector competence experiments

Mosquitoes were infected orally with an artificial feeder in 41 of the 45 studies, whereas four articles used interferon deficient mice infected with ZIKV to infect Cx. pipiens and Ae. triseriatus [39], Cx. quinquefasciatus [51,61], Cx. tarsalis [61] and Sabethes cyaneus [60]. The dose used to infect mosquitoes ranged from 4 to 8 log₁₀ PFU/ml. Vector competence studies are best done with field derived material rather than strains that have been colonized for many generations. Twenty-four studies included mosquitoes captured from field populations recently from generations F₁-F₃ and three studies from F₄-F₉. Of these, 10 studies also tested some colonized strains. Eighteen studies only used mosquitoes colonized for more than 10 generations and up to 50 years. In most cases, readers were able to infer or distinguish between if colonized or field derived mosquitoes were used, but many articles failed to clearly indicate the generations the mosquitoes had been in the laboratory.

Virus strains used

Studies using well described viral strains, including strain names, source and passage history were considered of high scientific quality. Study design elements including testing >1 strain, use of low passage strains (≤ 20 passages), and inclusion of strains from epidemiologically relevant endemic regions of the world were all items in our quality grading tool. Among the 45 articles included in our systematic review, we identified 27 ZIKV strains used in experiments (Table 3). The most used strain was PRVABC59 (GenBank accession numbers KU501215 or KX601168) isolated in 2015 used in 14 of the studies, followed by MR766 the prototype strain originally isolated from a sentinel monkey during YF surveillance studies in 1947 was included in seven studies. Virus strains used by researchers appear driven mostly by access, but details provided by investigators was not standardized. For example, not all investigators included GenBank accession numbers or detailed information on passage histories. It was clear, however, in 30 of 45 articles (67%) if the virus used had a low or high passage history, and 26 of 45 studies (58%) included a low passage virus in their experiments. Of the articles providing passage numbers, for most specific values that were generally < 10 passages for “low” passage, apart from one research group in Senegal reporting the use of 20 passages for strain MR766 [38]. This latter prototype strain was reported to have between 146–150 passages for most authors who used it in their experiments [37,43,46,53,78]. No recombinant viruses were used for experiments in the studies reviewed. Additionally, 56% (25 of 45) included clear descriptions of both virus and mosquito strains used.

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Table 3. Virus strains used in 45 vector competence studies examining secondary mosquito vectors for Zika virus.

GenBank accession numbers were not included unless specified in original article.

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Vector competence parameters

The principal objective of the 45 studies evaluated was a straightforward evaluation of the ability of one or more mosquito species to become infected, disseminate, and transmit ZIKV at different viral titers and rearing temperatures. There were only a few exceptions where the authors objectives were focused on understanding the biology behind VC patterns observed [13,55]. Although, the main VC parameters (infection, dissemination, and transmission) were universally recognized, terminology used to describe these parameters was variable and more alarming, inconsistent; the terms dissemination rate (DR) and transmission rate (TR) had distinct definitions depending on the article. Fortunately, most authors provided definitions for the terms used, but unless readers are meticulous, mistakes in interpretation are inevitable. All 45 articles estimated infection rates either by testing mosquito bodies (usually the abdomen/thorax) or midguts for Zika virus or RNA. The denominator used was the total number mosquitoes tested which represented mosquitoes that were blood fed and held for 7–21 days. Dissemination rates were estimated in 37 of the 45 studies through testing heads, wings, legs, and in one case salivary glands and another ovaries/exoskeleton. Either the number of infected mosquitoes (stepwise approach), or alternatively, all mosquitoes tested (cumulative approach) were used in the denominator. The most relevant parameter, transmission was estimated in 39 of the articles. Two papers used salivary glands as a surrogate for virus transmission [31,54] whereas 37 articles tested saliva. In two articles, infected mosquitoes were observed to transmit Zika virus to neonatal mice [30,75]. As with dissemination, the denominator used to estimate transmission was the number of mosquitoes with disseminated infections (stepwise) or all the total number of mosquitoes tested (cumulative). Some authors use the term transmission efficiency and more recently dissemination efficiency [79] to make the distinction between stepwise and cumulative rates. Articles using the term “efficiency” distinguish between stepwise rates (e.g., dissemination rate [DR], transmission rate [TR]) from cumulative rates (e.g., dissemination efficiency [DE], transmission efficiency [TE]). The major cause of confusion is that among the 45 articles in our review, 22 used DR and TR to signify cumulative rates, whereas 17 articles used the same terminology, “DR” and “TR” to describe stepwise rates while sometimes also presenting cumulative rates using the terms “DE” and “TE”. Other authors avoided these terms and used their own well-defined definitions. Other terms used are dissemination infection rate (DIR) and transmission infection rate (TR[D]) to contrast these two different VC parameters.

Laboratory assays used to detect ZIKV or ZIKV RNA

Protocols employed to test mosquitoes for ZIKV or RNA varied across studies, but there is a clear distinction between studies that only used PCR to detect RNA as opposed to the more labor intensive and technical assay that directly measured infectious virus (Table 4). About 43% of the studies only used PCR assays to measure infection and dissemination, but for transmission 29% of the studies relied solely on PCR. For the identification of the transmission status of the tested mosquitoes a variation of laboratory assays was used. About 20% of the studies used a combined strategy of screening by either PCR or an infectious assay, followed by confirmation or titration with the other type of assay.

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Table 4. Laboratory Assays used to detect Zika virus or RNA for infection, dissemination, and transmission assays.

Number of studies (%).

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Studies using mice

In six studies, mice were utilized (either neonatal or genetically modified) to infect mosquitoes in a more natural and efficient manner or to confirm transmission of virus from the mosquito to a new host. Vector competence parameters were higher for mosquitoes fed on mice than those using artificial feeding strategies or direct testing of saliva, consistent with the observation that blood from viremic animals are typically more infectious for mosquitoes than artificial meals [80,81]. Four studies used interferon deficient mice infected with ZIKV to infect Cx. pipiens and Ae. triseriatus [39], Cx. quinquefasciatus [51], Cx. tarsalis [61] and Sa. cyaneus [60] mosquitoes experimentally, compared to Ae. aegypti. All species infected using mice were unable to transmit ZIKV, except for Sa. spp., where a single mosquito (11%) that fed on a mouse with 6.8 log₁₀ PFU/ml after being held for 21 days tested positive for ZIKV by plaque assay, compared to 70% of Ae. aegypti.

Two studies evaluated transmission in parallel with saliva testing for Cx. quinquefasciatus [30] and Ar. subalbatus [75]. The former study continues to be cited as the only credible evidence for the competence of Cx. quinquefasciatus for ZIKV transmission. Ten days after being fed on by orally infected mosquitoes, the brains of eight of nine neonatal mice tested positive for ZIKV RNA. Similarly, 8-day post exposure 80% of 10 mosquitoes had ZIKV RNA positive saliva, but only 1 of 3 mosquitoes >12 d post exposure had RNA positive saliva. Biologically, we would expect that once saliva tests positive, it would remain so; low sample numbers made clear evaluation difficult, but the infection of the neonatal mice is difficult to interpret as anything other than evidence of virus transmission. Using almost identical methodology for Ar. subalbatus, saliva tested positive for ZIKV RNA in 7–12% of the mosquitoes exposed and nine of ten of the neonatal mouse brains tested positive for RNA. Although both studies presented virus titers observed in the saliva samples, these were generated from standard curves developed in the laboratory, not based on the mosquito samples themselves. In both studies, the failure to use and infectious virus assay to test for ZIKV prevented unequivocal conclusions about each species as a vector.

Review of evidence for individual vector species other than Ae. aegypti and Ae. albopictus

Existing evidence for VC of vector species other than Ae. aegypti and Ae. albopictus for ZIKV can be divided into the following relevant epidemiological contexts: 1) African [38] and South American [60,64] forests cycles; 2) local vector species from areas directly affected by ZIKV transmission generally associated with Micronesia/Polynesia in island settings [37,45,57]; 3) local vector species in normally temperate areas where risk of ZIKV transmission was evaluated for outbreak readiness which included species in Australia [43,48], Mediterranean region [62,65,67], Europe [40,41,52,68,69], Canada [47], USA [39,50,51,56,61,71,77] and the examination of one cosmopolitan species Ae. japonicus [59,76]; and 4) in settings similar to endemic DENV/CHIKV transmission where Cx. species are potentially involved. The latter category resulted a considerable focus on Cx. species in areas with and without ZIKV transmission. Table 5 summarizes the existing evidence, by species and the four categories above, of VC by mosquito species.

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Table 5. Vector competence (VC) parameters of species investigated as possible vectors of Zika virus.

Infection Rate (IR, [#inf./#tested]) is defined as the percentage of mosquitoes contain virus in bodies or midguts (number positive/number tested). Terminology used for dissemination and transmission are expressed as cumulative (C) or stepwise (S) rates depending on the experimental design. For dissemination, we use cumulative dissemination rate (CDR, [#dissem./#tested]) or stepwise dissemination rate (SDR, [#dissem./#inf]), defined as the percentage of mosquitoes containing virus in head, legs+wings, or salivary glands/ovaries (number positive/number of engorged mosquitos tested for infection [CDR] or number positive /number of infected mosquitoes [SDR]). For transmission, we use cumulative transmission rate (CTR, [#transm./#tested]) or stepwise transmission rate (STR, [#transm./#dissem.]), defined as the number of mosquitos with virus in saliva or transmitting ZIKV to a mouse (number positive/number of engorged mosquitoes tested [CTR] or number positive/number disseminated infections [STR]). NT = not tested. Raw data presented in S1 Data.

https://doi.org/10.1371/journal.pntd.0011591.t005

Forest cycles

Laboratory incrimination studies for African forest vectors was limited to a single study conducted in Senegal [38]. The only forest species with laboratory evidence incriminating them as a ZIKV vectors were Ae. vittatus and Ae. luteocephalus, both able transmit virus with CDR of 1 and 17%, respectively. The same study did not find Ae. unilineatus to be competent, and the author’s pointed out the need for further studies on other important forest species that could be involved in transmission. For South American forest species, Ae. terrens, Ae. scapularis, Sa. identicus were completely refractory to infection, Sa. albiprivus had 0.5% infection rate, but Haemagogus leucocelaenus had infection rates of 14.8 to 40%, dissemination rates of 2.2 to 5% depending on day post infection but showed no evidence of transmission [64]. A laboratory study from a long-term colony of Sa. cyaneus had one mosquito with evidence of infection, dissemination, and transmission out of 69 tested.

Vectors associated with Micronesia/Polynesia in Island settings

Evidence for Ae. hensilli and Ae. polynesiensis as vectors for ZIKV was evaluated because of their high abundance at the time of ZIKV outbreaks. Infection of Ae. hensilli was demonstrated but no evaluation of transmission (saliva or salivary gland testing) was conducted [37]. Aedes polynesiensis showed disseminated infections with ZIKV under laboratory conditions but did not transmit the virus [45]. Dissemination was far less efficient than observed for Ae. aegypti. In contrast, a later study showed two populations of Ae. polynesiensis from French Polynesia and Wallis and Futuna had high infection rates (71%) that were less variable than Ae. aegypti populations from the same region and showed high dissemination rates (SDR = 45%, CDR = 39% and low transmission rates (STR = 3%, CDR = 1%) [57].

Local vectors not found to be competent to transmit Zika Virus

In Australia, there are many local Aedes species examined for their ability to transmit ZIKV [43]. Ae. vigilax, Ae. procax, and Ae. notoscriptus showed infection and dissemination rates consistent with Ae. aegypti, but all failed to transmit the virus. In contrast, a later study [48] found some transmission (<1%), but lower infection and dissemination rates than the previous study for both Ae. notoscriptus and Ae. camptorhynchus.

In Canada, of the three Culex species examined, Cx. sitiens and Cx. annulirostris were completely refractory to infection and only 2 out of 30 Cx. quinquefasciatus were infected but none developed disseminated infections. Similarly, in Europe, no evidence was found for transmission in Cx. pipiens pipiens or Cx. pipiens molestus [52,69], although both species did accumulate virus in saliva after intrathoracic injection. North American species Ae. triseriatus [39,71] and Ae. taeniorhynchus [51] were both found to be refractory to ZIKV infection. Ae. mediovittatus from Puerto Rico was studied [77] and found to be half less susceptible to oral infection than Ae. aegypti, indicating a more effective midgut infection barrier in Ae. mediovittatus. Dissemination rates were < 5% compared to 40–95% in Ae. aegypti. Insufficient saliva samples were obtained to evaluate transmission for this species. A single study [58] found laboratory colonies of An. gambiae and An. stephensi to be refractory to ZIKV infection.

One study from China investigated Ar. subalbatus, a species of interest because it developed in human waste lagoons where ZIKV could be shed in urine. Average infection rate for this species was 43% (measured in midguts) and 90% of the infant mice that were bitten by infectious mosquitoes had viral RNA in their brain although was not detected in mosquito saliva. Furthermore, Ar. subalbatus larvae reared in water containing ZIKV and human urine did not result in any adult mosquitoes with detectable ZIKV RNA in saliva, providing no evidence of transmission via this route. Ae. galloisi and Ae. punctor were evaluated in Japan, showing very low rates of susceptibility to ZIKV infection but dissemination and transmission were not evaluated. These species provide a good example of the methodological difficulties associated with evaluation when blood feeding rates are low, and the numbers of infected mosquitoes are so low a true evaluation of dissemination and transmission is difficult.

Species with evidence of very low transmission potential

Species with evidence of transmission, albeit at very low levels, were Ae. vexans, Ae. detritus, and Ae. japonicus (Table 5). Three studies indicated that, Ae. vexans is a competent vector for ZIKV [47,50,56] and while infection rates were high in some of these studies, all of them reported low dissemination and transmission rates. In Canada, of the 4 (13%) Ae. vexans positive for ZIKV RNA, 2 (50%) had ZIKV RNA detected in their legs for an overall dissemination rate of 6%. Twenty-three percent of Ae. vexans infected by intrathoracic injection had ZIKV RNA detected in their saliva. Evidence for Ae. vexans transmission was also found in Colorado, USA, with relatively high infection rates ranging from 66–91%, dissemination rates from 3–25%, but low transmission rates from 2–7%. Consistent with these finding was another study [56] from the great plains region that had high transmission rates in Ae. vexans, after intrathoracic infection, suggesting that the primary barrier in this species is midgut escape barrier. In the United Kingdom Ae. detritus (reported as Ochlerotatus detritus in original article) became infected and showed infectious virus in saliva at rates lower than Ae. albopictus, but saliva positivity increased at higher temperatures [70]. In the Mediterranean Region neither Ae. detritus nor Ae. caspius showed the ability to disseminate or transmit ZIKV [62,65,67]. Additionally, Ae. caspius had low infection rates. Multiple studies have shown that Ae. japonicus is a competent vector for ZIKV. However, the infection, dissemination, and transmission rates of the virus were found to be temperature-dependent, especially at temperatures above 27°C [59,68,71,73].

Possible role of Culex species

A total of 24 of the 45 studies identified in our systematic review tested Culex species; of these 22 included Cx. quinquefasciatus, and 14 used mosquito strains recently brought from the field to laboratory. Thus, the overwhelming weight of evidence is that Culex species are unable to transmit ZIKV. Culex quinquefasciatus species were refractory in 10 laboratory studies using mosquitoes from colonies (n = 5) and field (n = 5). Of the remaining 11 studies infection rates were very low, dissemination rare, and one report on infectious saliva in one mosquito infected at very high virus titer. Three studies make claims that Cx. quinquefasciatus can transmit ZIKV. Guedes et al. [31] showed viral particles in salivary glands by electron microscopy, suggesting this was evidence of transmission, but was not able to demonstrate infectious virus in saliva. A study from China [30] showed transmission of ZIKV from laboratory infected mosquitoes to mice, however saliva from the same mosquitoes, tested by PCR only, showed presence of RNA at 8 dpi (80%) and 12 dpi (10%) but not 16 dpi, a result consistent with the observed detection of decreasing levels of noninfectious pieces of RNA rather than infectious virus. Finally, infectious virus was detected on FTA cards exposed to laboratory-infected field mosquitoes, evidence of transmission [32]. In summary, the studies evaluating Culex. laboratory competence indicated that Culex species has poor VC for ZIKV overall, but the possibility of geographically isolated strains that are competent must be considered. Macleod and Dimopoulos [13] provide a comprehensive review and interpretation of the data available for Cx. quinquefasciatus VC to date.

Role of temperature

Four of the reviewed articles conducted VC experiments that asked specific questions about the impact of rearing temperatures on European Culex species [52], Ae. detritus [70] and Ae. japonicus [59,73]. Transmission increased with rearing temperature for both Aedes species tested and when Ae. japonicus was reared at more realistic fluctuating temperature scheme (daily variation between 14°C and 27°C compared 27°C) there was no impact of fluctuating temperatures on VC (Table 5).

Research interest and study design often driven by outbreaks and financial considerations

Interest in mosquito vectors of ZIKV, in particular secondary vectors, has been driven by outbreak response with limited publications until 2016, about one year into the ZIKV outbreak in the Western Hemisphere, especially its association with fetal abnormalities.

In the following paragraphs we first describe the characteristics of ZIKV VC studies conducted on 1) forest species, 2) in response to the Yap Island outbreak in 2014, 3) rapid publications during 2015–2017 ZIKV outbreak, and 4) 2018 to present where study quality has improved, and research has been more systematic with the aim to better understand and predict ZIKV disease transmission [7].

Prior to 2007, sylvatic YF surveillance studies isolated ZIKV from wild-caught African forest species, but laboratory-based VC studies to properly assess the competence of these species was not a research priority and limited to a single study included in our systematic review [38]. These studies identified several mosquito species of interest for further study. Unfortunately, technical and logistical challenges for forest species that cannot survive or feed on blood in laboratory conditions in sufficient numbers required to conduct VC studies represented a significant barrier to VC evaluation. These limitations, extend to two studies conducted on forest species from the western hemisphere [60,64].

A second torrent of research on mosquito vectors occurred between 2007–2014 in response to ZIKV outbreaks in the South Pacific, but studies were quite limited overall, one conducted as part of the outbreak response did not include evaluation of transmission [37]. Only two studies on the potential secondary vector Ae. polynesiensis showed mixed transmission results [45,57]. This observation highlights the potential for large genetic and VC differences among vector populations of the same species. Calvez et al. [57] did evaluate three Ae. aegypti and two Ae. polynesiensis populations with a single virus strain from New Caledonia. Ideally, more strains from more locations with distinct virus strains could be evaluated, but these experiments are labor intensive and costly.

At the initiation of the South American ZIKV outbreak, VC studies appeared to be designed based on what mosquito colonies and virus strains could be obtained rapidly. Many of these initial publications, were short communications, and in many ways appeared rushed. There was considerable concern in Europe and the US, that ZIKV could potentially be transmitted by local species, leading to a higher proportion of studies from these countries. Although assessing the risk of potential vectors in these locations made sense, early studies used mosquito strains that had been in colony often for decades, transitioning into more studies that collected mosquitoes directly from the field that could be brought back to the laboratory. Also, reading between the lines were studies that appeared to retrospectively verify they were detecting infectious virus rather than relying exclusively on PCR to detect ZIKV RNA. Thus, these initial publications often lacked detailed metadata that are important for future meta-analyses or full assessments of a particular species’ vector potential. These studies did provide needed quick assessments for making Public Health decisions. ZIKV represents a prototype of what occurs during the initial response to emerging Public Health Emergency, in which an unprecedented amount of research was published very quickly [83].

Research priorities should include a more systematic and comprehensive approach to understanding forest species. ZIKV has been isolated from 15 Ae. species as well as a few species from Culex, Eretmapodites, Mansonia, and Anopheles genera [4,5,23] where no laboratory studies have been conducted. Virus isolation from field-collected mosquitoes does not constitute evidence for their role as a vector, only that the mosquito fed on an infected host, or contamination occurring within a trap. Laboratory VC studies are required for vector implication to justify further studies on the role of these species in potential spill-over events as well as species whose ecology has changed and could have important implications for ZIKV transmission. For example, Ae. africanus and Ae. furcifur became important vectors of YF in villages where these species moved freely between the forest canopy and villages nearby where Ae. aegypti was absent. Theoretically, the same is possible for ZIKV.

There was significant proliferation of studies on Cx. quinquefasciatus based on a premature press release [85] and data from a single study [30], despite significant evidence otherwise. It was not until 2020, that a clear effort was made to replicate the results of the original study from China and evaluate the existing evidence for competence of this species [13]. At the time we had completed our search in March 2022, many groups had evaluated this species as well as many other species within the Culex genera. Many of these studies were a part of coordinated research effort by the Zika Alliance. Obadia et al. [79], a study published a few months after concluding our search, provides an illustration of a coordinated research effort using a unified protocol across a wide geographic distribution using strains of Ae. albopictus and Ae. japonicus directly from the field involving multiple research groups. These kinds of efforts allow experimental studies that would be otherwise logistically impossible for a single research group. A collaborative approach where multiple research groups evaluate one or two local mosquito strains and if possible, an epidemiologically relevant virus strain (e.g., in sites with endemic transmission a strain from the same location), provides more credible and robust conclusions. We now have interesting candidates for future study: Ae. vexans, Ae. japonicus, and Ae. detritus for more comprehensive studies examining the role of ambient temperature.

Variability in laboratory methodologies

VC studies were generally of high quality methodologically, but have highly variable study designs, using different mosquito and ZIKV virus strains (appropriate), different blood feeding techniques (different artificial feeders, animal blood sources, murine models), virus preparations (dose, frozen versus fresh virus), methods for evaluating infection (testing mosquito bodies versus midguts), dissemination (testing mosquito heads, legs, wings, or salivary glands), and transmission (direct saliva testing, saliva amplified in cell culture, saliva amplified in mosquitoes by intrathoracic inoculations, with mice to confirm transmission). This variability makes comparison across studies difficult, although not impossible. Unfortunately, details on experimental designs were inconsistent across the articles reviewed.

Recently, a minimum data standard for VC experiments was proposed [7] which suggested metadata reporting for mosquito and virus strains used and experimental details and outcomes. Most articles provided most of the necessary experimental metadata, but experimental outcomes were less consistent overall. Mosquito and virus strain metadata varied across the publications, and for some articles the reader was left to infer the age of mosquito colonies and virus passage histories. Some authors provided this information clearly in tables with clear virus passage histories and the number of generations a mosquito strain had been in the laboratory, whereas other articles implied that a strain was local and “recent” but did not provide this information. The best example of this was the HAI strain of Cx. quinquefasciatus from China originally found to transmit ZIKV to neonatal mice [30] and later evaluated by MacLeod and Dimopoulous [13]. Neither publication provides precise information on when that colony was established other than the year 2014.

During the review, our team attempted to extract all the metadata mentioned and would recommend that authors include it in a tabular format in the main body of the publication, rather than placing these details in supplementary information or referring readers to a previous publication. Even if “none” or “not known” is included for mosquito colony generations or virus passage history, excluding this information leaves the perception of a lack of transparency. In the following sections, we highlight the more prominent issues associated with interpreting the reviewed articles, but that apply to the VC literature in general.

Definitions of Infection, Dissemination, Transmission, and other terms

When assessing VC, the following barriers have been described: midgut infection and escape barriers, and the salivary gland infection and escape barriers. Infection implies that virus has passed the midgut infection barrier, whereas dissemination -where virus is found throughout the body of the mosquito- suggests the virus has made it at a minimum through the midgut escape barrier [6,7,82]. Virus in saliva means virus has escaped from the salivary glands. Understanding where the barriers to VC exist for individual species is important to delineate biological mechanisms underlying the ability of virus to make it from the midgut to the saliva at titers sufficient to infect another host. These processes are essential for predicting the potential for emergence events where critical mutations in either the virus or vector would result in a virus-vector pair switch from incompetent to competent. Thus, presentation of stepwise rates has value, but in the context of a Public Health Emergency, like observed with ZIKV, the critical question is “can this species serve as a vector?”. To answer this later question, cumulative rates are the most informative since they are using the total number of mosquitoes exposed and tested in the denominator. There is no technical reason, that both stepwise and cumulative rates cannot be presented side by side, and many of the manuscripts did this, but the terms used to present these rates were not consistent. A major concern remains that the terms dissemination rate (DR) and transmission rate (TR) have different definitions depending on the article. One alternative has been the introduction of the terms dissemination efficiency (DE) and transmission efficiency (TE), terminology used in 11 of the reviewed articles but clearly distinguishes between stepwise and cumulative rates [79]. Moving forward, our recommendation is the universal adoption of the terminology for cumulative and stepwise rates as we have presented in Table 5.

Both definitions are presented clearly in Wu et al. [7], who make the additional recommendation that authors include raw data from their experiments in a supplementary appendix so that those calculations can be made by anyone reading the manuscript. They point out the “original raw data may never be reported and is often impossible to reconstruct from provided bar or line charts” and “derived quantities often follow different calculations, with (usually intentional but) very different biological meaning (e.g., the difference between ‘dissemination rate and ‘disseminated infection rate’ are often used interchangeably)” [7]. Although, most articles clearly defined their parameters, the choice on how to present data was often driven by the desire to tell a story. Interpretation of these parameters are also hindered by the inevitable problem of low sample sizes for dissemination and transmission assessments. When infection and dissemination rates are low, transmission rates presented as a percentage are often based on 1–2 mosquitoes.

Finally, in the reviewed articles the number of days after mosquitoes were fed blood infected with ZIKV was reported in days post-infection (dpi) for most articles and as days post-exposure (dpe) in a few. Although less problematic than the terms dissemination and transmission rate having distinct definitions, the term dpe is more appropriate and should be adopted to standardize terminology in the VC literature.

Methods that dissect out body parts versus testing bodies, legs, wings, and heads compared to murine models

Although, most published articles use the methodology of testing mosquito bodies (abdomens) to indicate infection, either wing, legs, heads, thoraces, and sometimes other organs to indicate dissemination, there is at the very least qualitative differences when comparing to papers testing dissected midguts and salivary glands. Experiments directly comparing these methodologies would be helpful to the field. The use of genetically modified murine models showed higher infection, dissemination, and transmission rates than experiments using artificial feeding methods. The observation that using a viremic animal to infect mosquitos represents a more realistic model than artificial blood feeding methods is not a new concept [80,81,86]. The ability to implement more realistic and appropriate study designs for VC studies in low- and middle-income countries where ZIKV and other arboviruses are endemic may be limited due to financial and logistical restraints. Thus, more detailed head-to-head comparisons of results from studies using artificial blood feeding with those using animal models or direct human feeds need to be conducted. The same is true for testing transmission from mosquitoes to a new host, that is—head-to-head comparisons of virus detection in saliva to infection of neonatal mice, especially under circumstances when mice become infected in the absence of a clear presence of infectious virus in saliva.

Minimum quality standards for vector competence studies and how to evaluate them

There are many challenges to the implementation of minimal quality standards for VC studies and there is significant recognition that their absence is a significant problem, but at the same time reluctance to impose undue burden on investigators, especially in locations with limited resources and facilities [7,82]. Our strongest recommendation is the transparent and clear reporting of metadata associated with VC experiments, as suggested by Wu et al. [7], and reflected by our quality grading scale. We strongly support the recommendation to provide access to raw data sets. There is strong consensus that presentation of experimental results needs to clearly report total counts of tested and positive mosquitoes at each stage evaluated (infection, dissemination, and transmission). Also critical is that transmission competence should only indicate the presence of virus in saliva, and not just positive salivary glands [83]. Reliance on PCR assays only, with no component to verify that there is infectious virus present is becoming unacceptable, with the minority of studies in the review not including some recognition of this.

At present, there have been efforts or suggestions for more standardized reporting of experimental data, but not for experimental design or preferred study laboratory assays. Vector competence studies require a balance between well controlled experimental conditions with approximation of natural conditions which are often at odds with each other [7,82]. We found the application of our quality grading tool difficult because much of the metadata especially on virus and mosquito strains, denominators used for competence rate calculations are incomplete or difficult to extract. Also apparent was the lack of experimental replication, which can only be accomplished if sufficient insectary space, mosquitoes, and labor are available to run replicates at the same time, all of which presents numerous technical challenges of rearing enough mosquitoes of the same generation to conduct these experiments. Laboratory conditions cannot be 100% duplicated, running multiple replicates for distinct species/virus or mosquito strain may be less of a priority that other components on the quality grading list. Related to this issue, however, is the observation that few manuscripts provided measures of variability or conducted statistical analyses, a more serious issue. Because our review focused on secondary vectors, the use of multiple virus and mosquito strains was limited, but either additional geographical isolates or studies that include a wide range of geographic mosquito strains together with multiple virus strains will provide convincing evidence of a species VC [79].

We agree with Azar and Weaver [83] that adherence to reporting guidelines such as those suggested by Wu et al. [7], would improve our ability to conduct meta-analyses and draw conclusions from reviews including systematic reviews, but also point out the need for establishing some experimental standards as well. Their recommendations including the use of viral doses that are consistent with viremias observed in human patients that may be easier to achieve using animal models which are not available for ZIKV except for knockout mice with their innate immunity altered for which many research teams may not have access. Insect specific viruses (ISVs) and vector microbiome also play a role in VC, but neither of these parameters were mentioned in the 45 articles we included in our review with one exception.

MacLeod and Dimopolous [13] hypothesized that the virus particles observed in Cx. quinquefasciatus mosquitoes challenged with ZIKV [31] represent ISVs rather than ZIKV particles which would be consistent with the absence of viral RNA found in mosquito saliva. We also argue that future systematic reviews potentially exclude articles where no infectious assays are included. We recognize there may be limited circumstances where publication of studies that rely on PCR assays to detect viral RNA are justified, the reliability of this evidence is greatly reduced. Another area that requires input from subject matter experts is minimum number of mosquitoes per stage the require evaluation to have confidence in rate estimates.

Our quality grading tool scored the title of each article, providing a point if the “vector competence” appeared in the title. We do not recommend this moving forward but the appropriate use of key parameters such as susceptible, refractory, transmit deserve discussion. Another item in our grading tool was the issue of sample size. We set 30 mosquitos per virus-mosquito pair evaluated in our quality grading tool as a minimum standard, but this is another experimental component where guidelines would be appropriate. This would be particularly important for reporting results for species deemed negative. We included publications reporting results for refractory species, but this may be an underestimate since there is often a bias against publishing negative results. We recognize, along with other authors [7,82] that complete standardization of VC studies is not possible, but the next logical extension toward this goal is developing quality guidelines, like our quality grading tool to help evaluate the relative quality of manuscripts for inclusion in future metanalyses which are evidently needed to clearly define vector competent species. We view our tool as a good start that needs additional revision by experts before widespread application.